Iron Chelators for the Treatment of Cancer

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thiosemicarbazone iron chelator in terms of its assessment in humans is 3-aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP). Observations from these ...
Current Medicinal Chemistry, 2012, 19, 2689-2702

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Iron Chelators for the Treatment of Cancer Y. Yu, E. Gutierrez, Z. Kovacevic, F. Saletta, P. Obeidy, Y. Suryo Rahmanto and D.R. Richardson* Department of Pathology, Blackburn Building (D06), University of Sydney, Sydney, New South Wales, 2006, Australia Abstract: The study of iron chelators as anti-tumor agents is still in its infancy. Iron is important for cellular proliferation and this is demonstrated by observations that iron-depletion results in cell cycle arrest and also apoptosis. In addition, many iron chelators are known to inhibit ribonucleotide reductase, the iron-containing enzyme that is the rate-limiting step for DNA synthesis. Desferrioxamine is a well known chelator used for the treatment of iron-overload disease, but it has also been shown to possess anti-cancer activity. Another class of chelators, namely the thiosemicarbazones, have been shown to possess anti-cancer activity since the 1950’s, although their mechanism(s) of action have only recently been more comprehensively elucidated. In fact, the redox activity of thiosemicarbazone iron complexes is thought to be important in mediating their potent cytotoxicity. Moreover, unlike typical iron chelators which simply act to deplete tumors of iron, several thiosemicarbazones (i.e., Bp44mT and Dp44mT) do not induce this effect, their anti-cancer efficacy being due to other mechanisms e.g., redox activity. Other reports have also shown that some thiosemicarbazones inhibit topoisomerase IIα, demonstrating that this class of agents have multiple molecular targets and act by various mechanisms. The most well characterized thiosemicarbazone iron chelator in terms of its assessment in humans is 3-aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP). Observations from these clinical trials highlight the less than optimal activity of this ligand and several side effects related to its use, including myelo-suppression, hypoxia and methemoglobinemia. The mechanisms responsible for these latter effects must be elucidated and the design of the ligand altered to minimize these problems and increase efficacy. This review discusses the development of chelators as unique agents for cancer treatment.

Keywords: Iron chelation, DFO, 3-AP, Dp44mT, tachpyr, PIH, ribonucleotide reductase. 1. INTRODUCTION Iron is a critical component for important enzymes that play crucial biological roles [1]. For instance, iron is found in the prosthetic groups of proteins, including heme and iron-sulfur clusters [2]. Indeed, the iron within the heme moieties of cytochromes b, c and cytochrome P450 is required for oxidative phosphorylation and the detoxification of chemicals, respectively [3]. Iron-sulfur clusters are found in a number of enzymes, including complex I and II, which are important for redox reactions involved in respiration and ATP generation [2]. Iron also plays a role in other redox reactions within cells [4] and can participate in the Haber-Weiss reaction that leads to the generation of reactive oxygen species (ROS) [5]. The ability of iron to cycle between its di- or trivalent states permits it to gain and lose electrons which are crucial for electron transport. However, this same property can also cause cellular toxicity via the generation of cytotoxic ROS by donating electrons to oxygen. Therefore, intracellular iron levels must be tightly regulated, there being a balance between iron uptake, usage and storage [6]. Iron is transported in the circulation by binding to transferrin (Tf) [6, 7]. Each Tf molecule binds two atoms of iron(III) with high affinity [6] and it is internalized into cells by binding to the transferrin receptor 1 (TfR1) on the cell surface [8]. For a more detailed description of the mechanisms of cellular iron uptake, transport and its regulation, the reader is referred to comprehensive reviews on the subject [6, 9]. Iron chelators are commonly used for the treatment of iron overload diseases such as β -thalassemia where they are implemented to reduce tissue iron levels [10]. Some examples of these include the commercially available ligands: deferiprone (Ferriprox®), deferasirox (Exjade®) and desferrioxamine (Desferal®; Fig. 1) [11]. These agents are bidentate, tridentate and hexadentate ligands in which two, three, or six donor atoms, respectively, coordinate with iron [12, 13]. The electron donor atoms within chelators are commonly, oxygen, nitrogen or sulfur and in terms of chelator design, the choice of these can markedly influence the biological activity of the ligand [13].

*Address correspondence to this author at the Department of Pathology, University of Sydney, Sydney, New South Wales, 2006, Australia; Tel: +61-2-9036-6548; Fax: +612-9351-3429; E-mail: [email protected] -;/12 $58.00+.00

In addition to their application in the treatment of iron overload diseases, a variety of studies have demonstrated that iron chelators have the ability to inhibit the growth of aggressive tumors in vitro and in vivo [14-16]. Hence, iron chelation therapy is an exploitable therapeutic strategy that has potential for the treatment of tumors. In this review, we discuss: (i) the importance of iron in cellular proliferation; (ii) the rationale and potential use of chelators as chemotherapeutics for the treatment of cancer; (iii) the types of ligands currently being considered for anti-tumor therapy; and (iv) the potential side effects of chelation therapy in cancer patients. 2. RATIONALE FOR THE USE OF IRON CHELATORS AS ANTI-CANCER THERAPEUTICS 2.1. Iron in Neoplastic Cells The importance of iron in cellular proliferation is demonstrated by early observations that iron-deficiency reduces DNA synthesis [17] and that the growth of leukemic cells can be induced continuously in serum-free medium supplemented with either Tf or iron salts [18]. In fact, low molecular weight iron can substitute for Tf during cell growth in vitro [19, 20]. Neoplastic cells have a higher requirement for iron than normal cells due, in part, to their rapid rate of DNA synthesis and this is illustrated by the increased expression of a number of iron-binding and transport proteins [21], as discussed below. 2.2. Ribonucleotide Reductase Ribonucleotide reductase (RR) is a tetrameric enzyme involved in the rate-limiting step of DNA synthesis, providing the precursors for this pathway by converting ribonucleotides to their 2’deoxyribonucleotide counterparts [22]. It contains a di-iron center in its active site (R2), which stabilizes the tyrosyl radical needed for the reduction of ribonucleotides [22]. The ability of iron to cycle between its two stable states (ferrous and ferric) in the active site of RR enables it to act as an electron donor and acceptor [22]. Therefore, the activity of RR is directly dependent on intracellular iron levels. Importantly, RR plays a critical role in cellular proliferation due to its function. The rapid proliferation of neoplastic cells dictates that a large amount of deoxyribonucleotides are required during the S-phase of the cell cycle [23]. This is supported by the observation that the enzymatic activity of RR correlates to an increase in the © 2012 Bentham Science Publishers

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O

A O

NH3

H N

B (CH2)5 N

(CH2)5 N

(CH2)5 N

HO O

HO

O

OH O

N

O

HO

C

H N

DFO

Deferiprone

OH O N N N OH HO

Deferasirox Fig. (1). Chemical structures of: (A) desferrioxamine (DFO), (B) deferiprone (Ferriprox®) and (C) deferasirox (Exjade®).

potential for cell invasion [24, 25]. Hence, there is increased RR activity and subsequently, an increased requirement for iron in neoplastic cells [26, 27]. Currently, the RR inhibitor, hydroxyurea, is being used for the treatment of several cancers and its activity is mediated by scavenging the tyrosyl radical of RR [28]. However, hydroxyurea suffers from a short half-life, low affinity for RR and resistance against this drug is a problem [29]. Importantly, studies have shown that iron chelators can inhibit RR and overcome hydroxyurearesistance, due to their different mechanism of activity which involves the binding of iron rather than their ability to scavenge the tyrosyl radical [26]. 2.3. Transferrin Receptor 1 Expression When compared to normal cells, neoplastic cells demonstrate increased expression of TfR1, leading to an increased rate of iron uptake [30]. In studies that inhibited iron uptake using monoclonal antibodies to prevent Tf binding to the TfR1, there was observable inhibition of tumor growth in vitro [31]. Moreover, the administration of phosphorothiolated anti-sense TfR1 targeted to the sequences of TfR1 mRNA also showed selective anti-cancer activity. In fact, the IC50 of this agent was 30 times lower in tumor cells relative to normal cells [32]. These results demonstrate the importance of TfR1-mediated iron uptake in cancer cell growth. During incubation with iron chelators, cells respond to irondepletion by up-regulating TfR1 in order to increase iron uptake. However, at the same time, chelators prevent the uptake of iron by binding it [33, 34] and also target other molecular effectors which lead to inhibition of cell cycle progression and apoptosis [35]. 2.4. Cell Cycle Progression Cellular iron-depletion results in G1/S arrest through affecting the expression of several molecules critical for cell cycle progression and also causes apoptosis (for a more detailed discussion see [26]). A graphical summary of molecules affected by iron-depletion is shown in Fig. 2. For example, some of the cyclins which facilitate cell cycle progression, are expressed at lower levels

following iron-depletion, particularly cyclin D1, and to a lesser extent cyclins A and B [36]. Cyclin-dependent kinase 2 (cdk2), which forms a complex with cyclins A and D to perpetuate G1/S progression, was also decreased upon iron-depletion [36, 37]. Cyclin D binds to cdk4 and cdk2 to phosphorylate retinoblastoma protein (pRb) during the G1 phase, resulting in the release of transcription factors (e.g., E2F1) that promote gene expression and progression into the S phase [38, 39]. Thus, the decrease in cdk2 and cyclin D1 expression after iron chelation causes hypophosphorylation of pRb, leading to G1/S phase arrest [36]. Other molecules that can suppress growth and metastasis, such as p53 and N-myc downstream regulated gene-1 (Ndrg1), have also been shown to be up-regulated by cellular iron-depletion [26, 39, 40]. In fact, the phosphorylation of p53 at Ser-9, Ser-15, Ser-20 and Ser-37 was also found to be up-regulated, indicating transcriptional activation of this molecule after iron-depletion [41, 42]. The p53 tumor suppressor protein can initiate expression of various downstream genes that lead to commitment to cell cycle arrest and/or apoptosis [26]. These include the cyclin-dependent kinase inhibitors, p21 WAF1/CIP1 and p27Kip1, as well as growth arrest and DNA damage (GADD) family of genes, all of which are induced by cellular iron-depletion [33, 43, 44]. Recent studies suggest that the metastasis suppressor Ndrg1 increases the efficiency of the p53-dependent apoptosis pathway in lung and colon cancer [45]. Ndrg1 has been found to play an important role in the progression of many cancers, being correlated with better prognosis in prostate, breast, colon and pancreatic cancer patients [46-49]. Interestingly, Ndrg1 was also recently found to up-regulate the p53-inducible cyclin-dependent kinase inhibitor, p21 WAF1/CIP1 in a p53-independent manner in prostate and lung cancer cells, which may contribute to its anti-tumor activity [50]. Considering that iron chelators are also able to increase p21WAF1/CIP1 mRNA expression [43], its effects on the expression of this latter molecule may be mediated, at least in part, through Ndrg1. Cellular iron-depletion has also been shown to stimulate mitogen activated protein kinases (MAPK), particularly the stressactivated protein kinases, c-Jun amino-terminal kinase (JNK) and the p38 signaling transduction pathways [42]. The increased

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IRON DEPLETION

cyclin D1 cyclin A cyclin B cdk2

pRb

p21WAF1/CIP1 p27Kip1

Phosphop53

NDRG1

MAPK PhosphoJNK Phosphop38

GADD45

E2F1

CELL CYCLE ARREST Fig. (2). Summary of molecules affected by iron-depletion. These includes cell cycle-related molecules, particularly cyclin D1, cyclin A, cyclin B, cyclindependent kinase 2 (cdk2) and the cyclin-dependent kinase inhibitors, p21WAF1/CIP1 and p27Kip1p53. Other molecules affected include: p53, N-myc downstream regulated gene-1 (NDRG1), growth arrest and DNA damage (GADD45), the c-Jun amino-terminal kinase (JNK) and p38 mitogen activated protein kinases (MAPK).

phosphorylation of these kinases may be mediated by the upstream apoptosis signaling kinase 1 (ASK1), as this latter kinase was activated during iron-depletion [42]. The JNK and p38 pathways are largely tumor suppressive due to their positive roles in apoptosis and cell cycle arrest [51, 52]. In fact, p38 has been shown in some cell types to induce G1/S arrest through a variety of mechanisms, such as direct activation of p53 phosphorylation, stabilization of p21 WAF1/CIP1 and decreased cyclin D1 protein stability [52]. JNK is also known to lead to p53 phosphorylation [52]. Thus, activation of p38 and JNK may also play a role in iron-depletion-induced cell cycle arrest. Overall, these findings demonstrate that appropriate levels of iron are necessary for cell cycle progression and that iron chelators are able to target crucial iron-regulated molecules which contributes to their anti-proliferative activity. 3. IRON CHELATORS: THEIR POTENTIAL AS ANTI-CANCER AGENTS FROM CLINICAL TRIAL OBSERVATIONS 3.1. Desferrioxamine (Desferal®; DFO) Desferrioxamine (DFO; Fig. 1A) is a naturally occurring hexadentate siderophore from the bacterium, Streptomyces pilosus [53]. This iron chelator is deemed as the ‘gold-standard’ treatment for iron-loading observed in the hemoglobinopathy, β -thalassemia major [54]. It has a higher affinity for ferric iron than other cations due to its three hydroxamic acid iron-binding moieties. The iron(III)-containing complex of DFO shows very high stability (log K = 1031) [55] and forms a 1:1 complex leading to a fully occupied coordination sphere, inhibiting the direct access of hydrogen peroxide or oxygen with the iron centre [56]. DFO has poor membrane permeability, but is able to remove stored iron from the iron storage molecule, ferritin, probably by an indirect mechanism involving the binding of iron in transit between cellular compartments [57]. This ligand can also bind iron from Tf in the presence of labilizing agents [58]. DFO has been shown to inhibit iron uptake and promote iron release in many cells types [33, 34], as well as reducing iron stores in patients with iron overload conditions. However, in studies using mice, administration of DFO did not markedly affect hemoglobin levels [59, 60], but it

reduces liver iron stores in these studies. Notably, DFO is not able to remove iron from the porphyrin rings in hemoglobin, myoglobin or cytochromes [55]. The clinical use of DFO is limited by its poor bioavailability and short plasma half-life of ~ 12 minutes [11]. Thus, DFO therapy requires a rigorous regime of subcutaneous infusions which must be administered eight to twelve hours/day, three to seven days/week [61]. Early studies examined the anti-cancer efficacy of DFO in neuroblastoma cell lines where exposure to the ligand (60 µM) for 72 h resulted in approximately 90% cell death [14]. This cytotoxic effect could be prevented by co-incubation of the ligand with greater than stoichiometric amounts of ferric citrate [14], highlighting that iron chelation is a major mechanism of cytotoxicity. In addition, DFO also demonstrated irreversible inhibition of the proliferation and colony formation activity of HL60 leukemia cells [16]. In a clinical trial of patients with neuroblastoma, 7 of 9 patients responded to DFO (150 mg/kg/day) after a 5 day course with no apparent toxicity [62]. However, in other studies involving children with neuroblastoma, DFO (120-240 mg/kg/day as continuous i.v. infusion for five days every other week) failed to produce a response [63]. At 240 mg/kg/day, DFO had unacceptable short-term toxicity leading to lethargy, dizziness, blurred vision and leg cramps [63]. Combination therapy of DFO with other anti-cancer agents, including thio-TEPA, carboplatin, etoposide and cyclophosphamide using 57 advanced neuroblastoma patients resulting in 24 complete responses, 26 partial responses, 3 minor responses and 4 non-responses [64]. A complete response was defined as the disappearance of all evidence of disease for at least 4 weeks. A partial response was defined as greater than a 50%, but less than 90% reduction, in all measurable lesions and improvement of all pre-existing bone lesions for at least 4 weeks. A minor response was defined as less than a 50% reduction in any measurable lesion and less than a 50% reduction in any other lesions. No response was defined as less than a 50% reduction in any measurable lesion with greater than a 25% increase in any other lesions. Toxicity was moderate in this study and included reversible myelo-suppression [64]. Thus, DFO has some side effects and its

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development as an anti-cancer drug was limited by its poor potency that is mediated by its low membrane permeability and short plasma half-life [11, 61].

vivo [71, 76]. Importantly, 3-AP was also active against hydroxyurea-resistant cells, suggesting that 3-AP has a different mechanism of action than hydroxyurea [71]. However, it should be noted that other iron chelators such as DFO, hydroxypyridones and 2-hydroxy-1-naphthylaldehyde isonicotinoyl hydrazone (311 or NIH), also inhibit RR activity [75, 77]. Indeed, the absence of a constant supply of iron to RR results in the inhibition of this enzyme [75].

3.2. 5-Hydroxypyridine-2-carboxaldehyde Thiosemicarbazone (HPCT) and 3-aminopyridine-2-carboxaldehyde Thiosemicarbazone (3-AP or Triapine®) Thiosemicarbazones are another group of iron chelators that have been comprehensively examined as anti-cancer agents, although it is only recently that their mechanism of action has been understood in greater detail. Many studies have shown that thiosemicarbazones also exhibit anti-bacterial, anti-malarial and anti-viral activities [65]. This class of tridentate ligands possess a N,N,S tridentate “soft donor” coordination system [66, 67] and form redox-active iron complexes which generate ROS that play an important role in their anti-cancer activity [34, 68-70]. Unlike DFO, some thiosemicarbazones do not show a marked loss of antiproliferative activity upon forming iron complexes indicating they are cytotoxic [70-72].

A study of 3-AP as a single agent in patients with recurrent or metastatic head and neck squamous cell carcinoma at 96 mg/m2, daily for 4 days every 14 days showed that this dosing schedule was well tolerated [78]. However, 3-AP showed only minor activity in treating this aggressive condition [78]. In a phase I trial, 3-AP administered at a dose of 96 mg/m2 by a 2 h i.v. infusion daily for 5 days on an every-other-week schedule demonstrated an acceptable safety profile, although myelo-suppression was observed together with leukopenia which was the expected consequence of RR inhibition [79]. The safety profile obtained from this latter study supported the initiation of combination trials of 3-AP with other anti-cancer agents. A summary of the clinical dose and the outcome of 3-AP in the clinical trials against various cancers is shown in Table 1.

Early observations of the anti-tumor activity of these compounds appeared in 1956 where 2-formylpyridine thiosemicarbazone (Fig. 3A) was shown to have anti-leukemic activity in mice bearing leukemia xenografts [73]. The ligand, 5-hydroxypyridine-2carboxaldehyde thiosemicarbazone (HPCT) (Fig. 3B), was one of the first of this class of agents to be studied clinically in patients for toxicity [74]. The iron chelation efficacy of this ligand was apparent from the characteristic dark-green urine observed after administration to patients, which was likely to be due to the excretion of the iron(II)-HPCT complex [74]. Although transient decreases in blast counts were observed in three of five patients with acute leukemia, no remission or anti-tumor activity was noted in patients with solid tumors [74]. Unfortunately, some adverse effects of HPCT included gastrointestinal toxicity and mild myelosuppressant activity, as well as hemolysis [74].

Phase II clinical trials of 3-AP in combination with gemcitabine in non-small-lung cancer showed non-objective responses with some patients developing neutropenia, hypoxia and methemoglobinemia [80]. Similar side effects were also observed in another trial using 3-AP as a single agent for advanced renal carcinoma where neutropenia and the acute reactions of hypoxia, hypotension and methemoglobinemia were observed [81]. A more recent Phase II trial combining 3-AP and gemcitabine conducted by Traynor et al., for advanced non-small cell lung cancer patients also identified acute infusion reactions to 3-AP together with elevated methemoglobinemia [82]. Thus, the use of 3-AP in the clinics is limited by its low efficacy and considerable toxicity.

The ligand, 3-aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP also known as Triapine®; Fig. 3C), is one of the best characterized thiosemicarbazones that has undergone Phase I and II clinical trials for cancer treatment [11]. It was initially developed as a potent RR inhibitor, although the exact mechanism of inhibition remains elusive and may be caused by ROS generation via the 3-AP iron complex and/or depletion of cellular iron pools [69, 75]. In comparison to hydroxyurea, 3-AP exhibited greater antiproliferative activity in L1210 leukemia cells both in vitro and in

A N

4.1. Aroylhydrazones The aroylhydrazone series of chelators are orally-active synthetic ligands [11]. The first ligand of this series was pyridoxal isonicotinoyl hydrazone (PIH; Fig. 4A) which was reported to be a highly active iron chelator in 1979 [83]. A major advantage of this

B

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HO

S

NH2

N H

N

N

N H

NH2

5-HPCT

2-formylpyridine

C

NH2 N

N

S N H

NH2

3-AP Fig. (3). Chemical structures of thiosemicarbazones: (A) 2-formylpyridine thiosemicarbazone, (B) 5-hydroxypyridine-2-carboxaldehyde thiosemicarbazone (HPCT) and (C) 3-aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP or Triapine®).

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Table 1.

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Summary of Clinical Trials Using 3-AP

Treatment 3-AP

Dose 5 - 105 mg/m

2

Patients

Outcome

Reference

27 patients with histological proof of solid tumor cancers

Relevant tumor inhibitory concentrations at the highest doses were achieved in the serum without significant toxicity.

Feun, L. et al., Cancer Chemother Pharmacol 2002, 50:223

No anti-tumor responses were observed in heavily pretreated cancer patients, although 8 patients had stabilization of their disease. 3-AP

120 - 160 mg/m2

24 patients with refractory leukemia

No patient had an objective response. Over 70% patients had >50% reduction of WBC count.

Giles, F.J. et al., Leuk Res 2003, 27:1077

3-AP

2 - 96 mg/m2

32 patients with different cancers. (50% had metastatic gastrointestinal tumors)

No partial or complete responses were observed.

Murren, J. et al., Clin Cancer Res 2003, 9:4092

3-AP and gemcitabine

3-AP: 105 – 185 mg/m2

26 patients with advanced cancer

Among 22 evaluable patients: 1 complete response, 2 partial responses, 1 had prolonged stabilization of a large liver metastasis.

Yen, Y. et al., Cancer Chemother Pharmacol 2004, 54:331

gemcitabine: 600 – 1,000 mg/m2 3-AP

20 – 224 mg/m2

21 patients with advanced or metastatic cancer

No objective responses, but there was prolonged stabilization of disease or decreases in serum tumor markers in 4 patients.

Wadler, S. et al., J Clin Oncol 2004, 22:1553

3-AP and

3-AP: 105 mg/m2

cytarabine (ara-C)

ara-C: 100 – 800 mg/m2

31 patients with refractory acute leukemia and myelodysplastic syndrome

4 patients achieved a complete response after the first cycle of therapy, no partial responses.

Yee, K. et al., Leuk Res 2006, 30:813

3-AP

50 – 96 mg/m2

19 patients with metastatic renal

1 patient experienced a partial response lasting over 4 months.

Knox, J.J. et al., Invest New Drugs, 2007, 25: 471

cell carcinoma

Seven patients had stable disease (range 3.4–8.2 months). Seven patients had early progressive disease (≤ 8 weeks of therapy). 3-AP and gemcitabine

3-AP: 105 mg/m

2

gemcitabine : 1,000 mg/m2 2

3-AP

96 mg/m

3-AP and gemcitabine

3-AP: 105 mg/m2 gemcitabine: 1,000 mg/m2

No objective response. Four patients had stable disease and the median time to progression was 3 months.

Ma, B. et al., Invest New Drugs, 2008, 26 :169

32 patients with recurrent or metastatic head and neck squamous cell carcinoma

Well tolerated, but has only minor activity.

Nutting, C.M. et al., Ann Oncol, 2009, 20: 1275

18 patients with advanced

No objective anti-tumor responses were seen. 3 patients experienced stable disease with median overall survival of 5.4 months.

Traynor, A.M. et al., Invest New Drugs, 2010, 28: 91

12 patients with metastatic non smallcell lung cancer

non-small cell lung cancer

ligand over DFO was that it could be synthesized through a simple one-step Schiff base condensation of pyridoxal and isonicotinic acid hydrazide [83]. In order to study the structure-activity relationships of PIH, a number of its analogues were developed. In fact, PIH and many of its analogues can be divided into three major groups known as the pyridoxal benzoyl hydrazone analogues (100 series), salicylaldehyde benzoyl hydrazone analogues (200 series) and 2-hydroxy-1-naphthylaldehyde benzoyl hydrazone analogues (300 series; for review see [84]). Despite minor structural differences, PIH and its analogues are tridentate ligands that form 2:1 ligand to iron complexes [85]. The chelators bind iron through the carbonyl oxygen, imine nitrogen and phenolic oxygen atoms [85]. In vitro analysis of PIH in the human neuroepithelioma cell line, SK-N-MC, demonstrated that despite its marked iron chelation efficacy at preventing iron uptake from Tf and increasing cellular iron release, it had lower antiproliferative activity than DFO [86]. In vivo studies showed that oral administration of PIH to rodents at doses between 25 and 100 mg/kg led to increased fecal iron excretion of up to 8 times greater than the normal level [87]. Subsequently, more lipophilic derivatives of PIH were synthesized by changing the pyridoxal moiety to 2-hydroxy-1naphthaldehyde which led to the chelator 311 (Fig. 4B) that showed

far greater anti-proliferative activity [86]. Studies examining the mechanism of action of 311 demonstrated that redox cycling did not play a major role in the anti-proliferative activity of this ligand [88]. In fact, 311 prevented ascorbate oxidation and benzoate hydroxylation [68] and its complexation with iron prevented its anti-proliferative activity [68, 88]. This latter observation demonstrated that the ability of 311 to chelate iron plays an essential role in its cytotoxic activity More recent work examining the activity of the PIH series has assessed the effect of the addition of a caging group, namely 1-(2nitrophenyl)ethyl (2-NPE), leading to the “caged-iron chelator”, 2NPE-PIH [89] (Fig. 4C). These agents have been designed to treat skin cancer and essentially are pro-drugs that are rendered inactive by a photo-labile protecting group [89]. The latter is then released upon exposure to UV irradiation generating the active ligand. Such an approach can potentially be useful as it minimizes prolonged dosing of iron chelators, avoiding the removal of essential iron. However, in vivo studies with these agents have yet to be reported. 4.2. Deferasirox Another iron chelator with potential applications in oncology is deferasirox (Fig. 1C). This agent has been designed as a treatment for iron-overload disease and is considered to be a better alternative

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B

HO

N N

N

H N

N O

OH

C

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D

HO

N N

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OH

PIH

N

H N

O

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H N

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O

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mTOR

cell proliferation Fig. (4). Chemical structures of aroylhydrazones: (A) pyridoxal isonicotinoyl hydrazone (PIH), (B) 2-hydroxy-1-naphthylaldehyde isonicotinoyl hydrazone (311) and (C) 1-(2-nitrophenyl)ethyl PIH (2-NPE-PIH). (D) A diagram showing the effect of deferasirox on increasing the expression of regulated in development and DNA damage response (REDD1) and its downstream protein, tuberous sclerosis complex (TSC2, also known as tuberin) to down-regulate the mammalian target of rapamycin (mTOR) pathway which can promote cell proliferation.

to DFO due to its oral-activity [90]. In fact, deferasirox was found to be highly effective at reducing iron-loading in β -thalassemia patients when administered orally once a day over 5 years [10]. Moreover, the ligand was well tolerated, with no significant side effects being reported [10]. Considering its oral-activity and low toxicity, a number of recent studies have examined the potential of deferasirox as an anticancer agent. Initial studies found that deferasirox had high antiproliferative activity against human hepatoma cells [91]. In fact, deferasirox was able to significantly reduce cell viability and inhibit the cell cycle at the G0/G1 and S-phase in the human hepatoma cell line, HUH7 [91]. Moreover, the anti-tumor effects of deferasirox were more pronounced in hepatoma cells relative to primary cultures of human hepatocytes [91]. Hence, these results suggest that deferasirox is able to selectively target cancer cells and has potential for the treatment of this disease. More recently, further studies have identified some crucial molecular targets of deferasirox that may contribute to its antitumor activity. These include the oncogenic nuclear factor-κB (NFκB) pathway, which was found to be markedly inhibited by deferasirox in leukemia cells [92]. Deferasirox was demonstrated to inhibit NF-κB activity by sequestering its p65 subunit in the cytoplasm in an inactive form [92]. NF-κB consists of a small group of proteins that, when associated with the inhibitory complex IκB kinase, leads to the inactive state of the p65 subunit. Previous studies on p65 have also demonstrated that its nuclear localization and transcriptional activity occur in acute myeloid leukemia (AML) blasts, but not in normal erythroid precursors [93]. Hence, the anticancer activity of deferasirox could be linked, in part, to its effect on the NF-κB pathway. Moreover, other iron chelators including

DFO and deferiprone did not affect NF-κB signaling in leukemia cell lines [92]. Interestingly, the addition of the permeant iron complex, ferric hydroxyquinolone, did not reduce the NF-κB inhibition observed in response to deferasirox, suggesting that this effect was not dependent on iron-depletion [92]. In addition, this study also found that pre-incubation of K562 erythroleukemia cells with deferasirox for 18 h followed by treatment with etoposide for a further 72 h induced a significantly higher rate of apoptosis than when compared to either drug alone [92]. These latter studies highlight the potential of deferasirox in combination therapy with other anti-cancer agents. Another recent study demonstrated that deferasirox modulates multiple signaling pathways related to cell survival, including proteins related to iron metabolism and hypoxia, such as growth differentiation factor 15 (GDF-15) and regulated in development and DNA damage response (REDD1;[94]). This study conducted by Ohyashiki et al., showed that deferasirox-treated cells showed an enhanced expression of REDD1 and its downstream protein, tuberous sclerosis complex (TSC2, also known as tuberin), that down-regulates the mammalian target of rapamycin (mTOR) pathway (Fig. 4D) [94]. This is of interest considering the oncogenic activity of mTOR in promoting cell proliferation [95] and that its reduced expression by deferasirox may contribute to the drugs anti-cancer activity. Further in vivo studies examining the anti-tumor activity of deferasirox in a mouse model of human myeloid leukemia (U937 xenografts) demonstrated that it significantly reduced tumor volume and increased survival of mice relative to vehicle controls [94]. Therefore, considering its oral activity, low toxicity and anti-proliferative efficacy, deferasirox may be a novel therapeutic for the treatment of cancer. However,

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further in vivo studies examining this compound are required to better elucidate its anti-tumor activity.

synthesized. One approach involved linkage of tachpyr to biomolecules such as monoclonal antibodies [99]. In addition to increasing the half-life of the ligand, this strategy can also potentially enhance the selective targeting of tachpyr to tumor cells and lower its side effects. Previous studies on monoclonal antibody immune-conjugates have shown improvement regarding systemic toxicity, potency and half-life [100]. For the synthesis of these agents, a linker design incorporating bifunctional tachpyr possessing a maleimide group was prepared for conjugation with thiolated monoclonal antibodies [99] (Fig. 5B). However, the antitumor efficacy of these antibody conjugates remains to be examined.

4.3. Tachpyr Tachpyr, N,N´,N˝-tris(2-pyridylmethyl)-cis,cis-1,3,5-triaminocyclohexane, is a hexadentate chelator which uses three pyridyl nitrogens and three secondary amine nitrogens to bind iron ([96]; Fig. 5A). When tested using bladder cancer cells, tachpyr showed moderate cytotoxicity, having an IC50 value of 4.6 µM, as compared with DFO with an IC50 value of 70 µM [96]. Several metal complexes of tachpyr including iron(II), calcium(II), manganese(II), magnesium(II), copper(II), and zinc(II) complexes have been synthesized. Of these, the zinc(II), copper(II), and iron(II) complexes were non-toxic, whereas the manganese(II) and magnesium(II) complexes remained as cytotoxic as tachpyr [96]. This feature is similar to DFO, as the iron complexes of both these ligands possess limited cytotoxic effects. To assess whether iron chelation was involved in the cytotoxic mechanism of tachpyr, sterically-hindered tachpyr derivatives were prepared through Nalkylation of the ligand. These tachpyr derivatives were non-toxic, consistent with the chelation of metals being the cytotoxic mechanism of this agent [96]. Moreover, tachpyr caused inhibition of ferritin synthesis, suggesting that it depletes intracellular iron [96].

4.4. Thiosemicarbazones Novel thiosemicarbazone iron chelators with anti-cancer activity were recently developed and include the di-2-pyridylketone thiosemicarbazones (DpT) and 2-benzoylpyridine thiosemicarbazones (BpT) analogues (Fig. 6A). The majority of chelators in these series showed greater selectivity for neoplastic cells over normal fibroblasts [34, 72]. These chelators demonstrate better iron mobilization efficacy when compared to DFO or PIH [34, 72]. The importance of metal chelation for the anti-tumor efficacy of the DpT chelators was shown through the analog, Dp2mT (Fig. 6B), which demonstrated no activity due to the 2-methyl group that prevented electron delocalization and metal ion binding of the ligand [34].

The use of reverse-phase high-performance liquid chromatography to measure tachpyr and its metal complexes identified that the majority of the chelators in cells appeared either unbound or as zinc or iron complexes, indicating that these metal ions are its major targets [97]. Consistent with this, the cytotoxicity of tachpyr and its ability to activate apoptotic caspases-3 and -9 were blocked in cells pre-treated with either iron or zinc [97]. Hence, the ability of tachpyr to bind intracellular zinc and, more importantly, iron is an integral part of its anti-proliferative activity. Studies examining tachpyr-iron(II) chelates showed that tachpyr decreases, but does not block oxidation of iron(II) [98]. This allows superoxide radicalinduced or tachpyr-induced reduction of iron(III), which consequently promotes hydroxyl radical formation through the Haber-Weiss reaction [98]. Hence, in addition to iron-depletion, tachpyr exerts low-level oxidative stress which may be important for its cytotoxic activity.

Like 3-AP, both the DpT and BpT series form redox-active complexes, with the ability to bind either iron(II) or iron(III) [72]. Hence, these chelators are able to facilitate ROS generation under physiological conditions and mediate oxidative stress [70, 72]. Indeed, subsequent structure-activity relationship studies showed that the potent anti-proliferative activity of these thiosemicarbazones relative to their previous generation hydrazone chelators (i.e., di-2-pyridylketone isonicotinoyl hydrazone) can be ascribed, in part, to the redox potentials of their iron complexes which lead to ROS generation [70, 72]. One of the best characterized chelators of the DpT series is di2-pyridylketone-4,4,-dimethyl-3-thiosemicarbazone (Dp44mT) [34, 101]. This ligand shows pronounced anti-proliferative activity across a variety of cancer cell types, including neuroepithelioma, neuroblastoma, mesothelioma, prostate, hepatoma, lung, breast and leukemia with better activity than 3-AP in majority of these cells

In order to overcome the limitations of the relatively short biological half-life of tachpyr, derivatives of this ligand have been A N

2695

N H HN

N H

N

N

Tachpyr

B N

N H HN

N H

H N

N O

O

H N

N O

N

O

Bifunctional tachpyr with maleimide linker Fig. (5). Chemical structures of: (A) N,N´,N˝-tris(2-pyridylmethyl)-cis,cis-1,3,5-triaminocyclohexane (tachpyr) and (B) bifunctional tachpyr with maleimide linker for conjugation with thiolated monoclonal antibodies.

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A

N

R'

H N

N

N

N

R

R'

H N

N R S

S N

N

DpT series

BpT series

DpT: R = H, R‘ = H Dp4mT: R = H, R‘ = CH3 Dp4eT: R = H, R‘ = CH2CH3 Dp4aT: R = H, R‘ = CH2CHCH3 Dp4pT: R = H, R‘ = Ph Dp44mT: R = H, R‘ = CH3

BpT: R = H, R‘ = H Bp4mT: R = H, R‘ = CH3 Bp4eT: R = H, R‘ = CH2CH3 Bp4aT: R = H, R‘ = CH2CHCH3 Bp4pT: R = H, R‘ = Ph Bp44mT: R = H, R‘ = CH3

B N

CH3 N

NH2

N S

N

Dp2mT C

D

S

O

S

N

N

NH2

N

NQST

N

N H

N

TSC24

E S

N N H

N H O

OMe N H

NSC73306 Fig. (6). Chemical structures of thiosemicarbazones: (A) di-2-pyridylketone thiosemicarbazone (DpT) and 2-benzoylpyridine thiosemicarbazone (BpT) analogues, (B) di-2-pyridylketone 2-methyl-3-thiosemicarbazone (Dp2mT) which cannot bind metal ions, (C) 1,2-naphthoquinone thiosemicarbazone (NQTS), (D) α-heterocyclic carboxaldehyde thiosemicarbazone 24 (TSC24) and (E) NSC73306.

[101]. In vivo studies of this latter chelator using a panel of human tumor xenografts in CD2F1 hybrid and Balb/c nu/nu mice showed that Dp44mT had potent and selective anti-tumor activity [34, 101]. Interestingly, this chelator did not cause systemic iron-depletion in mice, probably as a result of the low doses (0.4-0.75 mg/kg/day, 5 times/week) necessary to elicit anti-tumor efficacy in xenograft models [101]. No significant differences in hematological indices were found between treated and control groups [101]. However, a higher non-optimal dose of Dp44mT (0.75 mg/kg, once/day, 5 times per week for 2 weeks) led to the development of cardiac fibrosis in nude mice [101]. More recently, studies have progressed to examining the in vivo anti-tumor efficacy and tolerability of a new generation BpT chelator, namely 2-benzoylpyridine-4,4-dimethyl-3-thiosemicarbazone (Bp44mT), administered via the oral or intravenous routes [102]. This agent has previously been shown to actively bind intracellular iron to form redox-active iron complexes, leading to marked and selective anti-tumor activity in vitro [72]. Administration of Bp44mT by both the intravenous and oral routes resulted in a marked dose-dependent inhibition of the growth of DMS-53 lung tumor xenografts. When administered at 50 mg/kg via oral gavage 3 times/week for 23 days, net tumor xenograft growth was inhibited by 75% as compared to vehicle-treated mice

[102]. Toxicological examination showed reversible alterations including a slight reduction of RBC count, with a decrease of liver and splenic iron levels which confirmed iron chelation in vivo. Interestingly, as found for Dp44mT [101], treatment of the animals with Bp44mT did not lead to any evidence of overt tumor irondepletion as measured by direct assessment of iron levels. In fact, molecular indices, namely the expression of TfR1 and ferritin, indicated that there was iron sequestration within the tumor, which was opposite to what was observed in the liver [102]. Importantly, in contrast to Dp44mT, the chelator-treated mice did not suffer cardiac histological abnormalities and there was also no significant weight loss, suggesting oral administration of Bp44mT was well tolerated [102]. However, Bp44mT treatment was associated with mild anemia and hepatotoxicity which were shown to be reversible after discontinuation of the agent. Collectively, these results demonstrate the substantial promise of this orally-active thiosemicarbazone for cancer therapy. Clearly, oral administration of an effective chemotherapeutic provides the benefits of convenience for chronic dosing regimens. Considering the redox activity of the Dp44mT and Bp44mT iron complexes [70, 72, 103], recent studies evaluated the effect of thiosemicarbazones on important cysteine-containing anti-oxidant systems in cells, as these may be targeted by these agents [104].

Iron Chelators for the Treatment of Cancer

Interestingly, Dp44mT and Bp44mT were found to elevate oxidized trimeric thioredoxin levels and decrease thioredoxin reductase activity. In addition, Dp44mT and Bp44mT also decreased the glutathione/oxidized-glutathione ratio and the activity of glutaredoxin that requires glutathione as a reductant [104]. These results were in agreement with the ability of N-acetylcysteine and buthionine sulfoximine, which supplement and deplete cellular GSH levels, to inhibit and potentiate the anti-proliferative activity of these chelators, respectively [104]. These cysteine-containing systems play crucial roles in redox reactions and regulate protein disulfide composition such as the disulfide bond in RR [105]. Thus, thiosemicarbazones may have an additional mechanism of RR inhibition via their effects on these cysteine-containing molecules, which probably mediate in part, their potent anti-proliferative activity. Other thiosemicarbazones which incorporate a naphthoquinone moiety such as 1,2-napthoquinone thiosemicarbazone (NQTS; Fig. 6C) and 4-hydroxy-3-methyl-1,2-naphthoquinone-1-thiosemicarbazone (HM-NQTS) have been synthesized and show anti-tumor activity against MCF-7 human breast cancer cells [106]. In fact, the anti-proliferative activity of NQST was comparable to that of the known anti-cancer agent, etoposide [106]. Although the iron chelation efficacy of these ligands has not been reported, other metal complexes have been described, including nickel, copper and palladium [106]. Interestingly, the metal complexes of these thiosemicarbazones, especially NQST, were found to be more potent than the parent ligands [106]. It has also been shown by Rao and colleagues [107] that thiosemicarbazones inhibit topoisomerase IIα. Indeed, these investigators demonstrated that Dp44mT causes selective poisoning of DNA topoisomerase IIα, as measured by an in vitro DNA cleavage assay and cellular topoisomerase-DNA complex formation in MDA-MB-231 breast cancer cells. Another recent investigation by Huang et al [108] examined a series of α-heterocyclic carboxaldehyde thiosemicarbazones; the most potent agent being TSC24 (Fig. 6D). This latter agent showed broad anti-proliferative activity in a panel of tumor cell types, including colon, stomach, breast, lung and liver. In vivo studies with this agent using S-180 sarcoma-bearing mice also demonstrated growth inhibitory activity [108]. Apart from being an iron chelator, the anti-tumor activity of TSC24 may also be mediated by topoisomerase IIα catalytic inhibition, as it was shown to directly interact with the ATPase domain of topoisomerase IIα, leading to a reduction in ATP hydrolysis [108]. Currently, it is unclear whether inhibition of this enzyme is related to the iron-binding activity of thiosemicarbazones. Another important property of thiosemicarbazones is the observation that some of these ligands are able to overcome chemoresistance. Previous studies on the efficacy of Dp44mT in chemoresistant cells showed that it was able to overcome multidrug-resistance mechanisms in etoposide-resistant MCF-7VP clone of MCF-7 breast cancer cells and the vinblastine-resistant KB-V1 clone of KB3-1 epidermoid carcinoma cells [101]. Furthermore, vinblastine-resistant KB-V1 cells were more susceptible to Dp44mT than the vinblastine-sensitive parental cell line, KB3-1 [101]. Hence, this observation indicates that the mechanism of resistance has resulted in these cells becoming more sensitive to Dp44mT. Interestingly, another thiosemicarbazone, namely NSC73306 (Fig. 6E), has also demonstrated promising activity against multidrug resistant cancers [109]. In fact, NSC73306 was shown to be effective at inhibiting the proliferation of human epidermoid, ovarian and colon cancer cell lines expressing various levels of the multi-drug efflux pump, P-glycoprotein, that is known to mediate resistance [109]. These studies demonstrated that the toxicity of NSC73306 was potentiated by P-gp [109]. However, the

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mechanism involved was unclear and the efficacy of this latter ligand as an iron chelator is not known. Further studies on the pharmacophores of these ligands highlighted the importance of aromatic/hydrophobic features at the N4 position of the thiosemicarbazone and the reliance on the isatin moiety as key bioisosteric contributors [110]. Hence, thiosemicarbazones represent a promising future class of chelators for cancer treatment. 5. POTENTIAL SIDE EFFECTS OF IRON CHELATION THERAPY IN CANCER PATIENTS Based on the clinical observations of iron chelation therapy with both DFO and 3-AP, there are a number of potential side effects which may be important to investigate when treatment with chelators is envisaged. These need to be considered when developing future novel iron chelators as anti-cancer agents and are discussed in detail below. 5.1. Myelo-Suppression A large number of investigations demonstrated that one of the most common side effects in patients treated with iron chelators is reversible myelo-suppression [111]. Unlike other side effects such as hypotension, cutaneous reactions and gastrointestinal disturbances that do not usually result in drug discontinuation, myelo-suppression can be a serious condition [111, 112]. Myelosuppression can lead to anemia, severe bleeding, granulocytopenia/ agranulocytosis and life-threatening infections due to the lack of circulating neutrophils [113]. In DFO- or deferiprone-treated patients, mild to moderate bone marrow failure has been observed in clinical trials with the onset of either thrombocytopenia [114] or neutropenia [115, 116]. Moreover, myelo-suppression was observed to be more likely a side effect of iron chelation in patients whose plasma iron levels were low [117]. This topic is particularly important to consider in cancer patients and in patients with myelo-proliferative disorders because their bone marrow is often already suppressed due to chemotherapy [117]. Deferiprone-related neutropenia is typically reversible upon discontinuation of the drug, but can re-occur if the chelator is reintroduced [118]. It has yet to be elucidated if the neutropenia and agranulocytosis is an idiosyncratic reaction or due to dose-related direct myelo-toxicity [119]. However, frequent monitoring of white blood cell counts is often recommended [120]. On the other hand, the ability of iron chelators to modulate the immune system has been successfully exploited in some studies. In particular, DFO has been used as an immune-suppressive agent in eliminating graft versus host disease [121] and its modulatory role as an anti-leukemic agent has been described in vitro [122] and in vivo [16]. The direct cause of myelo-suppression after iron chelation therapy is probably due to the inhibition of RR, thereby decreasing DNA synthesis and cell growth in a broad number of cell types [123]. 5.2. Hypoxia Hypoxia can occur in response to some iron chelating agents (also see Section 5.3) and it is known that cellular iron-deficiency mimics a hypoxic-like state. This is further verified by the activation of the transcription factor, hypoxia inducible factor-1 (HIF-1; [124]), that is up-regulated in response to hypoxia (Fig. 7). HIF-1 is composed of two sub-units, a constitutively expressed β subunit and an α subunit which is regulated by the hypoxic state [124]. Under conditions of normal oxygen and iron levels, HIF-1α is regulated by prolyl hydroxylases which allow its binding to the von Hippel-Lindau (VHL) protein. The binding of HIF-1α to VHL activates a ubiquitin E3 ligase leading to the degradation of HIF-1α [124, 125]. However, when either cellular oxygen or iron levels are low, prolyl hydroxylases, which require both iron and oxygen, fail

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Normoxia Prolyl hydroxylase _

OH

OH

Fe chelation Hypoxia

OH

HIF-1α

HIF-1α

pVHL HIF-1β Proteasome

HIF-1

p53 VEGF1 Ndrg1 EPO Arrest Apoptosis

TfR1

Vascularisation

DNA-repair

Oxygen Fe Metastasis uptake uptake suppressor

Fig. (7). The effect of iron chelation on hypoxia inducible factor-1α (HIF-1α) and its downstream targets. In normoxic conditions, HIF-1α undergoes hydroxylation by prolyl hydroxylase (PH) at proline-402 and -564. The hydroxylation allows the binding of von Hippel-Lindau protein (pVHL) to HIF-1α and subsequently results in ubiquitination of the protein which leads to proteasomal degradation. Iron chelation and hypoxia prevent the hydroxylation of HIF-1α leading to its stabilization and translocation into the nucleus, where it binds to HIF-1β to form HIF-1. This results in p53 stabilization and transactivation of various genes involved in cellular arrest, DNA repair and apoptosis. HIF-1 also initiates the transcription of various genes including vascular endothelial growth factor 1 (VEGF1), erythropoietin (EPO) and transferrin receptor-1 (TfR1) that are involved in vascularization, oxygen transport and iron uptake, respectively. Additionally, HIF-1 can also initiate the expression of N-myc downstream regulated gene-1 (Ndrg1), a metastasis suppressor gene.

to function, resulting in the accumulation of HIF-1α in the cytoplasm [124]. HIF-1α is then able to translocate to the nucleus where it binds to HIF-1β to form the transcriptionally active HIF-1 complex [126].

increasing HIF-1 activity and up-regulating its downstream targets such as Ndrg1 [129]. Moreover, the ability of these agents to mimic hypoxia and induce HIF-1 highlights their potential suitability for the treatment of other diseases such as myocardial infarction [133].

Once assembled, the HIF-1 complex can directly modulate a number of genes by binding to their hypoxia-responsive element (HRE) located in the promoter [127]. These genes include the angiogenic factor, vascular endothelial growth factor 1 (VEGF1; [128]), the metastasis suppressor Ndrg1 [129], and under conditions of severe hypoxia, HIF-1 can also activate apoptotic pathways via the stabilization of p53 [130] and increase the expression of proapoptotic BNIP3 [131]. Due to the ability of some iron chelators to effectively deplete cancer cells of iron, this often results in the activation of HIF-1 and its downstream effectors [130]. Indeed, many of these downstream effects, such as the increase in Ndrg1 and p53, may contribute to the anti-tumor activity of these ligands.

Interestingly, there have been reports of a novel iron chelator known as N-(2-hydroxybenzyl)-L-serine (HBSer) that does not induce HIF-1 activity [134] (Fig. 8A). This ligand is a tridentate iron chelator synthesized through condensation of salicylaldehyde with the L-amino acid, serine [135]. In vitro experiments in murine dermal fibroblasts showed that HBSer was able to suppress ironinduced hydroxyl radical formation via the Fenton reaction and it significantly reduced UV-induced lipid peroxidation through iron chelation [135]. In addition, similar to iron chelators such as DFO and salicylaldehyde isonicotinoyl hydrazone (SIH), HBSer was found to increase iron regulatory protein/iron responsive element (IRP/IRE) binding activity in primary skin fibroblast cells, confirming its ability to modulate iron levels in cells [134]. Importantly, when HBSer was tested for its ability to increase HIF1 DNA-binding using the same cell line, unlike DFO or SIH, this chelator did not increase HIF-1-DNA-binding or HIF-1 promoter activity [134].

Aside from the effect of iron chelation on HIF-1 expression and the activation of its downstream targets, a number of other important side effects may arise in patients treated with these compounds that are linked to their effects on hypoxia. Clinical trials using 3-AP against a number of neoplasms including pancreatic cancer have reported hypoxia as one of the key side effects [81, 132]. In addition, DFO has also been found to induce hypoxiarelated injury in the microvasculature of a rodent model which mimics the damage caused by hypoxia [133]. Interestingly, this latter study suggested that iron chelators may be useful as a hypoxia mimic that can offer protection against subsequent hypoxia in patients with myocardial infarction and stroke [133]. This is based on the fact that iron chelators up-regulate HIF-1 which functions to protect cells from further hypoxia induced by blood vessel occlusion. Hence, although iron chelation can lead to hypoxia, this side effect may in fact contribute to their anti-tumor activity by

A possible reason for the lack of activity of HBSer in inducing HIF-1 related activity when compared to DFO and SIH may be its lower affinity for iron [135]. In fact, the association constant of HBSer for iron(III) was calculated to be log K ~ 21-22, which is substantially lower than that of DFO (log K ~ 30) and SIH (log K ~ 29) [135]. Hence, the lower iron-binding affinity of HBSer may enable this ligand to acquire iron without activating HIF-1, similar to endogenous iron-binding proteins which do not necessarily affect HIF-1 activity [134]. The anti-proliferative activity of HBSer has not been reported, but it is of interest considering that HIF-1 activity may be important for the anti-tumor effects of iron

Iron Chelators for the Treatment of Cancer

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chelators. Nonetheless, studies using HBSer suggest that it is possible to modulate cellular iron levels without affecting HIF-1 activity.

normally reduced back to deoxygenated Hb by nicotinamide adenine dinucleotide (NADH)-MetHb reductase [139]. Thus, chemical inhibition of this system may increase MetHb levels and the effect of 3-AP on this enzyme is important to assess.

5.3. Methemoglobinemia

MetHb production can also increase if heme iron is directly oxidized, which involves the transfer of electrons from ferrous heme iron to the oxidizing compound in the absence of oxygen [143]. Heme iron can also be oxidized indirectly, which requires oxygenated Hb. As a result, superoxide and hydrogen peroxide are produced when the oxygen bound to Hb accepts electrons from iron(II). Furthermore, MetHb formation can be initiated by active intermediates derived from the bio-transformation of certain drugs or chemicals [143]. Hence, it is possible that the effect of 3-AP could be mediated by one of its intermediary metabolites and this requires further assessment.

Methemoglobinemia is a recognized complication of 3-AP administration observed in clinical trials [136]. It is a clinical syndrome that is the result of an increase in methemoglobin (MetHb) levels [137] and is of particular concern in patients with compromised cardiopulmonary function due to the inability of MetHb to transport oxygen to tissues [138]. Under physiological conditions, approximately 1 – 2% of total hemoglobin (Hb) is MetHb, as auto-oxidation of Hb occurs at a slow rate [138]. Indeed, this reaction occurs when iron(II) within Hb is oxidized to iron(III) to form MetHb (Fig. 8B) [138]. MetHb is not capable of transporting oxygen to the tissues since iron(III) is unable to reversibly bind oxygen, and instead, a water molecule binds to the sixth coordination position in heme [139]. Autooxidation also generates superoxide and, therefore, can also lead to redox stress [140]. When the proportion of MetHb as a fraction of total Hb approaches 15%, patients exhibit central cyanosis that is nonresponsive to oxygen administration [141]. At higher MetHb levels of 20-30%, neurological and cardiovascular symptoms become evident. Patients treated with 3-AP show a relatively high incidence of mild to moderate levels of symptomatic methemoglobinemia that ranges from 7.8-17.6% of the total Hb concentration [80]. Currently, the mechanism involved in inducing this effect remains unclear, but could be related to the redox activity of the 3-AP iron complex [68]. Considering this, it is of interest that there have been no reports of methemoglobinemia in subjects treated with DFO, deferiprone or deferasirox, which do not form redox-active complexes. Generally, it is well known that an increase in MetHb levels can occur secondary to both congenital changes in Hb synthesis or metabolism, or redox imbalances induced by exposure to oxidizing agents [142]. These latter agents accelerate Hb oxidation so that it eventually overwhelms the capacity of endogenous reducing systems [141]. Considering this, MetHb is

A

CH2OH

SUMMARY AND CONCLUSIONS While chelation therapy has been mainly implemented for the treatment of iron overload disease, many studies have observed the anti-tumor activity of various classes of iron chelators. Amongst these are chelators that were originally designed for the purpose of iron overload conditions i.e., DFO and deferasirox. One of the most active iron chelators designed for the treatment of cancer, namely the thiosemicarbazone, 3-AP, has entered clinical trials for a wide variety of tumors. However, the use of 3-AP in humans has demonstrated some side effects such as myelo-suppression, hypoxia and methemoglobinemia. It remains unclear to what extent these side effects are related to the ability of this chelator to deplete cellular iron stores and form redox-active iron complexes. Other thiosemicarbazones with potential anti-tumor activity have more recently emerged, including Dp44mT, Bp44mT, NSC00, TSC42 and NQST. While the use of iron chelation therapy for cancer treatment remains in its infancy, the anti-tumor efficacy shown by these ligands demonstrates their promise for future development. Finally, it should be noted that a number of these ligands (e.g., Dp44mT) do not lead to overall iron-depletion of the tumor in vivo in animal models and act to sequester intracellular iron to generate

COOH

NH OH

B

Fig. (8). (A) Chemical structure of N-(2-hydroxybenzyl)-L-serine (HBSer). (B) Auto-oxidation of oxygenated hemoglobin (oxyHb) to methemoglobin (MetHb). The ferrous iron (Fe2+) bound to heme is oxidized to ferric iron (Fe3+), resulting in the release of superoxide (O2.-). A water molecule then occupies the sixth coordination position in heme, forming MetHb.

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cytotoxic iron complexes. Thus, their mechanism of action is different to typical chelators such as DFO that act solely to deplete tumor cell iron stores and inhibit cancer growth by that mechanism. CONFLICT OF INTEREST None declared.

Yu et al.

[26] [27] [28]

ACKNOWLEDGMENTS

[29]

This work was supported by the National Health and Medical Research Council of Australia [Grant 570952 and Fellowship 571123]; and the Cancer Institute New South Wales [Research Scholar Awards 07/RSA/1-33, 06/RSA/1-12, 07/RSA/1-22, and Career Development Fellowship 08/ECF/1-36].

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Received: May 04, 2011

Revised: July 29, 2011

Accepted: August 23, 2011

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