Gallium and Its Competing Roles with Iron in Biological Systems

Biochimica et Biophysica Acta 1863 (2016) 2044–2053

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Biochimica et Biophysica Acta

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Review Gallium and its competing roles with iron in biological systems

Christopher R. Chitambar

Division of Hematology and Oncology, Department of Medicine, Medical College of Wisconsin, 9200 W. Wisconsin Avenue, Milwaukee, WI 53226, USA article info abstract

Article history: Gallium, a group IIIa metal, shares chemical properties with iron. Studies have shown that gallium-based Received 15 March 2016 compounds have potential therapeutic activity against certain cancers and infectious microorganisms. By Received in revised form 27 April 2016 functioning as an iron mimetic, gallium perturbs iron-dependent proliferation processes in tumor cells. Gallium's Accepted 30 April 2016 action on iron homeostasis leads to disruption of ribonucleotide reductase, mitochondrial function, and the Available online 03 May 2016 regulation of transferrin receptor and ferritin. In addition, gallium nitrate stimulates an increase in mitochondrial Keywords: reactive oxygen species in cells which triggers downstream upregulation of metallothionein and hemoxygenase-1. Gallium Gallium's anti-infective activity against bacteria and fungi results from disruption of microbial iron utilization Iron through mechanisms which include gallium binding to siderophores and downregulation of bacterial iron uptake. Metallodrug therapeutics Gallium compounds lack cross-resistance to conventional chemotherapeutic drugs and antibiotics thus making Cancer them attractive agents for drug development. This review will focus on the mechanisms of action of gallium Infection with emphasis on its interaction with iron and iron proteins. Ribonucleotide reductase © 2016 Elsevier B.V. All rights reserved.

1. Introduction animal studies to assess drug toxicity and establish safe dosing limits, gallium nitrate was deemed an investigational drug by the National Gallium compounds first entered medical application in the late Cancer Institute and was advanced to future evaluation in clinical 1960s when it was discovered that 67Ga citrate when injected into trials [8]. tumor-bearing animals concentrated in sites of actively growing tumors Human studies confirmed the antineoplastic activity of gallium [1]. Subsequent studies in humans confirmed these findings and the nitrate in non-Hodgkin's lymphoma and bladder cancer [9–11],howev- 67Ga scan emerged as a tool for imaging tumors in patients [2]. Follow- er, an understanding of gallium's mechanisms of action at the cellular ing evaluation in various malignancies, the 67Ga scan proved to be of and molecular level lagged behind its use in the clinic. As a result, the diagnostic value in lymphoma to determine whether masses that drug was evaluated in the clinic without a clear insight into its cellular persisted after completion of treatment represented viable or non- and molecular targets. Ongoing basic research however, has yielded im- viable tissue [3]. Metabolically active lymphomatous masses continue portant information regarding the interaction of gallium with cellular to take up 67Ga while non-viable tumor masses do not [3].Withthe iron metabolism. This is relevant to the advancement of a next genera- development of more sophisticated imaging technology in the tion of gallium compounds as therapeutic agents for certain cancers United States, the 67Ga scan has been replaced by the positron emission and, more recently, for infections. This review will focus on our current tomography (PET) scan which is based on 18F-fluorodeoxyglucose up- knowledge of gallium as a metal that competes with iron in biologic take by tumors [4], while scanning with 68Ga-labeled radiopharmaceu- systems and how this can be exploited for therapeutic purposes. ticals is being explored as a tool for molecular imaging of tumors [5,6]. However, the 67Ga scan remains an imaging modality in parts of the 2. Gallium — chemistry world where advanced scans are not available. The observation that 67Ga could be taken up by tumors prompted Credit for the discovery of gallium in 1875 is given to Paul-Emile the logical question as to whether non-radioactive gallium compounds Lecoq de Boisbaudran who noted its presence as two distinct bands on could also concentrate in tumors and inhibit their growth. Preclinical spectroscopy. Gallium is a group IIIA metal, atomic number 31 that studies that ensued revealed that gallium nitrate, when injected into exists in the earth's crust at a concentration of 5–15 mg/kg and is Sprague–Dawley rats and CDFI mice with subcutaneously implanted obtained as a byproduct of extraction of aluminum and zinc ores. tumors, suppressed tumor growth [7]. Following a series of preclinical It has a shiny, silvery white color with a melting temperature of 28.7646 °C (85.5763 °F) that renders it near-liquid at room temperature. It shares certain properties with iron (III) in that the octahedral Abbreviations: Tf, transferrin; TfR, transferrin receptor; RR, ribonucleotide reductase; 3+ MT-2A, metallothionein-2A; HO-1, heme oxygenase-1. ionic radius for Ga is 0.620 Å compared with 0.645 Å for high spin 3+ E-mail address: [email protected]. Fe . In addition, the tetrahedral ionic radius is 0.47 Å and 0.49 Å for

Ga3+ and Fe3+, respectively. The ionization potential and electron to transferrin (Tf), an 80 kDa protein containing two iron-binding sites, afﬁnity values for Ga3+ are 64 eV and 30.71 eV, respectively while for one at the carboxy and another at the amino terminal. Under physiolog- high spin Fe3+ they are 54.8 eV and 30.65 eV, respectively [12]. Gallium ical conditions, approximately one-third of circulating Tf is occupied by undergoes hydrolysis to yield a mixture of gallium hydroxides of iron (III) (Tf-Fe) while the remaining two-third of Tf is available to

Ga(OH)11 and Ga(OH)3 at pH ~ 4, and a mixture of Ga(OH)3 and Ga bind additional metals, or additional iron when plasma iron levels are Ga(OH)4 at physiologic pH 7.4 [13]. elevated in pathologic conditions. The Tf–TfR cycle is summarized in Fig. 1A. Tf-Fe is taken up by cells 3. Gallium as an iron mimetic in mammalian cells via cell surface Tf receptor1 (TfR1)-mediated endocytosis. Once inter- nalized, TfR1–Tf-Fe locates in an acidic endosome where iron is The properties that gallium share with iron permits it to bind with released, reduced to Fe(II) by a ferrireductase, and transported out of high avidity to certain iron-binding proteins. However, while the the endosome to the cytoplasm by a membrane-based divalent metal binding of iron to a protein promotes protein function, the substitution transporter-1 (DMT-1). The subsequent steps in iron trafﬁcking within of gallium for iron in a protein usually disrupts its function and may lead the cells are somewhat obscure, however it is generally agreed that to adverse downstream effects in cells. iron moves to a labile “pool” bound to low molecular weight iron chelates to maintain solubility. From this pool, iron is utilized for a 3.1. Iron transport and cellular uptake variety of critical cellular purposes including the functioning of the M2-subunit of ribonucleotide reductase (RRM2) (Fig. 1A), mitochondri- A brief overview of iron transport, cellular uptake, and storage is pro- al iron–sulfur cluster-containing proteins (Fig. 1B), and certain steps in vided to set the stage for discussing some of the similarities between the cell cycle regulation [14,15]. iron and gallium. The reader is referred to recent reviews on iron metab- Cellular iron homeostasis is tightly regulated and is balanced by the olism for a detailed insight into this topic [14,15]. Following the absorp- interplay of proteins responsible for iron import, storage, and export. tion of dietary iron in the duodenum, iron circulates in the blood bound A decrease in iron below a critical level can trigger processes leading

Fig. 1. A. An overview of the cellular handling of iron. Iron is bound to transferrin (Tf) in the circulation and is incorporated into cells by transferrin receptor1 (TfR1)-mediated endocytosis of Tf-Fe complexes. The Tf-Fe–TfR complex translocates from the cell surface to an intracellular acidic endosome where Fe(III) dissociates from Tf and is reduced to Fe(II) by STEAP3 (six membrane epithelial antigen of the prostate 3). Fe(II) exits the endosome through divalent metal transporter1 (DMT1, not shown) to a labile iron “pool.” From here, iron traffics to different compartments (mitochondria, ribonucleotide reductase, and others) presumably bound to low-molecular weight ligands. Excess iron is stored in ferritin. Iron exits from the cell through cell membrane-based ferroportin. Cytoplasmic iron regulatory proteins (IRPs) function as sensors of cellular iron status and regulate the synthesis of transferrin receptors, ferritin, and ferroportin at the mRNA translational level by interaction with iron response elements (IREs) present on the untranslated regions of their respective mRNAs. shows the known sites of interaction of gallium with cellular iron metabolism. B. Potential sites of interaction of gallium in the mitochondria. The iron–sulfur cluster (Fe–S) proteins in the citric acid cycle and mitochondrial complexes are potential targets for the cytotoxic action of gallium compounds. 2046 C.R. Chitambar / Biochimica et Biophysica Acta 1863 (2016) 2044–2053 to cell death. On the other hand, excess iron is hazardous as it can cata- endosome is an initial step in the cellular uptake of both gallium and lyze the production of damaging hydroxyl radicals and superoxide. Thus iron, a significant fraction of gallium appears to cycle back out of the as a cytoprotective process, excess iron not utilized for cellular function cell suggesting that all of the incorporated gallium is not unloaded to is stored in ferritin, a shell-shaped protein composed of 24 subunits of the cytoplasm [34]. This is in contrast to iron where the majority of H- and L-ferritin [16]. To control the iron entry and storage, cellular iron unloaded from Tf enters the cytoplasm. Furthermore, while DMT- iron status is sensed by cytoplasmic iron regulatory proteins (IRPs). 1 plays a role in transporting iron (II) out of the acidic endosome, it These proteins regulate the translation of ferritin and TfR1 mRNAs does not appear to be involved in the transport of gallium [35]. through an interaction with iron response elements present on the 5′ Proteins that modify TfR endocytosis may affect cellular 67Ga uptake. and 3′ untranslated regions of their respective mRNAs. Under condi- The wild-type (wt) hemochromatosis gene product (HFE protein) binds tions of iron excess, IRP–IRE interactions are suppressed resulting in to TfR at sites that overlap with Tf binding sites on the receptor [36,37]. an increase in ferritin synthesis and a decrease in TfR synthesis [14, In patients with hereditary hemochromatosis, an inherited iron over- 15]. The converse occurs with cellular iron deprivation; here, RNA–IRE load disease caused by a C282Y mutation of the HFE gene, the mutated interactions increase resulting in a decrease in ferritin and an increase HFE protein fails to associate with TfR. Thus, when HFE is mutated or in TfR synthesis [14,15]. when wt HFE is absent there is an increase in cellular Tf-Fe uptake Whereas TfR and ferritin play central roles in cellular iron uptake [38]. Conversely, when wt HFE is expressed it produces a decrease in and storage, iron balance in cells is also maintained by ferroportin, a Tf-Fe uptake relative to cells lacking wt HFE and a state of cellular iron membrane-based protein that exports iron from cells. The binding of deficiency [39]. 67Ga uptake studies conducted in HeLa cells containing ferroportin to hepcidin, a 25 amino acid peptide produced in the liver, a tet-off and tet-on HFE expression system showed that 67Ga-Tf uptake results in the internalization of the ferroportin–hepcidin complex and (like that of iron) was significantly reduced when the wt HFE gene was its degradation in the lysosome [17,18]. This results in a decrease in turned on relative to when HFE expression was turned off [40]. cellular iron efflux and an increase in cellular iron content. Gallium may also be incorporated into cells through a TfR- The ferroportin/hepcidin axis has clinical implications. Recent independent process akin to the Tf-independent uptake mechanism studies show that the downregulation of ferroportin coupled with an in- for iron [41]. However, it remains to be determined whether the crease in hepcidin in breast cancer tumors is associated with an adverse Tf-independent uptake of gallium and iron uptake occurs through preclinical outcome [19,20]. Earlier studies showed that TfR expression cisely the same transporter. Despite similarities between gallium and may be increased in certain malignancies such as lymphoma, bladder iron, it is likely that different cellular uptake systems are responsible cancer, glioblastoma, and breast cancer and that this is associated with for each metal. This is suggested by the finding that TfR-independent biologically aggressive tumor behavior [21–24]. Thus, a scenario of uptake or iron from Tf-Fe or Fe-citrate by cells involves the reduction increased iron import (elevated TfRs) and diminished iron export of Fe(III) to Fe (II) by a cell surface ferrireductase [42,43] and/or the gen- (decreased ferroportin) would elevate intracellular iron levels to meet eration of free radicals [44]. In contrast, gallium is not reduced from the high demands of tumor growth. Moreover, elevated intracellular Ga(III) to Ga(II) and would need to be transported into cells as Ga(III) iron could catalyze the formation of pro-carcinogenic reactive oxygen or a Ga (III)-chelate. species (ROS). Although both gallium and iron circulate in the blood bound to Tf and are taken up by cells via TfRs, they display different pharmacokinet- 3.2. Gallium transport and cellular uptake ics in humans. Following intravenous injection of 67Ga-citrate and 59Fe citrate in healthy individuals, the elimination rate constant of 67Ga cit- The human body appears to handle gallium as iron with regard to its rate is 50-times slower than that of 59Fe citrate, indicating that 59Fe is transport in the blood. Clues to similarities between gallium and iron cleared from the blood at a more rapid rate than 67Ga [45]. The volume were provided by early studies which demonstrated that 67Ga uptake of distribution of 67Ga in the body is approximately 6 times that of 59Fe by malignant cells in vitro could be increased by the addition of exoge- as the latter metal is largely confined to hematopoietic tissues for hemo- nous Tf to the culture medium [25,26]. The biologic relevance of this globin production. The uptake of 59Fe by the sacrum, liver, spleen, and observation was confirmed in animal studies where 67Ga injected intra- heart follows classically described ferrokinetics with uptake progres- venously in rabbits as 67Ga citrate was bound exclusively to Tf in the sively declining after achieving a peak uptake at 24 h following injection blood [27]. The studies of Harris and Pecararo clearly established that [45,46]. In contrast 67Ga rapidly accumulates in the same tissue in the gallium binds to Tf with high avidity [28]. Modeling data summarized first 24 h following injection but then progressively increases in these by Bernstein [12] suggests that at concentrations up to 50 μM gallium tissues with time [45]. in the blood binds entirely to Tf. At higher blood concentrations which exceed the metal-binding capacity of Tf, gallium's binding to Tf dimin- 3.3. Intracellular distribution of gallium ishes and it circulates as gallate [Ga(OH)4 −] [12]. Experiments with different cell lines in vitro indicated that 67Ga was taken up by cells via Whereas the similarities between gallium and iron with respect to TfR-mediated endocytosis of 67Ga–Tf [26,29–31]. The role of TfR in binding to Tf and uptake by the TfR have been described, much less is cellular 67Ga uptake was further supported by studies which showed understood about the intracellular trafficking of gallium. Initial studies that 67Ga uptake by human promyelocytic leukemic HL60 cells could conducted in the 1970s with a view to understanding the cellular be blocked by 42/6, an anti-TfR monoclonal antibody (MoAb) that localization of 67Ga following its uptake by normal and malignant cells blocks TfR internalization [30]. 67Ga uptake directly correlated with reported 67Ga to be bound to a 45 kDa protein in cell extracts from rat cell surface TfR levels [30]. The importance of TfR in 67Ga uptake was hepatoma [47,48]. Additional studies showed 67Ga to preferentially confirmed in a panel of lymphoid cell lines [31]. In a tumor-bearing locate in hepatic microvesicles and lysosomes [49–51]. Beyond being murine model, anti-TfR MoAb B3/25 produced a 75% reduction in 67Ga able to concentrate in metabolically active tumors in vivo, 67Ga also uptake by subcutaneously implanted melanoma cells, lending further concentrates in sites of inflammation and infection in vivo [52]. Here, support to the importance of TfR in gallium uptake [32]. 67Ga uptake 67Ga behaves like iron in that it binds strongly to the iron-binding by a mutant Chinese hamster ovary (CHO) cell line that does not protein lactoferrin present in neutrophils, an essential component of endogenously express the TfR increased significantly when these cells the inflammatory response. 67Ga binding to lactoferrin is 50-fold stron- were transfected to overexpress the TfR [33].Increased67Ga uptake ger than to Tf [53]. Apart from binding to lactoferrin, 67Ga may localize by these cells was seen both in vitro and in vivo when these cells in sites of infection by exchanging with iron in siderophores. The latter were grown as explants in nude mice [33]. However, although TfR- are low molecular weight iron chelating compounds that are released mediated endocytosis and the translocation of Tf-metal to an acidic by microorganisms to acquire iron for their growth [54]. Gallium and C.R. Chitambar / Biochimica et Biophysica Acta 1863 (2016) 2044–2053 2047 other trivalent metals are capable of displacing iron from siderophores chloride produced an increase in Tf-independent cellular 67Ga uptake under reducing conditions [55]. The decrease in pH that occurs in an [41]. The ability of gallium and iron to stimulate each other's uptake in- abscess at the site of infection would favor the exchange of gallium for dicates that they either use the same Tf-independent transport system iron in siderophores. for entry into HL60 cells or activate a process responsible for the uptake Gallium may also bind to ferritin. In an in vitro equilibrium dialysis of either metal. multi-chamber system, 70% of 67Ga bound to Tf transferred to horse In another study, Richardson investigated the TfR-independent up- spleen ferritin in the presence of ATP [56]. Ascorbate, citrate, and lactate take of Tf-iron by human SK-Mel-28 melanoma cells and demonstrated also facilitated 67Ga transfer to ferritin but to a far lesser extent than ATP that pre-incubation of the cells with gallium nitrate stimulated their [56]. Other studies show 67Ga to be associated with immunoprecipitable TfR-independent uptake of 59Fe from 59Fe-Tf [63]. Since Ga(III) is not ferritin in cells [30]. However, none of these studies demonstrate that redox-active, it was concluded that gallium-stimulated iron uptake did gallium is actually incorporated into the ferritin shell. In fact, it is more not require the generation of free radicals [63]. The effect of gallium likely that gallium, when associated with ferritin, is bound to a site on on Tf-independent iron uptake is not limited to malignant cell lines. the external surface of the protein, however this has not been studied. Sturm et al. showed that when primary hepatocytes isolated from Ferritin does not appear to play a role in modulating 67Ga uptake by Sprague–Dawley rats were pre-incubated with gallium nitrate there cells since a 20-fold increase in ferritin levels in HL60 cells was not was an increase in their uptake of non-transferrin-bound iron (NTBI) accompanied by a parallel increase in cellular 67Ga uptake [30]. in a time and concentration-dependent manner [64]. In contrast, differ- Interestingly though, exposure of CCRF-CEM cells to gallium nitrate in- ent divalent metals (Zn, Ni, Co, Cd, Mn, and Cu) had minimal influence duces H-ferritin mRNA expression [57]; the mechanisms responsible on iron uptake [64]. for this induction remain to be determined. The Tf-independent iron uptake system can deliver sufficient iron to support cellular viability and growth [65]. Theoretically therefore, if 3.4. Action of gallium on cellular iron-dependent processes certain cells are capable of bypassing the gallium-induced block in TfR-mediated iron uptake by activating TfR-independent iron uptake 3.4.1. Cellular iron uptake they might be less sensitive to the cytotoxicity of gallium. The rela- Gallium, as Tf-Ga, can compete with Tf-Fe for binding to TfR and tive contribution of TfR-dependent and TfR-independent iron uptake inhibit receptor-mediated iron uptake by cells [58].Thisgallium- pathways is likely to differ among cell types and may, in part explain induced block in cellular iron incorporation may also include a disrup- differences in cell sensitivity to gallium compounds. tion of endosome acidification which affects the dissociation of iron from Tf. As a result, iron cycles out of the cell rather than being 3.4.2. Ribonucleotide reductase (RR) transferred across the endosome to the cytoplasm [58]. The synthesis of ribonucleoside diphosphate to deoxyribonucleotide The gallium-induced interference in cellular iron uptake renders diphosphate, an essential step in DNA synthesis, is catalyzed by RR, cells relatively iron-deficient; this has adverse downstream effects on a heterodimer which consists of homodimeric M1 and homodimeric certain iron-dependent processes and is particularly striking in M2 subunits [66–68]. RRM1 and RRM2 are under the control of different erythroid cells that require iron for hemoglobin production. Tf-Ga genes and are expressed at different phases of the cell cycle. RRM1 is diminishes DMSO-induced hemoglobin production and 59Fe incorpora- present during all phases of the cell cycle while RRM2 appears in late tion into hemoglobin in Friend erythroleukemia cells [59].Theeffectof G1 as cells enter S-phase [69,70]. Both subunits come together to form gallium on hemoglobin production is independent of any effect on cell an actively functioning enzyme. RRM1 contains effector and substrate proliferation since other drugs that inhibit the growth of these cells do binding sites whereas the RRM2 subunit contains a binuclear iron not block hemoglobin synthesis [59]. The level of hemoglobin produc- center and a tyrosyl free radical (Fig. 1A). The tyrosyl radical signal in tion in gallium-treated cells can be restored to normal by the addition intact cells and cell lysates can be detected by electron paramagnetic of Tf-Fe or iron pyridoxal isonicotinyl hydrazone (Fe-PIH), thus indicat- resonance (EPR) spectroscopy; the amplitude of this signal corresponds ing that the block in hemoglobin production is due to a decrease in in- specifically to the activity of RRM2 and thus to overall RR enzyme activ- tracellular iron [59]. Cancer patients treated with gallium nitrate may ity [71]. Disruption of the tyrosyl radical by radical scavenging agents develop microcytic anemia and an elevated red cell zinc protoporphy- leads to loss of RR activity. The iron center is essential for RRM2 function rin; both these features occur with iron deficiency and provide evidence and is closely linked to the EPR signal of RRM2. Loss of iron from RRM2 that gallium nitrate interferes with iron metabolism in vivo [60]. Gallium results in a loss of the tyrosyl radical and RRM2 function. When iron sulfate has been shown to reduce the activity of delta-aminolevulinic availability is low, RRM2 protein is still present in cells; however, in acid (ALAD) dehydratase in the heme biosynthesis pathway in the this situation it exists as apoRRM2 (devoid of iron) that lacks a tyrosyl liver, kidney, and erythroid cells in vivo [61]. This could also contribute radical signal and is functionally inactive [72]. RRM2 protein has a to the development of anemia in patients treated with gallium nitrate. half-life of about 4 h and turns over frequently during cell proliferation As might be anticipated with cellular iron deficiency, murine L1210, [70]. To maintain the activity of newly synthesized M2 protein, cells human promyelocytic leukemia HL60, and Friend erythroleukemia cells require a constant supply of iron. The cellular requirement for iron to incubated with Tf-Ga display an increase in TfR mRNA and protein support RR activity is reflected by the increase in TfRs that occurs as expression [62]. However, for reasons not understood, this effect of cells enter the S-phase of the cell cycle [73–76]. gallium is not uniformly seen in other cells. In human lymphoma Gallium compounds inhibit the activity of RR by a dual mechanism. CCRF-CEM cells, Tf-Ga blocks iron uptake but does not produce an Early studies demonstrated that Tf-Ga produced a decrease in the upregulation of TfR expression [62]. RRM2 subunit tyrosyl radical signal within 6 h of incubation with intact In contrast to inhibiting TfR-mediated cellular iron uptake, gallium HL60 cells and that this signal was lost by 18 h [77]. Consistent with a nitrate stimulates the Tf-independent uptake of ferric nitriloacetic acid block in RR activity, cells incubated with Tf-Ga displayed a decrease in (Fe-NTA) by HL60 cells [41]. These experiments, conducted in serum- dNTP pools compared with untreated cells [77]. Co-incubation of cells free medium to ensure the absence of Tf, showed that an increase in with hemin as a source of iron delivered to cells independent of TfR Tf-independent Fe-NTA uptake occurred within 5 min of incubation of prevented the gallium-induced inhibition of the RRM2 tyrosyl radical cells with gallium nitrate and could be saturated with increasing con- signal in intact cells. Cytoplasmic extracts of 1210 cells prepared after centrations of Fe-NTA. The upregulation of Fe-NTA uptake persisted their incubation with gallium nitrate for 18 h displayed a significant for at least 24 h after removal of gallium from cells. These studies clearly decrease the RRM2 tyrosyl radical EPR signal [78].However,thetyrosyl indicated that gallium nitrate can upregulate a Tf-independent trans- radical was restored to normal within 10 min following the addition of porter for iron. It was also shown that incubation of HL60 cells to ferric ferrous ammonium sulfate to the extract [78]. These experiments 2048 C.R. Chitambar / Biochimica et Biophysica Acta 1863 (2016) 2044–2053 indicate that gallium does not block the synthesis of RRM2 protein but The magnitude of resistance to gallium nitrate may influence the interferes with the availability of iron to support RRM2 activity. While extent of compensatory changes in iron metabolism as cells adapt to the inhibition of iron availability for RR should itself be sufficient to in- gallium nitrate. In another clone of HL60 cells that was only 6.6-fold hibit DNA synthesis, gallium also blocks RR activity through an addition- more resistant to gallium nitrate than the gallium-sensitive cells, TfR al mechanism which involves competitive inhibition of substrate (CDP expression and Tf-iron uptake by gallium-resistant cells were de- and ADP) binding to the enzyme [79]. This potential mechanism of creased relative to gallium-sensitive cells [88].Furthermore, action of gallium has not been further elucidated; however, since galli- gallium-resistance was associated with distinct differences in iron um can form complexes with nucleotides [80], it is entirely likely that and gallium trafficking; in resistant cells 59Fe taken up by cells was Ga-ADP or Ga-CPD formed in vitro might compete with the substrates associated with a Tf–TfR1–HFE complex whereas this association ADP and CDP for binding to RR. Thus, gallium inhibition of RR appears was not seen in gallium-sensitive cells [88]. to occur through two distinct mechanisms: intracellular iron depriva- Studies with human CCRF-CEM lymphoma cells with acquired tion which limits iron availability for RRM2, and by competitive resistance to gallium nitrate have been more revealing as to changes inhibition of substrate binding to the enzyme. in iron homeostasis that occur with the development of gallium resistance. Here, gallium-resistant cells display a downregulation of 3.4.3. Action of gallium on the mitochondria TfR expression and a decrease in their accumulation of iron and gallium Given the importance of iron–sulfur cluster-containing proteins in over time [57,89]. Ferritin levels in resistant cells are six to ten-fold the citric acid cycle and the electron transport chain of the mitochon- lower in the resistant cells while the binding of IRP-1 to ferritin IRE dria, it appears logical to surmise that gallium might interact with mRNA is increased [57]. This indicates that the gallium-induced de- these proteins (Fig. 1A). Although Ga–S clusters have been chemically crease in ferritin in resistant cells is due to changes at the level of ferritin synthesized in vitro [81,82], there are few studies of gallium's action mRNA translation [57]. The cytotoxicity of gallium to gallium-resistant on the mitochondria. In lymphoma cell lines, gallium nitrate induces cells can be restored by increasing the level of exogenous apoTf in the apoptosis in CCRF-CEM cells primarily through the mitochondrial culture, thus strongly suggesting that the mechanism of drug resistance pathway. This involves the activation of Bax, a pro-apoptotic protein to gallium nitrate involves a decrease in gallium accumulation in cells in the cytoplasm, and its translocation to the mitochondria which [57]. While this could be solely due to changes in cellular TfR- leads to a loss of mitochondrial membrane potential, the release of mi- mediated gallium uptake, the possibility that an increase in gallium tochondrial cytochrome c and the downstream activation of execution- export from the cell via ferroportin or another pathway has not been er caspase-3 [83]. These events, which culminate in apoptosis occur examined. over a 24 h incubation of cells with gallium nitrate, and are preceded hours earlier by a decrease in cellular oxygen consumption rate [84]. 4.2. Metallothionein and heme oxygenase-1 The earliest measureable cellular changes that suggest that gallium acts on the mitochondria are seen after a 1–2 h incubation of cells A comparison of the expression of 96 genes related to metal metab- with gallium nitrate. At these early time-points, cells display a decrease olism in gallium-resistant and -sensitive CCRF-CEM cells revealed strik- in GSH/GSSG ratio and an increase in ROS indicating that gallium in- ing differences in an unforeseen area. Relative to gallium-sensitive cells, duces oxidative stress in cells [84]. This increase in cellular ROS can be resistant cells displayed a 32- and 28-fold upregulation of the genes for abrogated by mitoquinone, a mitochondria-targeted antioxidant [85]. metallothionein-2A and metallothionein-3, respectively. Also upregu- This suggests that ROS in gallium-treated cells originates from the mito- lated were the genes for zinc transporter-1 (ZnT-1) (7.6-fold increase) chondria and is the result of an action of gallium on mitochondria. It is and metal-responsive transcription factor-1 (MTF-1) (2.2-fold increase) known that blockade of mitochondrial complexes I, II, and III can result [90]. This upregulation of metallothionein in gallium-resistant cells can- in a leak of ROS from the mitochondria [86], but it remains to be deter- not be explained on the basis of iron mimicry by gallium since iron does mined whether the increase in mitochondrial ROS content in cells ex- not influence metallothionein levels. Metallothionein is a low molecular posed to gallium nitrate is due to inhibition of these mitochondrial weight protein that binds zinc and is involved in zinc metabolism [91, complexes. Studies are in progress to define the site of gallium's action 92]; it sequesters toxic divalent metals such as cadmium [93]. on the mitochondria. A role for metallothionein in modulating the cytotoxicity of gallium- sensitive cells was suggested by studies in which gallium-sensitive cells 4. Cellular adaptation to the cytotoxicity of gallium nitrate with zinc-induced upregulation of metallothionein displayed a decrease in the cytotoxicity of gallium nitrate. This protective effect of metallo- 4.1. Changes in iron metabolism thionein against gallium's cytotoxicity was lost when metallothionein levels returned to baseline [90]. A direct correlation between endoge- The development of cell lines with acquired resistance to the nous metallothionein expression in a panel of lymphoma cell lines and growth-inhibitory action of gallium nitrate has revealed insights into their sensitivity to growth inhibition by gallium nitrate could be the mechanisms of action of gallium. To study the basis for drug resis- drawn, further suggesting a connection between gallium's mechanism tance to gallium nitrate, human leukemic HL60 cells were continuously of action and changes in metallothionein [90]. exposed for 9–12 months to subtoxic concentrations of gallium nitrate. Although at first glance it would appear that gallium's interaction This resulted in the emergence of a clone of HL60 cells that were nearly with cellular metal metabolism involves both iron and zinc, further 29-fold more resistant to growth inhibition by gallium nitrate than the studies suggested that the induction of metallothionein expression by parental gallium-sensitive cell line; the concentration of gallium nitrate gallium nitrate may be mediated through an indirect mechanism. As that inhibited cell growth by 50% was ~80 μM in the parental gallium- stated earlier, exposure of gallium-sensitive CCRF-CEM cells to gallium sensitive line compared with ~2296 μM in the gallium-resistant cells nitrate produces an increase in intracellular mitochondrial ROS within [87]. Gallium uptake was similar in both sensitive and resistant cells 1–2h[84]. Additional studies showed that with continued exposure indicating that resistance did not involve a decrease in gallium uptake to gallium nitrate, these cells displayed increased levels of metallothio- by resistance cells [87]. However, TfR expression and ferric chloride nein and heme oxygenase-1 (HO-1) at 6 and 16 h, respectively [84].The uptake was increased in resistant cells relative to sensitive cells, gallium nitrate-induced increase in HO-1 involves the triggering the suggesting that the development of a high level of gallium resistance p38p MAP kinase pathway with downstream activation of Nrf2 a in HL60 cells involved changes in cellular iron incorporation to compen- leucine zipper transcription factor that interacts with antioxidant sate for the perturbation of intracellular iron homeostasis produced by response elements present in the promoter region of the HO-1 gene. gallium nitrate [87]. Both MT and HO-1 induction can be blocked by the antioxidant C.R. Chitambar / Biochimica et Biophysica Acta 1863 (2016) 2044–2053 2049 n-acetyl cysteine indicating that gallium-induced ROS production plays A number of investigations in animal models of infection have a central role in the upregulation of MT and HO-1 expression [84]. clearly indicated that gallium's ability to interfere with microbial iron The above described sequence suggests a model for gallium's utilization has therapeutic potential. Olakanmi et al. demonstrated that mechanisms of action as illustrated in Fig. 2. Here, following its cellular gallium nitrate displayed antimicrobial activity against Mycobacterium uptake, gallium induces the release of mitochondrial ROS which in turn tuberculosis and Mycobacterium avium residing inside and outside of leads to the sequential induction of MT2A and HO-1 gene expression. macrophages by disrupting their iron metabolism [97]. Further studies Both MT2A and HO-1 are likely to represent an initial cytoprotective showed that gallium nitrate inhibited the growth of M. tuberculosis in action by cells in response to gallium-induced stress. However, with a murine model and M. avium subspecies paratuberculosis in neonatal continued cellular uptake of gallium and an expansion of the intracellu- calves [96,98]. Kaneko et al. showed that gallium inhibited the growth lar gallium pool, these protective mechanisms are overwhelmed, iron of P. aeruginosa, inhibited blocked biofilm formation and killed delivery to RRM2 is blocked and cell death ensues. The sustained upreg- planktonic and biofilm bacteria in vitro, and was effective against ulation of MT in gallium-resistant cells even in the absence of exogenous this pathogen in murine lung infection models [96]. In another study, gallium nitrate suggest that these cells may have adapted to gallium by the topical delivery of gallium combined with desferrioxamine enhanced increasing their mitochondrial activity and basal level of ROS (Ahmed the antimicrobial action of gentamicin in a rabbit model of P. aeruginosa and Chitambar, unpublished observations). Further studies are in corneal infection [99]. In a murine model of P. pseudomonas-infected progress to elucidate this process. burn injury all the control animals treated with a vehicle died from infection whereas, 100% of the animals who received subcutaneously administered gallium maltolate were protected from systemic infection and 5. Gallium as an iron mimetic in microbial systems death [100]. Gallium maltolate significantly decreased the colonization of Staphyloccus aureus and Acinetobacter baumannii in the wounds of Microorganisms require iron for their survival and growth however thermally injured mice [100]. In a murine model, orally administered their mechanisms of iron acquisition are distinctly different from those gallium maltolate showed activity against Rhodococcus equi. This bacteri- of mammalian cells [54]. Bacteria and fungi secrete siderophores. These um can produce pneumonia in foals and immunocompromised individ- are low-molecular weight peptide-based catecholates, hydroxamates, uals, and multiply in macrophages resulting in granulomatous lesions or hydroxycarboxylates that chelate iron with high affinity and return [101]. Other studies demonstrated that gallium citrate applied topically it to the microorganism for its use [54]. In addition, certain bacteria to skin wounds infected with Klebsiella pneumoniae reduced bacterial may produce hemophores (analogous to siderophores) designed to ac- burden and biofilm formation [102]. quire iron from exogenous heme (iron-protoporphyrin). Other bacteria Beyond animal studies, a clinical trial of gallium nitrate for the may obtain iron from transferrin and lactoferrin through receptors for treatment of P. aeruginosa in patients with cystic fibrosis is in progress these proteins [54,94]. It is not unexpected therefore that gallium may and the results of this study are eagerly awaited. interpose itself in place of iron in these systems and interfere with Thus, a new direction for the use of gallium in medicine has microbial iron uptake. Gallium has been shown to exchange for iron in emerged. Given the growing concern over antibiotic resistance in siderophores [55]. The siderophore pyochelin has been shown to trans- pathogenic organisms and the rising cost of newer antibiotics that port gallium into Pseudomonas aeruginosa [95]. As with mammalian adds to the financial burden of developing countries, research to define cells, gallium cannot be utilized for essential iron-catalyzed reactions. the efficacy of gallium compounds as antimicrobial is a step in the right This leads to the death of the organism. In addition, gallium per se may direction. The readers are referred to recent reviews on gallium's perturb the regulation of iron acquisition systems as was shown in potential as an antimicrobial agent [94,103]. P. aeruginosa where gallium nitrate repressed pvdS, a transcriptional regulator for pyoverdine, a siderophore produced by this organism [96]. 6. Therapeutic gallium compounds

There are four main gallium compounds at the forefront of clinical application (Fig. 3).

6.1. Gallium nitrate

Gallium nitrate (Ganite™) was the ﬁrst compound to enter clinical use and undergo extensive evaluation in humans. As a result, there exists a considerable body of information on the efﬁcacy and toxicity of this gallium formulation [8,104]. Although initially evaluated as an antineoplastic drug, gallium nitrate was found to reduce blood calcium levels and decrease bone turnover in patients with hypercalcium and Paget's disease of the bone [105,106]. Gallium concentrates in the bone and reduces bone resorption by inhibiting osteoclast activity [107]. Early studies suggested that gallium's action on bone metabolism was independent of iron and Tf [107], but in light of recent advances in our knowledge of iron metabolism, this relationship might be worthy of reevaluation. Presently, gallium nitrate is FDA-approved for treatment Fig. 2. Mitochondria-related events triggered by gallium nitrate. An early event in the of hypercalcemia in cancer patients [108] but, it has also shown antineo- cellular response to gallium is an increase in intracellular reactive oxygen species (ROS) of mitochondrial origin. Changes in zinc homeostasis along with ROS generation induce plastic activity against bladder cancer and lymphoma in numerous the expression of metallothionein-2A (MT-2A) through increased binding of metal clinical trials [9–11]. It is worth noting that the solution of gallium transcription factor-1 (MTF-1) to metal response elements (MRE) on the MT-2A gene. nitrate marketed for clinical use is actually a mixture of 25 mg/ml ROS may also trigger MT2A gene expression via action on antioxidant response gallium nitrate and 28.75 mg/ml sodium citrate dehydrate. Based on elements (ARE). Heme oxygenase-1 (HO-1) gene expression is triggered by ROS via the the aqueous chemistry of gallium [13,109], it is likely that gallium p38 MAP kinase pathway and activation of transcription factor Nrf2. ROS and the downstream activation of MT-2A and HO-1 can be blocked by the antioxidant N- nitrate administered to patients consists of a mixture of gallium acetylcysteine (NAC). hydroxides [primarily Ga(OH)3 at neutral pH] and gallium citrate. 2050 C.R. Chitambar / Biochimica et Biophysica Acta 1863 (2016) 2044–2053

Fig. 3. Chemical structures of gallium nitrate (A), gallium maltolate (B), tris(8-quinolinolato)gallium(III) (D), and gallium citrate (D).

6.2. Gallium maltolate extensive preclinical testing and is in clinical trials as reported elsewhere [113]. Gallium maltolate can be considered a second generation gallium compound which, in preclinical studies, appears to have certain advan- 6.4. Gallium citrate tages over gallium nitrate. The compound consists of a central gallium atom bound to three maltol [tris(3-hydroxy-2methyl-pyrone)] ligands Gallium citrate (Panaecin™) is the newest addition to gallium drugs in a propeller-like arrangement [110]. Unlike gallium nitrate, gallium in development; it is being evaluated in clinical trials as a novel drug maltolate is an oral formulation that has good bioavailability in animal for the treatment of a variety of infections. and human studies. Serum gallium levels of 0.115 and 0.569 μg/ml In addition to the above compounds, a number of novel gallium- have been achieved following a single oral dose of 100–500 mg gallium containing compounds have been shown to have anti-proliferative maltolate; this is comparable to steady-state levels achieved with galli- activity against cell lines in vitro and are in development [114].Further um nitrate administered by continuous intravenous infusion [110].The evaluation of the therapeutic activities for these gallium-based agents majority of gallium appearing in the circulation after orally adminis- is awaited. tered gallium maltolate is bound to Tf [111]. Gallium maltolate appears to have mechanisms of cytotoxicity that are different from gallium 7. Conclusion nitrate. For example, gallium maltolate inhibits the growth of lymphoma cell lines with acquired resistance or endogenous resistance to the Gallium does not have a known role in normal cellular physiology. cytotoxicity of gallium nitrate [112]. Gallium maltolate is also cytotoxic However, the fascination with this metal in biology relates to the fact to WTK-1 lymphoma cells which possess a p53 mutation. In contrast, that it shares certain chemical properties with iron that enable it to be these cells are resistant to growth inhibition by gallium nitrate as well taken up by tumor cells and microorganisms as an iron mimetic. This as to radiation [112]. These observations support the notion that the has resulted in the development of gallium-based agents for use in biologic mechanisms for tumor cell resistance to gallium nitrate are diagnostic and therapeutic medicine. Studies with gallium have provid- different than gallium maltolate. ed insights into the cellular handling of metals and iron metabolism. Beyond the biological applications of gallium, it is worth noting that gallium displays semiconducting properties and is being used extensively 6.3. Tris(8-quinolinolato)gallium(III) as gallium arsenide in the electronic industry [115]. Although there has been progress in our understanding of the inter- Tris(8-quinolinolato)gallium(III) (KP46) is a gallium complex bound action of gallium with biologic systems, much remains to be learned to the organic ligand 8-quinolinol. This complex displays high thermo- about its interaction with other iron-dependent processes. For example, dynamic and kinetic stability and is stable in water for hours indicating iron plays a key role in cell signaling through the WNT and mTOR that it does not rapidly release gallium to Tf. The drug has undergone pathways [116–121]. Aberrant signaling through these pathways has C.R. Chitambar / Biochimica et Biophysica Acta 1863 (2016) 2044–2053 2051 been implicated in malignancy [122–124]. Could gallium compounds [22] I. Basar, A. Ayhan, K. Bircan, A. Ergen, C. Tasar, Transferrin receptor activity as a marker in transitional cell carcinoma of the bladder, Br. J. Urol. 67 (1991) 165–168. act on the iron-dependent steps in these pathways? If so, it could have [23] A. Calzolari, L.M. Larocca, S. Deaglio, V. Finisguerra, A. Boe, C. Raggi, L. Ricci-Vitani, F. implications for cancer therapy. Further research will be necessary to Pierconti, F. Malavasi, M.R. De, U. Testa, R. Pallini, Transferrin receptor 2 is fre- address these questions. Clinical trials of gallium nitrate in cancer quently and highly expressed in glioblastomas, Transl. Oncol. 3 (2010) 123–134. [24] H.O. Habashy, D.G. Powe, C.M. Staka, E.A. Rakha, G. Ball, A.R. Green, M. demonstrated responses to treatment in patients with non-Hodgkin's Aleskandarany, E.C. Paish, M.R. Douglas, R.I. Nicholson, I.O. Ellis, J.M. Gee, lymphoma and urothelial cancers. However, these trials were conduct- Transferrinreceptor(CD71)isamarkerofpoorprognosisinbreastcancer ed without an understanding of gallium nitrate's mechanisms of action and can predict response to tamoxifen, Breast Cancer Res. Treat. (2009). and gallium nitrate is often dismissed as an “old drug.” However, newer [25] R.P. Warrell Jr., M. Issacs, N.W. Alcock, R.S. Bockman, Gallium nitrate for treatment of refractory hypercalcemia from parathyroid carcinoma, Ann. Intern. Med. l07 gallium compounds have emerged that display greater antitumor (1987) 683–686. efficacy than gallium nitrate in preclinical testing. If the newer gallium [26] A.W. Harris, R.G. Sephton, Transferrin promotion of 67Ga and 59Fe uptake by – compounds are to be advanced for the treatment of malignancy and cultured mouse myeloma cells, Cancer Res. 37 (1977) 3634 3638. [27] S.R. Vallabhajosula, J.F. Harwig, J.K. Siemsen, W. Wolf, Radiogallium localization in infections, further investigation needs to focus on gaining a better tumors: blood binding and transport and the role of transferrin, J. Nucl. Med. 21 understanding of gallium's cellular targets and the molecular (1980) 650–656. determinants of sensitivity and drug resistance. [28] W.R. Harris, V.L. Pecoraro, Thermodynamic binding constants for gallium transferrin, Biochemistry 22 (1983) 292–299. [29] R.G. Sephton, N. Kraft, 67Ga and 59Fe uptake by cultured human lymphoblasts and – Transparency document lymphocytes, Cancer Res. 38 (1978) 1213 1216. [30] C.R. Chitambar, Z. Zivkovic, Uptake of gallium-67 by human leukemic cells: demonstration of transferrin receptor-dependent and transferrin-independent The Transparency document associated with this article can be mechanisms, Cancer Res. 47 (1987) 3929–3934. found, in the online version. [31] F. Nejmeddine, N. Caillat-Vigneron, F. Escaig, J.L. Moretti, M. Raphael, P. Galle, Mechanism involved in gallium-67 (Ga-67) uptake by human lymphoid cell lines, Cell. Mol. Biol. 44 (1998) (Dec 1998) 1215–1220. [32] S.M. Chan, P.B. Hoffer, P. Duray, Inhibition of gallium-67 uptake in melanoma by an Acknowledgements anti-human transferrin receptor monoclonal antibody, J. Nucl. Med. 28 (1987) 1303–1307. This work was supported by the Thomas A. and Lorraine M. Rosenberg [33] C.A. Luttropp, J.A. Jackson, B.J. Jones, M.H. Sohn, R.E. Lynch, K.A. Morton, Uptake of Award for Translational Cancer Research from the Froedtert Hospital gallium-67 in transfected cells and tumors absent or enriched in the transferrin receptor, J. Nucl. Med. 39 (1998) 1405–1411. Foundation and the Medical College of Wisconsin. [34] P.L. Goering, R.R. Maronpot, B.A. Fowler, Effect of intratracheal gallium arsenide administration on delta-aminolevulinic acid dehydratase in rats: relationship to urinary excretion of aminolevulinic acid, Toxicol. Appl. Pharmacol. 92 (1988) References 179–193. [35] A.C. Illing, A. Shawki, C.L. Cunningham, B. Mackenzie, Substrate profile and metal- [1] C.L. Edwards, R.L. Hayes, Tumor scanning with 67Ga citrate, J. Nucl. Med. 10 (1969) ion selectivity of human divalent metal-ion transporter-1, J. Biol. Chem. 287 (2012) 103–105. 30485–30496. [2] P. Hoffer, Status of gallium-67 in tumor detection, J. Nucl. Med. 21 (1980) 394–398. [36] J.N. Feder, D.M. Penny, A. Irrinki, V.K. Lee, J.A. Lebron, N. Watson, Z. Tsuchihashi, E. [3] D. Front, O. Israel, The role of Ga-67 scintigraphy in evaluating the results of Sigal, P.J. Bjorkman, R.C. Schatzman, The hemochromatosis gene product com- therapy of lymphoma patients, Semin. Nucl. Med. 25 (1995) 60–71. plexes with the transferrin receptor and lowers its affinity for ligand binding, [4] P. Seam, M.E. Juweid, B.D. Cheson, The role of FDG-PET scans in patients with Proc. Natl. Acad. Sci. U. S. A. 95 (1998) 1472–1477. lymphoma, Blood 110 (2007) 3507–3516. [37] J.A. Lebron, A.P. West Jr., P.J. Bjorkman, The hemochromatosis protein HFE [5] M.U. Khan, S. Khan, S. El-Refaie, Z. Win, D. Rubello, A. Al-Nahhas, Clinical competes with transferrin for binding to the transferrin receptor, J. Mol. Biol. 294 indications for gallium-68 positron emission tomography imaging, Eur. J. Surg. (1999) 239–245. Oncol. 35 (2009) 561–567. [38] C.N. Roy, D.M. Penny, J.N. Feder, C.A. Enns, The hereditary hemochromatosis [6] A. Al-Nahhas, Z. Win, T. Szyszko, A. Singh, C. Nanni, S. Fanti, D. Rubello, Gallium-68 protein, HFE, specifically regulates transferrin-mediated iron uptake in HeLa PET: a new frontier in receptor cancer imaging, Anticancer Res. 27 (2007) 4087–4094. cells, J. Biol. Chem. 274 (1999) 9022–9028. [7] M.M. Hart, C.F. Smith, S.T. Yancey, R.H. Adamson, Toxicity and antitumor activity of [39] B. Corsi, S. Levi, A. Cozzi, A. Corti, D. Altimare, A. Albertini, P. Arosio, Overexpression gallium nitrate and periodically related metal salts, J. Natl. Inst. 47 (1971) of the hereditary hemochromatosis protein, HFE, in HeLa cells induces an iron- 1121–1127. deficient phenotype, FEBS Lett. 460 (1999) 149–152. [8] B.J. Foster, K. Clagett-Carr, D. Hoth, B. Leyland-Jones, Gallium nitrate: the second [40] C.R. Chitambar, J.P. Wereley, Expression of the hemochromatosis (HFE) gene metal with clinical activity, Cancer Treat. Rep. 70 (1988) 1311–1319. modulates the cellular uptake of 67Ga, J. Nucl. Med. 44 (2003) 943–946. [9] D.J. Straus, Gallium nitrate in the treatment of lymphoma, Semin. Oncol. 30 [41] C.R. Chitambar, D. Sax, Regulatory effects of gallium on transferrin-independent (2003) 25–33. iron uptake by human leukemic HL60 cells, Blood 80 (1992) 505–511. [10] C.R. Chitambar, Gallium nitrate for the treatment of non-Hodgkin's lymphoma, [42] R.S. Inman, M.M. Coughlin, M. Wessling-Resnick, Extracellular ferrireductase activ- Expert Opin. Investig. Drugs 13 (2004) 531–541. ity of K562 cells is coupled to transferrin-independent iron transport, Biochemistry [11] L. Einhorn, Gallium nitrate in the treatment of bladder cancer, Semin. Oncol. 30 33 (1994) 11850–11857. (2003) 34–41. [43] I. Jordan, J. Kaplan, The mammalian transferrin-independent iron transport [12] L.R. Bernstein, Mechanisms of therapeutic activity for gallium, Pharmacol. Rev. 50 system may involve a surface ferrireductase activity, Biochem. J. 302 (1994) (1998) 665–682. 875–879. [13] B. Hacht, Gallium(III) ion hydrolysis under physiological conditions, Bull. Kor. [44] D.R. Richardson, E. Baker, The effect of desferrioxamine and ferric ammonium cit- Chem. Soc. 29 (2008) 372–376. rate on the uptake of iron by the membrane iron-binding component of human [14] M.W. Hentze, M.U. Muckenthaler, B. Galy, C. Camaschella, Two to tango: regulation melanoma cells, Biochim. Biophys. Acta 1103 (1992) 275–280. of mammalian iron metabolism, Cell 142 (2010) 24–38. [45] K.J. Logan, P.K. Ng, C.J. Turner, P.R. Schmidt, U.K. Terner, J.R. Scott, B.C. Lentle, A.A. [15] D.J. Lane, A.M. Merlot, M.L. Huang, D.H. Bae, P.J. Jansson, S. Sahni, D.S. Kalinowski, Noujaim, Comparative pharmacokinetics of 67Ga and 59Fe in humans, Int. J. Nucl. D.R. Richardson, Cellular iron uptake, trafficking and metabolism: key molecules Med. Biol. 8 (1981) 271–276. and mechanisms and their roles in disease, Biochim. Biophys. Acta 1853 (2015) [46] M. Pollycove, M. Tono, Studies of the erythron, Semin. Nucl. Med. 5 (1975) 11–61. 1130–1144. [47] R.L. Hayes, J.E. Carlton, A study of the macromolecular binding of 67Ga in normal [16] F.M. Torti, S.V. Torti, Regulation of ferritin genes and protein, Blood 99 (2002) and malignant animal tissues, Cancer Res. 33 (1973) 3265–3272. 3505–3516. [48] D. Lawless, D.H. Brown, K.F. Hubner, S.P. Colyer, J.E. Carlton, R.L. Hayes, Isolation [17] T. Ganz, E. Nemeth, Hepcidin and disorders of iron metabolism, Annu. Rev. Med. 62 and partial characterization of a 67Ga-binding glycoprotein from Morris 5123C (2011) 347–360. rat hepatoma, Cancer Res. 34 (1978) 4440–4444. [18] D.M. Ward, J. Kaplan, Ferroportin-mediated iron transport: expression and [49] E. Aulbert, U. Haubold, Isolation of the 67gallium accumulating fraction in normal regulation, Biochim. Biophys. Acta 1823 (2012) 1426–1433. rat liver, Nucl. Med. 13 (1974) 72–84. [19] Z.K. Pinnix, L.D. Miller, W. Wang, R. D'Agostino Jr., T. Kute, M.C. Willingham, H. [50] P.A.G. Hammersley, M.N. Cauchi, D.M. Taylor, Uptake of 67Ga in regenerating rat Hatcher, L. Tesfay, G. Sui, X. Di, S.V. Torti, F.M. Torti, Ferroportin and iron regulation liver and its relationship to lysosomal enzyme activity, Cancer Res. 35 (1975) in breast cancer progression and prognosis, Sci. Transl. Med. 2 (2010), 43ra56. ll54–ll58. [20] S. Zhang, Y. Chen, W. Guo, L. Yuan, D. Zhang, Y. Xu, E. Nemeth, T. Ganz, S. Liu, [51] D.H. Brown, B.L. Byrd, J.E. Carlton, D.C. Swartzendruber, R.L. Hayes, A quantitative Disordered hepcidin– ferroportin signaling promotes breast cancer growth, Cell. study of the subcellular localization of 67Ga, Cancer Res. 36 (1976) 956–963. Signal. 26 (2014) 2539–2550. [52] C.J. Palestro, The current role of gallium imaging in infection, Semin. Nucl. Med. 24 [21] S. Kvaloy, R. Langholm, O. Kaalhus, T. Michaelsen, S. Funderud, A.A. Foss, T. Godal, (1994) 128–141. Transferrin receptor and B-lymphoblast antigen—their relationship to DNA synthe- [53] W.R. Harris, Thermodynamics of gallium complexation by human lactoferrin, sis, histology and survival in B-cell lymphomas, Int. J. Cancer 33 (1984) 173–177. Biochemistry 25 (1986) 803–808. 2052 C.R. Chitambar / Biochimica et Biophysica Acta 1863 (2016) 2044–2053

[54] R. Saha, N. Saha, R.S. Donofrio, L.L. Bestervelt, Microbial siderophores: a mini [83] C.R. Chitambar, J.P. Wereley, S. Matsuyama, Gallium-induced cell death in lympho- review, J. Basic Microbiol. 53 (2013) 303–317. ma: role of transferrin receptor cycling, involvement of Bax and the mitochondria, [55] T. Emery, Exchange of iron by gallium in siderophores, Biochemistry 25 (1986) and effects of proteasome inhibition, Mol. Cancer Ther. 5 (2006) 2834–2843. 4629–4633. [84] M.Yang,C.R.Chitambar,Roleofoxidativestressintheinductionof [56] R.E. Weiner, G.J. Schreiber, P.B. Hoffer, In vitro transfer of Ga-67 from transferrin to metallothionein-2A and heme oxygenase-1 gene expression by the antineo- ferritin, J. Nucl. Med. 24 (1983) 608–614. plastic agent gallium nitrate in human lymphoma cells, Free Radic. Biol. Med. [57] C.R. Chitambar, J.P. Wereley, Resistance to the antitumor agent gallium nitrate in 45 (2008) 763–772. human leukemic cells is associated with decreased gallium/iron uptake, increased [85] G.F. Kelso, C.M. Porteous, C.V. Coulter, G. Hughes, W.K. Porteous, E.C. Ledgerwood, activity of iron regulatory protein-1, and decreased ferritin production, J. Biol. R.A. Smith, M.P. Murphy, Selective targeting of a redox-active ubiquinone to Chem. 272 (1997) 12151–12157. mitochondria within cells: antioxidant and antiapoptotic properties, J. Biol. [58] C.R. Chitambar, P.A. Seligman, Effects of different transferrin forms on transferrin Chem. 276 (2001) 4588–4596. receptor expression, iron uptake and cellular proliferation of human leukemic [86] D.F. Stowe, A.K. Camara, Mitochondrial reactive oxygen species production in HL60 cells: mechanisms responsible for the specific cytotoxicity of transferrin- excitable cells: modulators of mitochondrial and cell function, Antioxid. Redox gallium, J. Clin. Invest. 78 (1986) 1538–1546. Signal. 11 (2009) 1373–1414. [59] C.R. Chitambar, Z. Zivkovic, Inhibition of hemoglobin production by transferrin- [87] C.R. Chitambar, Z. Zivkovic-Gilgenbach, J. Narasimhan, W.E. Antholine, gallium, Blood 69 (1987) 144–149. Development of drug resistance to gallium nitrate through modulation of cellular [60] P.A. Seligman, P.L. Moran, R.B. Schleicher, E.D. Crawford, Treatment with gallium iron uptake, Cancer Res. 50 (1990) 4468–4472. nitrate: evidence for interference with iron metabolism in vivo, Am. J. Hematol. [88] N.P. Davies, Y.S. Rahmanto, C.R. Chitambar, D.R. Richardson, Resistance to the 41 (1992) 232–240. antineoplastic agent gallium nitrate results in marked alterations in intracellular [61] P.L. Goering, S. Rehm, Inhibition of liver, kidney, and erythrocyte δ-aminolevulinic iron and gallium trafficking: identification of novel intermediates, J. Pharmacol. acid dehydratase (porphobilinogen synthetase) by gallium in the rat, Environ. Res. Exp. Ther. 317 (2006) 153–162. 53 (1990) 135–151. [89] C.R. Chitambar, J.P. Wereley, Transferrin receptor-dependent and -independent [62] R.U. Haq, C.R. Chitambar, Modulation of transferrin receptor mRNA by transferrin- iron transport in gallium-resistant human lymphoid leukemic cells, Blood 91 gallium in human myeloid HL60 cells and lymphoid CCRF-CEM cells, Biochem. J. (1998) 4686–4693. 294 (1993) 873–877. [90] M. Yang, S.H. Kroft, C.R. Chitambar, Gene expression analysis of gallium-resistant [63] D.R. Richardson, Iron and gallium increase iron uptake from transferrin by human and gallium-sensitive lymphoma cells reveals a role for metal-responsive tran- melanoma cells: further examination of the ferric ammonium citrate-activated scription factor-1, metallothionein-2A, and zinc transporter-1 in modulating the iron uptake process, Biochim. Biophys. Acta 1536 (2001) 43–54. antineoplastic activity of gallium nitrate, Mol. Cancer Ther. 6 (2007) 633–643. [64] B. Sturm, U. Lassacher, N. Ternes, A. Jallitsch, H. Goldenberg, B. Scheiber- [91] S.R. Davis, R.J. Cousins, Metallothionein expression in animals: a physiological Mojdehkar, The influence of gallium and other metal ions on the uptake of non- perspective on function, J. Nutr. 130 (2000) 1085–1088. transferrin-bound iron by rat hepatocytes, Biochimie 88 (2006) 645–650. [92] M. Namdarghanbari, W. Wobig, S. Krezoski, N.M. Tabatabai, D.H. Petering, [65] P.A. Seligman, J. Kovar, R.B. Schleicher, E.W. Gelfand, Transferrin-independent iron Mammalian metallothionein in toxicology, cancer, and cancer chemotherapy, J. uptake supports B lymphocyte growth, Blood 78 (1991) 1526–1531. Biol. Inorg. Chem. 16 (2011) 1087–1101. [66] L. Thelander, P. Reichard, Reduction of ribonucleotides, Annu. Rev. Biochem. 48 [93] C.D. Klaassen, J. Liu, S. Choudhuri, Metallothionein: an intracellular protein to (1979) 133–158. protect against cadmium toxicity, Annu. Rev. Pharmacol. Toxicol. 39 (1999) [67] Role of ribonucleotide reductase in cell division, in: J.G. Cory, J.G.Cory, A.H.Cory 267–294. (Eds.), Inhibitors of Ribonucleoside Diphosphate Reductase Activity, Pergamon [94] A.B. Kelson, M. Carnevali, V. Truong-Le, Gallium-based anti-infectives: targeting Press, New York 1989, pp. 1–16. microbial iron-uptake mechanisms, Curr. Opin. Pharmacol. 13 (2013) 707–716. [68] Y. Aye, M. Li, M.J. Long, R.S. Weiss, Ribonucleotide reductase and cancer: biological [95] E. Frangipani, C. Bonchi, F. Minandri, F. Imperi, P. Visca, Pyochelin potentiates the mechanisms and targeted therapies, Oncogene (2014). inhibitory activity of gallium on Pseudomonas aeruginosa,Antimicrob.Agents [69] Y. Engstrom, S. Eriksson, I. Jildevik, S. Skog, L. Thelander, B. Tribukait, Cell cycle- Chemother. 58 (2014) 5572–5575. dependent expression of mammalian ribonucleotide reductase. Differential regula- [96] Y. Kaneko, M. Thoendel, O. Olakanmi, B.E. Britigan, P.K. Singh, The transition metal tion of the two subunits, J. Biol. Chem. 260 (1985) 9114–9116. gallium disrupts Pseudomonas aeruginosa iron metabolism and has antimicrobial [70] S. Eriksson, A. Gräslund, S. Skog, L. Thelander, B. Tribukait, Cell cycle-dependent and antibiofilm activity, J. Clin. Invest. 117 (2007) 877–888. regulation of mammalian ribonucleotide reductase. The S phase-correlated in- [97] O. Olakanmi, B.E. Britigan, L.S. Schlesinger, Gallium disrupts iron metabolism of crease in subunit M2 is regulated by de novo protein synthesis, J. Biol. Chem. mycobacteria residing within human macrophages, Infect. Immun. 68 (2000) 259 (1984) 11695–11700. 5619–5627. [71] A. Gräslund, M. Sahlin, B.-M. Sjöberg, The tyrosyl free radical in ribonucleotide [98] O.Olakanmi,B.Kesavalu,R.Pasula,M.Y.Abdalla,L.S.Schlesinger,B.E.Britigan,Gallium reductase, Environ. Health Perspect. 64 (1985) 139– 149. nitrate is efficacious in murine models of tuberculosis and inhibits key bacterial [72] S. Nyholm, G.J. Mann, A.G. Johansson, R.J. Bergeron, A. Gräslund, L. Thelander, Role Fe-dependent enzymes, Antimicrob. Agents Chemother. 57 (2013) 6074–6080. of ribonucleotide reductase in inhibition of mammalian cell growth by potent iron [99] E. Banin, A. Lozinski, K.M. Brady, E. Berenshtein, P.W. Butterfield, M. Moshe, M. chelators, J. Biol. Chem. 268 (1993) 26200–26205. Chevion, E.P. Greenberg, E. Banin, The potential of desferrioxamine-gallium as an [73] J.W. Larrick, P. Cresswell, Modulation of cell surface iron transferrin receptors anti-Pseudomonas therapeutic agent, Proc. Natl. Acad. Sci. U. S. A. 105 (2008) by cellular density and state of activation, J. Supramol. Struct. 11 (1979) 16761–16766. 579–586. [100] K. DeLeon, F. Balldin, C. Watters, A. Hamood, J. Griswold, S. Sreedharan, K.P. [74] C.R. Chitambar, E.J. Massey, P.A. Seligman, Regulation of transferrin receptor Rumbaugh, Gallium maltolate treatment eradicates Pseudomonas aeruginosa expression on human leukemic cells during proliferation and induction of infection in thermally injured mice, Antimicrob. Agents Chemother. 53 (2009) differentiation. Effects of gallium and dimethylsulfoxide, J. Clin. Invest. 72 (1983) 1331–1337. 1314–1325. [101] J.R. Harrington, R.J. Martens, N.D. Cohen, L.R. Bernstein, Antimicrobial activity of [75] E. Pelosi, U. Testa, F. Louache, P. Thomopoulos, G. Salvo, P. Samoggia, C. Peschle, gallium against virulent Rhodococcus equiin vitro and in vivo, J. Vet. Pharmacol. Expression of transferrin receptors in phytohemagglutinin-stimulated human Ther. 29 (2006) 121–127. T-lymphocytes, J. Biol. Chem. 261 (1986) 3036–3042. [102] M.G. Thompson, V. Truong-Le, Y.A. Alamneh, C.C. Black, J. Anderl, C.L. Honnold, R.L. [76] C.R. Chitambar, J.P. Wereley, Effect of hydroxyurea on cellular iron metabolism in Pavlicek, R. Abu-Taleb, M.C. Wise, E.R. Hall, E.J. Wagar, E. Patzer, D.V. Zurawski, human leukemic CCRF-CEM cells: changes in iron uptake and the regulation of Evaluation of gallium citrate formulations against a multidrug-resistant strain of transferrin receptor and ferritin gene expression following inhibition of DNA Klebsiella pneumoniae in a murine wound model of infection, Antimicrob. Agents synthesis, Cancer Res. 55 (1995) 4361–4366. Chemother. 59 (2015) 6484–6493. [77] C.R. Chitambar, W.G. Matthaeus, W.E. Antholine, K. Graff, W.J. O'Brien, Inhibition of [103] A. Rangel-Vega, L.R. Bernstein, E.A. Mandujano-Tinoco, S.J. Garcia-Contreras, R. leukemic HL60 cell growth by transferrin-gallium: effects on ribonucleotide Garcia-Contreras, Drug repurposing as an alternative for the treatment of recalci- reductase and demonstration of drug synergy with hydroxyurea, Blood 72 trant bacterial infections, Front. Microbiol. 6 (2015) 282. (1988) 1930–1936. [104] C.R. Chitambar, Medical applications and toxicities of gallium compounds, Int. J. [78] J. Narasimhan, W.E. Antholine, C.R. Chitambar, Effect of gallium on the tyrosyl Environ. Res. Public Health 7 (2010) 2337–2361. radical of the iron-dependent M2 subunit of ribonucleotide reductase, Biochem. [105] R.P. Warrell Jr., Clinical trials of gallium nitrate in patients with cancer-related Pharmacol. 44 (1992) 2403–2408. hypercalcemia, Semin. Oncol. 18 (1991) 26–31. [79] C.R. Chitambar, J. Narasimhan, J. Guy, D.S. Sem, W.J. O'Brien, Inhibition of ribonucle- [106] R.S. Bockman, F. Wilhelm, E. Siris, F. Singer, A. Chausmer, R. Bitton, J. Kotler, B.J. otide reductase by gallium in murine leukemic L1210 cells, Cancer Res. 51 (1991) Bosco, D.R. Eyre, D. Levenson, A multicenter trial of low dose gallium nitrate in 6199–6201. patients with advanced Paget's disease of bone, J. Clin. Endocrinol. Metab. 80 [80] L.G. Marzilli, B. de Castro, J.P. Caradonna, R.C. Stewart, C.P. Van Vuuren, Nucleoside (1995) 595–602. complexing. A Raman and 13C NMR spectroscopic study of the binding of hard and [107] R. Bockman, The effects of gallium nitrate on bone resorption, Semin. Oncol. 30 soft metal species, J. A. Chem. Soc. 102 (1980) 916–924. (2003) 5–12. [81] T.C. Pochapsky, M. Kuti, S. Kazanis, The solution structure of a gallium-substituted [108] B. Leyland-Jones, Treatment of cancer-related hypercalcemia: the role of gallium putidaredoxin mutant: GaPdx C85S, J. Biomol. NMR 12 (1998) 407–415. nitrate, Semin. Oncol. 30 (2003) 13–19. [82] T. Shibahara, S. Kobayashi, N. Tsuji, G. Sakane, M. Fukuhara, Sulfur-bridged cubane- [109] A.W. Harris, A.E. Martell, Aqueous complexes of gallium, Inorg. Chem. 15 (1976) type molybdenum–gallium clusters with Mo(3)GaS(4)(n)(+) (n = 5, 6) cores. 713–720. X-ray structures of [Mo(3)GaS(4)(H(2)O)(12)](CH(3)C(6)H(4)SO(3))(5)·14H(2)O [110] L.R. Bernstein, T. Tanner, C. Godfrey, B. Noll, Chemistry and pharmacokinetics of and [Mo(3)GaS(4)(H(2)O)(12)](CH(3)C(6)H(4)SO(3))(6)·17H(2)O, Inorg. Chem. gallium maltolate, a compound with high oral gallium bioavailability, Metal- 36 (1997) 1702–1706. Based Drugs 7 (2000) 33–47. C.R. Chitambar / Biochimica et Biophysica Acta 1863 (2016) 2044–2053 2053

[111] K.P. Allamneni, R.B. Burns, D.J. Gray, F.H. Valone, L.R. Bucalo, S.P. Sreedharan, Wnt/beta-catenin signaling and proliferation by a ferrous iron chelator with thera- Gallium maltolate treatment results in transferrin-bound gallium in patient peutic efﬁcacy in genetically engineered mouse models of cancer, Oncogene 31 serum, 2004 230 (abstract 1013). (2012) 213–225. [112] C.R. Chitambar, D.P. Purpi, J. Woodliff, M. Yang, J.P. Wereley, Development of [119] M.Ndong,M.Kazami,T.Suzuki,M.Uehara,S.Katsumata,H.Inoue,K. gallium compounds for treatment of lymphoma: gallium maltolate, a novel Kobayashi, T. Tadokoro, K. Suzuki, Y. Yamamoto, Iron deﬁciency down-regulates hydroxypyrone gallium compound induces apoptosis and circumvents lymphoma the Akt/TSC1-TSC2/mammalian Target of Rapamycin signaling pathway in rats cell resistance to gallium nitrate, J. Pharmacol. Exp. Ther. 322 (2007) 1228–1236. and in COS-1 cells, Nutr. Res. 29 (2009) 640–647. [113] A.R. Timerbaev, Advances in developing tris(8-quinolinolato)gallium(iii) [120] J.H. Ohyashiki, C. Kobayashi, R. Hamamura, S. Okabe, T. Tauchi, K. Ohyashiki, The as an anticancer drug: critical appraisal and prospects, Metallomics 1 (2009) oral iron chelator deferasirox represses signaling through the mTOR in myeloid 193–198. leukemia cells by enhancing expression of REDD1, Cancer Sci. 100 (2009) [114] C.R. Chitambar, Gallium-containing anticancer compounds, Future Med. Chem. 4 970–977. (2012) 1257–1272. [121] S.V. Torti, F.M. Torti, Iron and cancer: more ore to be mined, Nat. Rev. Cancer 13 [115] R.R. Moskalyk, Gallium: the backbone of the electronics industry, Miner. Eng. 16 (2013) 342–355. (2003) 921–929. [122] J.N. Anastas, R.T. Moon, WNT signalling pathways as therapeutic targets in cancer, [116] M.J. Brookes, J. Boult, K. Roberts, B.T. Cooper, N.A. Hotchin, G. Matthews, T. Iqbal, C. Nat. Rev. Cancer 13 (2013) 11–26. Tselepis, A role for iron in Wnt signalling, Oncogene 27 (2008) 966–975. [123] J.A. Engelman, J. Luo, L.C. Cantley, The evolution of phosphatidylinositol 3-kinases [117] S. Song, T. Christova, S. Perusini, S. Alizadeh, R.Y. Bao, B.W. Miller, R. Hurren, Y. as regulators of growth and metabolism, Nat. Rev. Genet. 7 (2006) 606–619. Jitkova, M. Gronda, M. Isaac, B. Joseph, R. Subramaniam, A. Aman, A. Chau, D.E. [124] N. Chapuis, J. Tamburini, A.S. Green, L. Willems, V. Bardet, S. Park, C. Lacombe, P. Hogge, S.J. Weir, J. Kasper, A.D. Schimmer, R. Al-awar, J.L. Wrana, L. Attisano, Wnt Mayeux, D. Bouscary, Perspectives on inhibiting mTOR as a future treatment inhibitor screen reveals iron dependence of beta-catenin signaling in cancers, strategy for hematological malignancies, Leukemia 24 (2010) 1686–1699. Cancer Res. 71 (2011) 7628–7639. [118] G.S. Coombs, A.A. Schmitt, C.A. Canning, A. Alok, I.C. Low, N. Banerjee, S. Kaur, V. Utomo, C.M. Jones, S. Pervaiz, E.J. Toone, D.M. Virshup, Modulation of