Biochimica et Biophysica Acta 1863 (2016) 2044–2053

Contents lists available at ScienceDirect

Biochimica et Biophysica Acta

j ourna l homepage: www.e lsev ie r .com/ locate /bbamcr

Review

Gallium and its competing roles with iron in biological systems

Christopher R. ChitambarDivision of Hematology and Oncology, Department of Medicine, Medical College of Wisconsin, 9200 W. Wisconsin Avenue, Milwaukee, WI 53226, USA

Abbreviations: Tf, transferrin; TfR, transferrin receptoMT-2A, metallothionein-2A; HO-1, heme oxygenase-1.

E-mail address: chitambr@mcw.edu.

http://dx.doi.org/10.1016/j.bbamcr.2016.04.0270167-4889/© 2016 Elsevier B.V. All rights reserved.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 15 March 2016Received in revised form 27 April 2016Accepted 30 April 2016Available online 03 May 2016

Gallium, a group IIIa metal, shares chemical properties with iron. Studies have shown that gallium-basedcompounds have potential therapeutic activity against certain cancers and infectious microorganisms. Byfunctioning as an ironmimetic, gallium perturbs iron-dependent proliferation processes in tumor cells. Gallium'saction on iron homeostasis leads to disruption of ribonucleotide reductase, mitochondrial function, and theregulation of transferrin receptor and ferritin. In addition, gallium nitrate stimulates an increase inmitochondrialreactive oxygen species in cellswhich triggers downstreamupregulation ofmetallothionein andhemoxygenase-1.Gallium's anti-infective activity against bacteria and fungi results from disruption of microbial iron utilizationthroughmechanismswhich include gallium binding to siderophores and downregulation of bacterial iron uptake.Gallium compounds lack cross-resistance to conventional chemotherapeutic drugs and antibiotics thus makingthem attractive agents for drug development. This review will focus on the mechanisms of action of galliumwith emphasis on its interaction with iron and iron proteins.

© 2016 Elsevier B.V. All rights reserved.

Keywords:GalliumIronMetallodrug therapeuticsCancerInfectionRibonucleotide reductase

1. Introduction

Gallium compounds first entered medical application in the late1960s when it was discovered that 67Ga citrate when injected intotumor-bearing animals concentrated in sites of actively growing tumors[1]. Subsequent studies in humans confirmed these findings and the67Ga scan emerged as a tool for imaging tumors in patients [2]. Follow-ing evaluation in various malignancies, the 67Ga scan proved to be ofdiagnostic value in lymphoma to determine whether masses thatpersisted after completion of treatment represented viable or non-viable tissue [3]. Metabolically active lymphomatous masses continueto take up 67Ga while non-viable tumor masses do not [3]. With thedevelopment of more sophisticated imaging technology in theUnited States, the 67Ga scan has been replaced by the positron emissiontomography (PET) scan which is based on 18F-fluorodeoxyglucose up-take by tumors [4], while scanning with 68Ga-labeled radiopharmaceu-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 theworld where advanced scans are not available.

The observation that 67Ga could be taken up by tumors promptedthe logical question as to whether non-radioactive gallium compoundscould also concentrate in tumors and inhibit their growth. Preclinicalstudies that ensued revealed that gallium nitrate, when injected intoSprague–Dawley rats and CDFI mice with subcutaneously implantedtumors, suppressed tumor growth [7]. Following a series of preclinical

r; RR, ribonucleotide reductase;

animal studies to assess drug toxicity and establish safe dosing limits,gallium nitrate was deemed an investigational drug by the NationalCancer Institute and was advanced to future evaluation in clinicaltrials [8].

Human studies confirmed the antineoplastic activity of galliumnitrate in non-Hodgkin's lymphoma and bladder cancer [9–11], howev-er, an understanding of gallium's mechanisms of action at the cellularand molecular level lagged behind its use in the clinic. As a result, thedrug was evaluated in the clinic without a clear insight into its cellularandmolecular targets. Ongoing basic research however, has yielded im-portant information regarding the interaction of gallium with cellulariron metabolism. This is relevant to the advancement of a next genera-tion of gallium compounds as therapeutic agents for certain cancersand, more recently, for infections. This review will focus on our currentknowledge of gallium as a metal that competes with iron in biologicsystems and how this can be exploited for therapeutic purposes.

2. Gallium — chemistry

Credit for the discovery of gallium in 1875 is given to Paul-EmileLecoq de Boisbaudran who noted its presence as two distinct bands onspectroscopy. Gallium is a group IIIA metal, atomic number 31 thatexists in the earth's crust at a concentration of 5–15 mg/kg and isobtained as a byproduct of extraction of aluminum and zinc ores.It has a shiny, silvery white color with a melting temperature of28.7646 °C (85.5763 °F) that renders it near-liquid at room tempera-ture. It shares certain properties with iron (III) in that the octahedralionic radius for Ga3+ is 0.620 Å compared with 0.645 Å for high spinFe3+. In addition, the tetrahedral ionic radius is 0.47 Å and 0.49 Å for

2045C.R. Chitambar / Biochimica et Biophysica Acta 1863 (2016) 2044–2053

Ga3+ and Fe3+, respectively. The ionization potential and electronaffinity values for Ga3+ are 64 eV and 30.71 eV, respectively while forhigh spin Fe3+ they are 54.8 eV and 30.65 eV, respectively [12]. Galliumundergoes hydrolysis to yield a mixture of gallium hydroxides ofGa(OH)11 and Ga(OH)3 at pH ~ 4, and a mixture of Ga(OH)3 and GaGa(OH)4 at physiologic pH 7.4 [13].

3. Gallium as an iron mimetic in mammalian cells

The properties that gallium share with iron permits it to bind withhigh avidity to certain iron-binding proteins. However, while thebinding of iron to a protein promotes protein function, the substitutionof gallium for iron in a protein usually disrupts its function andmay leadto adverse downstream effects in cells.

3.1. Iron transport and cellular uptake

Abrief overviewof iron transport, cellular uptake, and storage is pro-vided to set the stage for discussing some of the similarities betweeniron and gallium. The reader is referred to recent reviews on ironmetab-olism for a detailed insight into this topic [14,15]. Following the absorp-tion of dietary iron in the duodenum, iron circulates in the blood bound

Fig. 1.A. An overviewof the cellular handling of iron. Iron is bound to transferrin (Tf) in the circuof Tf-Fe complexes. The Tf-Fe–TfR complex translocates from the cell surface to an intracellu(six membrane epithelial antigen of the prostate 3). Fe(II) exits the endosome through divaleto different compartments (mitochondria, ribonucleotide reductase, and others) presumablythe cell through cell membrane-based ferroportin. Cytoplasmic iron regulatory proteins (IRreceptors, ferritin, and ferroportin at the mRNA translational level by interaction with iron re

shows the known sites of interaction of gallium with cellular iron metabolism. B. Poten

proteins in the citric acid cycle and mitochondrial complexes are potential targets for the cytot

to transferrin (Tf), an 80 kDa protein containing two iron-binding sites,one at the carboxy and another at the amino terminal. Under physiolog-ical conditions, approximately one-third of circulating Tf is occupied byiron (III) (Tf-Fe) while the remaining two-third of Tf is available tobind additional metals, or additional iron when plasma iron levels areelevated in pathologic conditions.

The Tf–TfR cycle is summarized in Fig. 1A. Tf-Fe is taken up by cellsvia cell surface Tf receptor1 (TfR1)-mediated endocytosis. Once inter-nalized, TfR1–Tf-Fe locates in an acidic endosome where iron isreleased, reduced to Fe(II) by a ferrireductase, and transported out ofthe endosome to the cytoplasm by a membrane-based divalent metaltransporter-1 (DMT-1). The subsequent steps in iron trafficking withinthe cells are somewhat obscure, however it is generally agreed thatiron moves to a labile “pool” bound to low molecular weight ironchelates to maintain solubility. From this pool, iron is utilized for avariety of critical cellular purposes including the functioning of theM2-subunit of ribonucleotide reductase (RRM2) (Fig. 1A),mitochondri-al iron–sulfur cluster-containing proteins (Fig. 1B), and certain steps inthe cell cycle regulation [14,15].

Cellular iron homeostasis is tightly regulated and is balanced by theinterplay of proteins responsible for iron import, storage, and export.A decrease in iron below a critical level can trigger processes leading

lation and is incorporated into cells by transferrin receptor1 (TfR1)-mediated endocytosislar acidic endosome where Fe(III) dissociates from Tf and is reduced to Fe(II) by STEAP3nt metal transporter1 (DMT1, not shown) to a labile iron “pool.” From here, iron trafficsbound to low-molecular weight ligands. Excess iron is stored in ferritin. Iron exits fromPs) function as sensors of cellular iron status and regulate the synthesis of transferrinsponse elements (IREs) present on the untranslated regions of their respective mRNAs.

tial sites of interaction of gallium in the mitochondria. The iron–sulfur cluster (Fe–S)

oxic 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-lyze the production of damaging hydroxyl radicals and superoxide. Thusas a cytoprotective process, excess iron not utilized for cellular functionis stored in ferritin, a shell-shaped protein composed of 24 subunits ofH- and L-ferritin [16]. To control the iron entry and storage, cellulariron status is sensed by cytoplasmic iron regulatory proteins (IRPs).These proteins regulate the translation of ferritin and TfR1 mRNAsthrough an interaction with iron response elements present on the 5′and 3′ untranslated regions of their respective mRNAs. Under condi-tions of iron excess, IRP–IRE interactions are suppressed resulting inan increase in ferritin synthesis and a decrease in TfR synthesis [14,15]. The converse occurs with cellular iron deprivation; here, RNA–IREinteractions increase resulting in a decrease in ferritin and an increasein TfR synthesis [14,15].

Whereas TfR and ferritin play central roles in cellular iron uptakeand storage, iron balance in cells is also maintained by ferroportin, amembrane-based protein that exports iron from cells. The binding offerroportin to hepcidin, a 25 amino acid peptide produced in the liver,results in the internalization of the ferroportin–hepcidin complex andits degradation in the lysosome [17,18]. This results in a decrease incellular iron efflux and an increase in cellular iron content.

The ferroportin/hepcidin axis has clinical implications. Recentstudies show that thedownregulation of ferroportin coupledwith an in-crease in hepcidin in breast cancer tumors is associated with an adverseclinical outcome [19,20]. Earlier studies showed that TfR expressionmay be increased in certain malignancies such as lymphoma, bladdercancer, glioblastoma, and breast cancer and that this is associated withbiologically aggressive tumor behavior [21–24]. Thus, a scenario ofincreased iron import (elevated TfRs) and diminished iron export(decreased ferroportin) would elevate intracellular iron levels to meetthe high demands of tumor growth. Moreover, elevated intracellulariron could catalyze the formation of pro-carcinogenic reactive oxygenspecies (ROS).

3.2. Gallium transport and cellular uptake

The human body appears to handle gallium as ironwith regard to itstransport in the blood. Clues to similarities between gallium and ironwere provided by early studies which demonstrated that 67Ga uptakeby malignant cells in vitro could be increased by the addition of exoge-nous Tf to the culture medium [25,26]. The biologic relevance of thisobservationwas confirmed in animal studies where 67Ga injected intra-venously in rabbits as 67Ga citrate was bound exclusively to Tf in theblood [27]. The studies of Harris and Pecararo clearly established thatgallium binds to Tf with high avidity [28]. Modeling data summarizedby Bernstein [12] suggests that at concentrations up to 50 μM galliumin the blood binds entirely to Tf. At higher blood concentrations whichexceed the metal-binding capacity of Tf, gallium's binding to Tf dimin-ishes and it circulates as gallate [Ga(OH)4−] [12]. Experiments withdifferent cell lines in vitro indicated that 67Ga was taken up by cells viaTfR-mediated endocytosis of 67Ga–Tf [26,29–31]. The role of TfR incellular 67Ga uptake was further supported by studies which showedthat 67Ga uptake by human promyelocytic leukemic HL60 cells couldbe blocked by 42/6, an anti-TfR monoclonal antibody (MoAb) thatblocks TfR internalization [30]. 67Ga uptake directly correlated withcell surface TfR levels [30]. The importance of TfR in 67Ga uptake wasconfirmed in a panel of lymphoid cell lines [31]. In a tumor-bearingmurine model, anti-TfR MoAb B3/25 produced a 75% reduction in 67Gauptake by subcutaneously implanted melanoma cells, lending furthersupport to the importance of TfR in gallium uptake [32]. 67Ga uptakeby a mutant Chinese hamster ovary (CHO) cell line that does notendogenously express the TfR increased significantly when these cellswere transfected to overexpress the TfR [33]. Increased 67Ga uptakeby these cells was seen both in vitro and in vivo when these cellswere grown as explants in nude mice [33]. However, although TfR-mediated endocytosis and the translocation of Tf-metal to an acidic

endosome is an initial step in the cellular uptake of both gallium andiron, a significant fraction of gallium appears to cycle back out of thecell suggesting that all of the incorporated gallium is not unloaded tothe cytoplasm [34]. This is in contrast to iron where the majority ofiron unloaded from Tf enters the cytoplasm. Furthermore, while DMT-1 plays a role in transporting iron (II) out of the acidic endosome, itdoes not appear to be involved in the transport of gallium [35].

Proteins thatmodify TfR endocytosismay affect cellular 67Ga uptake.Thewild-type (wt) hemochromatosis gene product (HFE protein) bindsto TfR at sites that overlap with Tf binding sites on the receptor [36,37].In patients with hereditary hemochromatosis, an inherited iron over-load disease caused by a C282Y mutation of the HFE gene, the mutatedHFE protein fails to associate with TfR. Thus, when HFE is mutated orwhen wt HFE is absent there is an increase in cellular Tf-Fe uptake[38]. Conversely, when wt HFE is expressed it produces a decrease inTf-Fe uptake relative to cells lacking wt HFE and a state of cellular irondeficiency [39]. 67Ga uptake studies conducted in HeLa cells containinga tet-off and tet-on HFE expression system showed that 67Ga-Tf uptake(like that of iron) was significantly reduced when the wt HFE gene wasturned on relative to when HFE expression was turned off [40].

Gallium may also be incorporated into cells through a TfR-independent process akin to the Tf-independent uptake mechanismfor iron [41]. However, it remains to be determined whether theTf-independent uptake of gallium and iron uptake occurs through pre-cisely the same transporter. Despite similarities between gallium andiron, it is likely that different cellular uptake systems are responsiblefor each metal. This is suggested by the finding that TfR-independentuptake or iron from Tf-Fe or Fe-citrate by cells involves the reductionof Fe(III) to Fe (II) by a cell surface ferrireductase [42,43] and/or the gen-eration of free radicals [44]. In contrast, gallium is not reduced fromGa(III) to Ga(II) and would need to be transported into cells as Ga(III)or a Ga (III)-chelate.

Although both gallium and iron circulate in the blood bound to Tfand are taken up by cells via TfRs, they display different pharmacokinet-ics in humans. Following intravenous injection of 67Ga-citrate and 59Fecitrate in healthy individuals, the elimination rate constant of 67Ga cit-rate is 50-times slower than that of 59Fe citrate, indicating that 59Fe iscleared from the blood at a more rapid rate than 67Ga [45]. The volumeof distribution of 67Ga in the body is approximately 6 times that of 59Feas the lattermetal is largely confined to hematopoietic tissues for hemo-globin production. The uptake of 59Fe by the sacrum, liver, spleen, andheart follows classically described ferrokinetics with uptake progres-sively declining after achieving a peak uptake at 24 h following injection[45,46]. In contrast 67Ga rapidly accumulates in the same tissue in thefirst 24 h following injection but then progressively increases in thesetissues with time [45].

3.3. Intracellular distribution of gallium

Whereas the similarities between gallium and iron with respect tobinding to Tf and uptake by the TfR have been described, much less isunderstood about the intracellular trafficking of gallium. Initial studiesconducted in the 1970s with a view to understanding the cellularlocalization of 67Ga following its uptake by normal and malignant cellsreported 67Ga to be bound to a 45 kDa protein in cell extracts from rathepatoma [47,48]. Additional studies showed 67Ga to preferentiallylocate in hepatic microvesicles and lysosomes [49–51]. Beyond beingable to concentrate in metabolically active tumors in vivo, 67Ga alsoconcentrates in sites of inflammation and infection in vivo [52]. Here,67Ga behaves like iron in that it binds strongly to the iron-bindingprotein lactoferrin present in neutrophils, an essential component ofthe inflammatory response. 67Ga binding to lactoferrin is 50-fold stron-ger than to Tf [53]. Apart from binding to lactoferrin, 67Ga may localizein sites of infection by exchanging with iron in siderophores. The latterare low molecular weight iron chelating compounds that are releasedby microorganisms to acquire iron for their growth [54]. Gallium and

2047C.R. Chitambar / Biochimica et Biophysica Acta 1863 (2016) 2044–2053

other trivalent metals are capable of displacing iron from siderophoresunder reducing conditions [55]. The decrease in pH that occurs in anabscess at the site of infection would favor the exchange of gallium foriron in siderophores.

Gallium may also bind to ferritin. In an in vitro equilibrium dialysismulti-chamber system, 70% of 67Ga bound to Tf transferred to horsespleen ferritin in the presence of ATP [56]. Ascorbate, citrate, and lactatealso facilitated 67Ga transfer to ferritin but to a far lesser extent than ATP[56]. Other studies show 67Ga to be associatedwith immunoprecipitableferritin in cells [30]. However, none of these studies demonstrate thatgallium is actually incorporated into the ferritin shell. In fact, it is morelikely that gallium, when associated with ferritin, is bound to a site onthe external surface of the protein, however this has not been studied.Ferritin does not appear to play a role in modulating 67Ga uptake bycells since a 20-fold increase in ferritin levels in HL60 cells was notaccompanied by a parallel increase in cellular 67Ga uptake [30].Interestingly though, exposure of CCRF-CEM cells to gallium nitrate in-duces H-ferritin mRNA expression [57]; the mechanisms responsiblefor this induction remain to be determined.

3.4. Action of gallium on cellular iron-dependent processes

3.4.1. Cellular iron uptakeGallium, as Tf-Ga, can compete with Tf-Fe for binding to TfR and

inhibit receptor-mediated iron uptake by cells [58]. This gallium-induced block in cellular iron incorporation may also include a disrup-tion of endosome acidification which affects the dissociation of ironfrom Tf. As a result, iron cycles out of the cell rather than beingtransferred across the endosome to the cytoplasm [58].

The gallium-induced interference in cellular iron uptake renderscells relatively iron-deficient; this has adverse downstream effects oncertain iron-dependent processes and is particularly striking inerythroid cells that require iron for hemoglobin production. Tf-Gadiminishes DMSO-induced hemoglobin production and 59Fe incorpora-tion into hemoglobin in Friend erythroleukemia cells [59]. The effect ofgallium on hemoglobin production is independent of any effect on cellproliferation since other drugs that inhibit the growth of these cells donot block hemoglobin synthesis [59]. The level of hemoglobin produc-tion in gallium-treated cells can be restored to normal by the additionof Tf-Fe or iron pyridoxal isonicotinyl hydrazone (Fe-PIH), thus indicat-ing that the block in hemoglobin production is due to a decrease in in-tracellular iron [59]. Cancer patients treated with gallium nitrate maydevelop microcytic anemia and an elevated red cell zinc protoporphy-rin; both these features occurwith iron deficiency and provide evidencethat galliumnitrate interfereswith ironmetabolism in vivo [60]. Galliumsulfate has been shown to reduce the activity of delta-aminolevulinicacid (ALAD) dehydratase in the heme biosynthesis pathway in theliver, kidney, and erythroid cells in vivo [61]. This could also contributeto the development of anemia in patients treated with gallium nitrate.

As might be anticipated with cellular iron deficiency, murine L1210,human promyelocytic leukemia HL60, and Friend erythroleukemia cellsincubated with Tf-Ga display an increase in TfR mRNA and proteinexpression [62]. However, for reasons not understood, this effect ofgallium is not uniformly seen in other cells. In human lymphomaCCRF-CEM cells, Tf-Ga blocks iron uptake but does not produce anupregulation of TfR expression [62].

In contrast to inhibiting TfR-mediated cellular iron uptake, galliumnitrate stimulates the Tf-independent uptake of ferric nitriloacetic acid(Fe-NTA) by HL60 cells [41]. These experiments, conducted in serum-free medium to ensure the absence of Tf, showed that an increase inTf-independent Fe-NTA uptake occurred within 5 min of incubation ofcells with gallium nitrate and could be saturated with increasing con-centrations of Fe-NTA. The upregulation of Fe-NTA uptake persistedfor at least 24 h after removal of gallium from cells. These studies clearlyindicated that gallium nitrate can upregulate a Tf-independent trans-porter for iron. It was also shown that incubation of HL60 cells to ferric

chloride produced an increase in Tf-independent cellular 67Ga uptake[41]. The ability of gallium and iron to stimulate each other's uptake in-dicates that they either use the same Tf-independent transport systemfor entry into HL60 cells or activate a process responsible for the uptakeof either metal.

In another study, Richardson investigated the TfR-independent up-take of Tf-iron by human SK-Mel-28 melanoma cells and demonstratedthat pre-incubation of the cells with gallium nitrate stimulated theirTfR-independent uptake of 59Fe from 59Fe-Tf [63]. Since Ga(III) is notredox-active, it was concluded that gallium-stimulated iron uptake didnot require the generation of free radicals [63]. The effect of galliumon Tf-independent iron uptake is not limited to malignant cell lines.Sturm et al. showed that when primary hepatocytes isolated fromSprague–Dawley rats were pre-incubated with gallium nitrate therewas an increase in their uptake of non-transferrin-bound iron (NTBI)in a time and concentration-dependentmanner [64]. In contrast, differ-ent divalent metals (Zn, Ni, Co, Cd, Mn, and Cu) had minimal influenceon iron uptake [64].

The Tf-independent iron uptake system can deliver sufficient iron tosupport cellular viability and growth [65]. Theoretically therefore, ifcertain cells are capable of bypassing the gallium-induced block inTfR-mediated iron uptake by activating TfR-independent iron uptakethey might be less sensitive to the cytotoxicity of gallium. The rela-tive contribution of TfR-dependent and TfR-independent iron uptakepathways is likely to differ among cell types and may, in part explaindifferences in cell sensitivity to gallium compounds.

3.4.2. Ribonucleotide reductase (RR)The synthesis of ribonucleoside diphosphate to deoxyribonucleotide

diphosphate, an essential step in DNA synthesis, is catalyzed by RR,a heterodimer which consists of homodimeric M1 and homodimericM2 subunits [66–68]. RRM1and RRM2 are under the control of differentgenes and are expressed at different phases of the cell cycle. RRM1 ispresent during all phases of the cell cycle while RRM2 appears in lateG1 as cells enter S-phase [69,70]. Both subunits come together to forman actively functioning enzyme. RRM1 contains effector and substratebinding sites whereas the RRM2 subunit contains a binuclear ironcenter and a tyrosyl free radical (Fig. 1A). The tyrosyl radical signal inintact cells and cell lysates can be detected by electron paramagneticresonance (EPR) spectroscopy; the amplitude of this signal correspondsspecifically to the activity of RRM2 and thus to overall RR enzyme activ-ity [71]. Disruption of the tyrosyl radical by radical scavenging agentsleads to loss of RR activity. The iron center is essential for RRM2 functionand is closely linked to the EPR signal of RRM2. Loss of iron from RRM2results in a loss of the tyrosyl radical and RRM2 function. When ironavailability is low, RRM2 protein is still present in cells; however, inthis situation it exists as apoRRM2 (devoid of iron) that lacks a tyrosylradical signal and is functionally inactive [72]. RRM2 protein has ahalf-life of about 4 h and turns over frequently during cell proliferation[70]. To maintain the activity of newly synthesized M2 protein, cellsrequire a constant supply of iron. The cellular requirement for iron tosupport RR activity is reflected by the increase in TfRs that occurs ascells enter the S-phase of the cell cycle [73–76].

Gallium compounds inhibit the activity of RR by a dual mechanism.Early studies demonstrated that Tf-Ga produced a decrease in theRRM2 subunit tyrosyl radical signal within 6 h of incubation with intactHL60 cells and that this signal was lost by 18 h [77]. Consistent with ablock in RR activity, cells incubated with Tf-Ga displayed a decrease indNTP pools compared with untreated cells [77]. Co-incubation of cellswith hemin as a source of iron delivered to cells independent of TfRprevented the gallium-induced inhibition of the RRM2 tyrosyl radicalsignal in intact cells. Cytoplasmic extracts of 1210 cells prepared aftertheir incubation with gallium nitrate for 18 h displayed a significantdecrease the RRM2 tyrosyl radical EPR signal [78]. However, the tyrosylradical was restored to normal within 10 min following the addition offerrous 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 butinterferes with the availability of iron to support RRM2 activity. Whilethe inhibition of iron availability for RR should itself be sufficient to in-hibit DNA synthesis, galliumalso blocks RR activity through an addition-al mechanism which involves competitive inhibition of substrate (CDPand ADP) binding to the enzyme [79]. This potential mechanism ofaction of gallium has not been further elucidated; however, since galli-um can form complexes with nucleotides [80], it is entirely likely thatGa-ADP or Ga-CPD formed in vitro might compete with the substratesADP and CDP for binding to RR. Thus, gallium inhibition of RR appearsto occur through two distinct mechanisms: intracellular iron depriva-tion which limits iron availability for RRM2, and by competitiveinhibition of substrate binding to the enzyme.

3.4.3. Action of gallium on the mitochondriaGiven the importance of iron–sulfur cluster-containing proteins in

the citric acid cycle and the electron transport chain of the mitochon-dria, it appears logical to surmise that gallium might interact withthese proteins (Fig. 1A). Although Ga–S clusters have been chemicallysynthesized in vitro [81,82], there are few studies of gallium's actionon the mitochondria. In lymphoma cell lines, gallium nitrate inducesapoptosis in CCRF-CEM cells primarily through the mitochondrialpathway. This involves the activation of Bax, a pro-apoptotic proteinin the cytoplasm, and its translocation to the mitochondria whichleads to a loss of mitochondrial membrane potential, the release of mi-tochondrial cytochrome c and the downstream activation of execution-er caspase-3 [83]. These events, which culminate in apoptosis occurover a 24 h incubation of cells with gallium nitrate, and are precededhours earlier by a decrease in cellular oxygen consumption rate [84].The earliest measureable cellular changes that suggest that galliumacts on the mitochondria are seen after a 1–2 h incubation of cellswith gallium nitrate. At these early time-points, cells display a decreasein GSH/GSSG ratio and an increase in ROS indicating that gallium in-duces oxidative stress in cells [84]. This increase in cellular ROS can beabrogated by mitoquinone, a mitochondria-targeted antioxidant [85].This suggests that ROS in gallium-treated cells originates from themito-chondria and is the result of an action of gallium on mitochondria. It isknown that blockade of mitochondrial complexes I, II, and III can resultin a leak of ROS from the mitochondria [86], but it remains to be deter-mined whether the increase in mitochondrial ROS content in cells ex-posed to gallium nitrate is due to inhibition of these mitochondrialcomplexes. Studies are in progress to define the site of gallium's actionon the mitochondria.

4. Cellular adaptation to the cytotoxicity of gallium nitrate

4.1. Changes in iron metabolism

The development of cell lines with acquired resistance to thegrowth-inhibitory action of gallium nitrate has revealed insights intothe mechanisms of action of gallium. To study the basis for drug resis-tance to gallium nitrate, human leukemic HL60 cells were continuouslyexposed for 9–12 months to subtoxic concentrations of gallium nitrate.This resulted in the emergence of a clone of HL60 cells that were nearly29-fold more resistant to growth inhibition by gallium nitrate than theparental gallium-sensitive cell line; the concentration of gallium nitratethat inhibited cell growth by 50% was ~80 μM in the parental gallium-sensitive line compared with ~2296 μM in the gallium-resistant cells[87]. Gallium uptake was similar in both sensitive and resistant cellsindicating that resistance did not involve a decrease in gallium uptakeby resistance cells [87]. However, TfR expression and ferric chlorideuptake was increased in resistant cells relative to sensitive cells,suggesting that the development of a high level of gallium resistancein HL60 cells involved changes in cellular iron incorporation to compen-sate for the perturbation of intracellular iron homeostasis produced bygallium nitrate [87].

The magnitude of resistance to gallium nitrate may influence theextent of compensatory changes in iron metabolism as cells adapt togallium nitrate. In another clone of HL60 cells that was only 6.6-foldmore resistant to gallium nitrate than the gallium-sensitive cells, TfRexpression and Tf-iron uptake by gallium-resistant cells were de-creased relative to gallium-sensitive cells [88]. Furthermore,gallium-resistance was associated with distinct differences in ironand gallium trafficking; in resistant cells 59Fe taken up by cells wasassociated with a Tf–TfR1–HFE complex whereas this associationwas not seen in gallium-sensitive cells [88].

Studies with human CCRF-CEM lymphoma cells with acquiredresistance to gallium nitrate have been more revealing as to changesin iron homeostasis that occur with the development of galliumresistance. Here, gallium-resistant cells display a downregulation ofTfR expression and a decrease in their accumulation of iron and galliumover time [57,89]. Ferritin levels in resistant cells are six to ten-foldlower in the resistant cells while the binding of IRP-1 to ferritin IREmRNA is increased [57]. This indicates that the gallium-induced de-crease in ferritin in resistant cells is due to changes at the level of ferritinmRNA translation [57]. The cytotoxicity of gallium to gallium-resistantcells can be restored by increasing the level of exogenous apoTf in theculture, thus strongly suggesting that themechanism of drug resistanceto gallium nitrate involves a decrease in gallium accumulation in cells[57]. While this could be solely due to changes in cellular TfR-mediated gallium uptake, the possibility that an increase in galliumexport from the cell via ferroportin or another pathway has not beenexamined.

4.2. Metallothionein and heme oxygenase-1

A comparison of the expression of 96 genes related to metal metab-olism in gallium-resistant and -sensitive CCRF-CEM cells revealed strik-ing differences in an unforeseen area. Relative to gallium-sensitive cells,resistant cells displayed a 32- and 28-fold upregulation of the genes formetallothionein-2A and metallothionein-3, respectively. Also upregu-lated were the genes for zinc transporter-1 (ZnT-1) (7.6-fold increase)andmetal-responsive transcription factor-1 (MTF-1) (2.2-fold increase)[90]. This upregulation ofmetallothionein in gallium-resistant cells can-not be explained on the basis of ironmimicry by gallium since iron doesnot influencemetallothionein levels. Metallothionein is a lowmolecularweight protein that binds zinc and is involved in zinc metabolism [91,92]; it sequesters toxic divalent metals such as cadmium [93].

A role formetallothionein in modulating the cytotoxicity of gallium-sensitive cells was suggested by studies in which gallium-sensitive cellswith zinc-induced upregulation ofmetallothionein displayed a decreasein the cytotoxicity of gallium nitrate. This protective effect of metallo-thionein against gallium's cytotoxicity was lost when metallothioneinlevels returned to baseline [90]. A direct correlation between endoge-nous metallothionein expression in a panel of lymphoma cell lines andtheir sensitivity to growth inhibition by gallium nitrate could bedrawn, further suggesting a connection between gallium's mechanismof action and changes in metallothionein [90].

Although at first glance it would appear that gallium's interactionwith cellular metal metabolism involves both iron and zinc, furtherstudies suggested that the induction of metallothionein expression bygallium nitrate may be mediated through an indirect mechanism. Asstated earlier, exposure of gallium-sensitive CCRF-CEM cells to galliumnitrate produces an increase in intracellular mitochondrial ROS within1–2 h [84]. Additional studies showed that with continued exposureto gallium nitrate, these cells displayed increased levels of metallothio-nein and heme oxygenase-1 (HO-1) at 6 and 16 h, respectively [84]. Thegallium nitrate-induced increase in HO-1 involves the triggering thep38p MAP kinase pathway with downstream activation of Nrf2 aleucine zipper transcription factor that interacts with antioxidantresponse elements present in the promoter region of the HO-1 gene.Both MT and HO-1 induction can be blocked by the antioxidant

2049C.R. Chitambar / Biochimica et Biophysica Acta 1863 (2016) 2044–2053

n-acetyl cysteine indicating that gallium-induced ROS production playsa central role in the upregulation of MT and HO-1 expression [84].

The above described sequence suggests a model for gallium'smechanisms of action as illustrated in Fig. 2. Here, following its cellularuptake, gallium induces the release of mitochondrial ROS which in turnleads to the sequential induction of MT2A and HO-1 gene expression.Both MT2A and HO-1 are likely to represent an initial cytoprotectiveaction by cells in response to gallium-induced stress. However, withcontinued cellular uptake of gallium and an expansion of the intracellu-lar gallium pool, these protective mechanisms are overwhelmed, irondelivery to RRM2 is blocked and cell death ensues. The sustained upreg-ulation ofMT in gallium-resistant cells even in the absence of exogenousgallium nitrate suggest that these cells may have adapted to gallium byincreasing their mitochondrial activity and basal level of ROS (Ahmedand Chitambar, unpublished observations). Further studies are inprogress to elucidate this process.

5. Gallium as an iron mimetic in microbial systems

Microorganisms require iron for their survival and growth howevertheir mechanisms of iron acquisition are distinctly different from thoseof mammalian cells [54]. Bacteria and fungi secrete siderophores. Theseare low-molecular weight peptide-based catecholates, hydroxamates,or hydroxycarboxylates that chelate iron with high affinity and returnit to the microorganism for its use [54]. In addition, certain bacteriamay produce hemophores (analogous to siderophores) designed to ac-quire iron from exogenous heme (iron-protoporphyrin). Other bacteriamay obtain iron from transferrin and lactoferrin through receptors forthese proteins [54,94]. It is not unexpected therefore that gallium mayinterpose itself in place of iron in these systems and interfere withmicrobial iron uptake. Gallium has been shown to exchange for iron insiderophores [55]. The siderophore pyochelin has been shown to trans-port gallium into Pseudomonas aeruginosa [95]. As with mammaliancells, gallium cannot be utilized for essential iron-catalyzed reactions.This leads to the death of the organism. In addition, gallium per se mayperturb the regulation of iron acquisition systems as was shown inP. aeruginosa where gallium nitrate repressed pvdS, a transcriptionalregulator for pyoverdine, a siderophore produced by this organism [96].

Fig. 2. Mitochondria-related events triggered by gallium nitrate. An early event in thecellular response to gallium is an increase in intracellular reactive oxygen species (ROS)of mitochondrial origin. Changes in zinc homeostasis along with ROS generation inducethe expression of metallothionein-2A (MT-2A) through increased binding of metaltranscription factor-1 (MTF-1) to metal response elements (MRE) on the MT-2A gene.ROS may also trigger MT2A gene expression via action on antioxidant responseelements (ARE). Heme oxygenase-1 (HO-1) gene expression is triggered by ROS via thep38 MAP kinase pathway and activation of transcription factor Nrf2. ROS and thedownstream activation of MT-2A and HO-1 can be blocked by the antioxidant N-acetylcysteine (NAC).

A number of investigations in animal models of infection haveclearly indicated that gallium's ability to interfere with microbial ironutilization has therapeutic potential. Olakanmi et al. demonstrated thatgallium nitrate displayed antimicrobial activity against Mycobacteriumtuberculosis and Mycobacterium avium residing inside and outside ofmacrophages by disrupting their iron metabolism [97]. Further studiesshowed that gallium nitrate inhibited the growth of M. tuberculosis ina murine model and M. avium subspecies paratuberculosis in neonatalcalves [96,98]. Kaneko et al. showed that gallium inhibited the growthof P. aeruginosa, inhibited blocked biofilm formation and killedplanktonic and biofilm bacteria in vitro, and was effective againstthis pathogen in murine lung infection models [96]. In another study,the topical delivery of gallium combinedwith desferrioxamine enhancedthe antimicrobial action of gentamicin in a rabbit model of P. aeruginosacorneal infection [99]. In a murine model of P. pseudomonas-infectedburn injury all the control animals treatedwith a vehicle died from infec-tion whereas, 100% of the animals who received subcutaneously admin-istered gallium maltolate were protected from systemic infection anddeath [100]. Gallium maltolate significantly decreased the colonizationof Staphyloccus aureus and Acinetobacter baumannii in the wounds ofthermally injured mice [100]. In a murine model, orally administeredgalliummaltolate showed activity against Rhodococcus equi. This bacteri-um can produce pneumonia in foals and immunocompromised individ-uals, and multiply in macrophages resulting in granulomatous lesions[101]. Other studies demonstrated that gallium citrate applied topicallyto skin wounds infected with Klebsiella pneumoniae reduced bacterialburden and biofilm formation [102].

Beyond animal studies, a clinical trial of gallium nitrate for thetreatment of P. aeruginosa in patients with cystic fibrosis is in progressand the results of this study are eagerly awaited.

Thus, a new direction for the use of gallium in medicine hasemerged. Given the growing concern over antibiotic resistance inpathogenic organisms and the rising cost of newer antibiotics thatadds to the financial burden of developing countries, research to definethe efficacy of gallium compounds as antimicrobial is a step in the rightdirection. The readers are referred to recent reviews on gallium'spotential as an antimicrobial agent [94,103].

6. Therapeutic gallium compounds

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

6.1. Gallium nitrate

Gallium nitrate (Ganite™) was the first compound to enter clinicaluse and undergo extensive evaluation in humans. As a result, thereexists a considerable body of information on the efficacy and toxicityof this gallium formulation [8,104]. Although initially evaluated as anantineoplastic drug, gallium nitrate was found to reduce blood calciumlevels and decrease bone turnover in patients with hypercalcium andPaget's disease of the bone [105,106]. Gallium concentrates in thebone and reduces bone resorption by inhibiting osteoclast activity[107]. Early studies suggested that gallium's action on bonemetabolismwas independent of iron and Tf [107], but in light of recent advances inour knowledge of ironmetabolism, this relationshipmight beworthy ofreevaluation. Presently, gallium nitrate is FDA-approved for treatmentof hypercalcemia in cancer patients [108] but, it has also shown antineo-plastic activity against bladder cancer and lymphoma in numerousclinical trials [9–11]. It is worth noting that the solution of galliumnitrate marketed for clinical use is actually a mixture of 25 mg/mlgallium nitrate and 28.75 mg/ml sodium citrate dehydrate. Based onthe aqueous chemistry of gallium [13,109], it is likely that galliumnitrate administered to patients consists of a mixture of galliumhydroxides [primarily Ga(OH)3 at neutral pH] and gallium citrate.

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

2050 C.R. Chitambar / Biochimica et Biophysica Acta 1863 (2016) 2044–2053

6.2. Gallium maltolate

Gallium maltolate can be considered a second generation galliumcompound which, in preclinical studies, appears to have certain advan-tages over gallium nitrate. The compound consists of a central galliumatom bound to three maltol [tris(3-hydroxy-2methyl-pyrone)] ligandsin a propeller-like arrangement [110]. Unlike gallium nitrate, galliummaltolate is an oral formulation that has good bioavailability in animaland human studies. Serum gallium levels of 0.115 and 0.569 μg/mlhave been achieved following a single oral dose of 100–500 mg galliummaltolate; this is comparable to steady-state levels achieved with galli-um nitrate administered by continuous intravenous infusion [110]. Themajority of gallium appearing in the circulation after orally adminis-tered gallium maltolate is bound to Tf [111]. Galliummaltolate appearsto have mechanisms of cytotoxicity that are different from galliumnitrate. For example, gallium maltolate inhibits the growth of lympho-ma cell lines with acquired resistance or endogenous resistance to thecytotoxicity of gallium nitrate [112]. Gallium maltolate is also cytotoxicto WTK-1 lymphoma cells which possess a p53 mutation. In contrast,these cells are resistant to growth inhibition by gallium nitrate as wellas to radiation [112]. These observations support the notion that thebiologic mechanisms for tumor cell resistance to gallium nitrate aredifferent than gallium maltolate.

6.3. Tris(8-quinolinolato)gallium(III)

Tris(8-quinolinolato)gallium(III) (KP46) is a gallium complex boundto the organic ligand 8-quinolinol. This complex displays high thermo-dynamic and kinetic stability and is stable in water for hours indicatingthat it does not rapidly release gallium to Tf. The drug has undergone

extensive preclinical testing and is in clinical trials as reportedelsewhere [113].

6.4. Gallium citrate

Gallium citrate (Panaecin™) is the newest addition to gallium drugsin development; it is being evaluated in clinical trials as a novel drugfor the treatment of a variety of infections.

In addition to the above compounds, a number of novel gallium-containing compounds have been shown to have anti-proliferativeactivity against cell lines in vitro and are in development [114]. Furtherevaluation of the therapeutic activities for these gallium-based agentsis awaited.

7. Conclusion

Gallium does not have a known role in normal cellular physiology.However, the fascination with this metal in biology relates to the factthat it shares certain chemical properties with iron that enable it to betaken up by tumor cells and microorganisms as an iron mimetic. Thishas resulted in the development of gallium-based agents for use indiagnostic and therapeutic medicine. Studies with gallium have provid-ed insights into the cellular handling of metals and iron metabolism.Beyond the biological applications of gallium, it is worth noting that gal-lium displays semiconducting properties and is being used extensivelyas gallium arsenide in the electronic industry [115].

Although there has been progress in our understanding of the inter-action of gallium with biologic systems, much remains to be learnedabout its interactionwith other iron-dependent processes. For example,iron plays a key role in cell signaling through the WNT and mTORpathways [116–121]. Aberrant signaling through these pathways has

2051C.R. Chitambar / Biochimica et Biophysica Acta 1863 (2016) 2044–2053

been implicated in malignancy [122–124]. Could gallium compoundsact on the iron-dependent steps in these pathways? If so, it could haveimplications for cancer therapy. Further research will be necessary toaddress these questions. Clinical trials of gallium nitrate in cancerdemonstrated responses to treatment in patients with non-Hodgkin'slymphoma and urothelial cancers. However, these trials were conduct-ed without an understanding of gallium nitrate's mechanisms of actionand gallium nitrate is often dismissed as an “old drug.”However, newergallium compounds have emerged that display greater antitumorefficacy than gallium nitrate in preclinical testing. If the newer galliumcompounds are to be advanced for the treatment of malignancy andinfections, further investigation needs to focus on gaining a betterunderstanding of gallium's cellular targets and the moleculardeterminants of sensitivity and drug resistance.

Transparency document

The Transparency document associated with this article can befound, in the online version.

Acknowledgements

Thisworkwas supported by the ThomasA. and LorraineM. RosenbergAward for Translational Cancer Research from the Froedtert HospitalFoundation and the Medical College of Wisconsin.

References

[1] C.L. Edwards, R.L. Hayes, Tumor scanning with 67Ga citrate, J. Nucl. Med. 10 (1969)103–105.

[2] P. Hoffer, Status of gallium-67 in tumor detection, J. Nucl. Med. 21 (1980) 394–398.[3] D. Front, O. Israel, The role of Ga-67 scintigraphy in evaluating the results of

therapy of lymphoma patients, Semin. Nucl. Med. 25 (1995) 60–71.[4] P. Seam, M.E. Juweid, B.D. Cheson, The role of FDG-PET scans in patients with

lymphoma, Blood 110 (2007) 3507–3516.[5] M.U. Khan, S. Khan, S. El-Refaie, Z. Win, D. Rubello, A. Al-Nahhas, Clinical

indications for gallium-68 positron emission tomography imaging, Eur. J. Surg.Oncol. 35 (2009) 561–567.

[6] A. Al-Nahhas, Z. Win, T. Szyszko, A. Singh, C. Nanni, S. Fanti, D. Rubello, Gallium-68PET: a new frontier in receptor cancer imaging, Anticancer Res. 27 (2007) 4087–4094.

[7] M.M. Hart, C.F. Smith, S.T. Yancey, R.H. Adamson, Toxicity and antitumor activity ofgallium nitrate and periodically related metal salts, J. Natl. Inst. 47 (1971)1121–1127.

[8] B.J. Foster, K. Clagett-Carr, D. Hoth, B. Leyland-Jones, Gallium nitrate: the secondmetal with clinical activity, Cancer Treat. Rep. 70 (1988) 1311–1319.

[9] D.J. Straus, Gallium nitrate in the treatment of lymphoma, Semin. Oncol. 30(2003) 25–33.

[10] C.R. Chitambar, Gallium nitrate for the treatment of non-Hodgkin's lymphoma,Expert Opin. Investig. Drugs 13 (2004) 531–541.

[11] L. Einhorn, Gallium nitrate in the treatment of bladder cancer, Semin. Oncol. 30(2003) 34–41.

[12] L.R. Bernstein, Mechanisms of therapeutic activity for gallium, Pharmacol. Rev. 50(1998) 665–682.

[13] B. Hacht, Gallium(III) ion hydrolysis under physiological conditions, Bull. Kor.Chem. Soc. 29 (2008) 372–376.

[14] M.W. Hentze, M.U. Muckenthaler, B. Galy, C. Camaschella, Two to tango: regulationof mammalian iron metabolism, Cell 142 (2010) 24–38.

[15] D.J. Lane, A.M. Merlot, M.L. Huang, D.H. Bae, P.J. Jansson, S. Sahni, D.S. Kalinowski,D.R. Richardson, Cellular iron uptake, trafficking and metabolism: key moleculesand mechanisms and their roles in disease, Biochim. Biophys. Acta 1853 (2015)1130–1144.

[16] F.M. Torti, S.V. Torti, Regulation of ferritin genes and protein, Blood 99 (2002)3505–3516.

[17] T. Ganz, E. Nemeth, Hepcidin and disorders of ironmetabolism, Annu. Rev. Med. 62(2011) 347–360.

[18] D.M. Ward, J. Kaplan, Ferroportin-mediated iron transport: expression andregulation, Biochim. Biophys. Acta 1823 (2012) 1426–1433.

[19] Z.K. Pinnix, L.D. Miller, W. Wang, R. D'Agostino Jr., T. Kute, M.C. Willingham, H.Hatcher, L. Tesfay, G. Sui, X. Di, S.V. Torti, F.M. Torti, Ferroportin and iron regulationin breast cancer progression and prognosis, Sci. Transl. Med. 2 (2010), 43ra56.

[20] S. Zhang, Y. Chen, W. Guo, L. Yuan, D. Zhang, Y. Xu, E. Nemeth, T. Ganz, S. Liu,Disordered hepcidin–ferroportin signaling promotes breast cancer growth, Cell.Signal. 26 (2014) 2539–2550.

[21] S. Kvaloy, R. Langholm, O. Kaalhus, T. Michaelsen, S. Funderud, A.A. Foss, T. Godal,Transferrin receptor and B-lymphoblast antigen—their relationship to DNA synthe-sis, histology and survival in B-cell lymphomas, Int. J. Cancer 33 (1984) 173–177.

[22] I. Basar, A. Ayhan, K. Bircan, A. Ergen, C. Tasar, Transferrin receptor activity as amarker in transitional cell carcinoma of the bladder, Br. J. Urol. 67 (1991) 165–168.

[23] A. Calzolari, L.M. Larocca, S. Deaglio, V. Finisguerra, A. Boe, C. Raggi, L. Ricci-Vitani, F.Pierconti, F. Malavasi, M.R. De, U. Testa, R. Pallini, Transferrin receptor 2 is fre-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.Aleskandarany, E.C. Paish, M.R. Douglas, R.I. Nicholson, I.O. Ellis, J.M. Gee,Transferrin receptor (CD71) is a marker of poor prognosis in breast cancerand can predict response to tamoxifen, Breast Cancer Res. Treat. (2009).

[25] R.P. Warrell Jr., M. Issacs, N.W. Alcock, R.S. Bockman, Gallium nitrate for treatmentof refractory hypercalcemia from parathyroid carcinoma, Ann. Intern. Med. l07(1987) 683–686.

[26] A.W. Harris, R.G. Sephton, Transferrin promotion of 67Ga and 59Fe uptake bycultured mouse myeloma cells, Cancer Res. 37 (1977) 3634–3638.

[27] S.R. Vallabhajosula, J.F. Harwig, J.K. Siemsen, W. Wolf, Radiogallium localization intumors: blood binding and transport and the role of transferrin, J. Nucl. Med. 21(1980) 650–656.

[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 andlymphocytes, 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-independentmechanisms, Cancer Res. 47 (1987) 3929–3934.

[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 celllines, Cell. Mol. Biol. 44 (1998) (Dec 1998) 1215–1220.

[32] S.M. Chan, P.B. Hoffer, P. Duray, Inhibition of gallium-67 uptake inmelanoma by ananti-human transferrin receptor monoclonal antibody, J. Nucl. Med. 28 (1987)1303–1307.

[33] C.A. Luttropp, J.A. Jackson, B.J. Jones, M.H. Sohn, R.E. Lynch, K.A. Morton, Uptake ofgallium-67 in transfected cells and tumors absent or enriched in the transferrinreceptor, J. Nucl. Med. 39 (1998) 1405–1411.

[34] P.L. Goering, R.R. Maronpot, B.A. Fowler, Effect of intratracheal gallium arsenideadministration on delta-aminolevulinic acid dehydratase in rats: relationship tourinary excretion of aminolevulinic acid, Toxicol. Appl. Pharmacol. 92 (1988)179–193.

[35] A.C. Illing, A. Shawki, C.L. Cunningham, B. Mackenzie, Substrate profile and metal-ion selectivity of human divalentmetal-ion transporter-1, J. Biol. Chem. 287 (2012)30485–30496.

[36] J.N. Feder, D.M. Penny, A. Irrinki, V.K. Lee, J.A. Lebron, N. Watson, Z. Tsuchihashi, E.Sigal, P.J. Bjorkman, R.C. Schatzman, The hemochromatosis gene product com-plexes with the transferrin receptor and lowers its affinity for ligand binding,Proc. Natl. Acad. Sci. U. S. A. 95 (1998) 1472–1477.

[37] J.A. Lebron, A.P. West Jr., P.J. Bjorkman, The hemochromatosis protein HFEcompetes with transferrin for binding to the transferrin receptor, J. Mol. Biol. 294(1999) 239–245.

[38] C.N. Roy, D.M. Penny, J.N. Feder, C.A. Enns, The hereditary hemochromatosisprotein, HFE, specifically regulates transferrin-mediated iron uptake in HeLacells, J. Biol. Chem. 274 (1999) 9022–9028.

[39] B. Corsi, S. Levi, A. Cozzi, A. Corti, D. Altimare, A. Albertini, P. Arosio, Overexpressionof the hereditary hemochromatosis protein, HFE, in HeLa cells induces an iron-deficient phenotype, FEBS Lett. 460 (1999) 149–152.

[40] C.R. Chitambar, J.P. Wereley, Expression of the hemochromatosis (HFE) genemodulates the cellular uptake of 67Ga, J. Nucl. Med. 44 (2003) 943–946.

[41] C.R. Chitambar, D. Sax, Regulatory effects of gallium on transferrin-independentiron uptake by human leukemic HL60 cells, Blood 80 (1992) 505–511.

[42] R.S. Inman, M.M. Coughlin, M. Wessling-Resnick, Extracellular ferrireductase activ-ity of K562 cells is coupled to transferrin-independent iron transport, Biochemistry33 (1994) 11850–11857.

[43] I. Jordan, J. Kaplan, The mammalian transferrin-independent iron transportsystem may involve a surface ferrireductase activity, Biochem. J. 302 (1994)875–879.

[44] D.R. Richardson, E. Baker, The effect of desferrioxamine and ferric ammonium cit-rate on the uptake of iron by the membrane iron-binding component of humanmelanoma cells, Biochim. Biophys. Acta 1103 (1992) 275–280.

[45] K.J. Logan, P.K. Ng, C.J. Turner, P.R. Schmidt, U.K. Terner, J.R. Scott, B.C. Lentle, A.A.Noujaim, Comparative pharmacokinetics of 67Ga and 59Fe in humans, Int. J. Nucl.Med. Biol. 8 (1981) 271–276.

[46] M. Pollycove, M. Tono, Studies of the erythron, Semin. Nucl. Med. 5 (1975) 11–61.[47] R.L. Hayes, J.E. Carlton, A study of the macromolecular binding of 67Ga in normal

and malignant animal tissues, Cancer Res. 33 (1973) 3265–3272.[48] D. Lawless, D.H. Brown, K.F. Hubner, S.P. Colyer, J.E. Carlton, R.L. Hayes, Isolation

and partial characterization of a 67Ga-binding glycoprotein from Morris 5123Crat hepatoma, Cancer Res. 34 (1978) 4440–4444.

[49] E. Aulbert, U. Haubold, Isolation of the 67gallium accumulating fraction in normalrat liver, Nucl. Med. 13 (1974) 72–84.

[50] P.A.G. Hammersley, M.N. Cauchi, D.M. Taylor, Uptake of 67Ga in regenerating ratliver and its relationship to lysosomal enzyme activity, Cancer Res. 35 (1975)ll54–ll58.

[51] D.H. Brown, B.L. Byrd, J.E. Carlton, D.C. Swartzendruber, R.L. Hayes, A quantitativestudy of the subcellular localization of 67Ga, Cancer Res. 36 (1976) 956–963.

[52] C.J. Palestro, The current role of gallium imaging in infection, Semin. Nucl. Med. 24(1994) 128–141.

[53] W.R. Harris, Thermodynamics of gallium complexation by human lactoferrin,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 minireview, J. Basic Microbiol. 53 (2013) 303–317.

[55] T. Emery, Exchange of iron by gallium in siderophores, Biochemistry 25 (1986)4629–4633.

[56] R.E. Weiner, G.J. Schreiber, P.B. Hoffer, In vitro transfer of Ga-67 from transferrin toferritin, J. Nucl. Med. 24 (1983) 608–614.

[57] C.R. Chitambar, J.P. Wereley, Resistance to the antitumor agent gallium nitrate inhuman leukemic cells is associated with decreased gallium/iron uptake, increasedactivity of iron regulatory protein-1, and decreased ferritin production, J. Biol.Chem. 272 (1997) 12151–12157.

[58] C.R. Chitambar, P.A. Seligman, Effects of different transferrin forms on transferrinreceptor expression, iron uptake and cellular proliferation of human leukemicHL60 cells: mechanisms responsible for the specific cytotoxicity of transferrin-gallium, J. Clin. Invest. 78 (1986) 1538–1546.

[59] C.R. Chitambar, Z. Zivkovic, Inhibition of hemoglobin production by transferrin-gallium, Blood 69 (1987) 144–149.

[60] P.A. Seligman, P.L. Moran, R.B. Schleicher, E.D. Crawford, Treatment with galliumnitrate: evidence for interference with iron metabolism in vivo, Am. J. Hematol.41 (1992) 232–240.

[61] P.L. Goering, S. Rehm, Inhibition of liver, kidney, and erythrocyte δ-aminolevulinicacid dehydratase (porphobilinogen synthetase) by gallium in the rat, Environ. Res.53 (1990) 135–151.

[62] R.U. Haq, C.R. Chitambar, Modulation of transferrin receptor mRNA by transferrin-gallium in human myeloid HL60 cells and lymphoid CCRF-CEM cells, Biochem. J.294 (1993) 873–877.

[63] D.R. Richardson, Iron and gallium increase iron uptake from transferrin by humanmelanoma cells: further examination of the ferric ammonium citrate-activatediron uptake process, Biochim. Biophys. Acta 1536 (2001) 43–54.

[64] B. Sturm, U. Lassacher, N. Ternes, A. Jallitsch, H. Goldenberg, B. Scheiber-Mojdehkar, The influence of gallium and other metal ions on the uptake of non-transferrin-bound iron by rat hepatocytes, Biochimie 88 (2006) 645–650.

[65] P.A. Seligman, J. Kovar, R.B. Schleicher, E.W. Gelfand, Transferrin-independent ironuptake supports B lymphocyte growth, Blood 78 (1991) 1526–1531.

[66] L. Thelander, P. Reichard, Reduction of ribonucleotides, Annu. Rev. Biochem. 48(1979) 133–158.

[67] Role of ribonucleotide reductase in cell division, in: J.G. Cory, J.G.Cory, A.H.Cory(Eds.), Inhibitors of Ribonucleoside Diphosphate Reductase Activity, PergamonPress, New York 1989, pp. 1–16.

[68] Y. Aye, M. Li, M.J. Long, R.S. Weiss, Ribonucleotide reductase and cancer: biologicalmechanisms and targeted therapies, Oncogene (2014).

[69] Y. Engstrom, S. Eriksson, I. Jildevik, S. Skog, L. Thelander, B. Tribukait, Cell cycle-dependent expression ofmammalian ribonucleotide reductase. Differential regula-tion of the two subunits, J. Biol. Chem. 260 (1985) 9114–9116.

[70] S. Eriksson, A. Gräslund, S. Skog, L. Thelander, B. Tribukait, Cell cycle-dependentregulation of mammalian ribonucleotide reductase. The S phase-correlated in-crease in subunit M2 is regulated by de novo protein synthesis, J. Biol. Chem.259 (1984) 11695–11700.

[71] A. Gräslund, M. Sahlin, B.-M. Sjöberg, The tyrosyl free radical in ribonucleotidereductase, Environ. Health Perspect. 64 (1985) 139–149.

[72] S. Nyholm, G.J. Mann, A.G. Johansson, R.J. Bergeron, A. Gräslund, L. Thelander, Roleof ribonucleotide reductase in inhibition of mammalian cell growth by potent ironchelators, J. Biol. Chem. 268 (1993) 26200–26205.

[73] J.W. Larrick, P. Cresswell, Modulation of cell surface iron transferrin receptorsby cellular density and state of activation, J. Supramol. Struct. 11 (1979)579–586.

[74] C.R. Chitambar, E.J. Massey, P.A. Seligman, Regulation of transferrin receptorexpression on human leukemic cells during proliferation and induction ofdifferentiation. Effects of gallium and dimethylsulfoxide, J. Clin. Invest. 72 (1983)1314–1325.

[75] E. Pelosi, U. Testa, F. Louache, P. Thomopoulos, G. Salvo, P. Samoggia, C. Peschle,Expression of transferrin receptors in phytohemagglutinin-stimulated humanT-lymphocytes, J. Biol. Chem. 261 (1986) 3036–3042.

[76] C.R. Chitambar, J.P. Wereley, Effect of hydroxyurea on cellular iron metabolism inhuman leukemic CCRF-CEM cells: changes in iron uptake and the regulation oftransferrin receptor and ferritin gene expression following inhibition of DNAsynthesis, Cancer Res. 55 (1995) 4361–4366.

[77] C.R. Chitambar, W.G. Matthaeus, W.E. Antholine, K. Graff, W.J. O'Brien, Inhibition ofleukemic HL60 cell growth by transferrin-gallium: effects on ribonucleotidereductase and demonstration of drug synergy with hydroxyurea, Blood 72(1988) 1930–1936.

[78] J. Narasimhan, W.E. Antholine, C.R. Chitambar, Effect of gallium on the tyrosylradical of the iron-dependent M2 subunit of ribonucleotide reductase, Biochem.Pharmacol. 44 (1992) 2403–2408.

[79] C.R. Chitambar, J. Narasimhan, J. Guy, D.S. Sem,W.J. O'Brien, Inhibition of ribonucle-otide reductase by gallium in murine leukemic L1210 cells, Cancer Res. 51 (1991)6199–6201.

[80] L.G. Marzilli, B. de Castro, J.P. Caradonna, R.C. Stewart, C.P. Van Vuuren, Nucleosidecomplexing. A Raman and 13C NMR spectroscopic study of the binding of hard andsoft metal species, J. A. Chem. Soc. 102 (1980) 916–924.

[81] T.C. Pochapsky, M. Kuti, S. Kazanis, The solution structure of a gallium-substitutedputidaredoxin mutant: GaPdx C85S, J. Biomol. NMR 12 (1998) 407–415.

[82] T. Shibahara, S. Kobayashi, N. Tsuji, G. Sakane, M. Fukuhara, Sulfur-bridged cubane-type molybdenum–gallium clusters with Mo(3)GaS(4)(n)(+) (n = 5, 6) cores.X-ray structures of [Mo(3)GaS(4)(H(2)O)(12)](CH(3)C(6)H(4)SO(3))(5)·14H(2)Oand [Mo(3)GaS(4)(H(2)O)(12)](CH(3)C(6)H(4)SO(3))(6)·17H(2)O, Inorg. Chem.36 (1997) 1702–1706.

[83] C.R. Chitambar, J.P. Wereley, S. Matsuyama, Gallium-induced cell death in lympho-ma: role of transferrin receptor cycling, involvement of Bax and the mitochondria,and effects of proteasome inhibition, Mol. Cancer Ther. 5 (2006) 2834–2843.

[84] M. Yang, C.R. Chitambar, Role of oxidative stress in the induction ofmetallothionein-2A and heme oxygenase-1 gene expression by the antineo-plastic agent gallium nitrate in human lymphoma cells, Free Radic. Biol. Med.45 (2008) 763–772.

[85] G.F. Kelso, C.M. Porteous, C.V. Coulter, G. Hughes, W.K. Porteous, E.C. Ledgerwood,R.A. Smith, M.P. Murphy, Selective targeting of a redox-active ubiquinone tomitochondria within cells: antioxidant and antiapoptotic properties, J. Biol.Chem. 276 (2001) 4588–4596.

[86] D.F. Stowe, A.K. Camara, Mitochondrial reactive oxygen species production inexcitable cells: modulators of mitochondrial and cell function, Antioxid. RedoxSignal. 11 (2009) 1373–1414.

[87] C.R. Chitambar, Z. Zivkovic-Gilgenbach, J. Narasimhan, W.E. Antholine,Development of drug resistance to gallium nitrate through modulation of cellulariron uptake, Cancer Res. 50 (1990) 4468–4472.

[88] N.P. Davies, Y.S. Rahmanto, C.R. Chitambar, D.R. Richardson, Resistance to theantineoplastic agent gallium nitrate results in marked alterations in intracellulariron and gallium trafficking: identification of novel intermediates, J. Pharmacol.Exp. Ther. 317 (2006) 153–162.

[89] C.R. Chitambar, J.P. Wereley, Transferrin receptor-dependent and -independentiron transport in gallium-resistant human lymphoid leukemic cells, Blood 91(1998) 4686–4693.

[90] M. Yang, S.H. Kroft, C.R. Chitambar, Gene expression analysis of gallium-resistantand gallium-sensitive lymphoma cells reveals a role for metal-responsive tran-scription factor-1, metallothionein-2A, and zinc transporter-1 in modulating theantineoplastic activity of gallium nitrate, Mol. Cancer Ther. 6 (2007) 633–643.

[91] S.R. Davis, R.J. Cousins, Metallothionein expression in animals: a physiologicalperspective on function, J. Nutr. 130 (2000) 1085–1088.

[92] M. Namdarghanbari, W. Wobig, S. Krezoski, N.M. Tabatabai, D.H. Petering,Mammalian metallothionein in toxicology, cancer, and cancer chemotherapy, J.Biol. Inorg. Chem. 16 (2011) 1087–1101.

[93] C.D. Klaassen, J. Liu, S. Choudhuri, Metallothionein: an intracellular protein toprotect against cadmium toxicity, Annu. Rev. Pharmacol. Toxicol. 39 (1999)267–294.

[94] A.B. Kelson, M. Carnevali, V. Truong-Le, Gallium-based anti-infectives: targetingmicrobial iron-uptake mechanisms, Curr. Opin. Pharmacol. 13 (2013) 707–716.

[95] E. Frangipani, C. Bonchi, F. Minandri, F. Imperi, P. Visca, Pyochelin potentiates theinhibitory activity of gallium on Pseudomonas aeruginosa, Antimicrob. AgentsChemother. 58 (2014) 5572–5575.

[96] Y. Kaneko, M. Thoendel, O. Olakanmi, B.E. Britigan, P.K. Singh, The transition metalgallium disrupts Pseudomonas aeruginosa iron metabolism and has antimicrobialand antibiofilm activity, J. Clin. Invest. 117 (2007) 877–888.

[97] O. Olakanmi, B.E. Britigan, L.S. Schlesinger, Gallium disrupts iron metabolism ofmycobacteria residing within human macrophages, Infect. Immun. 68 (2000)5619–5627.

[98] O. Olakanmi, B. Kesavalu, R. Pasula,M.Y. Abdalla, L.S. Schlesinger, B.E. Britigan, Galliumnitrate is efficacious in murine models of tuberculosis and inhibits key bacterialFe-dependent enzymes, Antimicrob. Agents Chemother. 57 (2013) 6074–6080.

[99] E. Banin, A. Lozinski, K.M. Brady, E. Berenshtein, P.W. Butterfield, M. Moshe, M.Chevion, E.P. Greenberg, E. Banin, The potential of desferrioxamine-gallium as ananti-Pseudomonas therapeutic agent, Proc. Natl. Acad. Sci. U. S. A. 105 (2008)16761–16766.

[100] K. DeLeon, F. Balldin, C. Watters, A. Hamood, J. Griswold, S. Sreedharan, K.P.Rumbaugh, Gallium maltolate treatment eradicates Pseudomonas aeruginosainfection in thermally injured mice, Antimicrob. Agents Chemother. 53 (2009)1331–1337.

[101] J.R. Harrington, R.J. Martens, N.D. Cohen, L.R. Bernstein, Antimicrobial activity ofgallium against virulent Rhodococcus equiin vitro and in vivo, J. Vet. Pharmacol.Ther. 29 (2006) 121–127.

[102] M.G. Thompson, V. Truong-Le, Y.A. Alamneh, C.C. Black, J. Anderl, C.L. Honnold, R.L.Pavlicek, R. Abu-Taleb, M.C. Wise, E.R. Hall, E.J. Wagar, E. Patzer, D.V. Zurawski,Evaluation of gallium citrate formulations against a multidrug-resistant strain ofKlebsiella pneumoniae in a murine wound model of infection, Antimicrob. AgentsChemother. 59 (2015) 6484–6493.

[103] A. Rangel-Vega, L.R. Bernstein, E.A. Mandujano-Tinoco, S.J. Garcia-Contreras, R.Garcia-Contreras, Drug repurposing as an alternative for the treatment of recalci-trant bacterial infections, Front. Microbiol. 6 (2015) 282.

[104] C.R. Chitambar, Medical applications and toxicities of gallium compounds, Int. J.Environ. Res. Public Health 7 (2010) 2337–2361.

[105] R.P. Warrell Jr., Clinical trials of gallium nitrate in patients with cancer-relatedhypercalcemia, Semin. Oncol. 18 (1991) 26–31.

[106] R.S. Bockman, F. Wilhelm, E. Siris, F. Singer, A. Chausmer, R. Bitton, J. Kotler, B.J.Bosco, D.R. Eyre, D. Levenson, A multicenter trial of low dose gallium nitrate inpatients with advanced Paget's disease of bone, J. Clin. Endocrinol. Metab. 80(1995) 595–602.

[107] R. Bockman, The effects of gallium nitrate on bone resorption, Semin. Oncol. 30(2003) 5–12.

[108] B. Leyland-Jones, Treatment of cancer-related hypercalcemia: the role of galliumnitrate, Semin. Oncol. 30 (2003) 13–19.

[109] A.W. Harris, A.E. Martell, Aqueous complexes of gallium, Inorg. Chem. 15 (1976)713–720.

[110] L.R. Bernstein, T. Tanner, C. Godfrey, B. Noll, Chemistry and pharmacokinetics ofgallium maltolate, a compound with high oral gallium bioavailability, Metal-Based Drugs 7 (2000) 33–47.

2053C.R. Chitambar / Biochimica et Biophysica Acta 1863 (2016) 2044–2053

[111] K.P. Allamneni, R.B. Burns, D.J. Gray, F.H. Valone, L.R. Bucalo, S.P. Sreedharan,Gallium maltolate treatment results in transferrin-bound gallium in patientserum, 2004 230 (abstract 1013).

[112] C.R. Chitambar, D.P. Purpi, J. Woodliff, M. Yang, J.P. Wereley, Development ofgallium compounds for treatment of lymphoma: gallium maltolate, a novelhydroxypyrone gallium compound induces apoptosis and circumvents lymphomacell resistance to gallium nitrate, J. Pharmacol. Exp. Ther. 322 (2007) 1228–1236.

[113] A.R. Timerbaev, Advances in developing tris(8-quinolinolato)gallium(iii)as an anticancer drug: critical appraisal and prospects, Metallomics 1 (2009)193–198.

[114] C.R. Chitambar, Gallium-containing anticancer compounds, Future Med. Chem. 4(2012) 1257–1272.

[115] R.R. Moskalyk, Gallium: the backbone of the electronics industry, Miner. Eng. 16(2003) 921–929.

[116] M.J. Brookes, J. Boult, K. Roberts, B.T. Cooper, N.A. Hotchin, G. Matthews, T. Iqbal, C.Tselepis, A role for iron in Wnt signalling, Oncogene 27 (2008) 966–975.

[117] S. Song, T. Christova, S. Perusini, S. Alizadeh, R.Y. Bao, B.W. Miller, R. Hurren, Y.Jitkova, M. Gronda, M. Isaac, B. Joseph, R. Subramaniam, A. Aman, A. Chau, D.E.Hogge, S.J. Weir, J. Kasper, A.D. Schimmer, R. Al-awar, J.L. Wrana, L. Attisano, Wntinhibitor screen reveals iron dependence of beta-catenin signaling in cancers,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

Wnt/beta-catenin signaling and proliferation by a ferrous iron chelator with thera-peutic efficacy in genetically engineered mouse models of cancer, Oncogene 31(2012) 213–225.

[119] M. Ndong, M. Kazami, T. Suzuki, M. Uehara, S. Katsumata, H. Inoue, K.Kobayashi, T. Tadokoro, K. Suzuki, Y. Yamamoto, Iron deficiency down-regulatesthe Akt/TSC1-TSC2/mammalian Target of Rapamycin signaling pathway in ratsand in COS-1 cells, Nutr. Res. 29 (2009) 640–647.

[120] J.H. Ohyashiki, C. Kobayashi, R. Hamamura, S. Okabe, T. Tauchi, K. Ohyashiki, Theoral iron chelator deferasirox represses signaling through the mTOR in myeloidleukemia cells by enhancing expression of REDD1, Cancer Sci. 100 (2009)970–977.

[121] S.V. Torti, F.M. Torti, Iron and cancer: more ore to be mined, Nat. Rev. Cancer 13(2013) 342–355.

[122] J.N. Anastas, R.T. Moon, WNT signalling pathways as therapeutic targets in cancer,Nat. Rev. Cancer 13 (2013) 11–26.

[123] J.A. Engelman, J. Luo, L.C. Cantley, The evolution of phosphatidylinositol 3-kinasesas regulators of growth and metabolism, Nat. Rev. Genet. 7 (2006) 606–619.

[124] N. Chapuis, J. Tamburini, A.S. Green, L. Willems, V. Bardet, S. Park, C. Lacombe, P.Mayeux, D. Bouscary, Perspectives on inhibiting mTOR as a future treatmentstrategy for hematological malignancies, Leukemia 24 (2010) 1686–1699.