Biochimica et Biophysica Acta 1863 (2016) 2044–2053 Contents lists available at ScienceDirect Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbamcr 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 tempera- ture. 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 http://dx.doi.org/10.1016/j.bbamcr.2016.04.027 0167-4889/© 2016 Elsevier B.V. All rights reserved. C.R. Chitambar / Biochimica et Biophysica Acta 1863 (2016) 2044–2053 2045 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].