586 A Ferrous-triapine complex mediates formation of reactive oxygen species that inactivate human ribonucleotide reductase Jimin Shao,1,2 Bingsen Zhou,1 Angel J. Di Bilio,3 cytotoxicity assays. These results indicate that Triapine- Lijun Zhu,1,2 Tieli Wang,1 Christina Qi,1 induced inhibition of ribonucleotide reductase is caused by Jennifer Shih,1 and Yun Yen1 ROS. We suggest that ROS may ultimately be responsible for the pharmacologic effects of Triapine in vivo. [Mol 1Department of Medical Oncology and Therapeutic Research, Cancer Ther 2006;5(3):586–92] City of Hope National Medical Center, Duarte, California; 2Department of Basic Medical Sciences, Zhejiang University School of Medicine, Hangzhou, Zhejiang, People’s Republic of Introduction 3 China; Department of Chemistry, California Institute of Ribonucleotide reductases catalyze the reduction of Technology, Pasadena, California ribonucleotides to deoxyribonucleotides, which are re- quired for DNA replication and repair (1, 2). Human Abstract ribonucleotide reductase is a protein tetramer featuring Ribonucleotide reductase plays a central role in cell two identical large (hRRM1) and two identical small proliferation by supplying deoxyribonucleotide precursors (hRRM2 or p53R2) subunits. hRRM1 harbors the sub- for DNA synthesis and repair. The holoenzyme is a protein strate-catalytic and allosteric regulation sites. The small tetramer that features two large (hRRM1) and two small subunit contains an oxygen-linked diferric cluster and a (hRRM2 or p53R2) subunits. The small subunit contains a stable tyrosyl radical that are needed for function (Fig. 1A; refs. 1–4). p53R2 is a newly identified p53-inducible di-iron cluster/tyrosyl radical cofactor that is essential for f enzyme activity. Triapine (3-aminopyridine-2-carboxalde- protein that has 80% sequence homology with hRRM2 hyde thiosemicarbazone, 3-AP) is a new, potent ribonu- (5, 6). It has been suggested that hRRM2/hRRM1 supplies cleotide reductase inhibitor currently in phase II clinical deoxynucleotide triphosphates for DNA replication in a trials for cancer chemotherapy. Ferric chloride readily cell cycle–dependent manner, whereas p53R2/hRRM1 reacts with Triapine to form an Fe(III)-(3-AP) complex, supplies deoxynucleotide triphosphates for DNA repair which is reduced to Fe(II)-(3-AP) by DTT. Spin-trapping in a p53-dependent manner (7–10). The discovery of experiments with 5,5-dimethyl-1-pyrroline-N-oxide prove p53R2 has created much interest because of its possible role in tumorigenesis and cancer treatment (4–8). In- that Fe(II)-(3-AP) reduces O2 to give oxygen reactive species (ROS). In vitro activity assays show that Fe(II)-(3- creased ribonucleotide reductase activity is observed in AP) is a much more potent inhibitor of hRRM2/hRRM1 and tumor formation and metastasis (11–13). Inactivation of p53R2/hRRM1 than Triapine. Electron paramagnetic res- ribonucleotide reductase stops DNA synthesis, which onance measurements on frozen solutions of hRRM2 and inhibits cell proliferation. Thus, the enzyme has long been p53R2 show that their tyrosyl radicals are completely considered an excellent target for cancer chemotherapy. quenched by incubation with Fe(II)-(3-AP). However, the Strategies for inhibiting ribonucleotide reductase include enzyme activity is maintained in protein samples supple- quenching the tyrosyl radical of the small subunit, use of mented with catalase alone or in combination with nucleoside analogues to inhibit the large subunit, pertur- superoxide dismutase. Furthermore, catalase alone or in bation of interactions between subunits, and suppression combination with superoxide dismutase markedly of gene expression (13–15). decreases the antiproliferative effect of Triapine in Triapine (3-aminopyridine-2-carboxaldehyde thiosemi- carbazone, 3-AP) is a new, potent inhibitor of ribonucleo- tide reductase currently in phase II clinical trials for cancer chemotherapy (Fig. 1B; refs. 16–19). Triapine belongs to a class of heterocyclic carboxaldehyde thiosemicarbazones Received 9/22/05; revised 12/6/05; accepted 1/10/06. (HCT) that are efficient iron chelators (16, 17). Ribonucleo- Grant support: National Cancer Institute grant R01 CA72767 and Sino tide reductases are the primary cellular target of Triapine. America Cancer Foundation. The presence of iron is required for effective enzyme The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked inhibition and cytotoxicity by this compound (3, 16, 17). advertisement in accordance with 18 U.S.C. Section 1734 solely to The well-known antitumor drug hydroxyurea is the only indicate this fact. prescribed ribonucleotide reductase inhibitor that targets Requests for reprints: Yun Yen, Department of Medical Oncology and the small subunits (4, 13). Triapine is a 100- to 1,000-fold Therapeutic Research, City of Hope National Medical Center, 1500 East Duarte Road, Duarte, CA 91010. Phone: 626-359-8111, ext. 62307; more potent inhibitor of both ribonucleotide reductase and Fax: 626-301-8233. E-mail: [email protected] tumor cell growth than hydroxyurea. The compound is fully Copyright C 2006 American Association for Cancer Research. active against hydroxyurea-resistant tumors and increases doi:10.1158/1535-7163.MCT-05-0384 the effectiveness of other DNA-damaging and cytotoxic Mol Cancer Ther 2006;5(3). March 2006 Downloaded from mct.aacrjournals.org on October 2, 2021. © 2006 American Association for Cancer Research. Molecular Cancer Therapeutics 587 cytotoxicity. Our data strongly suggest that ROS are ultimately responsible for the pharmacologic effects of Triapine. A better understanding of the mechanisms of action of Triapine could result in clinical applications and help design novel ribonucleotide reductase inhibitors for cancer chemotherapy. Materials and Methods Materials Triapine was a gift from Vion Pharmaceuticals, Inc. (New Haven, CT). DMSO, ferric chloride, DTT, superoxide dismutase (SOD, from bovine liver, 2,880 units/mg), catalase (from human erythrocytes, 59,500 units/mg), 5, 5-dimethyl-1-pyrroline-N-oxide (DMPO), and diethylene- triaminepentaacetic acid were supplied by Sigma (St. Louis, MO). Hydrogen peroxide was purchased from Mallinck- rodt Baker, Inc. (Paris, KY). Solutions of Triapine were prepared in neat DMSO and diluted with 50 mmol/L Tris-HCl buffer (pH 7.2) contain- ing 100 mmol/L KCl. Fe(III)-(3-AP) formed readily by mixing Triapine with ferric chloride in a 2:1 ligand to metal ratio at room temperature. Fe(II)-(3-AP) was generated by reduction of Fe(III)-(3-AP) with DTT (20, 21). Triapine is insoluble in water but soluble in aqueous DMSO. Thus, all solutions of Triapine and its iron complexes contained DMSO. UV-Visible Spectrum Measurements UV-visible spectra were measured with a Bio-Rad Figure 1. A, structural model for the di-iron cluster/tyrosyl radical SmartSpec 3000 Spectrophotometer. The samples of Tri- cofactor of p53R2. Protein atoms that bind to the irons are drawn in ‘‘ball- apine and its iron complexes were prepared as described 138 and-stick’’ style. Radical-harboring Tyr (176). The residue numbers in above. Three doses of Triapine (500, 100, and 20 Amol/L) parenthesis are for hRRM2. The model is based on an X-ray crystal structure of a murine R2 (PDB code 1W69) and was built with SYBYL were tried based on previous reports on other HCTs 6.9.2 (Tripos, Inc., St. Louis, MO). hRRM2 and p53R2 share 95.5 and (22, 23); 100 Amol/L Triapine was found to be the best dose 81.2% sequence identity with murine R2. B, structure of Triapine. C, UV- for UV-visible absorption detection. visible absorption spectra of Triapin, Fe(II)-(3-AP), and Fe(III)-(3-AP) in 50 mmol/L Tris-HCl buffer (pH 7.2) containing 100 mmol/L KCl. The Protein Expression concentrations are 100 Amol/L Triapine, 1% DMSO, 50 Amol/L FeCl3, hRRM2, p53R2, and hRRM1 were expressed using the and 5 mmol/L DTT. pET28-BL21(DE3) prokaryotic system and isolated to above 90% purity by immobilized-metal (Ni2+) affinity chroma- tography. Proteins were dialyzed against 50 mmol/L Tris- agents (16, 17). Clinical trials suggest that Triapine is a HCl buffer (pH 7.4) containing 100 mmol/L KCl and stored promising cancer chemotherapeutic drug. However, ad- at À70jC (3). verse effects that include methemoglobinemia and hypoxia In vitro Assays have been reported (18, 19). The enzymatic activity of recombinant ribonucleotide The mechanism of ribonucleotide reductase inhibition reductase was measured using a previously reported by Triapine, the function of iron, and the correlation [3H]CDP reduction method (3, 4). Briefly, 100 ALof between the pharmacologic and side effects of the drug reaction mixture contained 0.125 Amol/L [3H]CDP (24 remain unclear. We report an investigation of the Ci/mmol), 50 mmol/L HEPES (pH 7.2), 6 mmol/L DTT, inhibitory effects of free Triapine and when bound to 4 mmol/L MgOAc, 2 mmol/L ATP, 0.05 mmol/L CDP, ferric and ferrous ions on recombinant human ribonucle- 100 mmol/L KCl, and 0.25 Amol/L ribonucleotide otide reductases by in vitro activity assays, antioxidant reductase (5 Ag of hRRM1 and 2.5 Ag of hRRM2 or protection analysis, and electron paramagnetic resonance p53R2). The molar amount of holoenzyme was calculated (EPR) spectroscopy. Our spin-trapping experiments show using the molecular weight of the tetramer. Where that Fe(II)-(3-AP) reacts with dioxygen to give reactive indicated, up to 60 Amol/L FeCl3 was added. Samples oxygen species (ROS). We found that the species Fe(II)- were analyzed by high-performance liquid chromatogra- (3-AP) is a much more potent inhibitor than free Triapine phy and liquid scintillation counting after incubation at and Fe(III)-(3-AP). Cell proliferation assays indicate that 37jC for 30 minutes and dephosphorylation. The specific the redox-active Triapine-Fe complex plays a role in enzymatic activity was 79.2 F 2.82 nmol of dCDP Mol Cancer Ther 2006;5(3).