A Ferrous-triapine complex mediates formation of reactive oxygen species that inactivate human ribonucleotide reductase Free

Ribonucleotide reductases catalyze the reduction of ribonucleotides to deoxyribonucleotides, which are required for DNA replication and repair (1, 2). Human ribonucleotide reductase is a protein tetramer featuring two identical large (hRRM1) and two identical small (hRRM2 or p53R2) subunits. hRRM1 harbors the substrate-catalytic and allosteric regulation sites. The small subunit contains an oxygen-linked diferric cluster and a stable tyrosyl radical that are needed for function (Fig. 1A; refs. 1–4). p53R2 is a newly identified p53-inducible protein that has ∼80% sequence homology with hRRM2 (5, 6). It has been suggested that hRRM2/hRRM1 supplies deoxynucleotide triphosphates for DNA replication in a cell cycle–dependent manner, whereas p53R2/hRRM1 supplies deoxynucleotide triphosphates for DNA repair in a p53-dependent manner (7–10). The discovery of p53R2 has created much interest because of its possible role in tumorigenesis and cancer treatment (4–8). Increased ribonucleotide reductase activity is observed in tumor formation and metastasis (11–13). Inactivation of ribonucleotide reductase stops DNA synthesis, which inhibits cell proliferation. Thus, the enzyme has long been considered an excellent target for cancer chemotherapy. Strategies for inhibiting ribonucleotide reductase include quenching the tyrosyl radical of the small subunit, use of nucleoside analogues to inhibit the large subunit, perturbation of interactions between subunits, and suppression of gene expression (13–15).

Triapine (3-aminopyridine-2-carboxaldehyde thiosemicarbazone, 3-AP) is a new, potent inhibitor of ribonucleotide reductase currently in phase II clinical trials for cancer chemotherapy (Fig. 1B; refs. 16–19). Triapine belongs to a class of heterocyclic carboxaldehyde thiosemicarbazones (HCT) that are efficient iron chelators (16, 17). Ribonucleotide reductases are the primary cellular target of Triapine. The presence of iron is required for effective enzyme inhibition and cytotoxicity by this compound (3, 16, 17). The well-known antitumor drug hydroxyurea is the only prescribed ribonucleotide reductase inhibitor that targets the small subunits (4, 13). Triapine is a 100- to 1,000-fold more potent inhibitor of both ribonucleotide reductase and tumor cell growth than hydroxyurea. The compound is fully active against hydroxyurea-resistant tumors and increases the effectiveness of other DNA-damaging and cytotoxic agents (16, 17). Clinical trials suggest that Triapine is a promising cancer chemotherapeutic drug. However, adverse effects that include methemoglobinemia and hypoxia have been reported (18, 19).

The mechanism of ribonucleotide reductase inhibition by Triapine, the function of iron, and the correlation between the pharmacologic and side effects of the drug remain unclear. We report an investigation of the inhibitory effects of free Triapine and when bound to ferric and ferrous ions on recombinant human ribonucleotide reductases by in vitro activity assays, antioxidant protection analysis, and electron paramagnetic resonance (EPR) spectroscopy. Our spin-trapping experiments show that Fe(II)-(3-AP) reacts with dioxygen to give reactive oxygen species (ROS). We found that the species Fe(II)-(3-AP) is a much more potent inhibitor than free Triapine and Fe(III)-(3-AP). Cell proliferation assays indicate that the redox-active Triapine-Fe complex plays a role in 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.

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 diethylenetriaminepentaacetic acid were supplied by Sigma (St. Louis, MO). Hydrogen peroxide was purchased from Mallinckrodt Baker, Inc. (Paris, KY).

Solutions of Triapine were prepared in neat DMSO and diluted with 50 mmol/L Tris-HCl buffer (pH 7.2) containing 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 spectra were measured with a Bio-Rad SmartSpec 3000 Spectrophotometer. The samples of Triapine and its iron complexes were prepared as described above. Three doses of Triapine (500, 100, and 20 μmol/L) were tried based on previous reports on other HCTs (22, 23); 100 μmol/L Triapine was found to be the best dose for UV-visible absorption detection.

hRRM2, p53R2, and hRRM1 were expressed using the pET28-BL21(DE3) prokaryotic system and isolated to above 90% purity by immobilized-metal (Ni²⁺) affinity chromatography. Proteins were dialyzed against 50 mmol/L Tris-HCl buffer (pH 7.4) containing 100 mmol/L KCl and stored at −70°C (3).

The enzymatic activity of recombinant ribonucleotide reductase was measured using a previously reported [³H]CDP reduction method (3, 4). Briefly, 100 μL of reaction mixture contained 0.125 μmol/L [³H]CDP (24 Ci/mmol), 50 mmol/L HEPES (pH 7.2), 6 mmol/L DTT, 4 mmol/L MgOAc, 2 mmol/L ATP, 0.05 mmol/L CDP, 100 mmol/L KCl, and 0.25 μmol/L ribonucleotide reductase (5 μg of hRRM1 and 2.5 μg of hRRM2 or p53R2). The molar amount of holoenzyme was calculated using the molecular weight of the tetramer. Where indicated, up to 60 μmol/L FeCl₃ was added. Samples were analyzed by high-performance liquid chromatography and liquid scintillation counting after incubation at 37°C for 30 minutes and dephosphorylation. The specific enzymatic activity was 79.2 ± 2.82 nmol of dCDP formed/min/mg protein for hRRM2/hRRM1 and 51.2 ± 1.39 nmol of dCDP formed/min/mg protein for p53R2/hRRM1. For inhibition assays, serially diluted Triapine, Fe(II)-(3-AP), and Fe(III)-(3-AP) were incubated with 0.25 μmol/L holoenzyme at room temperature for 30 minutes. The IC₇₅ of Triapine for hRRM2/hRRM1 and p53R2/hRRM1 determined by the in vitro ribonucleotide reductase assay was used for antioxidant protection analysis. Triapine, Fe(II)-(3-AP), and Fe(III)-(3-AP) were initially incubated with SOD, catalase, or both at 4°C for 30 minutes and then incubated with holoenzyme for additional 30 minutes at room temperature. Samples for negative control contained 0.25% DMSO (this was the content of DMSO in the reaction mixture for the highest Triapine concentration). The extent of enzyme inhibition is expressed as a percentage of the negative control value (relative ribonucleotide reductase activity).

ROS are short-lived and present at very low concentrations. Spin-trapping EPR spectroscopy is the most sensitive method thus far for the detection of ROS (24). Solutions for spin-trapping experiments contained Triapine or its iron complexes (0.001, 0.01, 0.1, 1, and 10 mmol/L), DMSO, and DMPO in 50 mmol/L HEPES buffer (pH 7.5; refs. 20, 21). Experiments were also conducted on samples supplemented with 1 mg/mL SOD, 50 μg/mL catalase, or both. H₂O₂ was used as a positive control. The EPR spectrum of each test solution was measured soon after mixing the reagents or after incubation at room temperature for up to 60 minutes. X-band EPR spectra were measured with a Bruker EMX spectrometer. A cylindrical cavity (Bruker, ER 4103TM) and a quartz flat cell were used for room temperature (∼22°C) measurements. The room temperature EPR spectra were simulated with the program WINEPR Simfonia (version 1.25, Bruker Analytische Messtechnik GmbH, Karlsruhe, Germany).

Samples of hRRM2 and p53R2 (35 μmol/L) in 50 mmol/L Tris-HCl (pH 7.4) buffer containing 100 mmol/L KCl were incubated separately at room temperature for 30 minutes with each of the following reagents (2, 10, and 50 μmol/L): Triapine, Fe(III)-(3-AP), and Fe(II)-(3-AP). The samples were frozen and analyzed by EPR spectroscopy. The content of DMSO for the highest Triapine concentration (50 μmol/L) was 0.5%. For antioxidant protection analysis, Triapine, Fe(III)-(3-AP), and Fe(II)-(3-AP) were incubated with SOD (500 μg/mL), catalase (25 μg/mL), or both at 4°C for 30 minutes and then incubated with hRRM2 and p53R2 at room temperature for additional 30 minutes. Samples were transferred to standard EPR tubes and frozen using liquid nitrogen. Frozen solution EPR spectra were measured using a high-sensitivity cavity (ER 4119HS) and an Oxford helium cryostat.

Human oropharyngeal carcinoma KB cells from the American Type Culture Collection (Manassas, VA) were cultured in RPMI 1640 supplemented with 10% fetal bovine serum in a 5% CO₂ humidified atmosphere at 37°C. KB cells in exponential growth were seeded at a density of 20,000/mL in 96-well plates. After 24 hours, solutions of Triapine in the complete culture media were added to cells in the presence or absence of SOD or/and catalase. Cells were incubated with treatments for 72 hours at 37°C. Antiproliferative activity of Triapine was then examined with the CellTiter 96 AQueous One Solution Cell Proliferation Assay kit (Promega, Madison, WI) following the user's protocol.

Triapine (UV-visible spectrum, λ_max = 360 nm) reacts readily with ferric chloride (2:1 molar ratio) to give Fe(III)--(3-AP) (λ_max = 410 nm; Fig. 1C). The latter is reduced to Fe(II)-(3-AP) (λ_max = 605 nm) by DTT. The absorption spectra of these species are similar to those for the ferric and ferrous chelates of other HCTs (22, 23). The absorption bands for both complexes did not shift for at least 60 minutes at room temperature.

Room temperature EPR measurements on solutions containing Fe(II)-(3-AP) and the spin trap DMPO reveal the formation of the methyl radical-DMPO spin adduct in the presence of DMSO (20). The methyl radical-DMPO spin adduct is characterized by a six-line EPR spectrum with hyperfine coupling constants A^N = 16.3 G and β-H A^H = 23.3 G (Fig. 2A). No EPR signals were detected from solutions of DMPO with Triapine or Fe(III)-(3-AP). The intensity of the EPR signals increased with the incubation time and concentration of Fe(II)-(3-AP). These results strongly suggest that the ferrous complex of Triapine activates molecular oxygen via a Fenton-like reaction (Eq. A).

The first step for oxygen activation is the one-electron reduction of oxygen to the superoxide anion (Eq. B). Addition of a second electron reduces superoxide to hydrogen peroxide (Eq. C).

The short-lived ·OH radicals react with the hydroxyl radical scavenger DMSO to form ·CH₃, which with excess DMPO gives the methyl DMPO spin adduct (Eq. D).

Catalase blocked the formation of the spin adducts by converting H₂O₂ to H₂O and O₂ (Fig. 2A). SOD did not have an effect on the formation of the spin adducts. The latter is expected because SOD catalyzes the conversion of superoxide to H₂O₂, which can then be reduced to hydroxyl radicals according to Eq. A.

H₂O₂ was used as a positive control (Fig. 2B). H₂O₂ and Fe(II) generated ·OH from the Fenton reaction. Spin trapping of ·OH by DMPO generated the hydroxyl radical-DMPO spin adduct featuring a four-line signal (20). Catalase prevented the reaction. Fe(III) or Fe(II) gave no radical siganals (data not shown).

The inhibitory properties of Triapine, Fe(III)-(3-AP), and Fe(II)-(3-AP) toward hRRM2/hRRM1 and p53R2/hRRM1 were tested by the in vitro assays described above. We found that Fe(II)-(3-AP) is a potent inhibitor of ribonucleotide reductases (the effect is greater for p53R2/hRRM1; Fig. 3A). Triapine shows low activity. However, inhibition is reversible with excess FeCl₃ in the reaction mixture; enzyme regeneration was more significant for p53R2/hRRM1 compared with hRRM2/hRRM1. Inactivation of hRRM2 and p53R2 by Fe(II)-(3-AP) is blocked by catalase or by a mixture of catalase and SOD (Fig. 3B).

EPR measurements on frozen samples of hRRM2 and p53R2 show that the tyrosyl radical is efficiently quenched by Fe(II)-(3-AP) (Fig. 4). Interestingly, the EPR signal of the tyrosyl radical is absent from samples of 35 μmol/L hRRM2 or p53R2 that were incubated with only 2 μmol/L Fe(II)-(3-AP). Catalase alone or in combination with SOD prevented quenching of the radical. Up to 50 μmol/L, Triapine alone did not show a significant effect on the tyrosyl radical EPR signal.

To investigate whether ROS is associated with the antiproliferation effect of Triapine in cells, antioxidants were examined for their abilities to decrease the cytotoxicity of Triapine (Fig. 5). SOD had no significant effect on the cytotoxicity of Triapine compared with Triapine alone. In contrast, catalase alone or in combination with SOD markedly decreased the antiproliferative activity of Triapine.

Triapine reacts readily with ferric chloride to form a stable Fe(III)-(3-AP) species, which is reduced to Fe(II)-(3-AP) by DTT. The formation of these iron-Triapine species was confirmed by UV-visible absorption measurements (Fig. 1). By using the spin-trapping technique and EPR spectroscopy, we established that Fe(II)-(3-AP) but not Triapine and Fe(III)-(3-AP) activates O₂ to give reactive oxygen species (Fig. 2). We then tested the effect of each species on the activity of ribonucleotide reductase by in vitro assays and found that Fe(II)-(3-AP) is a significantly more active inhibitor of the enzyme than free Triapine (Fig. 3). Furthermore, incubation of the small subunits with Fe(II)-(3-AP) results in quenching of the tyrosyl radical (Fig. 4). Of particular interest is that with excess DTT, iron-Triapine is continuously re-reduced, such that only catalytic amounts are required to completely quench the tyrosyl radical (Eqs. A-C, Fig. 4). By converting H₂O₂ to H₂O and oxygen, catalase exerts protection against ferrous-Triapine (Eqs. A-C). Samples of ribonucleotide reductase supplemented with catalase are active. In addition, the tyrosyl radicals are not quenched in samples of p53R2 and hRRM2 that were supplemented with catalase. Cell proliferation assays show that the cytotoxicity of the drug is related with the intracellular generation of ROS (Fig. 5).

Triapine causes cytotoxicity by inhibiting ribonucleotide reductase activity (16, 17). The iron complex of 1-formylisoquinoline thiosemicarbazone has been shown to quench the tyrosyl radical of hRRM2 in a reaction that requires oxygen (25). In in vitro experiments, Triapine increases ascorbate oxidation and benzoate hydroxylation. The compound causes marked plasmid DNA degradation in the presence of H₂O₂ and Fe(II) (26). Incubation of cells with Triapine reduces the cellular reductant reduced glutathione level, decreases the tyrosyl radical EPR signal of ribonucleotide reductase, and causes precipitation of cellular DNA, suggesting that a redox-active Triapine-iron complex may produce cytotoxicity in vivo (26). The DNA damage effects may be similar to that of Adriamycin and bleomycin. These antitumoral drugs produce damage to DNA via free radical chemistry upon binding of transition metal ions (27). In the present study, we provide direct evidence to confirm the generation of ROS by Fe(II)-(3-AP), which is involved in the quenching of the tyrosyl radical of ribonucleotide reductase small subunits, enzyme inactivation, and ultimately the antiproliferation activity of the drug. The effects of Triapine-induced ribonucleotide reductase inhibition (inhibit DNA synthesis and prevent DNA repair) and DNA damage may combine synergistically to cause cell cycle arrest and apoptosis.

HCTs are efficient iron chelators. Inhibition could occur either by coordination of iron in the metal bound enzyme or by the prior formation of the iron-HCT chelate, which then interacts with the enzyme (16). Our work shows that Triapine does not significantly remove active site iron from hRRM2 or p53R2 protein in vitro. In vivo, iron in complete cell culture medium, intracellular pools, or serum is available to combine with Triapine. Triapine affects cellular iron metabolism (26). Triapine can act as a chelator to inhibit Fe uptake from transferrin and increase Fe release from cells. Clinical studies on patients undergoing Triapine treatment indicate that Triapine causes transient increments in serum iron and ferritin but does not cause a net loss of body iron (19).

The effects of ROS on DNA, proteins, and other biomolecules are well documented (28). In vivo, ROS are toxic to proteins other than ribonucleotide reductase causing side effects, such as methemoglobinemia and hypoxia, which result in acute symptoms in patients with limited pulmonary or cardiovascular reserve (18). Oxidation of ferrous hemoglobin by ROS blocks oxygen binding and delivery.

Hydroxyurea, Triapine, and desferrioxamine are representative small-subunit inhibitors that perturb the di-iron cluster/tyrosyl radical cofactor. However, their efficacy and mechanism of action are different. Hydroxyurea inactivates ribonucleotide reductase by directly reducing the tyrosyl radical (13, 29). In the absence of iron, hydroxyurea is about equally active toward both hRRM2/hRRM1 and p53R2/hRRM1. However, with excess iron, hydroxyurea is less active and shows selectivity for hRRM2/hRRM1 over p53R2/hRRM1 (4). Triapine is much more potent than hydroxyurea and fully active against hydroxyurea-resistant cell lines (16, 17). Fe(II)-(3-AP) shows selective inhibition for p53R2/hRRM1 in this study. We suggest that this may be due to different sensitivity of hRRM2 and p53R2 to ROS (30). However, addition of excess iron significantly regenerates p53R2/hRRM1 resulting in a similar inhibitory potency of Fe(II)-Triapine toward both ribonucleotide reductases. Desferrioxamine forms a redox-inactive ferric complex (31). However, neither desferrioxamine nor its iron complex has a direct effect on the tyrosyl radical (32). Instead, desferrioxamine inhibits ribonucleotide reductase by sequestering environmental iron and thus preventing the small-subunit reconstitution reaction (31, 32).

Small-subunit inhibitors have important clinical value for cancer treatment either as single agents or in combination with other anticancer drugs (13, 16–19). However, the efficiency of ribonucleotide reductase small subunit inhibitors is affected by the regenerability of the iron/tyrosyl radical cofactor. The design of novel small-subunit inhibitors that target the iron/tyrosyl radical cofactor, but with alternative mechanisms of action, is a challenge. hRRM2 and p53R2 play different roles in cells and respond differently to small molecule inhibitors (1–8, 13). Development of small-subunit–specific inhibitors for different clinical applications is an attractive direction for research. Our findings shed light into the mechanism of action of Triapine in human cancer treatment.

We thank Dr. Leila Su (City of Hope) for helpful comments on the article.