Lactoperoxidase-Catalyzed Oxidation of the Anticancer Agent

Mitoxantrone [1,4-dihydroxy-5,8-bis[[2-[(2-hydroxyethyl)amino]ethyl]amino]-9,10-anthracene- dione, MXH2] is a novel anticancer agent frequently employ...
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Chem. Res. Toxicol. 1997, 10, 1325-1330

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Articles Lactoperoxidase-Catalyzed Oxidation of the Anticancer Agent Mitoxantrone by Nitrogen Dioxide (NO2•) Radicals Krzysztof J. Reszka,* Zenon Matuszak,† and Colin F. Chignell Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences/NIH, Research Triangle Park, North Carolina 27709 Received March 14, 1997X

Mitoxantrone [1,4-dihydroxy-5,8-bis[[2-[(2-hydroxyethyl)amino]ethyl]amino]-9,10-anthracenedione, MXH2] is a novel anticancer agent frequently employed in the chemotherapy of leukemia and breast cancer. Earlier studies have shown that metabolic oxidation to reactive 1,4-quinone or/and 5,8-diiminequinone intermediates may be an important mechanism of activation of this agent, pertinent to its cytotoxic action in vivo. Here we report that in the presence of nitrite ions (NO2-), MXH2 undergoes oxidation by the mammalian enzyme lactoperoxidase (LPO) and hydrogen peroxide and that the process proceeds at a rate that is proportional to NO2concentration. In contrast, when MXH2 was exposed to LPO/H2O2 in the absence of nitrite, oxidation of the drug was either completely absent or markedly inhibited. These experiments were carried out using concentrated solutions of MXH2 (∼100 µM) at near neutral pH where dimers of the drug predominate. We propose that oxidation of MXH2 is mediated by an LPO/ H2O2 metabolite of NO2-, most likely the •NO2 radical. Because in mitoxantrone therapy the drug is administered intravenously, it is directly exposed to nitrogen oxides and other free radicals produced by blood components. It is therefore possible that the ability of mitoxantrone to react with the nitrogen dioxide radical may be relevant to the biological action of the drug in vivo.

Introduction Mitoxantrone [1,4-dihydroxy-5,8-bis[[2-[(2-hydroxyethyl)amino]ethyl]amino]-9,10-anthracenedione, MXH21) (Scheme 1) is a novel anticancer agent useful in the chemotherapy of breast cancer and leukemia (1, 2). The exact mechanisms of the biological action of MXH2 have not been fully determined, but it has been shown that the compound inhibits several processes which are important in cancer development and dissemination such as angiogenesis (3, 4), lipid peroxidation (5, 6), platelet aggregation, and in vitro prostaglandin E2 production (7). In vitro and in vivo studies have also revealed that MXH2 undergoes metabolic oxidation to the highly electrophilic transients 1,4-quinone, MX(O), and/or 5,8-diiminoquinone, MX(N) (Scheme 1). In this oxidized form the drug reacts with physiological electron donors such as ascorbic acid, forms conjugates with reduced glutathione (8, 9), and binds covalently to DNA (10-13). In the absence of suitable substrates or electron donors, the oxidized drug can undergo intramolecular rearrangement to a cyclized product, hexahydronaphtho[2,3-f]quinoxaline-7,12-dione, MH2, (Scheme 1), which is also redox-active due to the presence of the hydroquinone moiety in the metabolite’s structure (14, 15). * To whom correspondence should be addressed. Phone: (919) 5414751. Fax: (919) 541-5737. E-mail: [email protected]. † On leave from the Department of Biophysics, Institute of Molecular Biology, Jagiellonian University, Krakow, Poland. X Abstract published in Advance ACS Abstracts, November 15, 1997. 1 Abbreviations: MXH , mitoxantrone; MXH•, mitoxantrone-derived 2 radical; MX(O) and MX(N), oxidized (two-electron) mitoxantrone-1,4quinone and -5,8-diiminoquinone forms, respectively; MH2 and MH22+, reduced and oxidized forms of the cyclized metabolite of mitoxantrone; LPO, lactoperoxidase.

S0893-228x(97)00039-8

Scheme 1. Structures of Mitoxantrone and Its Metabolites

Recently, we have reported that MXH2 scavenges nitrous acid (HNO2) and suggested that the drug may

This article not subject to U.S. Copyright.

Published 1997 by the American Chemical Society

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also interact with other reactive nitrogen species such as nitrogen dioxide (•NO2) radicals (16). The aim of the present study was to test this hypothesis. Here we report that when MXH2 was exposed to the mammalian enzyme lactoperoxidase (LPO), hydrogen peroxide, and nitrite ions (NO2-), the drug underwent oxidation and the process proceeded at a rate that was proportional to NO2concentration. In contrast, when MXH2 was exposed to LPO/H2O2 in the absence of nitrite, oxidation of the drug did not occur. We conclude that oxidation of MXH2 is mediated by an LPO/H2O2 metabolite of NO2-, most likely the •NO2 radical (17-21), and suggest that the ability to react with this nitrogen oxide may be relevant to the biological action of the drug in vivo.

Experimental Procedures MXH2 (the dihydrochloride form, NSC 301739) was obtained from the Drug Synthesis and Chemistry Branch, Developmental Therapeutic Program, Division of Cancer Treatment, NCI (Bethesda, MD). Sodium nitrite (99.9%) and LPO (EC 1.11.1.7, 80 units of activity/mg of solid) were from Sigma Chemical (St. Louis, MO), and sodium ascorbate was from Aldrich Chemical Co. (Milwaukee, WI). H2O2 (30%; Fisher Scientific, Fair Lawn, NJ) was diluted, and its concentration was determined spectrophotometrically at 240 nm using a molar absorptivity of 39.4 M-1 cm-1. The concentration of LPO was determined by measuring its absorption at 414 nm [ ) 1.12 × 105 M-1 cm-1 (22)], and that of MXH2 was determined at 682 nm [ ) 8.36 × 103 M-1cm-1 (23)]. Stock solutions of MXH2 (ca. 10 mM) and nitrite (1 M) were prepared in deionized water (HPLC reagent; Baker). Measurements were performed in 50 mM acetate buffer (pH 5.3) and 50 mM phosphate buffers (pH 7.4 and 8.1). Absorption spectra were measured in quartz cuvettes (0.4- and 1-cm light path) using a Hewlett-Packard diode array spectrophotometer model 8451A (Palo Alto, CA). To obtain MXH2 monomers, sodium dodecyl sulfate (SDS) (Schwarz/Mann Biotech, Cleveland, OH) was added to the drug dissolved in pH 7.4 buffer. Unless otherwise stated, reactions were initiated by the addition of LPO to samples containing all necessary components. When required, samples were bubbled continuously with a stream of air or nitrogen during measurements. The reaction of MXH2 with LPO/H2O2/NO2- was measured at pH 7.4 using MXH2 concentrations of ∼100 µM and [H2O2] >>> [LPO]. Under these conditions LPO metabolism of MXH2 in the absence of nitrite was either completely absent or markedly inhibited.

Results In aqueous solutions MXH2 exists as a mixture of monomers and dimers or even higher aggregates (2326). The dimerization constant has been determined to be 3 × 104 M-1 based on spectrophotometric measurements (24) and 2.7 × 104 M-1 using the fluorescence technique (26). The extent of aggregation depends on drug concentration, pH, and ionic strength of buffer. The visible absorption spectrum of MXH2 (ca. 47 µM) in phosphate buffer, pH 7.4, shows a maximum at 612 nm and a shoulder at 668 nm (Figure 1, spectrum a). In this sample ca. 44% of the drug molecules exist as monomers and the rest mostly as dimers. In the presence of the detergent SDS (5 mM) the absorption spectrum (Figure 2, spectrum b) exhibits an intense peak at the longer wavelength of 668 nm and a weaker one at 614 nm. These features are characteristic of a sample consisting mostly of the monomeric form of the drug (24) and suggest that SDS might be used to prevent drug aggregation. In experiments carried out in the present study, the drug exists primarily in the dimerized form.

Figure 1. Absorption spectra of mitoxantrone (∼47 µM) at pH 7.4. Spectrum a was obtained in phosphate buffer (50 mM) and is characteristic of a mixture of the drug’s monomers and dimers. Spectrum b was observed upon the addition of SDS (5 mM) to sample a and is characteristic of a solution containing MXH2 monomers.

Based on the reports that horseradish peroxidase (HRP) catalyzes oxidation of MXH2 by H2O2 (14, 15) and the known similarity of action between HRP and LPO toward polyphenolics, it was expected that LPO also should support oxidation of MXH2 by H2O2. However, we found that when [H2O2] . [LPO] and [MXH2] was 50 µM or higher, then oxidation of the drug did not occur or was markedly inhibited,2,3 as judged by the lack of any changes in the absorption spectrum of the drug during several minutes of incubation (Figure 2, spectrum a and panel B). Neither the order of addition of the reactants nor exposure of MXH2 to LPO/H2O2 at a more acidic pH (5.3), or a more alkaline pH (8.1), affected the absorption spectrum of the drug, suggesting that under these conditions LPO/H2O2 does not metabolize the drug. We attribute the inability of LPO/H2O2 to oxidize MXH2 to the drug aggregation,4 which hinders approach of the drug molecules to the active site of LPO since it is known that the heme pocket of LPO is narrower than that of HRP (19, 22, 27). In subsequent experiments we took advantage of the above observations and employed conditions under which LPO/H2O2 alone did not metabolize the drug. When MXH2 was exposed to the LPO/H2O2 system in the presence of nitrite, rapid changes in the absorption spectrum were observed, indicating that drug oxidation had occurred (Figure 2A, spectra b-d). The changes consisted of a blue shift of the peak at 610 nm to a new position at 590 nm, followed by its rapid decrease and the concomitant formation of a new broad absorption band centered around 732 nm (Figure 2A, spectrum d). 2 Compounds II and III of LPO oxidize mitoxantrone monomers, and LPO II oxidizes mitoxantrone dimers (Matuszak, Chignell, and Reszka, unpublished results). 3 This high [H O ]/[LPO] ratio favors the formation of LPO III, 2 2 especially in the absence of substrate (20). We found that MXH2 does not prevent the formation of LPO III, nor does it react with independently prepared LPO III. Because LPO III is formed from LPO II and H2O2, this result suggests that H2O2 is a better substrate for LPO II than are MXH2 dimers. However, when nitrite was added, oxidation of MXH2 did occur (Figure 3), implying that then the reaction of LPO/ H2O2 with NO2- is preferred over that with MXH2. 4 In the model of the mitoxantrone dimer, proposed based on the molecular exciton theory, the dimer has a parallel configuration with alkyl chains in one molecule oriented in a direction opposite to that in the second molecule (26). In such a configuration access to the drug’s redox groups might indeed be restricted.

Oxidation of Mitoxantrone by Nitrogen Dioxide

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Figure 2. Oxidation of mitoxantrone by LPO/H2O2/NO2- in a pH 7.4 phosphate buffer (50 mM). (A) Absorption spectra: a, spectrum of MXH2 (118 µM) in the presence of H2O2 (5 mM), no change in the spectrum was observed when LPO (0.27 µM) was added; b-d, same as above but in the presence of LPO and NO2- (23 mM) recorded after 60 s (b), 120 s (c), and 150 s (d) of incubation; e, spectrum observed after addition of ascorbate (excess) to sample d. (B) Time course of oxidation of MXH2 measured as decrease of absorbance at 610 nm. Arrows indicate time points of the addition of LPO (0.27 µM) and NaNO2 (17 mM). H2O2 was present from the beginning. The initial slow and the following fast decreases of the absorbance at 610 nm are assigned to oxidation of MXH2 (sector a) and the metabolite MH2 (sector b), respectively. The solution was purged with a stream of air during the measurement.

These new absorption bands are very close to those determined earlier for MH2, the cyclized metabolite of MXH2 (590 nm), and its oxidized form MH22+ (732 nm) (14-16). This suggestion is corroborated by the observation that upon the addition of ascorbate, the spectrum with the peak at 732 nm, tentatively assigned to MH22+, changed to that with the peak at 590 nm (Figure 2A, spectra d and e, respectively) in agreement with earlier reports (14-16). Thus it would appear that products of oxidation of MXH2 by LPO/H2O2/NO2-, HRP/ H2O2, MPO/H2O2, and HNO2 are similar (14-16, 28). The observation that oxidation of MXH2 by LPO/H2O2 is dependent upon NO2- suggests that a product of NO2metabolism, most likely the •NO2 radical, is directly involved in drug oxidation. The time course of drug oxidation was measured following the decrease of its absorbance at 610 nm (Figure 2B). It was observed that the absorbance remained unchanged upon the addition of H2O2 and LPO to the drug, but it started to decrease immediately following the addition of NO2-. Figure 2B also shows that the process has two kinetic components that produce a slow decrease in absorbance (sector a), which can be associated with the oxidative transformation of MXH2 to an intermediate metabolite, most likely the cyclized product MH2 (cf. Scheme 1), followed by a faster decrease (sector b) which can be assigned to further oxidation of the intermediate to MH22+. Effect of NaNO2 Concentration. The effect of NO2concentration (4.3, 8.6, 12.9, and 25 mM) on the rate of MXH2 oxidation was measured by following the time course of the decrease of the absorbance at 610 nm (Figure 3A). Relative rates of the oxidation of MXH2 and its presumed metabolite MH2 increase linearly with [NaNO2] (Figure 3B), which further supports the hypothesis that a product of NO2- metabolism is directly

involved in the oxidation process. Oxidation of MXH2 in aerated and air-free samples proceeded at very similar rates (not shown), indicating that dissolved oxygen does not participate in the reaction. Effect of pH. The addition of nitrite to MXH2 in an acidic buffer (pH 5.3) caused slow oxidation of the drug, even in the absence of LPO/H2O2 (Figure 4). This observation agrees with our earlier finding that nitrous acid, HNO2, which is formed from the nitrite at acid pH [pKa(HNO2/NO2-) ) 3.35 (29)], can oxidize the drug (16). However, the process was markedly accelerated by the addition of LPO and H2O2 to the sample (Figure 4). Thus, at acid pH oxidation of MXH2 can be accomplished via two pathways: one involving HNO2 and the other involving an LPO/H2O2 metabolite of the nitrite. At pH 8.1, oxidation of MXH2 proceeded similarly to that at pH 7.4.

Discussion We report that nitrite catalyzes oxidation of MXH2 by LPO and H2O2 and that the rate of this process is proportional to nitrite concentration. These results strongly suggest that a product of nitrite metabolism by LPO/H2O2, presumably the •NO2 radical, is involved in drug oxidation. Oxidation of nitrite by hemoprotein peroxidase enzymes and H2O2 has been studied in the past. In 1952, Chance (17) reported that HRP compound I, a product of two-electron oxidation of the native (ferric) form of the enzyme by H2O2, reacts with nitrite in a one-electrontransfer process forming compound II. This reaction requires that the complementary metabolite be a free radical, in this case •NO2. This observation has been later confirmed by other researchers (18). Because most hemoproteins in the presence of H2O2 show similar reactivity (19, 30), it seems reasonable to assume that

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Figure 3. Oxidation of mitoxantrone by LPO/H2O2: dependence on nitrite concentration. (A) Time course of the oxidation of MXH2 (114 µM) was measured by following the decrease of the absorbance at 610 nm in the presence of H2O2 (3.5 mM) and NaNO2. The reaction was initiated by the addition of LPO (0.2 µM) as indicated by the arrow V. Scans a-d were obtained at [NO2-] ) 4.3, 8.6, 12.9, and 25.0 mM, respectively. All measurements were carried out in a pH 7.4 phosphate buffer (50 mM) during continuous bubbling with air. (B) Relative rates of the oxidation of MXH2 (b) and the presumed cyclic metabolite MH2 (2) versus nitrite concentrations (from panel A). The rates were determined as slopes of the sectors a and b, respectively, as illustrated in Figure 2B.

Scheme 2. Proposed Mechanism of Oxidation of Mitoxantrone by LPO/H2O2 in the presence of NO2- a

a The process is initiated by oxidation of NO - to •NO by the LPO/H O system and subsequent reaction of the •NO radicals with 2 2 2 2 2 MXH2. MXH•, MX, MH2, and MH22+ represent MXH2-derived radical, oxidized (two-electron) MXH2 [corresponding to MX(O) and MX(N) in Scheme 1], and the reduced and oxidized forms of the cyclized metabolite of MXH2, respectively. MH2 may also undergo oxidation by •NO to MH 2+, after which it can be recovered by reaction with ascorbate, AH-. 2 2

LPO (Por-Fe3+) + H2O2 f LPO compound I (Por•+-FeIVdO) (1) LPO compound I + NO2- f LPO compound II (Por-FeIVdO) + •NO2 (2) LPO compound II + NO2- f LPO (Por-Fe3+) + •NO2 (3)

Figure 4. Oxidation of MXH2 by LPO/H2O2/NO2- in acetate buffer, pH 5.3. Arrows indicate time points of the addition of NaNO2 (13 mM), H2O2 (3.4 mM), and LPO (0.16 µM).

oxidation of NO2- by LPO/H2O2 will proceed according to the same peroxidase mechanism yielding •NO2 radicals (eqs 1-3).

where Por designates the porphyrin moiety of the enzyme. This mechanism has been corroborated by recent studies on the peroxidase-dependent antimicrobial activity and degradation of chlorophyll by nitrite (31, 20). Also the recent report showing that in the presence of NO2-, LPO/H2O2 converts tyrosine into nitrotyrosine is consistent with the generation of •NO2 radicals by this enzymatic system (21). This supports the conclusion that oxidation of MXH2 by LPO/H2O2/NO2- is mediated by •NO radicals. 2 Oxidation of MXH2 by •NO2 may involve the drug’s phenolic and/or aromatic amine functions because it is

Oxidation of Mitoxantrone by Nitrogen Dioxide

known that •NO2 oxidizes polyphenols and aromatic amines (32-34).5 Because •NO2 is a one-electron oxidant, the primary product of its reaction with MXH2 would appear to be a semiquinone radical, MXH• (Scheme 2). Attempts to detect this radical at near neutral pH by applying static EPR failed, presumably because of its rapid disproportionation to MXH2 and MX (Scheme 2) or because of its reaction with another molecule of •NO2 to form a fully oxidized compound, MX. We wish to emphasize that in the proposed mechanism (Scheme 2), MXH2 does not operate as a scavenger because it does not remove the •NO2 radicals from the system but converts them back into nitrite anions which then may re-enter the LPO metabolic pathway.6 Our spectrophotometric data suggest that oxidation of MXH2 by LPO/H2O2/NO2- leads to the formation of MH2, the primary stable product of the drug oxidation (Figure 2A, lines b, c, e). This is possible because transformation of the oxidized mitoxantrone (MX) to MH2 (Scheme 1) is a spontaneous process and occurs readily, especially when no suitable nucleophiles (thiols) are available. In the presence of LPO/H2O2/NO2-, MH2 was oxidize to MH22+ (Figure 2A, line d), and we tentatively attribute this process to the reaction of MH2 with •NO2 radicals (Scheme 2). Upon the addition of ascorbate, MH22+ was reduced back to MH2 (Figure 2, line e) in agreement with earlier reports (14-16). •NO is a highly cytotoxic species. It causes oxidation 2 of lipids and thiols and damages proteins by nitration of tyrosine residues (32-36). •NO2 is a common air pollutant and may be present in inhaled air. The radical has been shown to cause oxidative damage to blood plasma and depletion of blood antioxidants (36). Other sources of •NO2 radicals in vivo are enzymatic oxidation of nitrite, similar to that described in this work, and air oxidation of nitric oxide, •NO (37). It is known that in vivo •NO, which is produced by vascular endothelium and many other cell types, is the natural precursor of all other reactive nitrogen species (38, 39). These nitrogen oxides can be protective; for example, they participate in the bactericidal and anticancer action of macrophages (3841). However, the same species, if produced in excess, can be harmful to host tissues (42). We have shown that mitoxantrone reacts with metabolically generated •NO2 radicals in vitro, which opens the possibility that a similar reaction might occur in vivo, especially since the drug is administered intravenously (2) where it is directly exposed to reactive nitrogen oxides produced by blood components (43). In conclusion, the major finding of this study is the observation that mitoxantrone undergoes oxidation to the biologically active quinone/diiminoquinone form of the drug by a nitrite-derived metabolite of LPO/H2O2, presumably the •NO2 radical. Because in mitoxantrone therapy the drug is administered intravenously, it is directly exposed to nitrogen oxides and other free radicals produced by blood components. It is therefore possible that the ability of mitoxantrone to react with the nitrogen dioxide radical may be relevant to the biological action of the drug in vivo. 5 Although in this study the drug was oxidized by •NO reacting 2 with mitoxantrone dimers/aggregates, reaction of •NO2 with drug monomers or with mitoxantrone bound to proteins also should result in oxidation. 6 Preliminary results of MS measurements did not show the formation of nitrated/nitrosated products derived from MXH2.

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Acknowledgment. We would like to thank Dr. Kenneth Tomer, LPC/NIEHS, for providing the mass spectral data and Dr. Ann Motten for help in the preparation of this manuscript.

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