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Experimental Therapeutics |
Imperial Cancer Research Fund Molecular Pharmacology Unit, Biomedical Research Centre, University of Dundee, Ninewells Hospital and Medical School, Dundee, DD1 9SY, United Kingdom [A. P. G., M. J. I. P., C. R. W.]; Department of Pharmaceutical Chemistry, School of Pharmacy, University of California, San Francisco, California 94143-0446 [I. R-C.]; School of Pharmacy and Pharmaceutical Sciences, The University of Manchester, Oxford Road, Manchester, M13 9PL, United Kingdom [E. C. C., I. J. S.]; and SmithKline Beecham Research, King of Prussia, Pennsylvania 19406-0939 [D. G. T.]
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ABSTRACT |
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This study suggests that the NOSs could play a key role in the therapeutic effects of tirapazamine, particularly because NOS activity is markedly increased in several human tumors. In addition, the presence of NOS in the heart indicates that these enzymes may contribute to the cardiotoxicity of redox cycling drugs, such as doxorubicin.
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The NOS reductase domain shares sequence homology with the flavoenzyme, NADPH-cytochrome P450 oxidoreductase (EC 1.6.2.4), the activating coenzyme of the cytochrome P450 monooxygenase complex (9) . In addition to its role in P450-mediated phase I metabolism of xenobiotic compounds, P450 reductase also has the ability to reduce a range of one-electron acceptors. Importantly, these include the anticancer drugs doxorubicin (Adriamycin; Ref. 10 ) and tirapazamine (3-amino-1,2,4-benzotriazine-1,4-di-N-oxide, SR 4233; Ref. 11 ) as well as the model oxidative stress inducing compound menadione (2-methyl-1,4-naphthoquinone; Ref. 12 ).
Menadione is a simple quinoid compound that inhibits the growth of a number of tumor cells in vitro and has been used in combination therapies with warfarin and 5-fluorouracil to treat cancer cells (13) . Doxorubicin is a more complex anthracycline antibiotic with a broad spectrum of antitumor clinical activity, which has proved effective in the treatment of acute leukemias and malignant lymphomas as well as a number of solid tumors (14) . However, the clinical use of doxorubicin is severely limited due to its potentially lethal cardiotoxicity, which is probably the consequence of free radical formation (15) . Oxygen- and hydroxyl-free radicals may be generated by flavoenzyme-catalyzed redox cycling reactions of doxorubicin (16 , 17) . Thus, reducing enzymes, such as P450 reductase, may contribute significantly to doxorubicin-induced cardiomyopathy (18 , 19) .
In the absence of oxygen, P450 reductase interacts with doxorubicin leading to cleavage of the anthracycline glycosidic bond, producing a molecule with no antitumor activity (20) . Regions of low oxygen tension (hypoxia) can exist in many human solid tumors (21 , 22) , making this reaction of clinical importance. In addition, hypoxic cells are known to be radiation resistant and can adversely influence the outcome of radiation treatment in cancer therapy (23) . A recent study of cervical cancer indicated that the oxygen status of a tumor is the single most important prognostic factor (24) . Consequently, compounds that display a selective toxicity toward resistant hypoxic cells are, therefore, of potential clinical benefit in the treatment of solid tumors.
The benzotriazine di-N-oxide, tirapazamine, is the lead compound in a new class of potent hypoxic cell cytotoxins that exhibit selective hypoxic cell toxicity (25) . Currently, the use of tirapazamine for combination therapy with cisplatin and ionizing radiation is being clinical investigated.
Tirapazamine hypoxic selectivity is thought to be a consequence of enzyme-mediated, one-electron, reductive bioactivation of tirapazamine to a cytotoxic species. This reduction can be catalyzed by various reductase enzymes, including P450 reductase (11) . One-electron reduction of tirapazamine yields a nitroxide radical (26) , which reacts with DNA to cause deoxyribose fragmentation with the concomitant formation of the stable, inactive two-electron reduced product, SR 4317. The initial DNA damage subsequently leads to strand breakage and chromosome aberrations (27) . Cytotoxicity is minimal under aerobic conditions due to the rapid reoxygenation of the nitroxide radical, and this is thought to be the molecular basis for the unusually high selective tumor toxicity of tirapazamine (26) .
P450 reductase has been shown to activate tirapazamine in vitro to a DNA-damaging species under anaerobic conditions (28) . Also, breast cancer cell lines engineered to increase expression of P450 reductase show elevated sensitivity to tirapazamine (29) . P450 reductase is widely expressed in many tissue types (30) , and although its expression in tumors is poorly characterized, it is considered to be a major enzyme involved in both the bioactivation of tirapazamine and the redox cycling of quinones.
Due to the widespread expression and the structural similarities of NOS with P450 reductase, it may be expected that it may also play a similarly important role in the metabolism of such compounds. Using high-purity recombinant NOS isoforms, we, therefore, studied the role of the three major classes of NOS in the bioactivation of tirapazamine and also the redox cycling reactions of both doxorubicin and menadione.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Enzyme Preparation.
Purified recombinant rat NOS I (32)
, mouse NOS II (33)
, and bovine NOS III (34)
were obtained as indicated previously and were used throughout this study. cDNA encoding the bovine NOS III reductase domain (residues 489–1205) fused to a NH2-terminal poly-His-tag was cloned downstream of the TacTac promoter in the expression vector pCWori (35)
. Briefly, a new NdeI site was created by PCR so that a methionine residue was introduced into the NOS III cDNA at a position that corresponds to residue 488, just before the consensus sequence of the CaM recognition sequence. Hence, the new NH2-terminal sequence of the CaM recognition sequence and reductase domain was MGAGITRKK. The PCR product with the entire reductase cDNA was ligated into the pGEMT vector (Promega), and the cDNA was completely sequenced. A double NdeI-XbaI digest was then performed, and the excised cDNA was ligated into the previously described poly-His pCWori vector (34)
. The vector was used to transform competent cells containing the CaM coexpression plasmid (36
, 37)
. The cells were grown as described (32)
, except that the temperature was 30°C. The protein was purified by affinity chromatography using nickel agarose followed by 2',5'-ADP agarose columns, as described previously (35)
. To keep the CaM bound, we carried out the purification in the presence of 100 mM Ca2+. The resulting recombinant protein was expressed in 2x YT media at 30°C for 20 h and purified as described previously (34)
. In all cases, stabilizing L-arginine was removed by dialysis against 50 mM Tris-HCl (pH 7.5) followed by passing the sample through a Dowex 50X8-200 mini-column. Protein concentrations were determined by Bradford analysis using BSA as a protein standard.
Anaerobic Reduction of Tirapazamine and Doxorubicin.
The activity of NOSs in the reductive metabolism of the antitumor drug was determined in sealed septa under hypoxic conditions generated using zero-grade N2 gas passed through an oxytrap (Alltech, Camforth, United Kingdom), as described by Fitzsimmons et al. (28)
. All tirapazamine reactions were performed at 37°C for 10 min, during which time the rate of SR 4317 was found to be linear. Incubations contained 50 mM Tris-HCl buffer (pH 7.5)-1 mM NADPH in a total volume of 500 µl. After preincubation for 10 min (to remove virtually all of the residual oxygen), reactions were initiated by the addition of tirapazamine in 25 µl of DMSO to give final concentrations between 0.2 and 2 mM. Aliquots of 200 µl were removed after 3 and 6 min and added to 400 µl of methanol containing an internal standard (4-nitroquinoline N-oxide, 45 µg/ml). Prior to analysis, samples were centrifuged at 4°C (5000 x g for 5 min), and 20 µl were analyzed by HPLC (see below). Control reactions were performed in the presence of air, nitro-L-arginine (500 µM), Ca2+/CaM (250 µM, 400 units/ml), and boiled enzyme or in the absence of NADPH. Addition of Ca2+/CaM had no effect on rates of redox cycling.
Doxorubicin reductive cleavage assays were carried out essentially as above with 50 pmol of enzyme and a final doxorubicin concentration of 75 µM. Reactions were carried out for 10 min before being stopped by the addition of methanol. Samples were then centrifuged prior to 20 µl being analyzed by HPLC.
HPLC Analysis.
Tirapazamine and its stable metabolites were analyzed by isocratic reverse-phase HPLC. Chromatography was carried out using a Hewlett Packard 1100 system, with a Spherisorb ODS2 semi-preparative column (250 mm x 8 mm; Jones Chromatography). Substrate and metabolites were eluted from the column using 18% methanol in water at a flow rate of 2 ml/min. Metabolites were detected by absorbance at 254 nm. Peaks were identified and quantified by comparison with authentic standards.
Doxorubicin metabolites were analyzed by isocratic reverse-phase HPLC using a Hewlett Packard 1050 HPLC. Products were separated using a Hypersil ODS analytical column (250 mm x 4 mm; Hewlett Packard) and eluted with 60% 5 mM phosphate (pH 3)-28.6% acetonitrile in methanol at 1 ml/min. Metabolites were identified by fluorescence (Ex = 254 nm, Em = 560 nm) and compared to authentic standards.
Aerobic Enzyme Assays.
The reduction of doxorubicin and menadione was carried out in 50 mM Tris-HCl (pH 7.5)-1 mM NADPH and various substrate concentrations at 37°C. The total incubation volume was 500 µl. Reactions were initiated by the addition of 10 µg of NOS enzyme. The oxidation of NADPH was then monitored at 340 nm using a Shimadzu UV 2000 spectrophotometer. Final doxorubicin concentrations ranged from 20 to 100 µM, and menadione concentrations ranged from 10 to 22.5 µM. Control reactions were carried out in the absence of active enzyme and NADPH essentially as described above.
DNA Damage Assays.
The ability of NOS-mediated metabolism of tirapazamine to cause DNA damage was examined as described by Fitzsimmons et al. (28)
. Briefly, the conversion of supercoiled (form I) pBluescript (Stratagene) to the relaxed, circular conformation (form II) or to the linearized conformation (form III) was monitored by gel electrophoresis. Two µg of pBluescript DNA with 1 mM NADPH in 50 mM Tris-HCl (pH 7.5) were incubated with varying concentrations of enzyme (25–100 pmol) and tirapazamine (0.125–2 mM) in a final volume of 120 µl under both aerobic and hypoxic conditions. Superoxide dismutase (600 µg/ml) and catalase (600 µg/ml) were also added to some reactions. After 20 min at 37°C, incubations were stopped by the addition of 30 µl of 5 mM EDTA, 0.5% (w/v) SDS, 60% (v/v) glycerol, and 0.1% (w/v) bromphenol blue. Aliquots (25 µl) were then analyzed by gel electrophoresis using 1% (w/v) agarose gels and the DNA fragments identified by ethidium bromide staining (0.5 µg/ml). Quantification of the DNA band intensities was carried out by densitometric analysis using Molecular Analyst software using correction factors to account for the differential staining of form I DNA by ethidium bromide (27)
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NOS Inhibition Assay.
NOS activity was measured by monitoring the conversion of [14C]arginine to [14C]citrulline, as described by Bredt and Snyder (38)
. Assays were performed in 150 µl of 50 mM Tris-HCl (pH 7.5) containing 1 mM DTT, 1.25 mM CaCl2, 400 units/ml CaM, 1 mM NADPH, and 10 µML-arginine (0.5 µCi). Incubations were carried out in the presence of doxorubicin (20 or 60 µM), menadione (20 or 60 µM), or tirapazamine (600 or 1000 µM). Inhibition over a range of substrate concentrations was tested initially, and the two concentrations used in these experiments were those that gave low and high levels of inhibition. Reactions were initiated by the addition of 15 pmol of NOS and incubated at 37°C for 10 min. Reactions were stopped by the addition of 5 ml of 50% (v/v) Dowex-50X800 resin in water to absorb the L-arginine. The resin was allowed to settle before the supernatant was removed and analyzed by scintillation counting.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and 2
) showed that NOS III had the highest affinity for both doxorubicin and menadione with Kms of 40.6 and 22.7 µM, respectively. NOS II had the lowest affinity for both compounds with Kms of 210.5 and 44.1 µM, respectively. Interestingly, the maximal velocity of NOS II for both doxorubicin and menadione was considerably higher than for NOS I and III. This was reflected in the Kcats of NOS II for both quinone compounds, which were 5–10-fold higher than the other isoforms.
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and 2
).
NOS-mediated Doxorubicin Hypoxic Cleavage.
Under hypoxic conditions, all NOS isoforms and the NOS III reductase domain reduced doxorubicin to yield 7-deoxydoxorubicin aglycone (Fig. 1A)
. In all cases, the reaction was dependent on the presence of NADPH and active enzyme and the absence of oxygen (data not shown).
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. No product was formed in the presence of oxygen or in the absence of NADPH and active enzyme. NOS-mediated SR 4317 formation followed Michaelis-Menten kinetics (Table 3)
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Effect of NOS-mediated Tirapazamine Reduction on DNA.
The ability of the NOSs and NOS III reductase domain to metabolize tirapazamine via a product that damages DNA was investigated by incubating tirapazamine (2 mM) and plasmid DNA with increasing NOS concentrations. Reactions were carried out under both aerobic and hypoxic conditions (Figs. 2
and 3
). Under aerobic conditions, NOS isoforms metabolized tirapazamine to products that were able to damage DNA (Fig. 2A)
. Fig. 2B
shows the densitometric analysis of the DNA damage. The maximum quantity of DNA converted to the nicked form was 50% using enzyme concentrations of up to 100 pmol. However, under hypoxic conditions the level of DNA damage was markedly increased (Fig. 3B)
. In the presence of high concentrations of enzyme (100 pmol), over 85% of plasmid DNA was converted to the relaxed form (Fig. 3B)
. NOS II did not show an obvious dose response, possibly due to a higher affinity for tirapazamine.
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. Under aerobic conditions, even at the highest tirapazamine concentrations, the extent of the damage was limited to <40% (Fig. 4C)
. Under hypoxic conditions, the degree of single-strand breaks was markedly increased, with evidence of double-strand breaks occurring at high tirapazamine concentrations. This indicates the presence of a more damaging species under hypoxic conditions.
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. DNA damage under hypoxic conditions was unaffected by the presence of these enzymes. This suggests the presence of two different DNA-damaging species, depending on the presence of oxygen.
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. The quinone compounds, doxorubicin, and menadione, appeared to be the most potent NOS inhibitors. NOS III was the most sensitive to inhibition by all three compounds tested, and NOS I was the most resistant.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), which is marginally lower than that reported for P450 reductase (6.34 s-1). The use of a recombinant, truncated NOS III protein demonstrated that the reductase portion of NOS was sufficient to carry out the one-electron reduction of tirapazamine. Tirapazamine’s selective hypoxic toxicity is a consequence of its flavoprotein-catalyzed, one-electron reduction, which generates a reactive intermediate (28 , 38) . We have shown that NOS can catalyze the formation of DNA-damaging products from tirapazamine. The predominant form of plasmid DNA observed after incubation with NOS and tirapazamine under hypoxic conditions was that of the relaxed circular conformation (form II). The conversion of DNA, from form I to form II, is indicative of DNA single-strand breaks, thus demonstrating that NOS may play a significant role in the activity of tirapazamine and related compounds.
The addition of superoxide dismutase and catalase significantly inhibited tirapazamine-mediated lesions in the presence of oxygen but had no effect on DNA damage under hypoxic conditions. Thus, at least two different DNA-damaging species are formed. Under hypoxic conditions, P450 reductase is able to form the tirapazamine nitroxide radical, whereas under aerobic conditions, it forms the superoxide radical (26) , this is probably also true of NOS. The resistance of the nitroxide radical to detoxification by defense enzymes may enhance tirapazamine’s antitumor toxicity.
Thus, NOS, like P450 reductase, is able to carry out the one-electron reduction of tirapazamine to toxic products. Previous in vitro studies have suggested that P450 reductase is the primary enzyme responsible for the metabolic activation of tirapazamine (29 , 39) . However, the expression of P450 reductase within tumors is poorly characterized. Unlike P450 reductase, elevated NOS expression has been found in several tumor types (40) . In addition, the presence of a hypoxia response element within the NOS II promoter (41) suggests that its expression may be markedly increased within the hypoxic regions of tumors. Thus, NOS-mediated tirapazamine bioactivation may be relevant to the therapeutic activity of this drug.
Tirapazamine and other substrates subject to one-electron reduction can also inhibit the generation of NO, presumably by diverting electrons from the reductase domain. NO has been implicated in tumor vascularization by increasing blood flow and oxygen supply to tumor cells (42 , 43) . The administration of NOS inhibitors in an experimental mouse tumor model restricts tumor blood flow, leading to an increase in hypoxia (44 , 45) . This finding has been exploited with RB6145, a bioreductive drug whose tumor toxicity was enhanced by the application of nitro-L-arginine due to the increase in the number of susceptible hypoxic cells (46) . The possibility that tirapazamine is able to inhibit the formation of NO in vivo, thereby leading to a reduction in tumor blood flow, leads to the interesting possibility that tirapazamine will potentiate its own toxicity by increasing the degree of hypoxia within tumors.
Doxorubicin and menadione are structurally related quinone-containing compounds that are capable of undergoing redox cycling reactions. The reductase domain of NOS III can produce superoxide (47) , and work by Vásquez-Vivar et al. (48) has shown that NOS III is able to undertake doxorubicin redox cycling to produce superoxide. We have extended this by showing that all three isoforms of NOS, as well as the NOS III reductase domain, are able to catalyze these reactions. Kinetic analysis of this reaction revealed that NOS III had the highest binding affinity for both compounds. In all cases, NOS exhibited a higher binding affinity for menadione compared with doxorubicin. The finding that the flavoprotein domain of NOS III displayed similar kinetic properties to the holoenzyme supported the model proposed by Vásquez-Vivar et al. that the oxygenase domain plays no part in this reaction (48) .
The by-product of NOS-mediated redox cycling, superoxide radical, can be further transformed into the highly toxic hydroxyl radical that is thought to play a pivotal role in doxorubicin-induced cardiotoxicity (18 , 49) . Because the heart is deficient in several key free radical detoxifying enzymes, such as superoxide dismutase and catalase, it is left particularly vulnerable to doxorubicin induced-oxidative injury (50 , 51) . Because NOSs have been identified in cardiac myocytes, endocardium, and vascular smooth muscle cells (52) , this work and that of Vásquez-Vivar et al. (48) raise the possibility that NOS is the major enzyme involved in the cardiotoxicity of redox cycling drugs, such as doxorubicin.
We have shown that the capacity of NOS to produce NO is reduced by doxorubicin, with NOS III being particularly susceptible to this doxorubicin-mediated inhibition. In vivo, this reduction in NO concentration may be compounded by the production of superoxide formed by redox cycling because superoxide is able to quench NO extremely efficiently to form peroxynitrite (53) . The resulting reduced levels of NO in the heart has important clinical implications with respect to cardiac stress because NO is a key regulator of vascular tone and an important mediator in the myocardial contractile response (54 , 55) . Consequently, the early reversible electrocardiograph changes that occur upon doxorubicin administration may be attributed to this reduction in NO concentration.
The absence of oxygen found within tumors can lead to alternative metabolic pathways of compounds. Under hypoxic conditions, all NOS isoforms are able to carry out the reductive cleavage of doxorubicin to yield the clinically inactive 7-deoxydoxorubicin aglycone. Thus NOS may be the enzyme responsible for the reductive glycosidic cleavage of anthracycline antibiotics, a major metabolic pathway in mammalian systems (10) .
It is evident from this study that the reductase domain of NOS is capable of activating many compounds, including chemical toxins and drugs. Although the isoforms tested were of different origin (rat, mouse, and bovine), there is a very high species conservation among NOSs (5) ; thus, any general trends observed should be relevant to human NOSs. This study, therefore, widens the accepted role of NOS to include a function as an important drug metabolism enzyme.
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FOOTNOTES |
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1 This study was jointly supported by grants from the Medical Research Council and SmithKline Beecham. 
2 To whom requests for reprints should be addressed, at Imperial Cancer Research Fund Molecular Pharmacology Unit, Biomedical Research Center, University of Dundee, Ninewells Hospital and Medical School, Dundee, DD1 9SY, United Kingdom. Phone: 44 (0)1382 632 621; Fax: 44 (0)1382 668278; E-mail: rooney{at}dundee.ac.uk
3 The abbreviations used are: NO, nitric oxide; NOS, NO synthase; CaM, calmodulin.
Received 10/26/98. Accepted 2/18/99.
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REFERENCES |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and 
, NOS I; 
and •, NOS II; 
and 
, NOS III; 
and 
, NOS III reductase domain. 
), 20 and 60 µM menadione (
), or 250 and 1000 µM tirapazamine (
). Columns, mean (n = 3) percentages of control values; bars, SD (some errors were too small to plot).