Chalcone Inhibition of Anthracycline Secondary Alcohol Metabolite

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Chem. Res. Toxicol. 2006, 19, 1518-1524

Chalcone Inhibition of Anthracycline Secondary Alcohol Metabolite Formation in Rabbit and Human Heart Cytosol Andrea Silvestrini,† Elisabetta Meucci,† Alberto Vitali,‡ Bruno Giardina,† and Alvaro Mordente*,† Institute of Biochemistry and Clinical Biochemistry, Institute of Chemistry of Molecular Recognition CNR, UniVersita` Cattolica del “Sacro Cuore”, Largo F. Vito 1, 00168 Roma, Italy ReceiVed July 13, 2006

Antineoplastic therapy with anthracyclines like doxorubicin (DOX) and daunorubicin (DNR) is limited by the possible development of a dose-related cardiomyopathy. Secondary alcohol metabolites like doxorubicinol (DOXol) and daunorubicinol (DNRol), formed by cytoplasmic two-electron reductases, have been implicated as potential mediators of anthracycline-induced cardiomyopathy. In the present study, we characterized the effects of 12 chalcones on the formation of anthracycline secondary alcohol metabolites by rabbit or human heart cytosol and compared them with those of quercetin and other flavonoids. Both chalcones and flavonoids inhibited DOXol or DNRol formation in isolated rabbit heart cytosol. Structure-activity relationships showed that inhibition by chalcones was determined primarily by the position of hydroxyl groups in their phenolic A and B rings. In particular, the presence of a hydroxyl group at C-4′ in the A ring was an important determinant of the inhibitory activity of chalcones. Among chalcones, 2′,4′,2-trihydroxychalcone exhibited the highest inhibition of both DOXol and DRNol formation, but it proved less efficient than quercetin. Different results were obtained with isolated human heart cytosol: in the latter, 2′,4′,2-trihydroxychalcone and other hydroxychalcones inhibited both DOXol and DNRol formation, whereas quercetin and other flavonoids inhibited DNRol formation but failed to inhibit or slightly stimulated DOXol formation. These results identify chalcones as versatile inhibitors of the cytoplasmic reductases that convert anthracyclines to cardiotoxic secondary alcohol metabolites. Introduction The anthracyclines doxorubicin (DOX)1 and daunorubicin (DNR) are highly effective anticancer agents, but their clinical use is limited by a cumulative dose-limiting cardiotoxicity (1, 2). Anthracycline-induced cardiotoxicity is a multifactorial process in which drug metabolites and byproducts may be involved in mediating the acute and chronic phases of myocardial dysfunction. One-electron redox-cycling of the quinone moiety in the C ring of anthracyclines generates reactive oxygen species, while two-electron reduction of the side-chain C13 carbonyl group generates secondary alcohol metabolites (DOXol and DNRol) (3-6). The current thinking is that reactive oxygen species could mediate acute cardiotoxicity, while secondary alcohol metabolites might have a role in advancing the course of cardiotoxicity toward end-stage cardiomyopathy and heart failure (5-8). The involvement of secondary alcohol metabolites in anthracycline-induced cardiomyopathy is suggested by the following pharmacokinetic and functional evidence: (1) the development of chronic cardiomyopathy usually coincides with an accumulation of DOXol in the heart (8); (2) anthracyclines lacking the side chain carbonyl group or exhibiting a reduced affinity for C13 reductases induce less severe or progressive cardiomyopathy in laboratory animals (9-11); (3) cardiac-restricted over* Corresponding author. Tel.: +390630155135; fax: +390630154309; e-mail: [email protected]. † Institute of Biochemistry and Clinical Biochemistry. ‡ Institute of Chemistry of Molecular Recognition CNR. 1 Abbreviations: DOX, doxorubicin; DNR, daunorubicin; DOXol, doxorubicinol; DNRol, daunorubicinol; DTPA, diethylenetriaminepentaacetic acid.

expression of human carbonyl reductase accelerates the course of development of cardiomyopathy induced by DOX in laboratory animals (12); and (4) genetic deletion of one copy of carbonyl reductase decreases the enzymatic conversion of DOX to DOXol and protects laboratory animals from cardiotoxicity (13). From a mechanistic view point, secondary alcohol metabolites are 20-40 times more potent than their parent drugs at inactivating membrane ATPases (7, 8, 10, 14) and related contractile events (15); moreover, secondary alcohol metabolites have been shown to be more reactive than their parent drugs toward cytoplasmic aconitase/iron regulatory protein 1 (1618), although this finding remains a matter of some debate (19, 20). Interestingly, secondary alcohol metabolites do not always mediate but sometimes diminish the antitumor activity of anthracyclines: in some cell lines, the activity of DNR was diminished by an overexpression of the reductases that formed DNRol (21, 22), while in other cell types, the activity of DOX was increased by inhibitors that suppressed DOXol formation (23). It follows that secondary alcohol metabolites might be important and rather selective mediators of cardiotoxicity, provided that they were formed inside the heart: in fact, circulating secondary alcohol metabolites were shown to be too polar to partition into the heart and reach levels high enough to induce cardiac dysfunction (6, 24). The aforesaid reasoning anticipates that inhibitors of the intramyocardial formation of anthracycline secondary alcohol metabolites might prove useful to mitigate cardiotoxicity and improve the therapeutic index of these drugs. Chalcones (trans-1,3-diphenyl-2-propen-1-ones) are the biogenetic precursors of all known flavonoids and are abundant in edible plants (25). Chemically, chalcones consist of open-chain

10.1021/tx060159a CCC: $33.50 © 2006 American Chemical Society Published on Web 10/11/2006

Chalcones and Anthracycline Alcohol Metabolite Formation

flavonoids in which the two aromatic rings are joined by a threecarbon R,β-unsaturated carbonyl system. Chalcones exhibit antioxidant and anti-inflammatory properties and have recently attracted attention also for their antitumor activity in preclinical models (26, 27). Moreover, chalcones modulate the activity of a broad array of enzymes, including members of the aldo-keto reductase superfamily that have been implicated to catalyze the formation of anthracycline secondary alcohol metabolites (28, 29). The present study was therefore aimed at assessing whether chalcones inhibited the formation of anthracycline secondary alcohol metabolites in rabbit and/or human heart.

Experimental Procedures Chemicals. Doxorubicin, DOXol, DNR, and DNRol were kindly provided by Dr. Antonino Suarato (Nerviano Medical Sciences, Milan, Italy). Anthracycline stock solutions were prepared in 18.2 MΩ·cm double-distilled deionized water (Milli-Q, Millipore Co., Bedford, MA) and shown to be stable for at least 1 month if stored at +4 °C in the dark. Sodium dihydrogen phosphate monohydrate (NaH2PO4‚H2O) and 85% ortho-phosphoric acid were obtained from Fluka; NADP(H) (tetrasodium salt), HEPES, ammonium sulfate, DMSO, sodium chloride, DTPA, EDTA (disodium salt), chrysin (5,7-dihydroxyflavone), morin (2′,3,4′,5,7-pentahydroxyflavone), quercetin (3,3′,4′,5,7-pentahydroxyflavone), taxifolin (dihydroquercetin), and rutin hydrate were purchased from Sigma-Aldrich; HPLC-grade acetonitrile and chloroform and disodium hydrogen phosphate 12-hydrate (Na2HPO4‚12H2O) were from Merck; and 1-heptanol was from BDH. The bicinchoninic acid protein assay reagent kit was purchased from Pierce. Chalcones were a kind gift of Dr. G. Delle Monache (Dipartimento di Studi di Chimica e Tecnologia delle Sostanze Biologicamente Attive, Universita` La Sapienza, Roma, Italy). Chalcones were synthesized as described (30, 31), and their structures were confirmed using melting point, 1H and 13C NMR, and EI-MS spectra. Chalcones were recovered dry at 4 °C in the dark before use to prevent cis-form isomerization. All other chemicals were of the highest grade of purity. Preparation of Cytosolic Fractions. Male New Zealand white rabbits (aged 3-4 months and weighing 3500-4000 g/rabbit) were obtained from the animal breeding department of the Catholic University School of Medicine in Rome. Rabbits (n ) 20) were sacrificed by captive bolt discharge in accordance with the Declaration of Helsinki and the Guide for the Care and Use of Laboratory Animals as adopted by the National Institutes of Health. Hearts were quickly excised, carefully rinsed in ice-cold saline, and homogenized in 4 vol of ice-cold 10 mM HEPES buffer (pH 7.4), containing 0.3 M NaCl and 0.5 mM EDTA (homogenization buffer), using an Ultra Turrax and a glass-Teflon Potter-Elvehjem homogenizer. Cytosolic fractions were prepared by sequential homogenization, 20 min centrifugation at 8500g and 23 000g, and 90 min ultracentrifugation at 140 000g, all in 0.3 M NaCl-10 mM HEPES (pH 7.4). Next, 140 000g supernatants were stirred overnight with 65% ammonium sulfate and centrifuged at 10 000g for 20 min. Protein precipitates were resuspended in 5-6 mL of homogenization buffer, dialyzed against three 1 L changes of the same buffer added with 1 mM EDTA (to remove adventitious iron) and then against three 1 L changes of EDTA-free buffer (to remove EDTA and EDTA-iron complexes). After low-speed centrifugation to remove any insoluble material, cytosolic proteins were assayed by the bicinchoninic acid method (32) and stored in aliquots at -80 °C until used. No apparent loss of activity of cytoplasmic reductases involved in anthracycline metabolism was observed after storage. Where indicated, the same protocol was adopted to prepare cytosol from post-mortem human myocardial samples. The latter were obtained during authorized autopsies at the Department of Forensic Medicine of the Catholic University School of Medicine. Tissue removal and examination were in accordance with Institutional Ethical Guidelines for the use of human tissues for teaching and research purposes. Samples were derived from 25-40 year-

Chem. Res. Toxicol., Vol. 19, No. 11, 2006 1519 old male (n ) 2) or female (n ) 1) individuals with morphologically normal myocardium and no clinical history of acute myocardial infarction, severe cardiosclerosis, or other cardiomyopathies. All samples were collected 24 h after death and stored at -80 °C until use. In a previous study, we demonstrated that cytosolic fractions derived from post-mortem myocardial samples had essentially the same enzymatic activities of limited ex vivo samples (33). Assay for Anthracycline Secondary Alcohol Metabolites. Unless otherwise indicated, the formation of anthracycline secondary alcohol metabolites was assayed in 0.5 mL incubations containing 1 mg of cytosolic protein/mL, 50 µM DOX or DNR, and 250 µM NADP(H) in 0.3 M NaCl-10 mM HEPES (pH 7.4). After 4 h at 37 °C, the reaction was stopped by adding an equal volume (0.5 mL) of 0.2 M Na2HPO4 (pH 8.4), and samples were extracted with 4 mL of a 9:1 (v/v) chloroform/1-heptanol mixture. After 15 min vigorous shaking, samples were centrifuged for 10 min at 20 °C to separate an upper aqueous phase and a lower organic phase. The latter was reextracted with 250 µL of 0.1 M ortophosphoric acid and vortexed vigorously for 1 min at room temperature to obtain an upper aqueous layer from which 50 µL was eventually removed and used for HPLC analysis as described by Fogli et al. (34). Incubations lacking cytosol or NADP(H) served as controls. Whatever the experimental conditions employed, the formation of anthracycline alcohol metabolites linearly increased during the incubation period (240 min). The chromatographic apparatus consisted of a HPLC 1100 system (Hewlett-Packard) equipped with diode array and fluorescence detectors. Reversed-phase chromatography was performed with a Hewlett-Packard ZORBAX CN column (250 mm × 4.6 mm, 5 µm) protected by a ZORBAX CN analytical guard column (12.5 mm × 4.6 mm, 5 µm). Isocratic elution was performed with a freshly prepared mobile phase consisting of a 75:25 (v/v) mixture of 50 mM sodium dihydrogen phosphate/acetonitrile, adjusted to pH 4.0 with orthophosphoric acid and filtered through a 0.22 µm membrane (Millipore). The flow rate was 1 mL/min. C13 alcohol metabolites were detected fluorimetrically with excitation at 480 nm and emission at 560 nm and quantified against appropriate standard curves. Retention times were as follows: DOX 10 min, DOXol 6 min, DNR 18 min, and DNRol 9 min. Determination of IC50 Values. To determine the inhibitory potency of chalcones and flavonoids, anthracycline alcohol metabolite formation was measured in the absence or in the presence of different concentrations of the tested compound, dissolved in DMSO. The final DMSO concentration never exceeded 1%. The IC50 values (the concentration of the inhibitor required to produce 50% inhibition of anthracycline alcohol metabolite formation) were determined by nonlinear regression analysis of the dose-inhibition curves. Each curve was obtained using at least eight concentrations of inhibitor. All values were means ( SE of three separate experiments performed in triplicate. Of note, control experiments showed that the iron-chelator DTPA did not alter the relative potency of all chalcones tested against anthracycline metabolite formation: thus, differences between one chalcone and the other could not be attributed to different susceptibilities to metal-catalyzed oxidation and loss of stability during the course of experiments. Other experimental conditions are given in the legends of the figures and table. Determination of the Type of Inhibition. To identify the inhibition mechanism and to calculate the inhibition constants, the formation of anthracycline alcohol metabolites was measured in the absence or in the presence of different concentrations of chalcone by varying either anthracycline concentration (25-500 µM) at a fixed and saturating concentration of NADP(H) (250 µM) or NADP(H) concentration at a fixed concentration of anthracycline (50 µM). Results were presented as double-reciprocal LineweaverBurk plots. Inhibition constants (Ki) were determined by simultaneously fitting the untransformed data (i.e., control and inhibition data set) to competitive, uncompetitive, noncompetitive, and mixed enzyme inhibition equations using a nonlinear regression program

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Chart 1. Molecular Structure of Chalcones Tested in this Study

(GraphPad Prism 4.0, Graphpad Software, San Diego, CA). Values are means ( SE of three separate determinations in triplicate.

Results Effect of Chalcones and Flavonoids on the Formation of Anthracycline Secondary Alcohol Metabolites by Rabbit Heart Cytosol: Structure-Activity Study. Twelve chalcones (Chart 1) and four flavonoids (Chart 2) were tested for their ability to inhibit DOXol or DNRol formation. As shown in Figure 1, chalcones and flavonoids inhibited the formation of DOXol (Figure 1A) and DNRol (Figure 1B) in a concentrationdependent manner. The IC50 values determined for chalcones and flavonoids are reported in Table 1. With the possible exception of 4′,4dihydroxychalcone (4′,4-DHC), chalcones were more effective

at inhibiting DOXol formation, while flavonoids were more effective at inhibiting DRNol formation. Structure-activity analyses indicated that chalcones lacking hydroxyl groups (like the prototypic simple chalcone) or bearing one or two OH groups in only the B ring, as 2-hydroxychalcone (2-HC), 4-hydroxychalcone (4-HC), and 2,4-dihydroxychalcone (2,4DHC), were virtually devoid of effect on DOXol or DNRol formation (IC50 values >250 µM). Substitutions in the A ring had position-dependent effects: thus, whereas a hydroxyl group at C4′ rendered 4′-hydroxychalcone (4′-HC) an effective inhibitor of both DOXol and DNRol formation (IC50 values of 45.1 ( 3.8 and 109.7 ( 9.8 µM, respectively), the presence of a hydroxyl group at the C2′ position rendered 2′-hydroxychalcone (2′-HC) a weak inhibitor of DOXol or DNRol formation (only 35 and 20% inhibition at 250 µM, respectively). The presence

Chalcones and Anthracycline Alcohol Metabolite Formation

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Chart 2. Molecular Structure of Tested Flavonoids

Table 1. Inhibitory Effects of Chalcones and Flavonoids on DOXol and DNRol Production by Rabbit Heart compounda chalcone 2-hydroxychalcone (2-HC) 4-hydroxychalcone (4-HC) 2′-hydroxychalcone (2′-HC) 4′-hydroxychalcone (4′-HC) 2′,4′-dihydroxychalcone (2′, 4′-DHC) 4′,2-dihydroxychalcone (4′, 2-DHC) 4′,4-dihydroxychalcone (4′, 4-DHC) 2′,4′,2- trihydroxychalcone (2′, 4′, 2-THC) 2′,4′,3- trihydroxychalcone (2′, 4′, 3-THC) 2′,4′,2,3- tetrahydroxychalcone (2′, 4′, 2, 3-THC) 2′,4′,2,4-tetrahydroxychalcone (2′, 4′, 2, 4-THC) quercetin taxifolin morin chrysin

Figure 1. Effect of chalcones and flavonoids on the formation of anthracycline secondary alcohol metabolites by rabbit heart cytosol. Percent of inhibition of DOXol (A) and DNRol (B) production in the presence of 2′,4′,2-trihydroxychalcone (9), 4′,2-dihydroxychalcone (2), morin ([), and quercetin (b). Experiments were performed by preincubating rabbit heart cytosolic fractions (1 mg of protein/mL) in 10 mM HEPES-300 mM NaCl, pH 7.4, with each compound added 5 min before DOX or DNR (50 µM). Reactions were started by adding NADP(H) (250 µM). Alcohol metabolites were assayed after 240 min at 37 °C as described in the Experimental Procedures. Values are means ( SE of three separate determinations in triplicate.

of hydroxyl groups at both C2′ and C4′ positions (2′,4′dihydroxychalcone/2′,4′-DHC) slightly enhanced the inhibitory potency as compared with 4′-HC but strongly potentiated it as compared with 2′-HC. These results suggest that a hydroxyl group at C4′ might be an absolute requirement for chalcones to inhibit the formation of anthracycline secondary alcohol metabolites. We also evaluated the effects of concomitant substitutions in the A and B rings. When a second hydroxyl group was inserted in the B ring of 4′-HC at C2 or C4 to obtain 4′,2-

DOX DNR (IC50) (µM) (IC50) (µM) >250 >250 >250 >250 45.1 ( 3.8 39.1 ( 4.4 107.1 ( 8.7 71.0 ( 9.7 21.2 ( 3.6 30.9 ( 5.5 25.3 ( 5.6 32.8 ( 8.8 13.7 ( 2.8 48.9 ( 6.0 49.3 ( 6.3 >250

>250 >250 >250 >250 109.7 ( 9.8 88.8 ( 9.4 197.0 ( 13.8 56.0 ( 4.8 33.8 ( 4.4 83.5 ( 6.9 39.9 ( 4.5 59.1 ( 9.1 8.0 ( 2.0 46.3 ( 3.6 18.8 ( 4.1 32.5 ( 7.4

a Experiments were performed by preincubating rabbit heart cytosolic fractions (1 mg of protein/mL) in 10 mM HEPES-300 mM NaCl (pH 7.4) with different concentrations (n ) 8) of each chalcone added 5 min before DOX or DNR (50 µM). Reactions were started by adding NADP(H) (250 µM) and carried out at 37 °C. Alcohol metabolites were assayed after 240 min at 37 °C as described in the Experimental Procedures. The IC50 values are the means ( SE of three separate experiments performed in triplicate.

dihydroxychalcone (4′,2-DHC) or 4′,4-dihydroxychalcone (4′,4DHC), the potency of inhibition of DOXol formation decreased substantially; however, 4′,4-DHC was a better inhibitor of DNRol formation as compared with 4′-HC. 2′,4′,2-Trihydroxychalcone (2′,4′,2-THC) was the most potent inhibitor of DOXol and DNRol formation among all chalcones examined, and moving the OH group from C2 to C3 (2′,4′,3-trihydroxychalcone/2′,4′,3-THC) diminished its inhibitory activity. Finally, tetrahydroxychalcones (2′,4′,2,4-tetrahydroxychalcone/2′,4′,2,4THC and 2′,4′,3,4-tetrahydroxychalcone/2′,4′,3,4-THC) showed a lower inhibitory activity than 2′,4′,2-THC. As mentioned, all flavonoids (with the exception of chrysin) proved to inhibit both DOXol and DRNol formation (Table 1).

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Figure 2. Lineweaver-Burk plots for the inhibition of anthracycline secondary alcohol metabolite formation by 2′,4′,2-trihydroxychalcone. Experiments were performed by preincubating rabbit heart cytosolic fractions (1 mg of protein/mL) with 0 µM (9), 25 µM (b), and 100 µM (2) of 2′,4′,2-trihydroxychalcone in the presence of (A) increasing concentrations of DOX (25-500 µM) at a fixed (250 µM) concentration of NADP(H) or (B) increasing concentrations of NADP(H) (10-1000 µM) at fixed (50 µM) concentrations of DOX. Alcohol metabolites were assayed after 240 min at 37 °C as described in the Experimental Procedures. Values are means ( SE of three separate determinations in triplicate.

Quercetin, a flavonol, was the most potent inhibitor of rabbit heart anthracycline reductase activity: in particular, quercetin was more efficient than morin (which differs for the position of OH groups in the phenyl ring: 3′-4′ for quercetin vs 2′-4′ for morin) at inhibiting both DOXol and DNRol formation (IC50s ) 13.7 ( 2.8 vs 49.3 ( 6.3 µM and 8.0 ( 2.0 vs 18.8 ( 4.1 µM, respectively). Quercetin also proved more potent than taxifolin at lowering DOXol or DNRol formation (IC50s ) 13.7 ( 2.8 vs 48.9 ( 6.0 µM and 8.0 ( 2.0 vs 46.3 ( 3.6 µM, respectively). Taxifolin is a flavanonol with the same hydroxylation pattern of quercetin, but its C ring lacks the 2,3double bond that is known to affect the planarity of the molecule. Inhibition Mechanisms. Aldo-keto reductases and carbonyl reductases have been considered to catalyze formation of anthracycline secondary alcohol metabolites (35, 36). The catalytic mechanism of these reductases follows an ordered Bi Bi mechanism, in which NADP(H) binds the enzyme first and NADP+ leaves the enzyme last (37, 38). Inhibitors of aldoketo or carbonyl reductases were shown to act by binding preferentially to the enzyme/NADP+ complex (37, 38). Keeping these premises in mind, we characterized the kinetics with which 2′,4′,2-THC (i.e., the hydroxychalcone with a high inhibitory activity) inhibited anthracycline alcohol metabolite formation by rabbit heart cytosol. Double reciprocal plots showed that 2′,4′,2-THC inhibited anthracycline alcohol metabolite formation noncompetively (Ki ) 95.5 ( 4.5 µM) with respect to DOX (Figure 2A) but uncompetitively (Ki ) 18.3 ( 1.9 µM) with respect to NADP(H) (Figure 2B). The same inhibition pattern was observed in the presence of other chalcones (4′-HC, 2′,4′DHC or 2′,4′,2,4-THC) or when DNR was used in place of DOX (data not shown). Overall, these results suggest that hydroxychalcones may inhibit heart cytosolic reductases by binding preferentially to the enzyme/NADP+ binary complex. Effect of Chalcones on the Formation of Anthracycline Secondary Alcohol Metabolites by Human Heart Cytosol. We recently reported that human heart flavonoids were good

SilVestrini et al.

Figure 3. Effect of chalcones and flavonoids on the formation of anthracycline secondary alcohol metabolites by human heart cytosol. Experiments were performed by preincubating human heart cytosolic fractions (1 mg of protein/mL) in 10 mM HEPES-300 mM NaCl, pH 7.4, with each compound (50 µM) added 5 min before 50 µM DOX (A) or DNR (B). Reactions were started by adding NADP(H) (250 µM). Alcohol metabolites were assayed after 240 min at 37 °C as described in the Experimental Procedures. Values are means ( SE of three separate determinations in triplicate.

inhibitors of DNRol formation but failed to inhibit DOXol formation (36). The same finding was obtained in the present study; in fact, Figure 3A shows that neither morin nor quercetin or taxifolin diminished DOXol formation in human heart cytosol. If anything, flavonoids slightly stimulated DOXol formation, similar to what was reported previously (see also Figure 3A and ref 36). In the same preparations, hydroxychalcones (4′-HC, 2′,4′-DHC, and 2′,4′,2-THC) inhibited both DOXol and DNRol formation (Figure 3A,B).

Discussion Previous studies with rats or isolated rat heart showed that biochemical or functional indices of anthracycline-induced cardiotoxicity could be diminished by inhibitors of aldo-keto or carbonyl reductases like phenobarbital or rutin, respectively (39, 40). Unfortunately, however, the tolerability of either inhibitor in humans would be questionable or uncertain. An additional concern pertains to the heterogeneity of cytosolic reductases involved in anthracycline metabolism and to the different levels and expression of such reductases in humans versus laboratory animals or in a given animal species and strain versus another. In keeping with this concern, we previously reported that phenobarbital and rutin did not inhibit DOXol formation in human cardiac cytosol (36), a model that encircled the activity of all reductases potentially involved in anthracycline metabolism and consequently gained more relevance to the mechanisms and levels of metabolite formation during the course of clinical treatments. Here, we have shown that chalcones potently inhibit the formation of anthracycline secondary alcohol metabolites in isolated rabbit and human heart cytosol. Structure-activity relationships highlight that a hydroxyl group at the 4′ position in the A ring of the chalcone skeleton is essential for the inhibitory activity of these compounds. Furthermore, kinetic studies reveal that hydroxychalcones (4′-HC, 2′,4′-DHC,

Chalcones and Anthracycline Alcohol Metabolite Formation

2′,4′,2,-THC, and 2′,4′,2,4-THC) inhibit alcohol metabolite formation noncompetitevely with regard to anthracyclines but uncompetitively with regard to NADP(H), suggesting that chalcones act by binding to an enzyme/NADP+ complex. In this regard, it is worth of noting that the 4′-hydroxyl group of the chalcones has a pKa value of 7.47: this is considerably more acidic than the pKa of that of 2′-and 4-hydroxyl groups (pKa 10.05 and 8.44, respectively), but it is very similar to that of the structurally related 7-hydroxyl group of several benzopyran4-one derivatives (7.35 in the case of quercetin) (41). It follows that at physiological pH, 4′-hydroxyl-substituted chalcones would be partially dissociated in their anionic form and consequently exhibit a negative charge liable to electrostatic interaction with NADP+. This would be similar to what characterized for many known inhibitors of aldo-keto (37, 42) or carbonyl reductases (38, 43). Among chalcones, 2′,4′,2-THC proved to be the most effective inhibitor of both DOXol and DRNol formation. 2′,4′,2THC was nonetheless less efficient than quercetin at inhibiting DOXol or DNRol formation in rabbit heart cytosol (Table 1); however, quercetin and other flavonoids did not inhibit but actually enhanced DOXol formation in human heart cytosol, a model in which hydroxychalcones were good inhibitors of both DOXol and DNRol formation (ref (36) and Figure 3). These results are explained by the different enzymology of anthracycline metabolism, which may be mediated by aldehyde or carbonyl reductases that belong to distinct superfamilies of aldoketo or short-chain dehydrogenases, respectively (42, 43). As previously suggested, the rabbit heart probably forms both DOXol and DNRol through the action of carbonyl reductases, while the human heart employs aldehyde reductases to form DOXol and carbonyl reductases to form DNRol (36), respectively. Keeping these premises in mind, several considerations can be put forward. On the one hand, it would appear that chalcones were good inhibitors of both aldehyde and carbonyl reductases of the human heart. On the other, the observation that quercetin increased DOXol formation to some extent in the human heart suggests that it might promote undesired effects such as an activation of aldehyde reductases (44) or a diversion of DOX from carbonyl reductases toward aldehyde reductases (35, 36). One possible mechanism by which flavonoids increase DOXol formation may be related to their peculiar binding capacity to aldo-keto reductases. Indeed, quercetin binds very tightly to both the coenzyme- and the substrate-binding site of aldehyde dehydrogenase. Binding in the latter location, when the enzyme is in the form of the enzyme/NADH complex, accounts for the activation through enhancement of dissociation rate of NADH, which is normally the limiting step controlling the overall Vmax of the enzyme (44). Inasmuch as the latter effects were not observed with hydroxychalcones, we would tentatively conclude that hydroxychalcones were more versatile than flavonoids at suppressing the formation of anthracycline secondary alcohol metabolites in both human and rabbit heart. Flavonoids have been assessed quite extensively for their use as cardioprotectants against anthracyclines, and some of them have already entered clinical trials (45-47). In general, the protective action of flavonoids was attributed to their ability to scavenge free radicals or reduce their formation or their ability to induce a direct positive effect on myocardial contractility (45-47). Nonetheless, one of the most promising flavonoids, like monohydroxyethylrutoside (monoHER), did not inhibit the formation of secondary alcohol metabolites (46).

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Here, we have shown that hydroxychalcones inhibit formation of both DOXol and DNRol in the human heart cytosolic fractions. This action, along with their known antioxidant properties, might render chalcones an attractive alternative to flavonoids. Chalcones were also shown to inhibit the growth of human cancer cells in vitro (27). This promising profile of activities calls for further characterizations of chalcones in preclinical models of cardiotoxicity induced by anthracyclines. In particular, it will be important to assess whether chalcones reached plasma and tissue levels permissive to inhibition of heart cytosolic reductases involved in the anthracycline metabolism. Acknowledgment. This work was supported by grants from MIUR-FIRB 2003 (RBNE034XSW) and MIUR FIRB 2002 (RBNE014HJ3).

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