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[Cancer Research 61, 8422-8428, December 1, 2001]
© 2001 American Association for Cancer Research

Biochemistry and Biophysics

Doxorubicin Irreversibly Inactivates Iron Regulatory Proteins 1 and 2 in Cardiomyocytes

Evidence for Distinct Metabolic Pathways and Implications for Iron-mediated Cardiotoxicity of Antitumor Therapy1

Giorgio Minotti2, Raffaella Ronchi, Emanuela Salvatorelli, Pierantonio Menna and Gaetano Cairo

Department of Drug Sciences, G. D’Annunzio University School of Pharmacy, 66013 Chieti [G. M., E. S., P. M.], and Institute of General Pathology-Consiglio Nazionale delle Ricerche Center for Cell Pathology, University of Milan School of Medicine, 20133 Milan [R. R., G. C.], Italy


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Changes in iron homeostasis have been implicated in cardiotoxicity induced by the anticancer anthracycline doxorubicin (DOX). Certain products of DOX metabolism, like the secondary alcohol doxorubicinol (DOXol) or reactive oxygen species (ROS), may contribute to cardiotoxicity by inactivating iron regulatory proteins (IRP) that modulate the fate of mRNAs for transferrin receptor and ferritin. It is important to know whether DOXol and ROS act by independent or combined mechanisms. Therefore, we monitored IRP activities in H9c2 rat embryo cardiomyocytes exposed to DOX or to analogues which were selected to achieve a higher formation of secondary alcohol metabolite (daunorubicin), a concomitant increase of alcohol metabolite and decrease of ROS (5-iminodaunorubicin), or a defective conversion to alcohol metabolite (mitoxantrone). On the basis of such multiple comparisons, we characterized that DOXol was able to remove iron from the catalytic Fe-S cluster of cytoplasmic aconitase, making this enzyme switch to the cluster-free IRP-1. ROS were not involved in this step, but they converted the IRP-1 produced by DOXol into a null protein which did not bind to mRNA, nor was it able to switch back to aconitase. DOX was also shown to inactivate IRP-2, which does not assemble or disassemble a Fe-S cluster. Comparisons between DOX and the analogues revealed that IRP-2 was inactivated only by ROS. Thus, DOX can inactivate both IRP through a sequential action of DOXol and ROS on IRP-1 or an independent action of ROS on IRP-2. This information serves guidelines for designing anthracyclines that spare iron homeostasis and induce less severe cardiotoxicity.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Antitumor therapy with the anthracycline DOX3 is limited by acute and chronic toxicity to the heart. Whereas the acute toxicity is transient and clinically manageable, the chronic toxicity evolves into progressive cardiomyopathy, which limits clinical use of DOX (1) . Several lines of evidence indicate that alterations of iron homeostasis may contribute to both forms of cardiotoxicity (2 , 3) ; therefore, it has been suggested that DOX might alter the function of cytoplasmic aconitase/IRP-1. The role of this protein is to adapt the levels of iron to the metabolic needs of the cell while preventing the accumulation of potentially toxic excess iron. When the cell needs iron, IRP-1 binds to IRE in the mRNA for transferrin receptor and in the mRNA for ferritin, increasing stability of the former while decreasing translation of the latter (4, 5, 6, 7) . These divergent but coordinate processes make iron uptake prevail over sequestration and consequently produce a pool of iron that is available for metabolic use. When the cellular levels of iron are too high, IRP-1 assembles a [4Fe-4S] cluster that abolishes IRE-binding capacity but confers an aconitase activity similar to that of the mitochondrial enzyme of the Krebs cycle (5) . Under these conditions, iron sequestration prevails over uptake, and the cell is protected from the toxicity of excess iron. Two independent studies have shown that DOX was able to attack aconitase/IRP-1 through the action of its secondary alcohol metabolite DOXol (8) or by producing ROS (9) . Whether DOXol and ROS acted by mutually exclusive or combined mechanisms has nonetheless remained an unresolved issue. Because this information would be of value for designing less cardiotoxic analogues, we performed experiments to better define the roles of ROS and DOXol as modifiers of aconitase/IRP-1. We also determined whether DOX was able to target IRP-2, which is similar to IRP-1 in modulating the fate of mRNAs but is not regulated through cluster assembly or disassembly (10) and, hence, may exhibit different responses to ROS or DOXol.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Drugs and Chemicals.
DOX, DNR, and 5-iminoDNR were obtained through the courtesy of Dr. Antonino Suarato (Department of Chemistry, Pharmacia-Upjohn, Milan, Italy); the corresponding secondary alcohol metabolites were synthesized and purified by us, as described (11) . MITOX, cis-aconitate, bovine erythrocyte CuZn SOD (EC 1.15.1.1), and horse heart cytochrome c were from Sigma Chemical Co.-Aldrich (Milan, Italy). cys and FAS were products of Merck (Darmstadt, Germany).

Cell Culture, Treatment, and Preparation of Lysates.
We used the embryonic, rat heart-derived cell line H9c2 (American Type Culture Collection-CRL 1446), which has proven to be useful for characterizing the responses of cardiomyocytes to pathophysiological stimuli (12, 13, 14) . Cells were grown at 37°C under 5% CO2/air in DMEM adjusted to contain 4 mM glutamine, 18 mM sodium bicarbonate, 25 mM glucose, 1 mM sodium pyruvate, 100 units/ml penicillin, and 0.1 ng/ml streptomycin and supplemented with 10% heat-inactivated FCS. Subconfluent cells (4.5 x 106) were seeded in 75-cm2 flasks and incubated for 16 h with increasing concentrations of DOX, DNR, 5-iminoDNR, or MITOX. At the end of treatment, adherent cells were scraped, washed in PBS, and homogenized in 1 ml of Munro buffer [10 mM HEPES (pH 7.6), 3 mM MgCl2, 40 mM KCl, and 5% glycerol]. Aliquots (800 µl) were extracted with a 4-fold excess of (1:1) CHCl3/CH3OH, and the organic phases were used for drug analysis. The remaining 200 µl were centrifuged at 16,000 x g for 5 min at 4°C, and the supernatants were split in 100-µl aliquots. One aliquot was added with 1 mM DTT and stored frozen until assay for IRP. The other aliquot was used for aconitase assay and was added with the substrate-intermediate cis-aconitate (20 µM) to prevent cluster decay during storage.

RNA-Protein Gel Retardation Assay.
The probe for the bandshift assay was transcribed from the linearized pSPT-fer plasmid containing the IRE of human ferritin H chain (15) , using T7 RNA polymerase in the presence of {alpha}-32P UTP (Amersham-Pharmacia Biotech, Milan, Italy). Equal amounts of lysates (2 µg of protein) were incubated, in the absence or presence of 1% 2-ME, with a molar excess of IRE probe and treated sequentially with RNase T1 and heparin (16) . After separation on 6% nondenaturing polyacrylamide gels, complexes between radioactive RNA and IRP-1 or -2 were visualized by autoradiography and quantitated by direct nuclear counting using an Instant Imager (Packard Instruments Co., Milan, Italy).

Western Blot Analysis.
Aliquots of the lysates used for IRP determination, and containing equal amounts of proteins (80 µg), were electrophoresed in 10% acrylamide-SDS gels, electroblotted to Hybond PVDF membranes (Amersham-Pharmacia Biotech), and incubated with a 1:500 dilution of rabbit antiserum against human recombinant IRP-1. IRP-1 was detected by chemiluminescence using an immunodetection kit (ECL Plus; Amersham-Pharmacia Biotech), according to the manufacturer’s instructions.

Drug Uptake and Assay for Secondary Alcohol Metabolites.
Vacuum-dried organic extracts of cardiomyocytes were suspended in 10–20 µl of CH3OH and analyzed for DOX, DNR, and 5-iminoDNR or their secondary alcohol metabolites by two-dimensional TLC on 0.25 mm (20 x 20 cm) Silica Gel F254 Plates (Merck; Ref. 11 ). Mobile phases (volume for volume) and Rf values were: CHCl3/CH3OH/CH3COOH/H2O (80:20:14:6) for separation of DOX (0.61) and DOXol (0.43); CHCl3/CH3OH/CH3COOH (80:20:4) for separation of DNR (0.44) and its alcohol metabolite DNRol (0.28); and CHCl3/CH3OH/CH3COOH (100:20:2) for separation of 5-iminoDNR (0.71) and 5-iminoDNRol (0.59). After identification by cochromatography with authentic anthracyclines and secondary alcohol metabolites, DOX(ol), DNR(ol), and 5-iminoDNR(ol) were quantitated fluorometrically against appropriate standard curves. Other metabolites usually were very low or absent; therefore, total drug uptake was calculated as (parent drug + secondary alcohol metabolite). Recovery was >90% for all anthracyclines and their alcohol metabolites; the lowest detection limit was 0.01 nmol/mg protein. MITOX, which was not converted to a secondary alcohol metabolite, was measured by optical spectroscopy by taking advantage of its high absorbances at 608 and 658 nm ({epsilon} = 19.2 and 20.9 mM-1 cm-1, respectively; Ref. 17 ). For this purpose, vacuum-dried cell extracts were dissolved in spectroscopy-grade CH3OH and analyzed in a Hewlett Packard 8453 diode array spectrophotometer equipped with computer-assisted corrections for turbidity and scatter. Two-dimensional TLC in CHCl3/CH3OH/CH3COOH/H2O (50:30:35:15) confirmed that cell-extracted MITOX comigrated as a single band (Rf = 0.57) with an authentic standard.

ROS Formation.
Drug-induced ROS formation was determined by measuring O2.- production in cardiac microsomes. The latter were isolated from small samples of human myocardium disposed of during aorto-coronary bypass grafting, using established procedures (18) . After isolation, microsomes were solubilized with sodium deoxycholate, at the final ratio of 0.1% detergent:5 mg of protein, to obtain a fraction enriched in NADPH-cytochrome P-450 reductase (specific activity before and after solubilization: 11 versus 29 mU/mg protein). O2.- was measured through the SOD-inhibitable cytochrome c reduction assay in 1-ml systems containing solubilized microsomes (25 µg of protein), NADPH (0.1 mM), drugs (10 µM), and cytochrome c (25 µM), plus or minus SOD (200 units) in 0.3 M NaCl (pH 7.0), 37°C. Drug-induced O2.- formation was determined as the net increase over a basal rate of 0.6 nmol/mg protein/min. DOX was shown to induce the formation of 0.61 nmol O2.-/mg protein/min, whereas DNR, 5-iminoDNR, and MITOX induced the formation of 0.57, 0.1, and 0.31 nmol O2.-/mg protein/min, respectively. The expected yield of ROS in cardiomyocytes was determined based on the efficacy of each drug in producing O2.- in vitro and its uptake in H9c2 cells. ROS formation by DOX was assumed to be equal to 100%; ROS formation by the analogues was calculated according to the equation: ROSanalogue = [(Uptake x O2.- formation)analogue: (Uptake x O2.- formation)DOX] x 100. Although indirect, these calculations avoided pitfalls in measuring cellular production of ROS with the fluorescent dye 2',7'-dichlorofluorescein (19) .

Assay for Aconitase.
Aconitase activity was determined spectrophotometrically by monitoring the disappearance of cis-aconitate ({epsilon}240 = 3.6 mM-1 cm-1; Ref. 20 ). The incubations (1 ml of final volume) contained lysates (15–50 µg of protein) and 0.1 mM cis-aconitate in 0.3 M NaCl (pH 7.0), 37°C; one mU was defined as the amount of enzyme which consumed 1 nmol of cis-aconitate/min (20) . Where indicated, lysates were preincubated for 10 min at 37°C with cys and FAS at the final ratios of 1000 or 50 nmol:mg protein, respectively. These ratios were shown previously to be optimal for reconstituting [4Fe-4S] clusters and aconitase activity (8) .

Other Assays.
Proteins were measured by the Bio-Rad kit assay (Segrate, Milan, Italy). LDH release was measured by the Sigma Chemical Co. kit 228 UV and was expressed as a percentage of total cellular activity. Other conditions are indicated in the legends to figures and Table 1Citation .


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  		Table 1 Uptake and metabolism of DOX, DNR, 5-iminoDNR, and MITOX under conditions of half-maximal concentration

 

   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effects of DOX on IRP-1 and -2.
As shown in Fig. 1ACitation , H9c2 cells exhibited IRP-1 and -2 activities. Treatment with 1–5 µM DOX increased IRP-1 activity by a maximum of 91 ± 21% at 5 µM, but such increase fell to 35 ± 17% at 10 µM (P < 0.001, paired Student’s t test, n = 8); this corresponded to a ~57% loss in IRP-1 activation when DOX was increased from 5 to 10 µM. Treating the cell lysates with 2-ME before the gel shift analysis increased IRP-1 activity in all samples, a finding consistent with the ability of 2-ME to regenerate -SH groups involved in IRP-IRE interactions (21 , 22) ; however, 2-ME did not blunt the concentration-related, bell-shaped response induced by DOX. The effects of DOX were not attributable to modifications in the cellular levels of IRP-1, as Western blot analysis detected similar levels of this protein in both control cells and those exposed to 1–10 µM DOX (Fig. 1B)Citation . Because IRP-1 binds to IRE in its cluster-free form, we determined whether DOX acted by posttranslational mechanisms that altered the equilibrium between IRP-1 and its cluster-containing counterpart, aconitase. As shown in Fig. 1CCitation , 1–5 µM DOX caused a significant decrease in aconitase activity, which correlated with the increase in IRP-1 activity observed in the same range of concentrations. Treatment of the cell lysates with cys/FAS, a procedure known to reconstitute [4Fe-4S] clusters, increased aconitase activity and blunted differences between control and 1–5 µM DOX cells. Fig. 1CCitation also shows that 10 µM DOX caused the greatest loss of aconitase activity; however, treatment of the cell lysates with cys/FAS recovered only ~50% of the maximal aconitase activity seen in the other samples. Such a loss in cys/FAS-activatable aconitase was in good agreement with the ~57%, 2-ME-insensitive loss of IRP-1 activation under comparable experimental conditions. Taken as a whole, these findings demonstrated that cardiomyocytes contained a mixed pool of cluster-containing aconitase and cluster-free IRP-1 and that DOX was able to influence the ratio between the two forms. Concentrations of DOX <=5 µM converted aconitase to IRP-1 but did not preclude aconitase regeneration after cluster reassembly with cys/FAS. In contrast, 10 µM DOX seemed to convert ~50% of a pool of exchangeable aconitase/IRP-1 into a "null" protein, which could not be committed to recognize IRE or to recover enzymatic activity. In the case of IRP-2, DOX induced a concentration-dependent decrease in RNA binding, which was not rescued by 2-ME (Fig. 1A)Citation . This demonstrated that IRP-2 was irreversibly modified by DOX.



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  		Fig. 1. Effects of DOX on IRPs in H9c2 cells. A, effects of 1–10 µM DOX on IRP-1 and -2, measured with or without 2-ME. B, Western blot of IRP-1 in cells treated with 1–10 µM DOX; rIRP-1 indicates recombinant human IRP-1. C (left), aconitase activity in control and DOX-treated cells. C (right), aconitase activity after lysate preincubation with cys/FAS. Gels were representative of eight experiments; aconitase values were means ±SE of three experiments.

 
Differential Effects of Secondary Alcohol Metabolites and ROS on IRP.
As shown in Fig. 2Citation , DOX is composed of a quinone-containing tetracyclic ring, a short side chain with a carbonyl group and a primary alcohol terminus, and an aminosugar attached by a glycosidic bond to the tetracyclic ring. ROS, like O2.- and H2O2, are formed after one electron reduction of the quinone moiety by mitochondrial, nuclear, or microsomal NAD(P)H oxidoreductases, like NADPH-cytochrome P450 reductase, producing a semiquinone-free radical that regenerates its parent quinone by oxidizing with oxygen (23) . DOXol is formed by cytoplasmic aldo-keto or carbonyl reductases, which add two electrons to the side chain carbonyl group (-CO-CH2OH -> -CHOH-CH2OH; Refs. 24 and 25 ). To characterize the role of DOXol and ROS as modifiers of IRP-1 and -2, we compared the effects of DOX to those of the following analogues: DNR, an analogue that contains both quinone and carbonyl moieties and thus retains the ability to form ROS and a secondary alcohol metabolite; 5-iminoDNR, an analogue that forms less ROS because of the replacement of a quinone group with an imino moiety; and MITOX, an aglyconic anthraquinone, which lacks carbonyl groups in its side chains and consequently fails to form secondary alcohol metabolites (see Fig. 2Citation and its legend). These analogues were assessed over different ranges of concentrations to obviate individual differences in cellular uptake (1–25 µM DNR, 10–50 µM 5-iminoDNR, and 1–10 µM MITOX). Under conditions of half-maximal concentration in the incubation medium, however, the uptake of DOX and of the analogues was confined in a relatively narrow range, which allowed for direct comparisons of alcohol metabolite or ROS formation by each individual drug. As shown in Table 1Citation , DNR yielded ~1.9 times more ROS than DOX, which correlated with a 2-fold increase in uptake. Under the same conditions, however, DNR produced about four times more alcohol metabolite than DOX, indicating that it was intrinsically more sensitive to the action of carbonyl- or aldo-keto reductases in H9c2 cells. DNR was therefore characterized by a 2-fold increase in the ratio of alcohol metabolite:ROS as compared with DOX. 5-iminoDNR exhibited the same uptake as DOX but produced cellular levels of its secondary alcohol metabolite that were 2-fold those produced by DOX; at the same time, 5-iminoDNR yielded 82% less ROS than DOX, a finding consistent with the redox-impairment caused by substitution of a quinone moiety with an imino residue (26) . 5-iminoDNR was therefore characterized by a 10-fold increase in the ratio of alcohol metabolite:ROS as compared with DOX. Finally, a combination of 50% efficacy in O2.- formation and 60% increase in cellular uptake enabled MITOX to yield ~80% of ROS as DOX, but the absence of side chain carbonyl groups precluded formation of secondary alcohol metabolites by this drug. Thus, MITOX was used to expose cardiomyocytes to the action of ROS but not of alcohol metabolites. The effects of DNR, 5-iminoDNR, and MITOX on aconitase/IRP-1 are reported in Fig. 3Citation . As shown by a representative gel, and by titrations over a 1–25 µM range, DNR had the same bell-shaped effect as DOX in modulating IRP-1, the only difference being that maximal activation occurred at 15 µM DNR as opposed to 5 µM DOX (Fig. 3A)Citation . This response was not blunted by the addition of 2-ME before the gel shift analysis (data not shown), a finding in agreement with the similar behavior of DOX-treated samples. DNR was similar to DOX also in inducing a loss of aconitase activity, which was recovered with cys/FAS in all samples exposed to <=15 µM DNR (Fig. 3ACitation , bottom panel). Increasing DNR from 15 to 25 µM eventually caused a 2-ME-insensitive, ~56% loss in IRP-1 activation, which was accompanied by a cys/FAS-insensitive loss of ~45% of aconitase activity. Thus, approximately half of the exchangeable pool of aconitase/IRP-1 was converted into a null protein when DNR was increased from 15 to 25 µM, similar to what was observed when DOX was increased from 5 to 10 µM DOX. Replacing DOX with DNR, an analogue characterized by a 2-fold increase in the ratio of alcohol metabolite:ROS, therefore extended the range of anthracycline concentrations within which aconitase switched to IRP-1 before the formation of a null protein became evident. This suggested that secondary alcohol metabolites were involved in converting aconitase to IRP-1, whereas ROS probably were involved in producing a null protein. Support to this interpretation was provided by experiments with 5-iminoDNR, the analogue characterized by a much higher ratio of alcohol metabolite:ROS attributable to a concomitant increase of the former and decrease of the latter. In fact, 5-iminoDNR was able to switch aconitase to IRP-1 but was unable to induce the formation of a null protein, as evidenced by persistent elevation of IRP-1 and a complete recovery of cys/FAS-inducible aconitase activity, even at a concentration of 50 µM (Fig. 3B)Citation . Neither the switch of aconitase to IRP-1 nor the formation of a null protein was observed in experiments with MITOX, the analogue producing ROS but not secondary alcohol metabolites (Fig. 3C)Citation . This observation confirmed that secondary alcohol metabolites were required for switching aconitase to IRP-1 and revealed that ROS produced a null protein by attacking the IRP-1 produced by secondary alcohol metabolites.



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  		Fig. 2. Structures of DOX and of the analogues used in this study. Shaded and white boxes, quinone or carbonyl moieties required for formation of ROS or secondary alcohol metabolites, respectively. Relevant modifications in the analogues: DNR, substitution of the side chain primary alcohol terminus with a methyl; 5-iminoDNR, same as in DNR plus imino substitution of a quinone; MITOX, lack of side chain carbonyl groups.

 


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  		Fig. 3. Aconitase and IRP activities in H9c2 cells treated with DNR, 5-iminoDNR, or MITOX. IRP and aconitase were assayed after 16-h treatment of H9c2 cells with increasing concentrations of DNR (A), 5-iminoDNR (B), or MITOX (C). Columns are means of IRP-1 activities ±SE of three to four experiments; columns without bars are means of two experiments with 80–90% agreement. Aconitase activities were measured in duplicate before or after preincubation of lysates with cys/FAS; values were expressed as a percentage of controls to permit direct comparisons. Basal or cys/FAS-stimulatable aconitase activities in control samples ranged from 3.8 to 5.9 and 8.6 to 12.1 mU/mg protein, respectively.

 
Additional inspection of Fig. 3, A–CCitation shows that DNR and MITOX, but not the quinone-modified 5-imino DNR, were able to decrease IRP-2. This pattern reproduced the behavior of DOX, indicating that IRP-2 was affected by quinone-derived ROS, regardless of the concomitant formation of secondary alcohol metabolites. While inactivating IRP-2 in a concentration-dependent manner, DOX, DNR, and MITOX also caused an increasing damage to cardiomyocytes, which was evidenced by LDH release in the incubation medium (Fig. 4)Citation . This was not observed in the case of 5-iminoDNR, which both spared IRP-2 and failed to induce LDH release. Thus, ROS were important mediators of damage, and IRP-2 was a sensitive indicator of oxidative stress.



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  		Fig. 4. IRP-2 activity and LDH release in H9c2 cells treated with increasing concentrations of DOX, DNR, 5-iminoDNR, or MITOX. IRP-2 activity (A) and LDH release (B) were determined in H9c2 cells incubated for 16 h with increasing concentrations of anthracyclines. Values with bars are means ±SE of three experiments; values without bars are taken from duplicate experiments with >90% agreement.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In a previous study, conducted in cytosolic fractions derived from human myocardium, we demonstrated that DOX lacks direct "structural" reactivity with aconitase/IRP-1 (8) . However, replacing DOX with DOXol resulted in iron delocalization from aconitase, showing that the anthracycline may gain reactivity toward the Fe-S cluster of this enzyme after metabolic conversion of its side chain carbonyl group into a secondary alcohol moiety. Iron delocalization and consequent loss of aconitase activity were not accompanied by a gain of IRP-1 activity; instead, DOXol produced a null protein which did not recognize IRE nor switch back to aconitase (8) . The formation of a null protein was attributed to irreversible modifications of -SH residues mediating both the recognition of IRE and the reassembly of the cluster (e.g., cys437; Refs. 8 and 22 ). Interestingly, DOXol reactions did not appear to involve ROS, as evidenced by the lack of effect of added scavengers like SOD or catalase (8) . This picture has been extended and modified by other investigators, based on their studies in isolated cardiomyocytes derived from rat heart. In these cells, DOX was found to reduce aconitase activity by mechanisms that involved metabolic conversion of the quinone moiety to ROS, evidenced by the protective efficacy of cell-permeable mimetics of SOD or glutathione peroxidase (9) . Unfortunately, these latter studies did not elucidate whether the loss in aconitase activity was accompanied by a gain in IRP-1 activity or by formation of a null protein. Moreover, aconitase inactivation was observed after incubation of cardiomyocytes with 20 µM DOX, a concentration exceeding the plasma peaks observed in patients after standard doses of the anthracycline (7–10 µM; Ref. 27 ). This factor may have produced unusually high levels of DOX in cardiomyocytes, facilitating reactions of O2.- and/or H2O2 with aconitase. Similarly, the studies in human cardiac cytosol were performed with a bolus of DOXol (3–4 nmol/mg protein), which exceeded the steady-state levels of this metabolite in intact cardiomyocytes and consequently magnified its ability to target Fe-S clusters. In the present study, H9c2 cardiomyocytes were exposed to concentrations of DOX that never exceeded 10 µM. Moreover, we adopted the strategy of comparing DOX to analogues whose pharmacokinetic and biochemical behavior resulted in a greater formation of alcohol metabolite (DNR), in concomitant increase of alcohol metabolite and decrease of ROS (5-iminoDNR), or in selective deficiency of alcohol metabolite formation (MITOX). Comparing DOX to these analogues provided a mechanism-based tool for dissecting the roles of alcohol metabolites and ROS while also avoiding the need for perturbing the system with ROS scavengers or inhibitors of anthracycline metabolism. These experimental settings have allowed us to demonstrate that DOX modulates aconitase/IRP-1 by mechanisms that convert aconitase to IRP-1 and eventually produce a null protein. Comparisons between DOX and DNR or 5-iminoDNR demonstrate that aconitase is converted to IRP-1 by DOXol, not by ROS. This finding is explained by keeping in mind that aconitase switches to IRP-1 after a complete disassembly of its [4Fe-4S] cluster, an action exhibited by DOXol (8) but not by O2.- or H2O2, which, at their best, would remove only the fourth labile iron required for aconitase activity (28 , 29) . Comparisons between DOX and DNR or 5-iminoDNR show that the role of ROS is to produce a null protein, but the experiments with MITOX clearly demonstrate that such a process requires also the presence of a secondary alcohol metabolite. This latter finding is explained by our previous demonstration that O2.- and H2O2 lack sufficient reactivity for inducing irreversible modifications of cys437, even when they are produced at a much greater rate through the xanthine/xanthine oxidase reaction (30) . Nonetheless, O2.- and H2O2 might gain reactivity if they were converted by iron into more potent oxidants like hydroxyl radicals or iron-peroxo complexes. Secondary alcohol metabolites can facilitate such conversion by inducing cluster disassembly and switching aconitase to IRP-1, a process accompanied by iron release and formation of anthracycline-iron complexes in the closest proximity to cys437 (8) . The effects of clinically relevant concentrations of DOX may therefore be more complex than believed previously, as they involve the action of DOXol in converting aconitase to IRP-1 and the action of ROS in converting the newly formed IRP-1 into a null protein.

Unlike IRP-1, IRP-2 lacks the ability to form or disassemble Fe-S motifs. We were unable to detect IRP-2 in our preceding study in human cardiac cytosol (8) , nor was IRP-2 characterized in the studies of the effects of DOX in isolated rat cardiomyocytes (9) . We therefore exploited our present settings to assess whether IRP-2 was modulated by DOX in a fashion similar to, or different from, that described for IRP-1. Our results demonstrate that DOX induces irreversible inactivation of IRP-2, similar to what was observed for IRP-1; however, only ROS are required for inactivating IRP-2. Such findings are explained by a greater susceptibility of IRP-2 to oxidation, followed by proteasome-mediated degradation (31, 32, 33) . The observation that DOXol does not attack IRP-2 confirms that its action is targeted to Fe-S motifs and, hence, to aconitase/IRP-1.

Studies in isolated cardiomyocytes provide a good model for a rapid characterization of structure-activity relationships that may be important in the development of cardiotoxicity. Previous reports (reviewed in Ref. 3 ) have suggested that ROS mediate the acute phase of cardiotoxicity, which develops at the beginning of DOX regimens. Our present results lend support to this interpretation, showing that H9c2 cells release LDH after an acute exposure to quinone-active DOX, DNR, and MITOX but not after an exposure to the quinone-modified 5-iminoDNR. The decline of IRP-2 that occurs after treatment with the quinone-active drugs might perhaps be viewed as a protective stratagem to facilitate the rapid translation of latent ferritin mRNAs and sequester iron before it amplifies oxidative damage by converting O2.- and H2O2 into more potent oxidants (3 , 7) . On the other hand, several lines of evidence indicate that the chronic phase of cardiotoxicity, which develops after multiple doses of DOX, coincides with an accumulation of DOXol inside cardiomyocytes (3 , 25 , 34 , 35) . DOXol, alone or in concert with ROS, has therefore been implicated as a potential mediator of the chronic, irreversible cardiomyopathy induced by DOX. The conversion of aconitase/IRP-1 into a null protein, described in our present study, represents a novel example of how DOXol might contribute to chronic cardiotoxicity by interacting with ROS. In fact, the null protein makes cells unable to sense modifications in iron availability and develop coordinate adaptations of the levels of transferrin receptor and ferritin (36) . While amplifying oxidative damage induced by ROS, such disorders may also misplace iron at cellular sites that govern the contraction-relaxation cycle of the heart but lose their function after sterical occupation by iron (e.g., the ryanodine receptor 2/calcium release channel of sarcoplasmic reticulum; Ref. 37 ).

In conclusion, DOXol and ROS irreversibly inactivate IRP-1 and IRP-2 in cardiomyocytes exposed to DOX. The results modify current knowledge in this field, especially with regard to novel evidence for a sequential action of DOXol and ROS in converting aconitase/IRP-1 into a null protein. This information has been obtained in a cardiomyocyte model which, in principle, may be exploited for screening a broad repertoire of anthracyclines. In this study, we chose to focus on selected analogues that have already been assessed for cardiotoxicity; and, hence, may serve for probing relationships between anthracycline metabolism, IRP inactivation, and development of cardiotoxicity; e.g., both 5-iminoDNR and MITOX proved to be more cardiac tolerable than DOX in some preclinical or clinical settings of chronic cardiotoxicity (38 , 39) , a finding consistent with our demonstration that neither drug converted aconitase/IRP-1 into a null protein because of impaired production of ROS or secondary alcohol metabolites, respectively. Results described in this study, therefore, offer a rationale to design other analogues which also spare aconitase/IRP-1 by forming less ROS and/or alcohol metabolites than DOX. Additional guidelines in this setting are provided by the differential roles of anthracycline-derived ROS and alcohol metabolites in cancer cells. Evidence for a possible involvement of ROS in the antitumor activity of anthracyclines has, in fact, been reported (40 , 41) . In contrast, several lines of evidence demonstrate that secondary alcohol metabolites do not always contribute to, but sometime diminish, the antitumor activity of anthracyclines (42, 43, 44) . Analogues characterized by a selective impairment in alcohol metabolite formation might therefore prove to be more advantageous for sparing cardiac aconitase/IRP-1 while also retaining equal or improved therapeutic efficacy, serving better alternatives to DOX for use in cancer patients.


   ACKNOWLEDGMENTS
 
We thank Dr. Lukas Kuhn (Swiss Institute for Experimental Cancer Research, Epalinges, Switzerland) for providing recombinant human IRP-1 and anti-IRP-1 rabbit antiserum.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by Associazione Italiana Ricerca sul Cancro and Ministero dell’Universitèa e Ricerca Scientifica e Tecnologica (COFIN 1999 and 2000) (to G. M. and G. C.), and by Ministero dell’Universitèa e Ricerca Scientifica e Tecnologica "Center of Excellence in Aging at the University of Chieti" (to G. M.). Back

2 To whom requests for reprints should be addressed, at Department of Drug Sciences, G. D’Annunzio University School of Pharmacy, Via dei Vestini, 66013 Chieti, Italy. Phone: 011-39-0871-3555237; Fax: 011-39-0871-3555315; E-mail: gminotti{at}unich.it Back

3 The abbreviations used are: DOX, doxorubicin; IRP, iron regulatory protein; IRE, iron responsive elements; DOXol, doxorubicinol; ROS, reactive oxygen species; O2.-, superoxide anion; H2O2, hydrogen peroxide; SOD, superoxide dismutase; (5-imino)DNR, (5-imino)daunorubicin; MITOX, mitoxantrone; cys, cysteine; FAS, ferrous ammonium sulfate; 2-ME, 2-mercaptoethanol; LDH, lactate dehydrogenase. Back

Received 4/26/01. Accepted 10/ 3/01.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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