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Originally published In Press as doi:10.1074/jbc.M310106200 on January 22, 2004

J. Biol. Chem., Vol. 279, Issue 14, 13738-13745, April 2, 2004
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Doxorubicin Paradoxically Protects Cardiomyocytes against Iron-mediated Toxicity

ROLE OF REACTIVE OXYGEN SPECIES AND FERRITIN*

Gianfranca Corna{ddagger}§, Paolo Santambrogio¶, Giorgio Minotti§||, and Gaetano Cairo{ddagger}§**

From the {ddagger}Institute of General Pathology, University of Milan, Via Mangiagalli 31, 20133 Milan, Protein Engineering Unit Dibit, IRCCS H. S. Raffaele, Via Olgettina 58, 20132 Milan, and ||Department of Drug Sciences and Centro Studi sull'Invecchiamento, G. D'Annunzio University School of Medicine, Chieti, Via dei Vestini, 66013 Chieti Italy

Received for publication, September 11, 2003 , and in revised form, January 22, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The cardiotoxicity induced by the anticancer anthracycline doxorubicin (DOX) is attributed to reactions between iron and reactive oxygen species (ROS) that lead to oxidative damage. We found that DOX forms ROS in H9c2 cardiomyocytes, as shown by dichlorodihydrofluorescein oxidation and the expression of stress-responsive genes such as catalase or aldose reductase. DOX also increased ferritin levels in these cells, particularly the H subunit. A considerable increase in ferritin mRNA levels showed that DOX acted at transcriptional level, but an additional potential mechanism was identified as the down-regulation of iron regulatory protein-2, post-transcriptional inhibitor of ferritin synthesis. Pretreatment with DOX protected H9c2 cells against the damage induced by subsequent exposure to ferric ammonium citrate, and experiments with 55Fe revealed that the protection was due to the deposition of iron in ferritin. Cytoprotection was also observed when DOX was replaced by glucose/glucose oxidase, a source of H2O2, thus suggesting that DOX increases ferritin synthesis through the action of ROS. This concept was supported by three more lines of evidence. (i) DOX-induced ferritin synthesis was blocked by N-acetylcysteine, a scavenger of ROS. (ii) Mitoxantrone, a ROS-forming analogue, similarly induced ferritin expression and protected the cells against iron toxicity. (iii) 5-Iminodaunorubicin, an analogue lacking ROS-forming activity, did not induce ferritin synthesis or protect the cells against iron toxicity. These results characterize a paradoxically beneficial link between anthracycline-derived ROS, increased ferritin synthesis, and resistance to iron-mediated damage. The role of iron and ROS in anthracycline-induced cardiotoxicity may, therefore, be more complex than previously believed.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Doxorubicin (DOX)1 is an anticancer anthracycline whose therapeutic efficacy is limited by the possible development of severe cardiotoxicity. It has been suggested that both iron and reactive oxygen species (ROS) mediate the cardiotoxicity induced by DOX, but the mechanisms through which iron and ROS interact and damage cardiac cells are still debated. It has long been known that one-electron redox cycling of a quinone moiety in the tetracyclic ring of DOX is accompanied by the formation of ROS, similar to superoxide () and hydrogen peroxide (H2O2). Iron could act by converting these ROS into more potent and damaging oxidants such as hydroxyl radicals (·OH) (1). On the other hand, we have demonstrated that both DOX-derived ROS and other anthracycline metabolites such as the side chain secondary alcohol metabolite doxorubicinol (DOXol) may act by altering the function of the cytoplasmic iron regulatory proteins (IRP) that govern iron homeostasis by binding to iron-responsive elements in the untranslated regions of mRNAs for transferrin receptor and ferritin (1). When activated, IRPs enhance transferrin receptor mRNA stability and block ferritin mRNA translation, thus favoring iron uptake over sequestration and forming a pool of iron available for metabolic use. Conversely, the down-regulation of IRP activity allows ferritin synthesis to proceed and reduces transferrin receptor expression, thus preventing an accumulation of potentially toxic excess iron (2, 3). Studies of cell-free systems and isolated cardiomyocytes show that the secondary alcohol moiety of DOXol oxidizes with the [4Fe-4S] cluster of cytoplasmic aconitase, a process that regenerates DOX while also inducing cluster disassembly and the consequent change of aconitase into active IRP-1 (4, 5). Subsequent interactions of DOX with cluster-released iron form an anthracycline-iron complex that irreversibly oxidizes the newly formed IRP-1, thus giving a "null" protein that lacks RNA binding activity even in the presence of a reducing agent such as 2-mercaptoethanol (4, 5). Quinone-derived ROS synergize with anthracycline-iron complexes in promoting the oxidation of IRP-1 to a null protein (5); at the same time ROS play an independent role in promoting oxidative modifications in a clusterless IRP-2, thus priming it to ubiquitination and proteasome-mediated degradation (5, 6). DOX therefore seems to alter the normal functioning of both IRPs through a sequential action of DOXol and ROS on aconitase/IRP-1 or an independent action of ROS on IRP-2. These mechanisms may act as important links between anthracyclines and ROS and iron-mediated toxicity. It is worth noting that a number of chemical and physical or biological sources of ROS have been shown to induce transcriptional activation of ferritin synthesis, an effect that could be of benefit if the ferritin sequestered iron before it reacted with ROS to generate potent cell oxidants (7). Whether this occurs in cells exposed to DOX-derived ROS has not been formally established. To improve our understanding of the role of iron and ROS in anthracycline-induced cardiotoxicity and given the central role that ferritin may play, we examined the expression of ferritin and its influence on iron-mediated toxicity in H9c2 rat cardiomyocytes exposed to DOX.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture—The H9c2 embryonic rat heart-derived cell line was obtained from The American Type Culture Collection (CRL 1446), grown at 37 °C in 5% CO2 in Dulbecco's-modified minimum Eagle's medium adjusted to contain 4 mM glutamine, 1.5 g/liter sodium bicarbonate, 4.5 g//liter glucose, 1 mM sodium pyruvate, 100 units/ml penicillin, 0.1 ng/ml streptomycin, and supplemented with 10% heat-inactivated fetal calf serum. Subconfluent cells were treated for 24 h with various concentrations of DOX, 5-iminodaunorubicin (5-i-DNR) (Amersham Biosciences), or mitoxantrone (Mitox) (Sigma) or incubated for various periods of time with 5 milliunits of glucose oxidase (Sigma) in the presence of 25 mM glucose in complete growth medium. In some experiments 10 mM N-acetylcysteine (Sigma) was added to the culture medium 2 h before DOX treatment. When appropriate, after exposure to anthracyclines or glucose oxidase, the cells were washed and treated for 16 h with increasing concentrations of ferric ammonium citrate in complete medium. At the end of the various treatments, the medium was removed, and the cells were washed with phosphate-buffered saline, collected, and homogenized as described below.

Preparation of Cell Lysates—The cells were homogenized in 10 mM Hepes, pH 7.6, 3 mM MgCl2, 40 mM KCl, 5% glycerol, 0.2% Nonidet P 40 (Sigma), and a protease inhibitor mixture (Sigma). After the addition of dithiothreitol to make a 1 mM final concentration, the lysate was centrifuged at 16.000 x g for 5 min at 4 °C. Aliquots of the supernatant were taken for ELISA or immunoblot analysis and the determination of IRP activity.

RNA-Protein Gel Retardation Assay—The probe for the band-shift assay was transcribed from the linearized pSPT-fer plasmid containing the iron-responsive elements of the human ferritin H chain (8) using T7 RNA polymerase in the presence of [{alpha}-32P]UTP in a commercially available kit (Promega Corp., Milan, Italy). Equal amounts of protein (2 µg, as determined using the Bio-Rad protein assay kit) from the cell lysates were incubated with a molar excess of an iron-responsive elements probe and sequentially treated with RNase T1 and heparin as previously described (9). After separation on 6% non-denaturing polyacrylamide gels, the RNA-protein complexes were visualized by autoradiography and quantitated by means of direct nuclear counting using an InstantImager (Packard Instruments Co.).

Western Blot Analysis—Aliquots of the cytosolic extracts containing equal amounts of proteins were electrophoresed in acrylamide-SDS gels and electroblotted to Hybond ECL membranes (Amersham Biosciences). After assessing the transfer by means of Ponceau S staining, the membranes were saturated in 4 mM Tris-HCl, pH 7.6, 30 mM NaCl (Tris-buffered saline) containing 20% nonfat milk and 0.1% Tween 80 and incubated with rabbit antiserum to IRP-2 (raised against a conserved sequence in the degradation domain of IRP2, 1:100 dilution), catalase (Sigma,1:5,000 dilution), and {beta}-actin (Sigma, 1:10,000 dilution) to control equal protein loading. To detect ferritin, the proteins were separated on 7.5% native polyacrylamide gels, and the blots were probed with a 1:1000 dilution of rabbit polyclonal antibodies raised against recombinant mouse H ferritin subunit (10). After incubation with the appropriate secondary antibodies and extensive washing with Tris-buffered saline containing Tween 80, the proteins were detected by means of chemiluminescence using an immunodetection kit (ECL Plus, Amersham Biosciences) according to the manufacturer's instructions. The proteins were quantified by means of densitometric scanning of the blots, making sure that all of the signals were in the linear range. All of the data were calculated by comparing the intensity of the bands within the same film exposure. The values were calculated after correction for the amount of {beta}-actin.

Northern Blot Analysis—Total cellular RNA was isolated as previously described (11), and equal amounts of RNA were electrophoresed under denaturing conditions. To confirm that each lane contained equal amounts of total RNA, the ribosomal RNA content in each lane was estimated in ethidium bromide-stained gels by means of laser densitometry. The RNA was transferred to Hybond-N filters (Amersham Biosciences) that were hybridized with the 32P-labeled DNA probes rat ferritin H and L subunit cDNAs (12, 13) and human aldose reductase cDNA (14). Quantitative determination was obtained by means of direct nuclear counting using an InstantImager (Packard Instruments Co.), and the values were calculated after normalization to the amount of ribosomal RNA.

Determination of Ferritin Subunit Content—Ferritin concentrations were determined in cell lysates by means of ELISA using polyclonal antibodies raised against mouse recombinant H and L ferritin subunits, and calibrated using the corresponding recombinant homopolymers (10). The specificity of the antibodies and the absence of cross-reactivity have been previously described (10). The microtiter plates were coated with 1 µg of polyclonal antibody specific for mouse H or L ferritin. Soluble tissue homogenates or standard ferritins were diluted in 50 mM sodium phosphate, pH 7.4, 150 mM NaCl, 0.05% (v/v) Tween 20, 1% bovine serum albumin and added to the plates. The presence of ferritin was revealed by means of incubation with the same antibody labeled with horseradish peroxidase. Peroxidase activity was developed using o-phenylenediamine dihydrochloride (Sigma).

Measurement of Oxidative Stress—ROS production in untreated or anthracycline-treated cells was monitored using the DCFH-diacetate fluorescent probe (Sigma), a cell-permeable indicator of ROS (15). DCFH-diacetate is activated by cellular esterases, and then the DCFH is converted by H2O2 and peroxidases to the dichlorofluorescein fluorescent derivate. After washing with phosphate-buffered saline, the cells were loaded with DCFH-diacetate (20 µM) for 20 min at 37 °C, thoroughly washed, trypsinized, and resuspended in phosphate-buffered saline. Fluorescence intensity was measured by means of FACScan flow cytometry on the FLH-1 channel.

MTT Assay—H9c2 cells were seeded in quadruplicate in 24-well plates and then left untreated or treated with DOX or DOX analogues for 24 h. They were subsequently exposed to increasing amounts of ferric ammonium citrate (FAC) (Sigma) for 16 h. At the end of the treatments cell viability was measured using thiazolyl blue (MTT, Sigma) as an indicator of mitochondrial function (16). Briefly, 50 µl of MTT solution (5 mg/ml) were added to each well with 450 µl of medium. After incubation at 37 °C for 2–3 h, formazan crystals were dissolved by adding 500 µl of the MTT solubilization solution and thorough pipetting up and down. Absorbance was read at 570 nm, and the background absorbance at 690 nm was subtracted.

Analysis of 55Fe-labeled Ferritin—H9c2 cells were incubated in the absence or presence of anthracyclines for 24 h at 37 °C in serum-free Dulbecco's-modified minimum Eagle's medium plus 0.5% bovine serum albumin added with 2 µCi/ml [55Fe] ferric iron citrate ({cong}2 µM iron), which was prepared by mixing 55FeCl3 (PerkinElmer Life Sciences) with citric acid in a 1:2 molar ratio. At the end of the incubation period, the medium was removed, and the cells were washed three times with cold phosphate-buffered saline and homogenized in the same lysis buffer as that used for the immunoblot analysis (see above). An aliquot was taken for protein determination, and another was mixed with Ultima Gold (Packard Instrument Co.) to measure the amount of cellular 55Fe by means of liquid scintillation counting. To evaluate 55Fe incorporation into ferritin, the lysates were centrifuged, and equal amounts of proteins from the supernatants were analyzed by means of the non-denaturing PAGE system used for the immunoblotting followed by Coomassie Blue staining (to assess equal protein loading), autoradiography, and InstantImager counting. The ferritin band was identified by comigration with recombinant mouse H ferritin


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
DOX Induces Ferritin Expression—We measured ferritin levels in H9c2 cardiomyocytes exposed for 24 h to 5–10 µM DOX, concentrations reproducing the plasma peak reached by standard infusions in patients (17). Western blot analyses performed using an antibody against the H subunit showed that DOX increased the amount of ferritin (Fig. 1A). The antibodies against the murine ferritin subunits were also used in an ELISA to quantify the increase in ferritin and ascertain whether DOX enhanced the levels of the H and/or L subunits. Fig. 1B shows that both ferritin subunits were increased by DOX, although there was a preferential induction (2.5–3-fold) of the H chain. The increase in the H subunits in the cells treated with 10 µM DOX was similar to that determined in cells exposed to iron, but the latter showed more evident accumulation of the L subunit.



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  		FIG. 1.
Effect of DOX on ferritin content of H9c2 cells. A, equal amounts of proteins (50 µg) from cells exposed for 24 h to increasing concentrations of DOX were loaded on non-denaturing polyacrylamide gels. Ferritin was immunoblotted with an antibody against mouse H ferritin and visualized by means of chemiluminescence. The same antibody recognized mouse recombinant H ferritin (rMHF), which migrated faster due to its homopolymer composition. The gel shown is representative of four independent experiments. B, quantification of H and L ferritin subunits in cytoplasmic extracts by ELISA. H9c2 cells were treated with different doses of DOX or 30 µg/ml FAC as a positive control. Results are mean values ± S.D. of three-five experiments. *, p < 0.001; **, p < 0.005; ***, p < 0.01; ****, p < 0.05 versus controls.

 
Mechanisms of Ferritin Induction—Fig. 2A shows that the IRP-1 activity measured using a band-shift assay increased in cells treated with 5 µM DOX. This result is consistent with the metabolization of DOX to DOXol and the ability of the latter to induce the complete disassembly of the Fe-S clusters associated with cytoplasmic aconitase, thus making the enzyme switch to IRP-1 (5). However, at 10 µM DOX, IRP-1 activity returned to control levels (Fig. 2A), and the same was also observed when the samples were treated with 1% 2-mercaptoethanol just before the band-shift assay (not shown). This finding was consistent with the fact that DOXol oxidized back to DOX, which then damaged IRP-1 by forming a complex with iron released from the cluster (4). As the redox activity of DOX-Fe complexes increased with the DOX:Fe ratio (18), raising DOX from 5 to 10 µM favored the formation of DOX-Fe complexes that redox-coupled with ROS and caused the irreversible oxidation of IRP-1 into a null protein (5). In addition to altering IRP-1 activity, DOX diminished the binding activity and immunodetectable levels of IRP-2 in a concentration-dependent manner. At 5 µM DOX, IRP-2 binding activity decreased by ~70% (Fig. 2A), which was consistent with the 60% decrease in immunodetectable protein (Fig. 2B). The DOX-induced reductions in IRP-2 activity and levels were, therefore, consistent with the concentration-dependent increase in ferritin levels. This was less evident in the case of IRP-1, whose activation or inactivation at 5 or 10 µM DOX should have been accompanied by, respectively, lower or higher ferritin levels. It, therefore, seems that IRP-2, but not IRP-1, is involved in modulating ferritin levels after DOX treatment. However, Fig. 2C shows that DOX also increased the steady-state levels of mRNAs for the ferritin H and L subunits, with a preferential (3-fold) induction of H subunit mRNA. This suggests that ferritin levels increased not only because of translational activation linked to IRP-2 down-regulation but also because DOX increases net ferritin mRNA levels.



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  		FIG. 2.
Effect of DOX on IRP binding activity, IRP-2 content, and ferritin mRNA levels. A, band-shift assay of IRP activity. Cytoplasmic extracts of cells exposed to increasing concentrations of DOX were incubated with an excess of a 32P-labeled iron-responsive element probe; RNA-protein complexes were separated on non-denaturing 6% polyacrylamide gels and revealed by means of autoradiography. The autoradiogram is representative of three independent experiments. B, immunoblot analysis of IRP-2 content. Equal amounts of proteins (80 µg) from cytoplasmic extracts were loaded on SDS gels. IRP-2 was detected using a specific polyclonal antibody and visualized by means of chemiluminescence. The same antibody recognized histidine-tagged rat recombinant IRP-2 (rIRP-2). The blots were reprobed with an antibody against {beta}-actin as a loading control. The gel shown is representative of three independent experiments. C, Northern blot analysis of ferritin mRNAs. A filter with equal amounts of total cellular RNA, as revealed by the ethidium bromide fluorescence of rRNA, was hybridized with rat H and L ferritin cDNAs. The autoradiograms are representative of three independent experiments.

 
ROS Formation in DOX-treated Cells—Because an increase in the transcription of the H ferritin subunit gene has been demonstrated under conditions of oxidative stress in a variety of experimental systems (7), we investigated whether ROS were involved in mediating ferritin induction in H9c2 cells exposed to DOX. We first assessed whether DOX causes oxidative stress in H9c2 cells. As shown in Fig. 3A, treatment with 5 and 10 µM DOX led to increases of, respectively, 2.2- and 3.1-fold in the levels of mRNA for aldose reductase, a well known stress-responsive gene (19). Once again, the effect was similar to that observed when treating cells with iron, a specific inducer of aldose reductase (14). Treatment with 5 and 10 µM DOX also increased the immunodetectable levels of catalase, another well known stress-responsive gene (20), by respectively, 1.5 and 2.5 times (Fig. 3B). Measurements of the oxidation of DCFH (a widely used marker of intracellular ROS formation) showed that ROS production increased 2.5–3 times in the DOX-treated cells (Fig. 3C). Taken together, these data indicate that DOX-dependent ferritin induction occurs under conditions of increased ROS production. To investigate the role of oxidative stress in ferritin induction, we tested whether N-acetylcysteine (a known ROS scavenger) prevented ferritin accumulation. Fig. 3C shows that the oxidation of DCFH is inhibited in H9c2 cells treated with N-acetylcysteine before the addition of DOX. Under these conditions, DOX did not increase ferritin levels, a finding that is consistent with the role of ROS in this setting (Fig. 3D).



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  		FIG. 3.
The induction of stress genes and antioxidant-inhibitable ferritin synthesis by DOX-derived ROS. A, Northern blot analysis of aldose reductase expression. A filter with equal amounts of total cellular RNA, as revealed by the ethidium bromide fluorescence of rRNA, was hybridized with a human aldose reductase probe. The autoradiogram is representative of three independent experiments. B, immunoblot analysis of catalase. Equal amounts of proteins (50 µg) from cytoplasmic extracts were loaded on SDS gels. Catalase was detected using a specific polyclonal antibody and visualized by means of chemiluminescence. The blots were reprobed with an antibody against {beta}-actin as a loading control. The results shown are representative of three independent experiments. C, evaluation of oxidative stress. DCFH oxidation was measured by means of fluorescence-activated cell sorter analysis, monitoring fluorescence at 522 nm. Results are mean values ± S.D. of three experiments *, p < 0.005; **, p < 0.01 versus control. D, quantification of H and L ferritin subunits in cytoplasmic extracts by ELISA. H9c2 cells were treated with different doses of DOX in the presence or absence of N-acetylcysteine (NAC). Results are mean values ± S.D. of three experiments. *, p < 0.001; **, p < 0.005 versus control.

 
ROS-mediated Ferritin Accumulation Protects against Iron Toxicity—The fact that ferritin has a cytoprotective effect in a number of experimental models (7) prompted us to investigate whether pre-exposure to DOX could protect H9c2 cells against the damage caused by an iron load. MTT assays showed that 16 h of iron loading caused a dose-dependent decrease in the number of viable H9c2 cells, but pretreatment with DOX significantly improved the resistance of cells to iron-induced cell death (Fig. 4). These results indicate that the increased ferritin levels induced by DOX as a result of ROS-dependent gene activation (and perhaps IRP-2 down-regulation) protect against iron toxicity.



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  		FIG. 4.
Effect of pretreatment with DOX on iron-mediated toxicity in H9c2 cells. The cells were left untreated or exposed for 24 h to 5 µM DOX. After the removal of the drug and extensive washing, the cells were challenged with increasing concentrations of FAC for 16 h. DOX treatment alone caused the death of 30–40% of the cells. Toxicity was evaluated as the percentage of viable cells after exposure to iron, as measured by MTT assay. Results are mean values ± S.D. of three experiments.

 
To assess the role of ROS further while re-addressing the role of secondary alcohol metabolites reactive to aconitase/IRP-1, we treated the H9c2 cells with anthracycline analogues characterized by their capacity to induce the formation of ROS or secondary alcohol metabolites to a different extent in comparison with DOX. The role of ROS was probed using 5-i-DNR, an analogue that contains the side-chain carbonyl group required for alcohol metabolite formation but is less than 20% as effective as DOX in generating ROS because of the presence of an imino group in place of the quinone (Fig. 5). The role of secondary alcohol metabolites was probed using mitoxantrone (Mitox), an analogue that contains the quinone but lacks a carbonyl group in its side chain (Fig. 5). The experiments were performed bearing in mind that the generation of ROS and alcohol metabolites depended not only on the intrinsic biochemical behavior of these molecules but also on the extent to which they entered the cardiomyocytes.



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  		FIG. 5.
Structures of DOX and the analogues used in this study. The arrows indicate biochemically important modifications in 5-I-DNR (the replacement of a quinone by an imino group precludes ROS formation) or Mitox (the lack of a carbonyl group in the side chain precludes the formation of a secondary alcohol metabolite).

 
On the basis of the results of our previous pharmacokinetic studies (5), 5-i-DNR and Mitox were used at concentrations of, respectively, 20 and 2.5 µM, which lead to the same level of uptake in cardiomyocytes as 5 µM DOX (5). As shown in Fig. 6A, measurements of DCFH oxidation confirmed that 5-i-DNR was unable to form ROS, which were formed by Mitox but apparently to a lesser extent than by DOX (Fig. 6A), probably because Mitox is an excellent reducing substrate for H2O2-activated peroxidases (21). It is quite possible that Mitox generated greater amounts of ROS than those detected by us, but its ability to react with peroxidases eventually diminished the yields of ROS, which were measured on the basis of DCHF oxidation by the same peroxidases.



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  		FIG. 6.
Effect of pretreatment with DOX and DOX analogues on ROS production, ferritin content, and iron-mediated toxicity in H9c2 cells. The cells were left untreated or exposed to 5 µM DOX, 20 µM 5-i-DNR, or 2.5 µM Mitox for 24 h. A, ROS were measured by means of the oxidation of DCFH as described in the legend to Fig. 3. Results are mean values ± S.D. of three experiments. *, p < 0.005; **, p < 0.01 versus control. B, quantification of H and L ferritin subunits using cytoplasmic extracts and an ELISA assay as described in the legend to Fig. 1. Results are mean values ± S.D. of three experiments. *, p < 0.001; **, p < 0.005; ***, p < 0.01; ****, p < 0.05 versus control. N.S., not significant. C, toxicity was evaluated as the post-treatment percentage of viable cells as evaluated by MTT assay. Results are mean values ± S.D. of three experiments.

 
Having obtained information on ROS formation, we next determined whether 5-i-DNR and Mitox increased ferritin content and protected cells from iron toxicity. ELISA experiments showed that Mitox increases cell levels of ferritin (particularly the H subunit) to an extent that is similar to the increase induced by 5 µM DOX, a finding that is consistent with its ability to reach the same cell levels as DOX and to generate ROS (Fig. 6B). On the contrary, 5-i-DNR had essentially no effect on ferritin levels, which is consistent with its ineffectiveness in producing ROS. The data concerning ROS formation and ferritin levels were mirrored by those relating to iron-mediated toxicity. Fig. 6C shows that 5-i-DNR (which did not form ROS or increase ferritin levels) did not protect cardiomyocytes against the damage induced by subsequent exposure to an iron load, whereas the cells pretreated with Mitox could withstand iron toxicity in a manner that was not significantly different from that of DOX (p = 0.180), which is in line with the fact that Mitox generates ROS and increases ferritin levels. Finally, Mitox had essentially the same effects as DOX despite its inability to form a secondary alcohol metabolite, thus confirming that the changes in IRP-1 activity induced by secondary alcohol metabolites are of little importance in mediating ferritin up-regulation and the consequent protection against iron toxicity.

To obtain further evidence that DOX-dependent ferritin induction was mediated by ROS and protected the cells from toxic iron, cardiomyocytes were exposed to a source of ROS other than DOX before undergoing iron treatment. We used the glucose/glucose oxidase reaction, which generates a constant flux of H2O2. As shown in Fig. 7, A–C, glucose/glucose oxidase led to the robust oxidation of DCFH, increased the levels of the L and (especially) H ferritin subunits, and protected the cardiomyocytes against the damage induced by a subsequent iron exposure.



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  		FIG. 7.
Effect of treatment with glucose/glucose oxidase on ROS production, ferritin content, and iron-mediated toxicity in H9c2 cells. The cells were left untreated or exposed to glucose/glucose oxidase (GOX) for the indicated times (panel A) or 7 h (panels B and C). ROS production, ferritin subunits content, and cytotoxicity were evaluated as described in the legend to Fig. 6. Results are mean values ± S.D. of three experiments *, p < 0.001; **, p < 0.005; ***, p < 0.01.

 
Effect of DOX on Intracellular Iron Distribution—To obtain more direct correlations between ferritin induction and cytoprotection, we examined the fate of the radioactive iron taken up by H9c2 cells during the course of exposure to a trace amount of 55Fe in the presence or absence of DOX. Autoradiography of non-denaturing gels of soluble cell homogenates showed that ferritin accounted for the vast majority of the 55Fe-labeled proteins (Fig. 8). Ferritin-associated radioactivity increased if the cells were also treated with DOX or Mitox but was not significantly enhanced by 5-i-DNR. In comparison with the cells exposed to 55Fe alone, those exposed to 55Fe plus DOX or the other drugs did not show any significant increase in the total cell content of 55Fe determined by means of the liquid scintillation counting of aliquots of cell homogenates (not shown). This indicates that the different abilities of DOX, Mitox, and 5-i-DNR to increase the incorporation of 55Fe into ferritin was not mediated by enhanced cellular iron uptake and/or reduced iron release but reflected the different extents to which the drugs up-regulate ferritin levels.



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  		FIG. 8.
Effect of exposure to DOX or DOX analogues on ferritin incorporation of 55Fe. The H9c2 cells were incubated with 55Fe iron citrate in the presence or absence of 5 µM DOX, 20 µM 5-i-DNR, or 2.5 µM Mitox for 24 h, and the cell extracts were analyzed by means of native PAGE. Ferritin (Ft) was revealed by autoradiography and identified on the basis of the migration of purified recombinant mouse H ferritin. The autoradiogram is representative of three independent experiments. c, control.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The cardiac toxicity of DOX has been attributed to its ability to generate ROS and promote iron-catalyzed oxidative damage. The results of the present study show that DOX produces ROS in H9c2 cardiomyocytes, as indicated by the increased levels of stress-responsive genes such as aldose reductase and catalase and by direct measurements of DCFH oxidation (Fig. 3). However, our results also show that DOX up-regulates ferritin levels in cardiomyocytes and that this offers excellent protection against the toxicity induced by subsequent iron exposure. The ferritin-inducing effect of DOX consisted of a preferential increase in the H subunits (Fig. 1), a finding that is consistent with those of previous studies showing that H ferritin increases under conditions of ROS generation (9, 22, 23). The role of ROS in inducing the synthesis of H ferritin and that of the H subunits in protecting against iron toxicity was demonstrated by various lines of evidence. First, ferritin synthesis was blocked by N-acetylcysteine, a ROS scavenger (Fig. 3). Second, neither H ferritin synthesis nor cytoprotection against iron was observed when DOX was replaced by 5-i-DNR, an analogue lacking ROS-forming activity, whereas both were seen when DOX was replaced by Mitox, an analogue that is still capable of producing ROS (Fig. 6). Third, DOX-induced cytoprotection did not correlate with any substantial change in cellular iron uptake or release but did correlate with the facilitated sequestration of iron inside ferritin (Fig. 8). Finally, anthracycline treatment led to a concomitant increase in L-type subunits, but this was small in comparison with the increase in H-type subunits and did not correlate with the ability of the analogue to provide protection against iron; in fact, the increase in L-type subunits was similar with all three drugs (Fig. 6).

We also found that 2.5 µM Mitox and 5 µM DOX are comparably taken up by cardiomyocytes and consistently induce similar increases in H ferritin, although ROS generation seemed to be less with Mitox. This last finding may introduce a caveat if ROS is involved in mediating the anthracycline-induced increase in ferritin anthracyclines. As mentioned above, this inconsistency may reflect the interference of Mitox with the DCFH assay for ROS, thus diminishing the apparent yield of detectable ROS. Another possibility is that the induction of H ferritin may require a threshold level of ROS formation above which ferritin synthesis continues regardless of any additional formation. Finally, it is possible that many genes may be activated by DOX and contribute to mediating protection against iron.

The protective efficacy of anthracycline pretreatment against subsequent iron loading is reminiscent of the preconditioning seen with H2O2, a process in which exogenous H2O2 or hypoxia-induced mitochondrial release of H2O2 protects cells against subsequent damage induced by, for example, ischemia-reperfusion (24, 25). We found that glucose oxidase (a source of H2O2) can replace DOX in increasing ferritin levels and protecting against iron (Fig. 7), finding that is important in two respects; (i) it confirms that the effects of DOX are mediated by ROS, and (ii) it indicates similarities between the protection induced by DOX and that induced by H2O2 preconditioning.

We investigated whether ferritin synthesis was enhanced by DOX as a result of changes in IRP-1 or IRP-2 activities. Our data support the possible role of ROS-dependent IRP-2 degradation in relieving a translational block of ferritin synthesis, as shown by the comparisons of the concentration-dependent patterns of ROS formation, IRP-2 degradation, and the increase in ferritin after treatment with 5 and 10 µM DOX as well as the comparisons of the effects of DOX, Mitox, and 5-i-DNR. On the contrary, neither the DOX/analogue comparisons nor the bell-shaped concentration-dependent pattern of IRP-1 activation or inactivation by 5 or 10 µM DOX seem to correlate with the pattern of enhanced ferritin synthesis (Figs. 1, 2, 3 and 6). However, we acknowledge that it is important to understand how ferritin synthesis increased after treatment with 5 µM DOX, when the degradation of IRP-2 was counteracted by the activation of IRP-1. In a previous study (26), we found that cytokine-treated macrophages underwent a similarly converse up-regulation of IRP-1 and down-regulation of IRP-2, whereas the level of ferritin synthesis increased as if the degradation of IRP-2 somehow served the prevailing mechanisms regulating it. All of these findings may be reconciled by the fact that the iron-responsive elements of ferritin mRNA are preferentially bound by IRP-2 (27). In any case, the ability of anthracyclines to protect against iron (DOX = Mitox >> 5-i-DNR) correlates with ROS formation, IRP-2 degradation and increased ferritin synthesis (5).

IRP-2 degradation is, therefore, a possible determinant of increased ferritin synthesis in H9c2 cardiomyocytes, but we also found that DOX considerably increased steady-state ferritin mRNA levels. Although we did not directly demonstrate increased ferritin transcription, we suggest that such a remarkable increase reflects an action at transcriptional level mediated by the direct targeting of the 5' sequences that are conserved in the regulatory regions of H and L ferritin genes as well as many other anti-oxidant response genes (23). Transcriptional activation, therefore, seems to be a prevalent mechanism of anthracycline-induced ferritin synthesis, which is possibly further accelerated as a result of IRP-2 degradation relieving a translational block. The transcriptional activation of ferritin expression and the consequent up-regulation of steady-state mRNA levels would also be sufficient to overcome a translational block possibly caused by any transient activation of IRP-1. We are currently identifying and characterizing ROS-sensitive ferritin regulatory sequences.

Other investigators have obtained similar evidence supporting the preferential accumulation of iron in ferritin after the exposure of cancer or cardiac cells to DOX; however, increased ferritin iron sequestration occurred in the absence of ferritin induction, and the overall mechanism of the anthracycline/iron/ferritin interactions remained uncertain, partially because the anthracycline concentrations were often too high to be of pathophysiological relevance (e.g. 20 µM) (28). In our study we found that anthracycline-derived ROS were related to ferritin induction and that increased ferritin levels were related to the ability of cardiac cells to withstand iron toxicity. Our results, therefore, offer the basis for a reappraisal of the role of ROS and iron in the cardiac damage induced by anthracyclines and suggest that there may be conditions under which anthracycline-derived ROS do not exacerbate iron toxicity but actually improve cell defenses against iron. This may be the case when chemotherapy increases the plasma levels of non-transferrin-bound iron, an effect that is due to the suppression of erythropoietic activity or the leakage of iron from necrotic tumoral foci (29, 30). This iron pool may well contribute to inducing cardiotoxicity if it enters cardiomyocytes and reacts with ROS, but our results suggest that one such mechanism of toxicity may be prevented by the sequestration of iron in the ferritin shells formed by the prior redox activation of anthracyclines. This concept is supported by the fact that plasma anthracycline levels peak within min of drug administration (17), whereas it takes days before the peak plasma levels of non-transferrin-bound iron are reached (29). There is, therefore, sufficient time for anthracylines to induce ferritin synthesis and prevent the toxicity induced by subsequent exposure of the heart to iron, a pathological condition that we have mimicked by treating isolated cardiomyocytes with anthracyclines before an iron load.

In conclusion, we have characterized a paradoxical "antioxidant" mode of action of anthracyclines, which is mediated by ferritin synthesis and leads to a long-lasting protection that persists after removal of DOX from the medium or after the decay or clearance of the drugs from biological fluids or tissues. These results raise some doubts against the widely accepted concept of the oxidative nature of cardiotoxicity but support the idea that antioxidants do not always protect laboratory animals against cardiotoxicity or mitigate or delay cardiotoxicity in patients. It clearly remains to be established what the precise mechanism of cardiotoxicity is and how it may be prevented or mitigated by the use of iron chelators such as dexrazoxane (31). Our results raise the possibility that iron and ROS become toxic as a result of reactions that extend beyond canonical oxidative damage and, among other mechanisms, may involve alterations in the metabolic use of iron (1) or its recruitment in pro-apoptotic signals that have not yet been characterized (32).


   FOOTNOTES
 
* This study was supported by grants from Associazione Italiana per la Ricerca sul Cancro, Ministero dell'Istruzione, dell'Università e della Ricerca (MURST) Fondo Investimento Ricerca di Base, COFIN 2001 and 2002 (to G. C. and G. M.), and from the MURST Center of Excellence on Aging at the University of Chieti (to G. M.). 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. Back

§ Contributed equally to this paper. Back

** To whom correspondence should be addressed. Tel.: 39-0250315350; Fax: 39-0250315338; E-mail: gaetano.cairo{at}unimi.it.

1 The abbreviations used are: DOX, doxorubicin; DOXol, doxorubicinol; IRP, iron regulatory protein; 5-i-DNR, 5-iminodaunorubicin; ELISA, enzyme-linked immunosorbent assay; FAC, ferric ammonium citrate; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; ROS, reactive oxygen species; DCFH, dichlorodihydrofluorescein; Mitox, mitoxantrone. Back


   ACKNOWLEDGMENTS
 
We thank Dr. M. Locati and Dr. D. Besusso for help with the fluorescence-activated cell sorter analysis, Dr. D. Barisani for the kind gift of human aldose reductase cDNA, and Dr. E. Leibold for the plasmid encoding IRP-2.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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