Doxorubicin Paradoxically Protects Cardiomyocytes against Iron-mediated ToxicityThe 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. 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. Doxorubicin (DOX) 1The 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. 1The 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. 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 ( O2˙-) and hydrogen peroxide (H2O2). Iron could act by converting these ROS into more potent and damaging oxidants such as hydroxyl radicals (·OH) (1Minotti G. Cairo G. Monti E. FASEB J. 1999; 13: 199-212Google Scholar). 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 (1Minotti G. Cairo G. Monti E. FASEB J. 1999; 13: 199-212Google Scholar). 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 (2Hentze M.W. Kuhn L.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8175-8182Google Scholar, 3Cairo G. Pietrangelo A. Biochem. J. 2000; 352: 241-250Google Scholar). 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 (4Minotti G. Recalcati S. Mordente A. Liberi G. Calafiore A.M. Mancuso C. Preziosi P. Cairo G. FASEB J. 1998; 12: 541-552Google Scholar, 5Minotti G. Ronchi R. Salvatorelli E. Menna P. Cairo G. Cancer Res. 2001; 61: 8422-8428Google Scholar). 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 (4Minotti G. Recalcati S. Mordente A. Liberi G. Calafiore A.M. Mancuso C. Preziosi P. Cairo G. FASEB J. 1998; 12: 541-552Google Scholar, 5Minotti G. Ronchi R. Salvatorelli E. Menna P. Cairo G. Cancer Res. 2001; 61: 8422-8428Google Scholar). Quinone-derived ROS synergize with anthracycline-iron complexes in promoting the oxidation of IRP-1 to a null protein (5Minotti G. Ronchi R. Salvatorelli E. Menna P. Cairo G. Cancer Res. 2001; 61: 8422-8428Google Scholar); 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 (5Minotti G. Ronchi R. Salvatorelli E. Menna P. Cairo G. Cancer Res. 2001; 61: 8422-8428Google Scholar, 6Iwai K. Drake S.K. Wehr N.B. Weissman A.M. LaVaute T. Minato N. Klausner R.D. Levine R.L. Rouault T.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4924-4928Google Scholar). 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 (7Torti F.M. Torti S.V. Blood. 2002; 99: 3505-3516Google Scholar). 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. 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 × 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 (8Mullner E.W. Neupert B. Kuhn L.C. Cell. 1989; 58: RNA in the presence of in a available of protein as the protein assay from the cell were incubated with a excess of an iron-responsive elements probe and treated with and as previously described G. G. E. A. A. J. Scholar). After on the complexes were by and by of an of the containing of proteins were in and to (Amersham After the by of the were in 4 mm pH 7.6, mm containing and and incubated with to against a in the degradation of catalase and to protein To the proteins were on and the were with a of against H ferritin P. A. S. E. A. P. 2000; Scholar). After with the secondary and with containing the proteins were by of an to the The proteins were by of of the that of the were in the of the were by the of the the same The were after for the of RNA was isolated as previously described A. E. G. G. A. E. Cairo G. and of RNA were To that of the RNA in was in by of The RNA was to (Amersham that were with the rat ferritin H and K. Proc. Natl. Acad. Sci. U. S. A. Scholar, N. J. and human aldose R. E. C. 2000; Scholar). determination was obtained by of an and the were after to the of of concentrations were in cell by of ELISA against H and ferritin and the P. A. S. E. A. P. 2000; Scholar). The of the and the of have been previously described P. A. S. E. A. P. 2000; Scholar). The were with 1 of for H or ferritin. or were in mm sodium pH mm and added to the The presence of ferritin was revealed by of with the same with activity was (Sigma). of in or cells was the probe (Sigma), a of ROS J. A. J. 1998; Scholar). is by and the is by and to the After with phosphate-buffered saline, the cells were with for min at 37 and in phosphate-buffered was by of on the cells were in in and or treated with DOX or DOX for 24 were exposed to increasing of ferric ammonium citrate (Sigma) for 16 At the end of the cell was as an of function P. A. A. Cancer Res. Scholar). of were added to with of medium. After at 37 °C for were by of the and and was at and the at was of cells were incubated in the or presence of anthracyclines for 24 h at 37 °C in Dulbecco's-modified minimum Eagle's medium added with 2 ferric iron citrate which was by with in a At the end of the the medium was removed, and the cells were washed three with phosphate-buffered and homogenized in the same as that used for the immunoblot analysis was taken for protein and was with to the of 55Fe by of To 55Fe into the were and of proteins from the were by of the used for the by protein and The ferritin was identified by with H ferritin DOX ferritin levels in H9c2 cardiomyocytes exposed for 24 h to DOX, concentrations the by in A. G. A. E. G. J. Scholar). an against the H showed that DOX increased the of ferritin The against the ferritin were also used in an ELISA to the increase in ferritin and DOX the levels of the H that both ferritin were increased by DOX, was a of the H The increase in the H in the cells treated with 10 DOX was similar to that in cells exposed to but the showed more accumulation of the subunit. of that the IRP-1 activity a band-shift assay increased in cells treated with 5 DOX. This is with the of DOX to DOXol and the of the to induce the complete disassembly of the with cytoplasmic aconitase, thus the to IRP-1 (5Minotti G. Ronchi R. Salvatorelli E. Menna P. Cairo G. Cancer Res. 2001; 61: 8422-8428Google Scholar). at 10 DOX, IRP-1 activity to levels and the same was also observed when the were treated with 2-mercaptoethanol before the band-shift assay This was with the that DOXol to DOX, which IRP-1 by forming a complex with iron from the cluster (4Minotti G. Recalcati S. Mordente A. Liberi G. Calafiore A.M. Mancuso C. Preziosi P. Cairo G. FASEB J. 1998; 12: 541-552Google Scholar). the redox activity of complexes increased with the G. Biochem. DOX from 5 to 10 the formation of complexes that with ROS and the oxidation of IRP-1 into a null protein (5Minotti G. Ronchi R. Salvatorelli E. Menna P. Cairo G. Cancer Res. 2001; 61: 8422-8428Google Scholar). In addition to altering IRP-1 activity, DOX the binding activity and levels of in a At 5 DOX, binding activity by which was with the in protein The DOX-induced in activity and levels therefore, with the increase in ferritin This was in the of IRP-1, whose activation or at 5 or 10 DOX have been accompanied or ferritin therefore, seems that IRP-2, but not IRP-1, is in ferritin levels after DOX treatment. that DOX also increased the levels of mRNAs for the ferritin H and with a of H This that ferritin levels increased not of activation to down-regulation but also DOX increases ferritin mRNA ROS in an increase in the of the H ferritin has been demonstrated of oxidative in a of systems (7Torti F.M. Torti S.V. Blood. 2002; 99: 3505-3516Google we ROS were in ferritin in H9c2 cells exposed to DOX. We DOX oxidative in H9c2 shown in with 5 and 10 DOX to increases and in the levels of mRNA for aldose a known stress-responsive S. A. FASEB J. Scholar). the effect was similar to that observed when cells with a of aldose R. E. C. 2000; Scholar). with 5 and 10 DOX also increased the levels of known stress-responsive G. A. Cairo G. 1996; by 1.5 and of the oxidation of used of ROS showed that ROS increased in the cells these that ferritin occurs of increased ROS To the role of oxidative in ferritin we N-acetylcysteine known ROS ferritin that the oxidation of is in H9c2 cells treated with N-acetylcysteine before the addition of DOX. these DOX did not increase ferritin a that is with the role of ROS in this against Iron that ferritin has a effect in a number of (7Torti F.M. Torti S.V. Blood. 2002; 99: 3505-3516Google to to DOX could protect H9c2 cells against the damage by an iron showed that 16 h of iron a in the number of H9c2 cells, but with DOX the resistance of cells to cell These results that the increased ferritin levels induced by DOX as a of activation protect against iron toxicity. To the role of ROS while the role of secondary alcohol metabolites reactive to we treated the H9c2 cells with anthracycline by to induce the formation of ROS or secondary alcohol metabolites to a in with DOX. The role of ROS was 5-i-DNR, an analogue that the for alcohol metabolite formation but is than as as DOX in ROS of the presence of an in of the quinone The role of secondary alcohol metabolites was mitoxantrone an analogue that the quinone but lacks a in its side chain The experiments were in that the of ROS and alcohol metabolites not on the of these but also on the to which the On the of the results of our (5Minotti G. Ronchi R. Salvatorelli E. Menna P. Cairo G. Cancer Res. 2001; 61: 8422-8428Google and were used at concentrations and which lead to the same of uptake in cardiomyocytes as 5 DOX (5Minotti G. Ronchi R. Salvatorelli E. Menna P. Cairo G. Cancer Res. 2001; 61: 8422-8428Google Scholar). shown in of oxidation that was to form ROS, which were formed by but to a than by DOX is an reducing for Res. Scholar). It is possible that of ROS than by but its to with the of ROS, which were on the of oxidation by the same obtained on ROS we and increased ferritin and protected cells from iron toxicity. ELISA experiments showed that increases cell levels of ferritin the H to an that is similar to the increase induced by 5 DOX, a that is with its to the same cell levels as DOX and to generate ROS On the effect on ferritin which is with its in ROS. The ROS formation and ferritin levels were by to iron-mediated toxicity. that did not form ROS or increase ferritin did not protect cardiomyocytes against the damage induced by subsequent exposure to an iron the cells with could iron toxicity in a that was not from that of DOX which is in line with the that ROS and increases ferritin the same as DOX its to form a secondary alcohol thus that the in IRP-1 activity induced by secondary alcohol metabolites are of in ferritin and the consequent protection against iron toxicity. To that ferritin was by ROS and protected the cells from toxic cardiomyocytes were exposed to a source of ROS other than DOX before iron treatment. We used the glucose/glucose oxidase which a of shown in glucose/glucose oxidase to the oxidation of DCFH, increased the levels of the and H ferritin and protected the cardiomyocytes against the damage induced by a subsequent iron of DOX on Iron more between ferritin and we examined the of the iron taken by H9c2 cells the of exposure to a of 55Fe in the presence or of DOX. of of cell showed that ferritin for the of the proteins increased if the cells were also treated with DOX or but was not by In with the cells exposed to 55Fe exposed to 55Fe DOX or the other did not show increase in the cell of 55Fe by of the of of cell This that the of DOX, Mitox, and to increase the of 55Fe into ferritin was not by iron uptake iron but the to which the ferritin The cardiac toxicity of DOX has been attributed to its to generate ROS and oxidative damage. The results of the show that DOX ROS in H9c2 cardiomyocytes, as by the increased levels of stress-responsive genes such as aldose and catalase and by of oxidation our results also show that DOX ferritin levels in cardiomyocytes and that this protection against the toxicity induced by subsequent iron The effect of DOX of a increase in the H a that is with of that H ferritin increases of ROS G. G. E. A. A. J. Scholar, Recalcati S. A. Cairo G. Scholar, Torti S.V. Torti F.M. Cell 2000; Scholar). The role of ROS in inducing the synthesis of H ferritin and that of the H in against iron toxicity was demonstrated by various lines of evidence. ferritin synthesis was blocked by N-acetylcysteine, a ROS scavenger H ferritin synthesis against iron was observed when DOX was replaced by 5-i-DNR, an analogue lacking ROS-forming activity, both were when DOX was replaced by Mitox, an analogue that is still of ROS DOX-induced did not with change in iron uptake or but did with the sequestration of iron ferritin anthracycline to a increase in but this was in with the increase in and did not with the of the analogue to protection against in the increase in was similar with three We also found that and 5 DOX are taken by cardiomyocytes and induce similar increases in H ROS to be with This may a if ROS is in the anthracycline-induced increase in ferritin this may the of with the assay for ROS, thus the of ROS. is that the of H ferritin may a of ROS formation which ferritin synthesis of additional it is possible that genes may be by DOX and to protection against The efficacy of anthracycline against subsequent iron is of the with H2O2, a process in which or of cells against subsequent damage induced for C. J. 1998; Scholar, A. E. J. Scholar). We found that glucose oxidase source of DOX in increasing ferritin levels and against iron that is important in (i) it that the of DOX are by ROS, and (ii) it between the protection induced by DOX and that induced by We ferritin synthesis was by DOX as a of in IRP-1 or the possible role of degradation in a block of ferritin synthesis, as shown by the of the of ROS and the increase in ferritin after with 5 and 10 DOX as as the of the of DOX, Mitox, and On the the the of IRP-1 activation or by 5 or 10 DOX to with the of ferritin synthesis 3 and we that it is important to ferritin synthesis increased after with 5 DOX, when the degradation of was by the activation of In a S. Cairo G. Blood. 1998; we found that a similarly of IRP-1 and down-regulation of IRP-2, the of ferritin synthesis increased as if the degradation of the mechanisms of these may be by the that the iron-responsive elements of ferritin mRNA are by J. J. 1998; Scholar). In the of anthracyclines to protect against iron with ROS degradation and increased ferritin synthesis (5Minotti G. Ronchi R. Salvatorelli E. Menna P. Cairo G. Cancer Res. 2001; 61: 8422-8428Google Scholar). degradation therefore, a possible of increased ferritin synthesis in H9c2 cardiomyocytes, but we also found that DOX increased ferritin mRNA we did not increased ferritin we that such a increase an action at transcriptional by the of the that are in the regulatory regions of H and ferritin genes as as other genes Torti S.V. Torti F.M. Cell 2000; Scholar). therefore, seems to be a mechanism of anthracycline-induced ferritin synthesis, which is as a of degradation a The transcriptional activation of ferritin expression and the consequent of mRNA levels also be to a block by activation of We are and ferritin regulatory have obtained similar the accumulation of iron in ferritin after the exposure of or cardiac cells to increased ferritin iron sequestration in the of ferritin and the mechanism of the interactions the anthracycline concentrations were to be of 2002; Scholar). In our we found that anthracycline-derived ROS were to ferritin and that increased ferritin levels were to the of cardiac cells to iron toxicity. therefore, the for a of the role of ROS and iron in the cardiac damage induced by anthracyclines and that may be which anthracycline-derived ROS not iron toxicity but improve cell against This may be the when increases the levels of an effect that is due to the of activity or the of iron from S. J. 99: Scholar, B. G. A. Scholar). This iron pool may to inducing cardiotoxicity if it cardiomyocytes and with ROS, but our results that such mechanism of toxicity may be by the sequestration of iron in the ferritin formed by the redox activation of This concept is supported by the that anthracycline levels min of A. G. A. E. G. J. it before the levels of iron are S. J. 99: Scholar). therefore, time for to induce ferritin synthesis and the toxicity induced by subsequent exposure of the to a that we have by isolated cardiomyocytes with anthracyclines before an iron In we have a of action of which is by ferritin synthesis and to a protection that after of DOX from the medium or after the or of the from biological or These results some against the concept of the oxidative of cardiotoxicity but the that not protect against cardiotoxicity or or cardiotoxicity in It to be the mechanism of cardiotoxicity is and it may be or by the of iron such as C. 1998; Scholar). results the that iron and ROS toxic as a of reactions that oxidative damage other may in the metabolic of iron (1Minotti G. Cairo G. Monti E. FASEB J. 1999; 13: 199-212Google or its in that have not been B. J. S. S. E. S. Cell Biochem. 2002; Scholar). We and for with the cell for the of human aldose and E. for the plasmid IRP-2.