Introduction

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Anthracyclines [e.g., doxorubicin (DOX); Fig. 1] are highly effective agents for the treatment of a wide variety of tumors. However, the major limitation of anthracyclines is cardiotoxicity (Gianni and Myers, 1992; Gerwirtz, 1999), which is thought to be associated with its marked ability to bind Fe (Garnier-Suillerot, 1988; Gianni and Myers, 1992).

View larger version (14K): [in this window] [in a new window]   Fig. 1.   Chemical structure of the anthracycline, doxorubicin (DOX), and its analogs daunorubicin (DAU) and epirubicin (EPI).

The fact that DOX can avidly bind Fe suggests that it may act as a chelator to perturb intracellular Fe pools. Certainly, DOX binds Fe from ferritin, transferrin, and microsomal membranes (Gianni and Myers, 1992). Moreover, Fe loading potentiates anthracycline toxicity, and the clinically used chelator desferrioxamine (DFO) reduces the toxic effects of DOX in vitro (Hershko et al., 1993; Link et al., 1996) and in vivo (Herman et al., 1994; Saad et al., 2001). Another Fe chelator known as ICRF-187 (also called dexrazoxane) reduces anthracycline-induced cardiotoxicity in many experimental models and clinical trials (Gerwirtz, 1999). These studies indicate that DOX cardiotoxicity is due, in part, to its interaction with Fe. However, there have been few studies examining the effects of anthracyclines on Fe metabolism of myocardial and neoplastic cells.

Cellular Fe homeostasis is largely regulated post-transcriptionally by iron-regulatory proteins (IRPs) (Hentze and Kühn, 1996). The mRNAs of certain molecules involved in Fe metabolism, including ferritin and transferrin receptor (TfR), contain hairpin loop structures in their 5'- or 3'-untranslated regions (UTRs), called iron-responsive elements (IREs) (Hentze and Kühn, 1996). The IRPs bind to IREs and either stabilize the mRNA against degradation or inhibit translation (Hentze and Kühn, 1996). In ferritin mRNA, the IRE is in the 5'-UTR, and IRP binding inhibits translation, thereby decreasing Fe storage. However, in TfR mRNA the IREs are in the 3'-UTR, and IRP binding confers mRNA stability, enhancing translation and Fe uptake via the TfR (Hentze and Kühn, 1996). Thus, the IRP-RNA-binding mechanism is crucial in regulating Fe homeostasis, and it is therefore the focus of this investigation.

The mRNA-binding activity of IRP1 is regulated by the presence of an [4Fe-4S] cluster within the protein (Hentze and Kühn, 1996; Theil and Eisenstein, 2000). When cells are Fe-depleted, the [4Fe-4S] cluster is absent (apo-IRP1), allowing for increased IRP1-IRE binding affinity. Conversely, when Fe levels are high, an [4Fe-4S] cluster becomes incorporated into the protein (holo-IRP1), preventing IRP1-IRE binding (Hentze and Kühn, 1996). Interestingly, the [4Fe-4S] cluster of IRP1 confers the molecule with aconitase activity. In fact, IRP1 is the cytoplasmic aconitase (c-acon), which is capable of converting citrate to isocitrate, with cis-aconitate (cis-acon) being an intermediate (Beinert and Kennedy, 1993). Apart from IRP1, a related RNA-binding molecule called IRP2 has been described that does not contain an [4Fe-4S] cluster. In Fe-replete cells, IRP2 is degraded by a proteasome-dependent mechanism (Guo et al., 1995).

The effect of DOX on IRP1 has been studied using heart tissue lysates in which the [4Fe-4S] cluster had been artificially reconstituted in vitro using Fe salts and cysteine (Minotti et al., 1995, 1998). The DOX metabolite, doxorubicinol (DOXol), in the presence of cis-acon, was suggested to interact with the [4Fe-4S] cluster of IRP1 to decrease IRP1-RNA-binding activity (Minotti et al., 1995, 1998). In contrast, in a more recent investigation using a rat heart-derived cell line, DOXol increased IRP1-RNA binding activity (Minotti et al., 2001). However, these latter experiments were performed in a cell line that has lost many differentiated features of myocardial cells (Hescheler et al., 1991). Hence, the relevance of these studies to normal heart cells was uncertain. Our current study is the first to examine the effect of anthracyclines on IRP-RNA-binding activity using intact and differentiated myocardial cells. Furthermore, the effects of anthracyclines on the IRP-RNA-binding activity of tumor cells were examined because this has not been assessed previously.

Our experiments using intact tumor cells show that anthracyclines induce a significant decrease in active IRP-RNA-binding activity after 6 h and a decrease in total IRP-RNA-binding activity after 24 h. Unexpectedly, these effects were not due to DOXol or the generation of free radicals by anthracyclines. Rather, the Fe or Cu complexes with DOX decreased active IRP-RNA-binding activity, suggesting that the formation of these compounds may result in the initial decrease in active IRP-RNA-binding. In primary cultures of cardiomyocytes, DOX had an effect on IRP1 similar to that described for neoplastic cells, and caused a decrease in IRP2-RNA-binding activity. Our results may be vital for understanding the effects of anthracyclines on Fe metabolism and the generation of novel treatment strategies.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Cell Treatments and Reagents. Desferrioxamine was obtained from Novartis (Basel, Switzerland). Catalase, cis-acon, ebselen, ferric ammonium citrate (FAC), N-ethylmaleimide (NEM), and superoxide dismutase (SOD) were obtained from Sigma-Aldrich (St. Louis, MO). The Mn(III) tetrakis (4-benzoic acid) porphyrin complex (MnTBAP) was obtained from ICN Pharmaceuticals Biochemicals Division (Aurora, OH). ICRF-187 was purchased from Chiron B.V. (Paasheuvelweg, Amsterdam, The Netherlands). DOX, daunorubicin (DAU), and epirubicin (EPI) were obtained from Pharmacia (Sydney, Australia). DOXol was kindly provided by Dr. G. Minotti, Department of Drug Sciences, G. D'Annunzio University School of Medicine, Chieti, Italy. The anthracycline-metal complexes were prepared as described previously (Garnier-Suillerot, 1988; Gianni and Myers, 1992).

Cell Culture. The human SK-Mel-28 melanoma, human SK-N-MC neuroepithelioma, and rat L8 skeletal muscle cell lines were obtained from the American Type Culture Collection (Manassas, VA). The M15 mouse kidney cell line was obtained from Dr. S. Wardrop, Institute of Molecular Biosciences, The University of Queensland, Brisbane, Australia. The human BE-2 neuroblastoma cell line was a gift from Dr. G. Anderson, Queensland Institute of Medical Research, Brisbane, Australia. Cells were grown and subcultured as described previously (Richardson and Baker, 1992a,b).

Preparation and Treatment of Cytosolic Extracts. Cellular extracts were prepared by incubating cells in culture with medium alone (control) or medium containing DFO (100 µM), FAC (100 µg/ml), or the anthracyclines (0.5-20 µM). The concentrations of anthracyclines examined were within the range used in previous studies (Minotti et al., 1995, 1998; DeAtley et al., 1999; Konorev et al., 1999; Sawyer et al., 1999). In experiments where Fe-depleted or Fe-loaded lysates were required, cells were incubated for 24 h at 37°C with the above concentrations of DFO or FAC, respectively. This incubation procedure has been shown, respectively, to deplete and load cells with Fe, as indicated by TfR mRNA levels, transferrin-bound ⁵⁹Fe uptake, IRP-IRE binding, c-acon activity, and the susceptibility of IRP-RNA binding to the sulfhydryl alkylating agent, NEM (Wardrop et al., 2000). Using the protocol with DFO and FAC, we showed in this study and previous investigations (Wardrop et al., 2000) that IRP1 was predominantly present without or with the [4Fe-4S] cluster, respectively. Indeed, after a 24-h incubation with control media, DFO (100 µM), or FAC (100 µg/ml), the c-acon activities were 12.3 ± 1.9 U/mg, 3.4 ± 0.3 U/mg, and 11.6 ± 1.0 U/mg (three experiments), respectively. These studies demonstrate that the control cells used in our experiments were Fe-replete. Approximately 2 to 5 × 10⁶ cells were lysed at 4°C in ice-cold Munro extraction buffer and processed (Leibold and Munro, 1988). In some experiments, the cytosolic extracts (2 µg) were directly incubated with the anthracyclines and other agents for 3 h at 4°C or 1 h at 37°C in 25 mM Tris/40 mM KCl (pH 8) (Wardrop et al., 2000).

RNA-Protein Gel Retardation Assays. The gel retardation assay was used to measure the interaction between IRPs and IREs via established techniques (Leibold and Munro, 1988; Wardrop et al., 2000). A ³²P-labeled pGL66 RNA transcript containing 118 base pairs of the ferritin H-chain (pGL66 was kindly provided by Dr. E. Leibold, University of Utah, Salt Lake City, UT) was transcribed in vitro using the Promega Riboprobe In Vitro Transcription Kit (Promega, Madison, WI). The riboprobe was then purified on a 6% urea/polyacrylamide gel.

View larger version (35K): [in this window] [in a new window]   Fig. 2.   Effect of DOX on cellular IRP-RNA-binding activity as a function of incubation time with SK-Mel-28 melanoma cells. A, SK-Mel-28 cells were treated with or without 20 µM DOX for 0.5 to 30 h at 37°C, and IRP-RNA-binding activity was assessed by the gel retardation assay. B, densitometric analysis of A. Results are a representative experiment of four performed.

Cytoplasmic Aconitase Activity Determination. Cytoplasmic aconitase activity was measured using well established procedures (Drapier and Hibbs, 1996). Briefly, cells were treated with the detergent digitonin, which selectively permeabilizes the plasma membrane and leaves the inner mitochondrial membrane intact (Drapier and Hibbs, 1996). Samples were spun at 1,800g for 8 min at 4°C. The supernatant was then ultracentrifuged at 230,000g for 20 min at 4°C to remove mitochondria. The cytosolic supernatant was subsequently desalted and concentrated using an Amicon concentrator (M_r cutoff = 30,000; Millipore Corporation, Bedford, MA) so that the protein concentration was 3 to 4 mg/ml. The c-acon activity of extracts was determined spectrophotometrically by measuring the disappearance of cis-acon at 240 nm as described previously (Drapier and Hibbs, 1996; Wardrop et al., 2000). Units represent nanomoles of substrate consumed per minute at 37°C (_{240 nm} = 3.6 mM¹ cm¹) (Drapier and Hibbs, 1996; Wardrop et al., 2000).

Western Blot Analysis. Western blot analysis was performed essentially as described previously (Gao and Richardson, 2001). Briefly, for cytoplasmic extracts, cells were collected and incubated on ice for 20 min with the lysis buffer [20 mM HEPES, pH 7.6, 20% glycerol, 10 mM NaCl, 1.5 mM MgCl₂, 0.2 mM EDTA, 0.1% Triton X-100, 1 mM dithiothreitol, 10 mM NaCl, and Complete protease inhibitors (Roche Diagnostics, Mannheim, Germany)] and centrifuged at 14,000 rpm/45 min at 4°C. The protein concentrations of lysates were assessed by the Bio-Rad protein assay kit (Bio-Rad, Hercules, CA).

Statistical Analysis. Experimental data were compared using Student's t test. Results were considered statistically significant when p < 0.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

Effect of Anthracyclines on IRP-RNA-Binding Activity of Neoplastic Cells as a Function of Incubation Time and Concentration. The cardiotoxic effects of anthracyclines have been linked to their ability to perturb cellular Fe metabolism (Garnier-Suillerot, 1988; Gianni and Myers, 1992). Considering the ability of anthracyclines to bind Fe (Garnier-Suillerot, 1988; Gianni and Myers, 1992), a potential target for these cytotoxic agents may be the IRPs that are crucial in controlling cellular Fe metabolism (Hentze and Kühn, 1996). Therefore, the effect of DOX on IRP-RNA-binding activity in human SK-Mel-28 melanoma cells was examined after incubation times of 0.5 to 30 h (Fig. 2). This cell type was assessed initially because its Fe metabolism has been well characterized (Richardson and Baker, 1992a,b), and the proliferation of these cells was sensitive to anthracyclines (data not shown). In the current investigation, the gel retardation assay was used to examine IRP-RNA binding. The active IRP-RNA-binding activity represents the proportion of IRP in the high affinity form that can spontaneously bind mRNA. Total IRP-RNA binding represents all cellular IRP present (both high and low affinity forms) that can be activated to bind mRNA after incubation with -ME and FeCN treatment (Hentze and Kühn, 1996) (see Materials and Methods).

View larger version (32K): [in this window] [in a new window]   Fig. 3.   The anthracyclines, DOX, DAU, and EPI, all affect IRP-RNA-binding in a similar manner in neoplastic cells after a 6- or 24-h incubation. A, SK-Mel-28 cells were incubated with control media or the anthracyclines (20 µM) for 6 h at 37°C, and IRP-RNA-binding activity was assessed. B, densitometric analysis of the results in A. C, SK-Mel-28 cells were incubated with either control media, DFO (100 µM), FAC (100 µg/ml), or anthracycline (0.5-20 µM) for 24 h at 37°C, and the IRP-RNA-binding activity was assessed. D, densitometric analyses of the results in C. Results are a representative experiment of three performed.

Effect of Anthracyclines on IRP-RNA-Binding Activity of Cardiomyocytes as a Function of Concentration and Incubation Time. Rodent IRP1 and IRP2 migrate at different rates in native polyacrylamide gels (Chitambar and Wereley, 1995), and in rat cardiomyocytes, the mRNA-binding activity of both proteins was observed (Fig. 4). Considering the results above in tumor cells (Figs. 2 and 3), the effects of DOX (1-20 µM) were assessed in cardiomyocytes after a 6-h (Fig. 4, A and B) and 24-h (Fig. 4, C and D) incubation in comparison with DFO (100 µM).

View larger version (32K): [in this window] [in a new window]   Fig. 4.   Effect of DOX on cellular IRP-RNA-binding activity after a 6- or 24-h incubation with cardiomyocytes. Primary cultures of cardiomyocytes were treated with or without 1 to 20 µM DOX or DFO (100 µM) for 6 h (A) or 24 h (C) at 37°C, and the IRP-RNA-binding activity was assessed by the gel retardation assay. B and D are densitometric analyses of A and C, respectively. Results are a representative experiment of four performed.

Role of Free Radicals in Anthracycline-Mediated Alterations in IRP-RNA-Binding Activity. Numerous studies have implicated a role for free radical production in anthracycline-mediated cardiotoxicity (Minotti et al., 1998; Konorev et al., 1999; Kotamraju et al., 2002). To determine whether free radicals mediate anthracycline-induced changes in IRP-RNA-binding activity in tumor cells, SK-Mel-28 cells were treated with or without DOX in the presence or absence of a range of free radical scavengers for 6 or 24 h (Fig. 5, A and B). Free radical scavengers examined included the cell-impermeable agents, SOD (1000 U/ml) and catalase (1000 U/ml), the cell-permeable SOD mimetic, MnTBAP (200 µM), and the cell-permeable glutathione peroxidase mimetic, ebselen (15 µM) (Konorev et al., 1999). The concentrations of free radical scavengers examined were within the effective range used in the literature (Minotti et al., 1998; Konorev et al., 1999; Kotamraju et al., 2002). None of the radical scavengers had any significant effect on IRP-RNA-binding activity in SK-Mel-28 cells after a 6-h (Fig. 5A) or 24-h incubation (Fig. 5B) in the presence or absence of DOX. Furthermore, whereas MnTBAP markedly protected tumor cells against DOX-induced cytotoxicity after 24 h (data not shown), total IRP-RNA-binding activity remained depressed (Fig. 5B). These results suggested that the effect of DOX on IRP-RNA-binding activity after 6 or 24 h was not mediated by free radical production.

View larger version (33K): [in this window] [in a new window]   Fig. 5.   Free radical scavengers do not protect cells against the DOX-induced changes in IRP-RNA-binding activity. SK-Mel-28 cells were treated simultaneously with control media or 20 µM DOX and the free radical scavengers, catalase (1000 U/ml), SOD (1000 U/ml), ebselen (15 µM), MnTBAP (200 µM), or the combination of ebselen and MnTBAP, for 6 h (A) or 24 h (B) at 37°C; and IRP-RNA-binding activity was assessed. These results are a representative experiment of three performed.

The Effect of Doxorubicinol and Cis-Aconitate on IRP-RNA-Binding and Cytoplasmic Aconitase Activity. Anthracyclines are metabolized in vivo to form a number of intermediates that have been shown to possess important biological effects (Takanashi and Bachur, 1976; Minotti et al., 1995, 1998). It was therefore important to further examine which intracellular metabolite was directly affecting IRP-RNA-binding activity. To investigate this, cell lysates were prepared and treated directly with the metabolites of interest, and the IRP-RNA-binding activity was assessed (see Materials and Methods).

View larger version (42K): [in this window] [in a new window]   Fig. 6.   Doxorubicinol (DOXol) and cis-aconitate have little effect on IRP-RNA-binding activity in Fe-loaded lysates from SK-Mel-28 melanoma cells. Lysates from Fe-loaded cells were prepared as described under Materials and Methods and then incubated for 1 h at 37°C with DOXol (1.6, 5, and 20 µM), cis-acon (20 or 40 µM), or combinations of both, and IRP-RNA-binding activity was assessed. These results are a representative experiment of three performed.

The Effect of Anthracyclines and Their Metal Ion Complexes on IRP-RNA-Binding Activity and Cytoplasmic Aconitase Activity. Because anthracyclines are capable of readily forming Fe and Cu complexes (Garnier-Suillerot, 1988; Gianni and Myers, 1992), and because both metal ions are found at relatively high levels in cells, it was important to determine the effects of anthracycline-Fe or -Cu complexes directly on IRP-RNA-binding activity (Fig. 7). Indeed, DOX-metal ion complexes can catalyze a range of redox reactions (Gianni and Myers, 1992), and their effects were important to assess. In all studies, the complexation of anthracyclines with metal ions was ensured by examination using UV-visible spectrophotometry (Garnier-Suillerot, 1988; Gianni and Myers, 1992).

View larger version (44K): [in this window] [in a new window]   Fig. 7.   The Fe and Cu complexes of anthracyclines cause a reversible decrease in active IRP-RNA-binding activity. A, SK-Mel-28 cells were treated with control media, DFO (100 µM), or FAC (100 µg/ml) for 24 h at 37°C, and cell lysates were prepared (see Materials and Methods). Lysates were then treated with NEM (1 mM), anthracycline (20 µM), anthracycline-Fe (3:1) complex, or anthracycline-Cu (2:1) complex (metal complexes at 20 µM anthracycline equivalent) for 3 h at 4°C, and IRP-RNA-binding activity was assessed. B, lysates prepared from control SK-Mel-28 cells were treated with NEM (1 mM), the anthracyclines, DOX (20 µM), DAU (20 µM), and EPI (20 µM), and the respective anthracycline-Fe complexes (at 20 µM anthracycline equivalent as in A) for 3 h at 4°C; and IRP-RNA-binding activity was assessed. Results are a representative experiment of three performed. C, densitometric analysis of B.

Doxorubicin Prevents the Chelator-Mediated Increase in IRP-RNA-Binding Activity. Cellular Fe sequestration by DFO and ICRF-187 results in activation of IRP-RNA-binding activity (Weiss et al., 1997). Having demonstrated that anthracyclines decrease total IRP-RNA-binding activity in SK-Mel-28 cells after 24 h (Figs. 2; 3, C and D; and 5B), we examined the ability of melanoma cells to respond to changes in Fe status at the same time as DOX treatment (Fig. 8). The SK-Mel-28 cells were simultaneously incubated with DOX (5 or 10 µM) and either DFO (6.25-25 µM) or ICRF-187 (300 or 1000 µM) for 24 h, and the IRP-RNA-binding activity assessed (Fig. 8).

View larger version (45K): [in this window] [in a new window]   Fig. 8.   Cells can no longer respond to cellular Fe deficiency by increasing active IRP-RNA-binding activity after treatment with DOX. A, SK-Mel-28 cells were treated with control media, DFO (6.25-25 µM), DOX (5 and 10 µM), ICRF-187 (300 and 1000 µM), and DFO or ICRF-187 plus DOX for 24 h at 37°C; and IRP-RNA-binding activity was assessed. (B) Densitometric analyses of A. Results are a representative experiment of three performed.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The current investigation is the first to assess the effects of anthracyclines on IRP-RNA-binding activity in both tumor cells and differentiated cardiomyocytes. We show using these cells that anthracyclines interfere with Fe metabolism by affecting both the active mRNA-binding activity of IRP and the total cellular IRP pool (Fig. 2). Short-term incubation with DOX (3-6 h) resulted in a dramatic decrease in IRP-RNA-binding activity, with a less pronounced effect on total IRP pools (Fig. 2). This may be due to the intracellular formation of anthracycline-Fe and/or -Cu complexes. Interestingly, DOX had a similar effect on IRP-RNA-binding activity in cardiomyocytes and neoplastic cells. A notable effect of DOX in cardiomyocytes was the decrease in IRP2-RNA-binding activity, particularly after a 6-h incubation (Fig. 4).

Because DOX forms a number of metabolites that have been shown to be important in its biological effects (Takanashi and Bachur, 1976; Minotti et al., 1998), these were assessed to understand their roles in IRP-RNA-binding activity. Our experiments in cell lysates demonstrated that DOX alone or DOXol in the presence or absence of cis-acon had no influence on IRP-RNA-binding (Fig. 6) or c-acon activity. These results are important because they demonstrate that DOX and one of its critical metabolites are not directly affecting IRP-RNA-binding activity and cellular Fe metabolism. In contrast, the Fe and Cu complexes of DOX reversibly reduced active IRP-RNA-binding activity in cell lysates (Fig. 7), suggesting that these metal complexes may be responsible for the short-term cellular effects observed after a 6-h incubation (Fig. 2). The effect of the DOX-Fe complex on IRP-RNA-binding activity rather than DOX itself is interesting (Fig. 7). This is because previous studies have shown that loading cells with Fe potentiates the cytotoxic effects of DOX (Hershko et al., 1993; Link et al., 1996).

It is important to consider the mechanism of how the DOX-Fe complex reduces IRP-RNA-binding activity (Fig. 7). It is clear from our IRP-RNA-binding studies (Fig. 7A) and the lack of effect of the DOX-Fe complex on c-acon activity, that the effect of this compound was independent of the [4Fe-4S] cluster. The DOX-Fe complex can catalyze a range of redox reactions (Gianni and Myers, 1992). For instance, it reacts with a range of reductants including glutathione to yield oxidized thiols (Muindi et al., 1984; Gianni and Myers, 1992). Therefore, the DOX-Fe complex could oxidize critical sulfhydryl groups involved in the IRP-mRNA-binding mechanism (Hentze and Kühn, 1996). In fact, DOX may reversibly inhibit IRP-RNA-binding activity via a similar mechanism to the sulfhydryl-oxidizing agent, diamide (Philpott et al., 1993; Hirling et al., 1994). It has been shown that diamide catalyzes disulfide bridging between crucial thiol groups in IRP1, namely, the cysteine-437 residue with either cysteine-503 or -506, inhibiting mRNA-binding activity (Hirling et al., 1994). Similar to the DOX-Fe complex (Fig. 7), inhibition by diamide could be recovered by treatment with -ME that reduces disulfide bonds (Philpott et al., 1993; Hirling et al., 1994). Considering that DOX can bind cellular Fe pools to form the DOX-Fe complex (Gianni and Myers, 1992), this molecule may then oxidize crucial IRP thiol groups and thereby inhibit mRNA binding. Hence, this reaction could explain the decrease in active IRP-RNA-binding activity observed within the first 6 h of incubation with DOX (Fig. 2).

Apart from the ability of the DOX-Fe complex to decrease IRP-RNA-binding activity, similar activity was also found for the DOX-Cu complex (Fig. 7). The mechanism of this effect could be similar to that of the DOX-Fe complex discussed above. Alternatively, it has been suggested that non-Fe metals can be incorporated into the Fe-S cluster to decrease IRP1-RNA-binding and increase c-acon activity (Oshiro et al., 2002). Hence, it is possible to speculate that the DOX-Cu complex and DOX-Fe complex may be able to directly donate their metal ions to IRP1 for cluster assembly and thus decrease RNA-binding affinity. However, we found no change in c-acon activity after incubation with DOX-Fe or DOX-Cu, which argues against this hypothesis.

Another reaction of the DOX-Fe complex is the generation of free radicals that could affect IRP-RNA-binding activity (Gianni and Myers, 1992). However, a range of radical scavengers could not protect against the DOX-mediated decrease in active IRP-RNA-binding activity (Fig. 5A). Furthermore, the SOD mimetic, MnTBAP, provided protection against toxicity in melanoma cells without preventing the DOX-mediated decrease in the total IRP pool after 24 h (Fig. 5B). These results again demonstrate that free radical production by DOX or its Fe complex was not the mechanism responsible for the changes in IRP mRNA-binding activity. These observations in neoplastic cells are again different from that observed in a heart cell line, where the effect of DOXol and free radicals irreversibly inactivated both IRP1 and 2, but only at a DOX concentration of 10 µM (Minotti et al., 2001).

The effect of the anthracyclines on active and total IRP-RNA-binding activity as a function of time was complex (Fig. 2). We have suggested above that the initial decrease observed within 6 h (Fig. 2) may be due to the effect of the Fe or Cu complex of DOX (Fig. 7). However, after this initial response, active IRP-RNA-binding activity then increased back to control levels. The mechanism by which this restoration occurs is not clear, although it may be due to a compensatory response that involves conversion of holo-IRP1 to its active RNA-binding form. The reason for the decrease in the total IRP pool after longer incubations (18-24 h) with anthracyclines (Fig. 2) is also not understood at present, although we showed that it was not a free radical-mediated effect (Fig. 5).

Simultaneous treatment of cells with DOX and the Fe chelators, DFO and ICRF-187, did not prevent the decrease in total IRP-RNA-binding activity induced by DOX after 24 h (Fig. 8). In addition, the presence of DOX inhibited the DFO- and ICRF-187-mediated increase in IRP-RNA-binding activity (Fig. 8). This result indicates that in the presence of DOX, cells can no longer respond to changes in Fe status via the IRP-regulatory system.

Our current data are clearly different from those reported by others using heart homogenates (Minotti et al., 1995, 1998) and a heart cell line (Minotti et al., 2001). These studies suggested a role for DOXol in removing the [4Fe-4S] cluster from IRP1, thereby affecting cellular Fe metabolism (Minotti et al., 1995, 1998; 2001). These observations are not in agreement with our results in which the formation of Fe or Cu complexes with DOX, rather than DOXol itself, affects IRP1-RNA-binding activity. Furthermore, the effect of DOX in our primary cultures of cardiomyocytes (Fig. 4) was somewhat similar to the changes observed in tumor cells (Figs. 2 and 3). The different response of cardiomyocytes to DOX shown in this investigation compared with studies using a rat heart-derived cell line (Minotti et al., 2001) could be due to the dedifferentiated state of this latter cell type (Hescheler et al., 1991). Also, it is difficult to compare our studies using intact cells or cell lysates to previous work using heart tissue homogenates in which IRP1 was loaded with Fe by chemical means (Minotti et al., 1995, 1998).

The effects of DOX on IRP-RNA-binding activity observed in this study using neoplastic cells and myocardial cells may facilitate the design of anthracyclines that exhibit high antitumor activity. For example, in tumor cells, the ability of DOX to inhibit the response of the IRPs to cellular Fe chelation may be important (Fig. 8). Indeed, Fe chelators have been assessed for their potential to treat cancer (Richardson et al., 1995; Richardson and Milnes, 1997), and DFO can markedly enhance the antitumor effect of DOX (Blatt and Huntley, 1989). Therefore, the combination of anthracyclines with Fe chelators that possess potent antiproliferative activity in neoplastic cells (Richardson et al., 1995; Richardson and Milnes, 1997) could be beneficial. Using this regimen, the ability of anthracyclines to effectively prevent the response to Fe chelation via the IRP system would inhibit the subsequent compensatory increase in TfR levels and Fe uptake. Hence, this would potentiate the antitumor activity observed and may partly explain the greater activity of combining chelators and DOX than either agent alone (Blatt and Huntley, 1989). Furthermore, by screening a variety of anthracycline analogs in combination with Fe chelators and assessing their effects on antiproliferative activity and indices of Fe metabolism (e.g., IRP-RNA-binding activity, etc.), effective treatment regimens may be developed.

In summary, our experiments demonstrated for the first time that anthracyclines interfere with the Fe metabolism of tumor cells and myocardial cells by affecting both the active IRP-RNA-binding activity and the total cellular IRP pool. Furthermore, both the Fe and Cu complexes of anthracyclines could reversibly decrease active IRP-RNA-binding activity, and the intracellular formation of these complexes could be responsible for the initial response to these drugs. The fact that DOX inhibited the response of the IRP system to Fe chelation may be important in terms of designing novel antitumor therapies combining anthracyclines with potent Fe chelators. Our results add significant new information regarding the effects of anthracyclines on the regulation of cellular Fe metabolism by IRPs.