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Examination of the Mechanism(s) Involved in Doxorubicin-Mediated Iron Accumulation in Ferritin: Studies Using Metabolic Inhibitors, Protein Synthesis Inhibitors, and Lysosomotropic Agents

Children's Cancer Institute Australia for Medical Research, Iron Metabolism and Chelation Program, Randwick, Sydney, New South Wales, Australia

Received June 23, 2003; accepted October 1, 2003

	Abstract

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Anthracyclines are potent anticancer agents, but their use is limited by cardiotoxicity at high cumulative doses. The mechanisms involved in anthracycline-mediated cardiotoxicity are still poorly understood, but numerous investigations have indicated a role for iron in this process. Our previous studies using neoplastic and myocardial cells showed that anthracyclines inhibit iron mobilization from the iron storage protein, ferritin, resulting in marked accumulation of ferritin-iron. Although the process of ferritin-iron mobilization is little understood, catabolism of ferritin by lysosomes may be a likely mechanism. Because anthracyclines have been shown to accumulate in lysosomes, this latter organelle may be a potential target for these drugs. The present study demonstrated, using native polyacrylamide gel electrophoresis-⁵⁹Fe autoradiography, that ferritin-⁵⁹Fe mobilization is an energy-dependent process that also requires protein synthesis. Depression of lysosomal activity via the enzyme inhibitors E64d [(2S,3S)-trans-epoxysuccinyl-L-leucylamido-2-methylbutane ethyl ester] and leupeptin or the lysosomotropic agents ammonium chloride, chloroquine, and methylamine resulted in a 3- to 5-fold increase in ⁵⁹Feferritin accumulation compared with control cells. In addition, the proteasome inhibitors N-benzoyloxycarbonyl (Z)-Leu-Leuleucinal (MG132) and lactacystin also significantly increased ⁵⁹Fe-ferritin levels compared with control cells. These effects of lysosomotropic agents or inhibitors of lysosomal activity were comparable with that observed with the anthracycline doxorubicin. Collectively, our study indicates a role for lysosomes and proteasomes in ferritin-iron mobilization, and this pathway is dependent on metabolic energy and protein synthesis. Furthermore, the lysosome/proteasome pathway may be a novel anthracycline target, inhibiting iron mobilization from ferritin that is essential for vital iron-requiring processes such as DNA synthesis.

Under physiological conditions, iron is delivered to cells via the binding of transferrin (Tf) to the TfR1 located on the cell membrane. The Tf-TfR1 complex is internalized into the cell via receptor-mediated endocytosis (Kwok and Richardson, 2002b). A decrease in endosomal pH releases the iron from Tf. The iron is then transported across the membrane via the divalent metal ion transporter 1 and enters the intracellular labile iron pool (Kwok and Richardson, 2002b). From this latter compartment, iron can be incorporated into heme and non–heme iron-containing proteins or stored in the protein ferritin (Kwok and Richardson, 2002b). Ferritin consists of 24 subunits of two types, namely heavy and light chains. These subunits are symmetrically organized, generating a cavity within the protein for iron storage (Harrison and Arosio, 1996).

We previously demonstrated that after iron uptake into cells, iron is initially incorporated into ferritin, followed by iron mobilization from ferritin and its redistribution to other cellular compartments (Kwok and Richardson, 2003). This supports the theory that ferritin is not merely an iron-storage molecule, but it can act as an intermediate in iron transport (Speyer and Fielding, 1979). Currently, little is understood regarding the physiological mechanisms of ferritin-iron mobilization. In vitro studies demonstrated that biological reductants including reduced flavins (Funk et al., 1985), superoxide (Bolann and Ulvik, 1987), and ascorbate (Bienfait and Van der Briel, 1980), can promote iron release from ferritin. Studies using isolated ferritin showed that substitution of conserved amino acid residues in the 3-fold axes enhances iron release from ferritin (Jin et al., 2001). In addition, structural changes in ferritin may open channels to facilitate iron mobilization (Funk et al., 1985). However, the physiological significance of these experiments on purified ferritin is uncertain. Others have suggested that ferritin-iron release necessitates the degradation of ferritin within lysosomes (Roberts and Bomford, 1988; Radisky and Kaplan, 1998; Persson et al., 2001). Lysosomes are acidic organelles that contain hydrolases for the degradation and turnover of intracellular molecules (Pillay et al., 2002). Indeed, studies using cultured cells have demonstrated lysosomal degradation of ferritin (Hernandez-Yago et al., 1980; Radisky and Kaplan, 1998).

We showed previously in neoplastic and myocardial cells that anthracyclines inhibit iron mobilization from ferritin, resulting in marked accumulation of ferritin-iron (Kwok and Richardson, 2003). This consequently inhibited iron redistribution from ferritin to other vital iron-containing pathways. Interestingly, anthracyclines can localize in lysosomes (Hurwitz et al., 1997) and disrupt lysosomal morphology and enzyme activity (Singal et al., 1985). However, the possibility that lysosomes are cellular targets of anthracyclines, resulting in the inhibition of lysosome-mediated ferritin degradation and the redistribution of iron to vital cellular processes, has not been investigated.

In the present study we showed that inhibition of energy generation or protein synthesis inhibits the ferritin-iron mobilization pathway. Importantly, a range of lysosomal enzyme inhibitors, lysosomotropic agents, and proteasomal inhibitors resulted in marked ferritin-iron accumulation that was comparable with that found using the anthracycline doxorubicin (DOX). These data provide further evidence of a mechanism involving lysosomes in the degradation of ferritin that is required for the release and redistribution of metabolically useful iron. The inhibition of iron redistribution to other compartments may have detrimental effects on vital iron-dependent processes, and it is possible that the lysosomal degradation pathway represents a potential target for anthracycline cytotoxicity.

	Materials and Methods

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Cell Treatments and Reagents. Chloroquine, cycloheximide, E64d, horse spleen ferritin, lactacystin, leupeptin, methylamine, MG132, pepstatin A, puromycin, rotenone, sodium azide, and sodium cyanide were obtained from Sigma Chemical Co. (St. Louis, MO). DOX was obtained from Pharmacia (Sydney, Australia). The noncytotoxic DOX aglycone, doxorubicinone, was kindly provided by Drs. B. Binaschi and M. Berettoni (Menarini Ricerche S.p.A., Pomerzia, Italy). All other chemicals were of analytical reagent quality.

Cell Culture. Human SK-Mel-28 melanoma cells were obtained from the American Type Culture Collection (Manassas, VA). Briefly, cells were grown in Eagle's modified minimum essential medium (MEM) (Invitrogen, Sydney, Australia) containing 10% fetal calf serum (Invitrogen), 1% nonessential amino acids (Invitrogen), 100 µg/ml streptomycin (Invitrogen), 100 U/ml penicillin (Invitrogen), and 0.28 µg/ml Fungizone (Squibb Pharmaceuticals, Montréal, Canada). Cells were cultured in an incubator (Thermo Forma, Marietta, OH) at 37°C in a humidified atmosphere of 5% CO₂/95% air and subcultured as described previously (Richardson and Baker, 1990). Cellular growth and viability were assessed by phase-contrast microscopy and Trypan blue staining. The SK-Mel-28 cell type was used as the experimental model to study ferritin-iron accumulation because our previous investigation demonstrated that the same effect was observed with cardiomyocytes and a variety of tumor cell lines (Kwok and Richardson, 2003).

Preparation of ⁵⁹Fe-Transferrin. Human apotransferrin (Sigma) was labeled with ⁵⁹Fe (PerkinElmer Life and Analytical Sciences, Boston, MA) to produce ⁵⁹Fe₂-transferrin (⁵⁹Fe-Tf), as described previously (Richardson and Baker, 1990). Unbound ⁵⁹Fe was removed by vacuum dialysis against 0.15 M NaCl with 1.4% NaHCO₃, pH 7.4. Fully saturated diferric Tf was used in all experiments.

Determination of Intracellular Iron Distribution Using Native PAGE-⁵⁹Fe Autoradiography. Native PAGE-⁵⁹Fe autoradiography was performed using established techniques (Richardson and Milnes, 1997). Briefly, cells were labeled with ⁵⁹Fe-Tf ([protein] = 0.75 µM) and then lysed using 55 µl of ice-cold 1.5% Triton X-100 containing 2 mM phenylmethylsulfonyl fluoride (Sigma) followed by one freeze-thaw cycle. Samples were vortexed and centrifuged at 14,000 rpm for 45 min at 4°C to separate the stromal-mitochondrial membrane (SMM) fraction from the cytosolic fraction. Under these conditions, SMM contains the disrupted plasma and nuclear membranes, intracellular membranes, and a variety of organelles including mitochondria and lysosomes (Rickwood and Patel, 1995).

The SMM pellets were further disrupted to examine ⁵⁹Fe distribution in this fraction (Richardson and Milnes, 1997). The pellets were vigorously vortexed in the presence of 40 µl of ice-cold 1.5% Triton X-100 containing freshly prepared 2 mM phenylmethylsulfonyl fluoride and glass powder (Richardson and Milnes, 1997). Samples were then centrifuged at 14,000 rpm for 45 min at 4°C, and the supernatant was collected.

Cytosolic fractions and/or SMM fractions were loaded onto a 5% native PAGE gel to give equal counts of radioactive ⁵⁹Fe across all samples. Therefore, the autoradiograph compares the relative intracellular distribution of ⁵⁹Fe within each fraction, rather than the amount of ⁵⁹Fe/µg protein. Radioactive counts were determined using a -scintillation counter (Amersham Biosciences, Uppsala, Sweden). Similar experiments were also performed loading equal amounts of protein (100 µg) across all samples. Protein concentrations were determined using a protein assay (Bio-Rad, Hercules, CA). Experiments loading equal radioactive counts gave results similar to those obtained using equal protein loading. Electrophoresis was performed at 15 mA/gel for 2 to 3 h at 4°C. Gels were subsequently dried at 80°C, and autoradiography was performed. Bands on X-ray film were quantified by scanning densitometry using a Laser Densitometer and analyzed by Kodak Biomax I Software (Eastman Kodak, Rochester, NY).

[³H]Leucine Incorporation Assay. To assess cellular protein synthesis, [³H]leucine assays were performed. Cells were seeded onto 96-well plates at 15,000 cells/well and incubated overnight. Cells were then incubated with the agents of interest for 24 h at 37°C, followed by incubation with [³H]leucine (0.67 µCi) for 2 h at 37°C (Amersham Biosciences UK, Ltd., Little Chalfont, Buckinghamshire, UK). Cells were harvested using a MicroCell Harvester (Molecular Devices Skatron, Lier, Norway) onto filter mats, and radioactivity was measured on a scintillation counter (1205 Beta-plate liquid scintillation counter; PerkinElmer Wallac, Turku, Finland). Protein synthesis was determined by the incorporation of [³H]leucine into cells using standard techniques (Richardson and Milnes, 1997).

Lysosomal Enzyme Activity Assay. To determine the activity of a range of lysosomal enzymes, cells were incubated with control media or DOX (5 µM) for 24 h at 37°C. The cell pellets were then lysed by freeze-thawing, and protein concentration was determined using the Lowry method. Enzyme activity was assessed using fluorogenic 4-methyl umbelliferyl substrates at 37°C according to standard clinical procedures (Giljaard, 1980) used at the Department of Chemical Pathology, Women's and Children's Hospital (Adelaide, Australia). Results are expressed as nanomoles of substrate cleaved per minute per milligram of protein.

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

	Results

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Cellular ⁵⁹Fe Uptake and Intracellular ⁵⁹Fe Distribution into the Cytosol and Stromal Mitochondrial Membrane Fraction in the Presence and Absence of DOX. We showed previously that incubation of tumor cells and primary cardiomyocyte cultures with DOX resulted in a marked accumulation of ferritin-⁵⁹Fe compared with control cells (Kwok and Richardson, 2003). These experiments were confined to examining the cytosol, and there was no assessment of ⁵⁹Fe incorporation in the membrane fraction. It is possible that ⁵⁹Fe accumulating in ferritin in the presence of DOX was derived from the SMM fraction, a hypothesis that was not examined in our previous investigation (Kwok and Richardson, 2003). Therefore, initially in the present study, we examined the effect of DOX on the intracellular distribution of ⁵⁹Fe in the cytosolic and SMM fractions. This latter fraction contains disrupted plasma and nuclear membranes, intracellular membranes, and a variety of organelles, including mitochondria and lysosomes (Rickwood and Patel, 1995). These experiments were critical in terms of assessing the mechanism(s) involved in the DOX-mediated ferritin-⁵⁹Fe accumulation. Examination of ⁵⁹Fe distribution in the SMM was particularly important considering the role of the mitochondrion and lysosome in iron metabolism (Roberts and Bomford, 1988; Link et al., 1996; Radisky and Kaplan, 1998; Persson et al., 2001) and their potential to be affected by anthracyclines (Singal et al., 1985; Link et al., 1996).

In these experiments, SK-Mel-28 melanoma cells were used to assess the effects of DOX (20 µM) compared with control medium on ⁵⁹Fe uptake from ⁵⁹Fe-Tf (0.75 µM). Quantitative ⁵⁹Fe uptake from ⁵⁹Fe-Tf in control cells was linear as a function of incubation time (r = 0.99) (Fig. 1A), as observed in previous studies using this cell type (Richardson and Baker, 1990). Cells incubated with DOX (20 µM) also demonstrated linear (r = 0.97) ⁵⁹Fe uptake from ⁵⁹Fe-Tf as a function of incubation time (Fig. 1A). Furthermore, DOX (20 µM) had no significant effect on the rate of ⁵⁹Fe uptake compared with the control (Fig. 1A). Examining the amounts of intracellular ⁵⁹Fe between the cytosol and SMM fractions, it was evident that most ⁵⁹Fe was found in the cytosol for both control and DOX-treated cells (Fig. 1B). After 18 and 24 h, significantly (p < 0.03) more ⁵⁹Fe was detected in the cytosol of DOX-treated cells compared with cells incubated with control medium (Fig. 1B). Conversely, significantly (p < 0.04) less ⁵⁹Fe was found in the SMM fraction in DOX-treated cells compared with control cells, particularly after 24 h (Fig. 1B). These results suggested that for time points beyond 18 h, DOX increased the ratio of ⁵⁹Fe between the cytosol and SMM during ⁵⁹Fe uptake from ⁵⁹Fe-Tf compared with control cells.

View larger version (16K): [in this window] [in a new window]   Fig. 1. The effect of DOX on total cellular 59Fe (A) and the intracellular distribution of 59Fe (B) between the cytosol and SMM fractions during incubation of SK-Mel-28 melanoma cells with 59Fe-Tf. The SK-Mel-28 melanoma cells were labeled with 59Fe-Tf (0.75 µM) in the presence or absence of DOX (20 µM) for 3 to 24 h at 37°C and washed, and the intracellular distribution of 59Fe was determined in the cytosol and SMM (as described under Materials and Methods). Results shown are mean ± S.D. of three separate experiments performed.

The cellular distribution of ⁵⁹Fe within the cytosol (Fig. 2A) and SMM (Fig. 2C) was then assessed using the native PAGE-⁵⁹Fe autoradiography technique (Fig. 2, A and C). In these studies, equal amounts of radioactivity were added to each lane of the gel, and the distribution of ⁵⁹Fe between intracellular molecules was assessed. As described in Fig. 1B, the quantitative amount of ⁵⁹Fe in the cytosol was greater than that found in the SMM. Considering this, it should be noted that although the ferritin-⁵⁹Fe bands in Fig. 2, A and C, are of similar intensity, this is because of the different exposure periods used to optimize detection of ⁵⁹Fe-containing components.

View larger version (41K): [in this window] [in a new window]   Fig. 2. Doxorubicin results in marked accumulation of 59Fe in cytosolic and SMM ferritin of SK-Mel-28 melanoma cells during 59Fe uptake from 59Fe-Tf. SK-Mel-28 cells were incubated with 59Fe-Tf (0.75 µM) for 3 to 24 h at 37°C in the presence or absence of DOX (20 µM). Native PAGE-59Fe autoradiography was performed on the cytosolic fraction (A) and the SMM fraction (C) as described under Materials and Methods. B and D are densitometric analyses of A and C, respectively. Results shown are a typical experiment of three performed.

The distribution of cytosolic ⁵⁹Fe in Figs. 2A and 3A has been reported previously (Kwok and Richardson, 2003), but it was important to include in this study so as to compare the cytosolic and SMM fractions. It was evident that ⁵⁹Fe distribution was similar in both the cytosol and SMM fractions (Fig. 2, A and C). A large proportion of ⁵⁹Fe in control and DOX-treated cells was found in a band that comigrated with horse spleen ferritin (Fig. 2, A and C) and could be super-shifted with an antiferritin antibody, further confirming its identity (Kwok and Richardson, 2003). Several bands above ferritin and a low molecular mass band were found in both the cytosol and SMM (Fig. 2, A and C). As discussed previously (Kwok and Richardson, 2003), the exact molecular identity of these bands above and below ferritin remain unclear and were not the focus of this investigation.

View larger version (16K): [in this window] [in a new window]   Fig. 3. The effect of a pulse-chase incubation with DOX or control media on total cellular 59Fe (A) and the redistribution of 59Fe (B) between the cytosol and SMM fractions in SK-Mel-28 melanoma cells prelabeled with 59Fe-Tf. The SK-Mel-28 cells were prelabeled with 59Fe-Tf (0.75 µM) for 3 h at 37°C and washed. This was followed by a 0- to 24-h chase period at 37°C in control media or media containing DOX (20 µM). Results shown are mean ± S.D. of three separate experiments performed.

In both the cytosol and SMM of control cells, ⁵⁹Fe incorporation in ferritin increased up to 6 h and thereafter decreased to levels observed at the 3-h time point (Fig. 2, A and C, compare lanes 1, 3, 5, and 7; Fig. 2, B and D). In contrast, DOX resulted in marked accumulation of ⁵⁹Fe in ferritin compared with the control, particularly for incubations of 6 h or more (Fig. 2, A and C, compare lanes 2, 4, 6, and 8; Fig. 2, B and D). Indeed, we have shown previously that after an 18- or 24-h label with ⁵⁹Fe-Tf in the presence of DOX, ⁵⁹Feferritin levels were significantly (p < 0.004) higher compared with control cells (Kwok and Richardson, 2003). The ⁵⁹Fe-containing bands above ferritin in the SMM were more pronounced in control cells than in those incubated with DOX after incubations of 18 or 24 h (Fig. 2C; compare lanes 5 and 7 with lanes 6 and 8). This suggested that in contrast to control cells, DOX resulted in the majority of ⁵⁹Fe being incorporated into ferritin with very little being distributed to other compartments. The presence of ferritin in the SMM is of interest, because although ferritin is a cytosolic protein, there are reports of ferritin associated with lysosomes, where it could be catabolized (Roberts and Bomford, 1988; Radisky and Kaplan, 1998; Persson et al., 2001). Alternatively, recently a mitochondrial ferritin has been discovered that is believed to play a role in iron metabolism (Corsi et al., 2002). Therefore, we cannot discount that at least some of the ⁵⁹Fe detected in the SMM may be associated with this latter molecule.

Cellular ⁵⁹Fe Redistribution into the Cytosol and Stromal Mitochondrial Membrane Fraction in the Presence and Absence of DOX. Our previous study demonstrated that DOX inhibits the redistribution of ⁵⁹Fe from cytosolic ferritin, resulting in a marked accumulation within this molecule (Kwok and Richardson, 2003). We investigated using pulse-chase experiments if the cytosolic ferritin-⁵⁹Fe accumulation was caused by the redistribution of ⁵⁹Fe from the SMM to the cytosol. Cells were incubated with ⁵⁹Fe-Tf (0.75 µM) for 3 h at 37°C, washed, and reincubated in control media or media containing DOX (20 µM) for 0 to 24 h.

Quantitative analysis of ⁵⁹Fe levels demonstrated that the total ⁵⁹Fe in both control and DOX-treated cells decreased as a function of reincubation time, with there being no significant difference between the two groups (Fig. 3A). Examination of the cytosol and SMM demonstrated that ⁵⁹Fe levels in these fractions also decreased as a function of incubation time in both control and DOX-treated cells (Fig. 3B). However, there was no significant difference in ⁵⁹Fe levels in the cytosol and SMM between control and DOX-treated cells (Fig. 3B). The decrease in cellular ⁵⁹Fe levels was caused by the efflux of ⁵⁹Fe into the reincubation medium that occurs in the presence of 10% FCS but not in its absence (data not shown; Richardson and Baker, 1990). The reason for this considerable release of ⁵⁹Fe in the presence of serum is unclear but could be related to the presence of the ferroxidase ceruloplasmin that acts to mobilize ⁵⁹Fe from cells (Richardson, 1999).

The distribution of ⁵⁹Fe within the cytosol and SMM was then assessed after the initial 3-h labeling period with ⁵⁹Fe-Tf (Fig. 4, A and C). Reincubation of cells in control media for 3 or 6 h markedly increased the incorporation of ⁵⁹Fe in cytosolic and SMM ferritin (Fig. 4, A and C; compare lane 1 with lanes 2 and 4). However, after a longer chase period of 18 to 24 h in control media, the incorporation of ⁵⁹Fe in ferritin markedly decreased to levels comparable with that observed at the 0-h time point (Fig. 4, A and C; compare lanes 2 and 4 with lanes 6 and 8; Fig. 4, B and D). Notably, in control cells at the 18- and 24-h time points, ⁵⁹Fe could be detected in a range of diffuse higher molecular mass bands in the cytosol and SMM (Fig. 4, A and C, lanes 6 and 8). These results indicate that in control cells, ⁵⁹Fe was initially incorporated into ferritin, but it is later mobilized from this molecule and relocated to other compartments within both the cytosol and SMM. These experiments support our previous study examining cytosolic ⁵⁹Fe distribution (Kwok and Richardson, 2003).

View larger version (35K): [in this window] [in a new window]   Fig. 4. DOX prevents the redistribution of 59Fe from ferritin in the cytosol and SMM of SK-Mel-28 melanoma cells. Cells were prelabeled with 59Fe-Tf (0.75 µM) for 3 h at 37°C and then washed. This was then followed by reincubation in control media or media containing DOX (20 µM) for 0 to 24 h at 37°C. The cytosolic (A) and SMM (C) fractions were separated, and native PAGE-59Fe autoradiography was performed on each fraction (see Materials and Methods). B and D are densitometric analyses of A and C, respectively. The results are a typical experiment from three performed.

When DOX was present during the chase period, ⁵⁹Fe incorporation into ferritin continued to increase with reincubation time, resulting in a marked accumulation of ⁵⁹Feferritin in the cytosol and SMM (Fig. 4, A and C, lanes 3, 5, 7, and 9; Fig. 4, B and D). Indeed, our earlier studies have demonstrated that after an 18- or 24-h reincubation with DOX, a significant (p < 0.0003) accumulation of ⁵⁹Fe-ferritin was observed compared with control cells (Kwok and Richardson, 2003). In contrast to control cells, there was little evidence of ⁵⁹Fe localization in other bands above or below the ferritin band in DOX-treated cells (Fig. 4, A and C; compare lanes 6 and 7, lanes 8 and 9).

Effect of an Anthracycline Aglycone on ⁵⁹Fe Incorporation into Ferritin. We investigated whether the inhibition of iron mobilization from ferritin was a nonspecific cytotoxic response after anthracycline treatment. Because the aminosugar on the anthracycline molecule is believed to be an important structural requirement for the cytotoxic and DNA-binding activity of anthracyclines (Zunino et al., 2001), we examined the less cytotoxic DOX aglycone, doxorubicinone, that lacks the sugar moiety (Zunino et al., 2001). In these experiments, SK-Mel-28 cells were labeled with ⁵⁹Fe-Tf for 3 h, washed, and reincubated in control media, DOX (5 and 10 µM), or doxorubicinone (5 and 10 µM) for 18 h at 37°C.

Incubation of cells with the aglycone doxorubicinone (5 and 10 µM) resulted in no cytotoxicity, as demonstrated by the absence of cell death and morphological changes compared with control cells (data not shown). As observed previously, DOX at both 5 and 10 µM resulted in a marked 5- to 7-fold accumulation of ⁵⁹Fe in ferritin compared with control cells (Fig. 5A; compare lanes 1 and 2, lanes 4 and 5; Fig. 5B). Cells incubated with 5 µM doxorubicinone resulted in a 1.8-fold increase in ⁵⁹Fe-ferritin levels compared with the control (Fig. 5A; compare lanes 1 and 3; Fig. 5B). Higher concentrations of 10 µM doxorubicinone caused a 3-fold increase in ⁵⁹Fe-ferritin compared with control cells (Fig. 5A; compare lanes 3 and 6; Fig. 5B). However, the ability of doxorubicinone to accumulate ⁵⁹Fe in ferritin was less than that observed in DOX-treated cells (Fig. 5A; compare lanes 2 and 3, lanes 5 and 6; Fig. 5B). Hence, even the noncytotoxic aglycone resulted in an accumulation of iron in ferritin, suggesting that the effect of DOX on increasing ferritin iron levels was not caused by its cytotoxic effect. Moreover, the less pronounced effect of the doxorubicin aglycone on inducing ferritin iron accumulation compared with DOX suggests that the aminosugar residue of DOX is an active moiety that contributes to this effect.

View larger version (30K): [in this window] [in a new window]   Fig. 5. The anthracycline aglycone doxorubicinone results in 59Fe-ferritin accumulation, but to a much lesser extent than DOX. SK-Mel-28 cells were prelabeled with 59Fe-Tf (0.75 µM) for 3 h at 37°C, washed, and reincubated in control media, DOX (5 and 10 µM), or doxorubicinone (5 and 10 µM) for 18 h at 37°C. Native PAGE-59Fe autoradiography (A) and densitometric analyses (B) were performed. Results are a typical experiment from two performed.

Indeed, the ability of DOX to induce ⁵⁹Fe accumulation in ferritin has been observed even at 1 µM DOX, and this effect plateaued at approximately 5 µM DOX (Kwok and Richardson, 2003). After a 24-h incubation of cells with these lower concentrations of DOX, little cell death was observed (Kwok and Richardson, 2003). In addition, previous experiments with another commonly used cytotoxic agent, cisplatin, had far less effect on ⁵⁹Fe-ferritin accumulation compared with cells incubated with DOX. This was despite the marked cell death observed with cisplatin-treated cells compared with cells incubated with DOX (Kwok and Richardson, 2003). Hence, the ability of DOX to inhibit ⁵⁹Fe mobilization from ferritin does not seem to simply be a nonspecific cytotoxic effect.

Collectively, the above studies suggest that the presence of DOX inhibited the normal redistribution of ⁵⁹Fe from ferritin to other molecules in the cytosol and SMM. Considering the possible role of lysosomes in ferritin-iron release (Roberts and Bomford, 1988; Radisky and Kaplan, 1998; Persson et al., 2001), the accumulation of ferritin-⁵⁹Fe in the cytosol and SMM of DOX-treated cells, but not control cells, could suggest that DOX was inhibiting lysosomal catabolism of ferritin.

In the experiments described below, we investigated the effects of a variety of well-characterized metabolic inhibitors, protein synthesis inhibitors, lysosomal enzyme inhibitors, proteasome inhibitors, and lysosomotropic agents to further understand the mechanisms involved in the DOX-mediated accumulation of ferritin-⁵⁹Fe.

Metabolic Inhibitors Induce Ferritin-⁵⁹Fe Accumulation. We showed that DOX induces ferritin-⁵⁹Fe accumulation by preventing ⁵⁹Fe mobilization from cytosolic and SMM ferritin (Fig. 4, A and C) (Kwok and Richardson, 2003). At present, little is known about the mechanisms of iron release from ferritin, although the lysosome has been implicated in this process through its ability to catabolize the molecule (Roberts and Bomford, 1988; Radisky and Kaplan, 1998; Persson et al., 2001). Alternatively, iron mobilization may occur by the reduction of Fe(III) to Fe(II) in the ferritin core by an unknown reductase or reducing factor (Harrison and Arosio, 1996). Irrespective of the mechanism(s) involved, metabolic energy will probably be essential for the process. Considering this, the effect of three well-known metabolic inhibitors on the accumulation of ⁵⁹Fe in ferritin were examined (Fig. 6, A and B).

View larger version (43K): [in this window] [in a new window]   Fig. 6. Metabolic inhibitors prevent 59Fe mobilization from ferritin in SK-Mel-28 melanoma cells. The cells were prelabeled with 59Fe-Tf (0.75 µM) for 6 h at 37°C, washed, and then reincubated for 18 h at 37°C with control media, DOX (10 and 20 µM), or the metabolic inhibitors NaN3 (5 or 30 mM), NaCN (1 or 5 mM), or rotenone (10 or 50 µM). Cellular 59Fe distribution was assessed using native PAGE-59Fe autoradiography (A) (see Materials and Methods) and densitometric analysis (B) of A. Results shown are a typical experiment from two performed.

Pulse-chase experiments were performed by prelabeling cells with ⁵⁹Fe-Tf for 6 h, followed by washing and reincubation of cells for 18 h with control media, DOX (10 or 20 µM), NaN₃ (5 or 30 mM), NaCN (1 or 5 mM), or rotenone (10 or 50 µM). We have shown previously that these concentrations effectively decrease cellular ATP levels (Watts and Richardson, 2001; Kwok and Richardson, 2003). As found for DOX, all metabolic inhibitors induced ferritin-⁵⁹Fe accumulation compared with the control (Fig. 6, A and B). Unexpectedly, cyanide at the higher concentration of 5 mM was less effective than at 1 mM in terms of inducing ferritin-⁵⁹Fe accumulation. The reason for this is uncertain at present, but it is possible that 5 mM cyanide caused cytotoxicity before any changes in ⁵⁹Fe-ferritin levels occurred in the cell. Collectively, the results indicate that ferritin-⁵⁹Fe mobilization is an energy-dependent event.

Inhibition of Protein Synthesis by Cycloheximide Inhibits Iron Mobilization and Results in the Accumulation of ⁵⁹Fe in Ferritin. Anthracyclines are known to depress cellular protein synthesis (Gianni and Myers, 1992), and it is possible that this latter activity could prevent ferritin-⁵⁹Fe mobilization and induce iron accumulation in this molecule. Because lysosomes may be involved in ferritin-iron mobilization (Hernandez-Yago et al., 1980; Radisky and Kaplan, 1998) and because lysosomal function requires the continuous synthesis of lysosomal enzymes and proteins (Pillay et al., 2002), it was important to assess the effects of inhibiting protein synthesis on ferritin-⁵⁹Fe accumulation. In the experiments below, we investigated the effect of the well-known protein synthesis inhibitor cycloheximide on ferritin-⁵⁹Fe accumulation (Fig. 7).

View larger version (41K): [in this window] [in a new window]   Fig. 7. Inhibition of cellular protein synthesis by cycloheximide results in accumulation of 59Fe in ferritin in SK-Mel-28 melanoma cells, and this is reversible upon removal of cycloheximide. A, the cells were prelabeled for 6 h at 37°C with 59Fe-Tf (0.75 µM) and washed. The cells were then reincubated with control media, cycloheximide (20–140 µM) or DOX (5 µM) for 18 h at 37°C, and native PAGE-59Fe autoradiography was performed (see Materials and Methods). B, densitometric analysis of A. C, cells were prelabeled with 59Fe-Tf (0.75 µM) for 6 h at 37°C, washed, and then incubated for 18 h at 37°C with media containing cycloheximide (20 µM). This was followed by washing and a 0- to 24-h reincubation in cycloheximide-free media (MEM) before native PAGE-59Fe autoradiography. D, densitometric analysis of C. Results shown are a typical experiment from three performed.

Pulse-chase experiments were performed by incubating cells with ⁵⁹Fe-Tf (0.75 µM) for 6 h at 37°C, followed by washing and reincubation with control media, cycloheximide (20–140 µM), or DOX (5 µM) for 18 h at 37°C (Fig. 7, A and B). These concentrations were in the effective range used in the literature (Darnell and Richardson, 1999). Cycloheximide at all concentrations (20–140 µM) resulted in a marked 9- to 10-fold increase in ⁵⁹Fe-ferritin levels compared with cells incubated with control media alone (Fig. 7, A and B; compare lane 1 with lanes 2–4). This accumulation was comparable with the 8-fold accumulation of ⁵⁹Fe-ferritin caused by 5 µM DOX (Fig. 7, A and B; compare lanes 1 and 5). Another protein synthesis inhibitor, puromycin (5–200 µM), was also assessed, and like cycloheximide, it prevented ⁵⁹Fe mobilization and resulted in marked ferritin-⁵⁹Fe accumulation (data not shown). These studies showed that, similar to DOX, the inhibition of cellular protein synthesis by cycloheximide or puromycin could prevent ferritin-⁵⁹Fe mobilization.

To further confirm the role of protein synthesis in the redistribution of ⁵⁹Fe-ferritin, cells exposed to cycloheximide were allowed to recover by washing and reincubation in medium alone. In these studies, cells were labeled with ⁵⁹Fe-Tf for 6 h at 37°C, washed, and reincubated in cycloheximide (20 µM) for 18 h at 37°C. Cells were then washed again and reincubated for 0 to 24 h in media (MEM) alone before ⁵⁹Fe-PAGE autoradiography (Fig. 7, C and D). The ⁵⁹Fe-ferritin levels after a 3-h reincubation in cycloheximide-free media decreased slightly compared with cells not reincubated in media (Fig. 7C, compare lanes 1 and 2; Fig. 7D). However, after a 6-h reincubation in cycloheximide-free media, ⁵⁹Fe-ferritin levels decreased to 43% compared with the 0-h time point (Fig. 7, C, compare lanes 1 and 3, and D). After a 24-h reincubation in cycloheximide-free medium, 93% of ⁵⁹Fe had been mobilized from ferritin (Fig. 7, C, compare lanes 1 and 5, and D). These results demonstrate that after the removal of cycloheximide, cells were able to recover their ability to mobilize ⁵⁹Fe from ferritin.

Inability of Cells to Recover ⁵⁹Fe Mobilization from Ferritin after DOX Treatment. The above studies demonstrated that cycloheximide acts similarly to DOX to induce ferritin-⁵⁹Fe accumulation, and this was at least partially reversible when the inhibitor was removed (Fig. 7, C and D). Experiments were then performed to determine whether ferritin-⁵⁹Fe accumulation after exposure to DOX could be reduced upon reincubation with DOX-free media (i.e., MEM). Cells were prelabeled with ⁵⁹Fe-Tf, washed, and reincubated with control media, DOX (1–5 µM), or cycloheximide (20 µM) for 18 h at 37°C. In parallel samples, cells were washed and then further reincubated for 24 h in media to allow recovery from DOX or cycloheximide (Fig. 8).

View larger version (26K): [in this window] [in a new window]   Fig. 8. The accumulation of 59Fe in ferritin observed after incubation with DOX was irreversible despite removal of the drug. A, the SK-Mel-28 cells were prelabeled with 59Fe-Tf (0.75 µM) for 6 h at 37°C, washed, and then reincubated for 18 h at 37°C with control media, DOX (1–5 µM), or cycloheximide (20 µM). In parallel samples, cells were washed and then further reincubated with media alone (MEM) for 24 h at 37°C before native PAGE-59Fe autoradiography. B, densitometric analyses of A. Results are a representative experiment from four performed.

Low concentrations of DOX (1–2 µM) only slightly increased ⁵⁹Fe-ferritin levels compared with control cells (Fig. 8, A and B; compare lane 1 with lanes 2 and 3). However, DOX (5 µM), and to a greater extent cycloheximide (20 µM), markedly increased ⁵⁹Fe-ferritin levels compared with the control (Fig. 8, A and B, compare lane 1 with lanes 4 and 5). Despite a further 24-h reincubation with DOX-free media, ⁵⁹Fe-ferritin accumulation in DOX-treated cells decreased only slightly or remained unchanged compared with cells not reincubated in MEM (Fig. 8, A and B; compare lanes 2–4 with lanes 7–9). However, as seen previously (Fig. 7, C and D), reincubation of cycloheximide-treated cells in MEM markedly decreased ⁵⁹Fe accumulation in ferritin (Fig. 8, A and B; compare lanes 5 and 10). These results suggested that the mechanism by which DOX inhibits ⁵⁹Fe mobilization from ferritin was not reversible despite removal of the agent from the incubation media. It is likely that DOX remains trapped within the cell even upon reincubation in DOX-free media.

Recovery of Protein Synthesis Upon Removal of Cycloheximide or Doxorubicin. We demonstrated above that incubation of SK-Mel-28 melanoma cells with cycloheximide or DOX resulted in marked ferritin-⁵⁹Fe accumulation (Figs. 7 and 8). This inhibition could be relieved when cycloheximide was removed from the cells, resulting in mobilization of ⁵⁹Fe from ferritin (Figs. 7 and 8). However, ferritin-⁵⁹Fe accumulation observed after incubation with DOX could not be reversed by reincubation with DOX-free media (Fig. 8). To determine whether these results correlated to the recovery of protein synthesis after removal of the test agents, [³H]leucine assays were performed (Fig. 9).

View larger version (14K): [in this window] [in a new window]   Fig. 9. Protein synthesis in SK-Mel-28 melanoma cells can be partially recovered after removal of cycloheximide, but this does not occur after removal of DOX. The SK-Mel-28 cells were incubated with control media, cycloheximide (0.14–18 µM), or DOX (0.02–20 µM) for 18 h at 37°C, and [3H]leucine incorporation was measured (see Materials and Methods). In parallel experiments, cells incubated with these concentrations of cycloheximide or DOX were washed and then reincubated for 24 h at 37°C with media alone (MEM), and [3H]leucine incorporation was assessed. Results are mean ± S.D. from six separate experiments.

Cells were incubated with control media, cycloheximide (0.14–18 µM) or DOX (0.02–20 µM) for 18 h, and total [³H]leucine incorporation was assessed. In a parallel set of experiments, cells incubated with these concentrations of cycloheximide or DOX were washed and then reincubated for 24 h at 37°C with media alone (MEM) before assessing [³H]leucine incorporation (Fig. 9).

Figure 9 shows that cycloheximide and DOX were extremely effective at inhibiting cellular protein synthesis in a concentration-dependent manner (Fig. 9, A and B). Interestingly, when cycloheximide-treated cells were reincubated with MEM for 24 h, a significant (p < 0.003) recovery of protein synthesis was observed at concentrations greater than 4 µM (Fig. 9A). Hence, the recovery of protein synthesis (Fig. 9A) and ⁵⁹Fe mobilization from ferritin (Fig. 7, C and D) were correlated after the removal of cycloheximide. Despite reincubation in MEM, [³H]leucine incorporation in DOX-treated cells continued to decrease (Fig. 9B). This was significant even in cells previously incubated with only 1 µM DOX (p < 0.00004). The inability of cells to recover protein synthesis after treatment with DOX could explain the absence of ⁵⁹Fe mobilization from ferritin upon removal of DOX from cells (Fig. 8, A and B). Again, this suggests that DOX is still present within the cell despite removal of the agent from the reincubation media. Hence, these studies suggested that mobilization of ⁵⁹Fe accumulated in ferritin required the recovery of cellular protein synthesis. Furthermore, these results demonstrated that anthracyclines irreversibly inhibit protein synthesis and ⁵⁹Fe mobilization from ferritin.

Inhibition of Lysosomal Enzymes Inhibits Ferritin-⁵⁹Fe Mobilization. Previous studies have suggested that the lysosome plays a role in the release of iron from ferritin (Roberts and Bomford, 1988; Radisky and Kaplan, 1998; Persson et al., 2001). Hence, the decrease in ⁵⁹Fe-ferritin levels and its redistribution to other cellular compartments observed in control cells (Fig. 4, A and C) may be mediated by lysosomal degradation of ferritin (Roberts and Bomford, 1988; Radisky and Kaplan, 1998; Persson et al., 2001). It is also possible that the accumulation of ferritin-⁵⁹Fe observed in DOX-treated cells (Fig. 4, A and C) was caused by DOX-induced inhibition of lysosomal ferritin degradation. In fact, anthracyclines localize in lysosomes (Hurwitz et al., 1997) and disrupt their morphology and enzyme activity (Singal et al., 1985). Considering this, we assessed the effect of lysosomal enzyme inhibitors on the redistribution of ⁵⁹Fe from ferritin. Lysosomal enzymes are largely composed of cysteine, aspartic, and serine proteases (Pillay et al., 2002). Hence, we examined the following: pepstatin A, an aspartic protease inhibitor with high specificity for cathepsin D (Knight and Barrett, 1976); E64d, a cysteine protease inhibitor with high specificity for cathepsin B (McGowan et al., 1989); and leupeptin, a general cysteine and serine protease inhibitor (Pillay et al., 2002). In addition, because proteasome-mediated degradation of oxidized ferritin has been reported (Rudeck et al., 2000), we examined the well-characterized proteosome inhibitors MG132 and lactacystin (Lee and Goldberg, 1998).

In these studies, pulse-chase experiments were performed in which cells were labeled with ⁵⁹Fe-Tf for 6 h at 37°C, washed, and then reincubated for 18 h at 37°C with control media, pepstatin A (100 µM), E64d (100 µM), leupeptin (50 and 100 µM), MG132 (10 µM), lactacystin (10 and 25 µM), or DOX (5 µM). These concentrations were within the effective range used in the literature (Ohshita et al., 1992). Pepstatin A alone had little effect on cytosolic ⁵⁹Fe-ferritin levels compared with control cells (Fig. 10, A and B, compare lanes 1 and 2), suggesting that aspartic proteases were not involved in ferritin-⁵⁹Fe mobilization. In contrast, all other lysosomal or proteasomal inhibitors induced ⁵⁹Fe-ferritin accumulation by 3- to 5-fold compared with control cells (Fig. 10, A and B, compare lane 1 with lanes 3–8). Combination of pepstatin A and E64d gave the same result as treatment with E64d alone (data not shown). Considering these results, the ferritin-⁵⁹Fe accumulation observed after incubation with DOX could be caused by the inhibition of the lysosome-mediated ferritin degradation pathway.

View larger version (41K): [in this window] [in a new window]   Fig. 10. Inhibition of lysosomal or proteasomal activity results in marked accumulation of 59Fe-ferritin to an extent similar to that found for DOX. A, the SK-Mel-28 cells were labeled with 59Fe-Tf (0.75 µM) for 6 h at 37°C and washed. Cells were then reincubated for 18 h at 37°C in control media, pepstatin A (100 µM), E64d (100 µM), leupeptin (50 or 100 µM), MG132 (10 µM), lactacystin (10 or 25 µM), or DOX (5 µM). Native PAGE-59Fe autoradiography was then performed. B, densitometric analysis of A. C, the SK-Mel-28 melanoma cells were labeled with 59Fe-Tf (0.75 µM) for 6 h at 37°C, washed, and then reincubated with NH4Cl (1.5 or 15 mM), chloroquine (25 or 250 µM), methylamine (1.5 or 15 mM) or DOX (5 µM) for 18 h at 37°C. Native PAGE-59Fe autoradiography was then performed (see Materials and Methods). D, densitometric analysis of C. Results are representative of three or four experiments in A and C, respectively.

Inhibition of Lysosomal Acidification Inhibits Ferritin-⁵⁹Fe Mobilization. To further test the hypothesis that ⁵⁹Fe release from ferritin involved lysosomes, experiments were performed to inhibit lysosomal acidification that is required for optimal enzymatic activity (Pillay et al., 2002). Pulse-chase experiments were therefore performed using three well-known lysosomotropic agents (NH₄Cl, chloroquine, and methylamine) that increase intralysosomal pH (Ohkuma and Poole, 1978).

In these studies, cells were prelabeled with ⁵⁹Fe-Tf for 6 h at 37°C, washed, and reincubated with NH₄Cl (1.5 or 15 mM), chloroquine (25 or 250 µM), methylamine (1.5 or 15 mM), or DOX (5 µM) for 18 h at 37°C to assess the role of lysosomal activity in ferritin-⁵⁹Fe accumulation (Fig. 10, C and D). The lower concentration of each lysosomotropic agent had little effect on ⁵⁹Fe-ferritin levels compared with control cells (Fig. 10, C, compare lane 1 with lanes 2, 4, and 6, and D). However, at the higher concentrations, NH₄Cl (15 mM), chloroquine (250 µM), and methylamine (15 mM) markedly increased ⁵⁹Fe-ferritin levels to 3- to 4-fold of that of the control (Fig. 10, C, compare lane 1 with lanes 3, 5, and 7, and D), whereas DOX increased it to 5.5-fold of that of the control value (Fig. 10C, compare lanes 1 and 9). These results with lysosomotropic agents suggest that inhibition of intralysosomal acidification could prevent the mobilization of ferritin-⁵⁹Fe. These data support our experiments using lysosomal enzyme inhibitors (Fig. 10, A and B). It is of interest to note that the ⁵⁹Fe-ferritin band observed in cells treated with high concentrations of NH₄Cl, chloroquine, or methylamine was not a well-defined band as seen in control cells (Fig. 10C, compare lane 1 with lanes 3, 5, and 7). The reason for this observation remains unclear at present.

Effect of DOX on Lysosomal Enzyme Activity. Because DOX markedly inhibits cellular protein synthesis (Fig. 9B) (Gianni and Myers, 1992) and inhibition of lysosomal enzyme activity results in marked iron accumulation in ferritin (Fig. 10), it was possible to speculate that this drug targets lysosomal enzyme synthesis, resulting in the inhibition of ferritin degradation and the release of iron from the protein. Hence, the activities of eight lysosomal enzymes were assessed in cells after 24-h incubations with control media or DOX (5 µM). The enzymes examined were the following: -galactosidase, -glucuronidase, -fucosidase, -mannosidase, -hexosaminidase A, -N-acetyl-galactosaminidase, arylsulfatase A, and acid phosphatase.

A slight increase in -galactosidase activity was detected in DOX-treated cells (24 ± 1 nmol/min/mg protein) compared with control cells (20 ± 0.1 nmol/min/mg protein), as previously shown by others (Singal et al., 1985). However, DOX did not markedly affect the activity of the other lysosomal enzymes examined (data not shown). These results suggested that inhibition of these lysosomal enzymes was not the primary target of DOX that induces ferritin-⁵⁹Fe accumulation. However, we cannot discount that other lysosomal enzymes or molecules may be the targets of anthracyclines.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
We demonstrated previously that anthracyclines inhibit iron mobilization from ferritin in neoplastic cells and cardiomyocytes, resulting in a marked accumulation of iron in this molecule (Kwok and Richardson, 2003). We hypothesized that this may represent a potential mechanism of anthracycline cytotoxicity, because the lack of iron redistribution to vital cellular functions may have detrimental effects on the cell (Kwok and Richardson, 2003).

Currently, the mechanisms by which iron is released from ferritin for metabolic use are little understood, but it has been suggested to involve a lysosomal-mediated protein degradation pathway (Roberts and Bomford, 1988; Radisky and Kaplan, 1998; Persson et al., 2001). In the present study, we demonstrated for the first time that ferritin-⁵⁹Fe mobilization is dependent on metabolic energy and protein synthesis. Moreover, a variety of well-characterized lysosomal enzyme inhibitors, lysosomotropic agents, and proteasomal inhibitors resulted in marked accumulation of ferritin-⁵⁹Fe that was comparable with that found using DOX. These data provide further evidence that DOX inhibits the activity of lysosomes in the degradation of ferritin that is essential for the release and redistribution of metabolically useful iron.

It could be suggested that the effect of DOX on inducing ferritin-⁵⁹Fe accumulation is a nonspecific cytotoxic response. However, we have shown that the noncytotoxic doxorubicin aglycone doxorubicinone also resulted in accumulation of ⁵⁹Fe in ferritin (Fig. 5, A and B). In addition, as demonstrated in our previous study (Kwok and Richardson 2003), another commonly used antitumor agent, cisplatin, at 10 µM had little effect on ⁵⁹Fe accumulation in ferritin compared with control cells, despite being very cytotoxic. Furthermore, as shown in this latter study, low concentrations of DOX (1–5 µM) resulted in marked ferritin iron accumulation, although there was little cell death. Collectively, these results demonstrate that the effect of DOX on inducing ferritin-iron accumulation was not caused by nonspecific cytotoxicity.

The lysosome plays a major role in the normal degradation and recycling of cellular proteins for anabolic use. To date, no study has examined the effect of anthracyclines on ferritin-iron metabolism and the potential of lysosomes and proteasomes as targets for these drugs. However, it is of considerable interest that anthracyclines accumulate in acidic vacuoles such as lysosomes (Hurwitz et al., 1997) and vesicles of the Golgi apparatus (Klohs and Steinkampf, 1988). In their uncharged, hydrophobic state, anthracyclines diffuse across membranes, and within acidic compartments they become protonated and trapped (Hurwitz et al., 1997). In addition, early in vivo studies demonstrated that anthracyclines affect lysosomal morphology and enzyme activity (Singal et al., 1985). Furthermore, it has been shown in the mouse leukemia cell line L1210 that DOX has high affinity for the 20S subunit of the proteasome (Kiyomiya et al., 2001). Although interactions between anthracyclines and lysosomes or proteasomes have been described, precisely how these anticancer agents affect proteolytic systems remains to be further investigated. As shown in our current study, the activity of eight lysosomal enzymes was not affected by DOX. This observation suggested that the ferritin-⁵⁹Fe accumulation observed in the presence of this drug was not a general effect on lysosomal enzymes. However, DOX may inhibit other lysosomal proteases that are essential for ferritin turnover. Indeed, the precise identities of the proteases that degrade ferritin remain unknown.

The current study demonstrated that general depression of cellular protein synthesis inhibited ferritin degradation and the redistribution of metabolically useful iron (Figs. 7 and 8). The fact that DOX markedly and irreversibly inhibited protein synthesis (Fig. 9B) may be one mechanism by which it inhibits the renewal of essential proteolytic mechanisms. Certainly, some lysosomal enzymes such as sialidase have a short half-life of approximately 2.7 h (Vinogradova et al., 1998), and hence, continued protein synthesis is vital to renew lysosomal enzymatic activity. Other crucial molecules in the lysosomal pathway include the lysosome-associated membrane proteins and the membrane-bound transporter of cysteine (for review see Pillay et al., 2002). Inhibition of the synthesis of any of these essential molecules will disrupt the normal lysosomal degradation process. Moreover, recent studies have demonstrated that lysosomes are highly dynamic, capable of moving from the cell periphery to the perinuclear regions and vice versa (Radisky and Kaplan, 1998). Therefore, microtubules probably play a significant role in the stability and trafficking of lysosomes (Matteoni and Kreis, 1987) and could be a potential target for protein synthesis inhibition that can disrupt lysosomal activity.

There are more than 50 lysosomal hydrolases, and these can be classified into the aspartic, cysteine, and serine proteases (Pillay et al., 2002). Incubation of SK-Mel-28 melanoma cells with the aspartic protease inhibitor pepstatin A had little effect on ferritin-⁵⁹Fe accumulation (Fig. 10, A and B), suggesting that this class of enzymes is not primarily involved in ferritin-⁵⁹Fe mobilization. However, both E64d and leupeptin markedly increased ferritin-⁵⁹Fe accumulation, suggesting that cysteine and/or serine proteases may be responsible for ⁵⁹Fe mobilization from this molecule. In fact, it is possible that the localization of anthracyclines in lysosomes (Hurwitz et al., 1997) inhibits certain lysosomal enzymes (Singal et al., 1985), resulting in a marked accumulation of ferritin-iron.

Interestingly, the structurally distinct and widely used proteasome inhibitors MG132 and leupeptin also markedly prevented ferritin-⁵⁹Fe mobilization (Fig. 10, A and B). The reversible inhibitor, MG132, targets the chymotrypsin-like activity of the proteasome (Lee and Goldberg, 1998). However, it is not completely selective and has been demonstrated to also target certain lysosomal cysteine proteases and the calpains (Lee and Goldberg, 1998). Similarly, whereas lactacystin acts irreversibly and has higher specificity, it can also inhibit the lysosomal protease cathepsin A (Dick et al., 1996). Hence, it could be suggested that the accumulation of ferritin-⁵⁹Fe observed with the proteasome inhibitors MG132 and lactacystin were caused by the inhibition of lysosomal proteases. This could argue against a role for proteasomes in the degradation of ferritin-iron. Alternatively, because the ubiquitin-proteasome pathway also plays a major role in the selective degradation of intracellular proteins (Lee and Goldberg, 1998), it is not unreasonable to speculate that proteasomes may be involved in ferritin degradation. Indeed, various in vitro and cellular studies have demonstrated proteasome-mediated degradation of ferritin (Rudeck et al., 2000). It is also possible that the avid association of anthracyclines with the 20S proteasomal subunit (Kiyomiya et al., 2001) may prevent ferritin degradation. This would explain the marked accumulation of ferritin-⁵⁹Fe when cells were incubated with the proteasome inhibitors MG132 and lactacystin (Fig. 10, A and B).

It is relevant to discuss that the potent metabolic inhibitors cyanide, azide, and rotenone all prevented ⁵⁹Fe mobilization from ferritin in a manner similar to that of DOX (Fig. 6). These three agents have been shown to markedly reduce ATP levels in our previous studies over shorter or the same incubation period used in this investigation (Watts and Richardson, 2001; Kwok and Richardson, 2003), whereas DOX at the same concentration used in our experiments (i.e., 5–20 µM) did not (Kwok and Richardson, 2003). This suggests that the ferritin-⁵⁹Fe accumulation observed after incubation with DOX was not predominantly caused by a pronounced decrease in ATP levels and could be related to its other properties such as protein synthesis inhibition. However, the fact that marked metabolic depletion inhibits the ⁵⁹Fe-mobilization process from ferritin indicates that this mechanism is energy-dependent.

In our previous study, we showed that the redox-cycling drugs menadione and paraquat also resulted in ferritin-⁵⁹Fe accumulation (Kwok and Richardson, 2003). This effect could be partially reversed by the cell-permeable glutathione peroxidase mimetic ebselen and the membrane-permeable superoxide dismutase mimetic MnTBAP (Kwok and Richardson, 2003). However, it is important to note that these agents were less effective at reversing ferritin-⁵⁹Fe accumulation induced by DOX (Kwok and Richardson, 2003). One possible explanation consistent with the current investigation is that the free radicals generated by these redox cycling agents may affect lysosomal function, as proposed in other studies (Persson et al., 2001).

In summary, the current study demonstrates that DOX inhibits ⁵⁹Fe mobilization from ferritin in the cytosol and SMM. We showed that inhibition of energy generation or protein synthesis inhibits the ferritin-⁵⁹Fe mobilization pathway. A variety of lysosomal enzyme inhibitors, proteasomal inhibitors, and lysosomotropic agents resulted in marked accumulation of ferritin-⁵⁹Fe that was comparable with that found using DOX. These data provide further evidence of a mechanism involving lysosomes in the degradation of ferritin for the release and redistribution of metabolically useful iron. The inhibition of iron redistribution from ferritin may have detrimental effects on vital iron-dependent processes in the cell. Considering that anthracyclines accumulate in lysosomes, it is possible that the lysosomal degradation pathway represents a potential target for anthracycline cytotoxicity.

	Acknowledgements

 
We kindly thank the Department of Chemical Pathology, Women's and Children's Hospital, Adelaide, Australia, for measuring lysosomal enzyme activity using their established protocols.

	Footnotes

 
Children's Cancer Institute Australia for Medical Research is affiliated with the University of New South Wales and Sydney Children's Hospital. The work was supported by a Ph.D. scholarship (to J.C.K.) and grant (D.R.R.) from the National Heart Foundation and by a fellowship and grants from the National Health and Medical Research Council (to D.R.R.).

ABBREVIATIONS: TfR1, transferrin receptor 1; DOX, doxorubicin; E64d, (2S,3S)-trans-epoxysuccinyl-L-leucylamido-2-methylbutane ethyl ester; MG132, N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal; MEM, minimum essential medium; SMM, stromal-mitochondrial membrane; Tf, transferrin; PAGE, polyacrylamide gel electrophoresis; MnTBAP, Mn(III)tetrakis(4-benzoic acid)-porphyrin.

Address correspondence to: Dr. D.R. Richardson, Children's Cancer Institute Australia for Medical Research, Iron Metabolism and Chelation Program, P.O. Box 81, High Street, Randwick, Sydney, NSW 2031, Australia. E-mail: d.richardson{at}ccia.org.au

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