Iron supplementation ameliorates aloin-induced iron deficiency anemia in rats.

Aloin, an anthraquinone glycoside, is one of other C-glycosides found in the leaf exudate of Aloe plant. Aloin possesses several biologic activities, including antitumor activity in vitro and in vivo. However, aloin treatment has shown iron deficiency anemia and erythropoiesis in vivo. The present study was undertaken to verify if iron supplementation could alleviate these perturbations, compared to doxorubicin, an anthracycline analog. Oral iron supplementation (20.56 mg elemental Fe/kg bw) to aloin-treated rats normalized red blood corpuscles count, hemoglobin concentration, and serum levels of total iron binding capacity and saturated transferrin, as well as hepatic iron content, hepcidin level, and mRNA expression of ferritin heavy chain (Ferr-H) and transferrin receptor-1 (TfR-1) genes. Although, serum hyperferremia, and leukocytosis were maintained, yet the spleen iron overload was substantially modulated. However, combined aloin and iron treatment increased iron storage levels in the heart and bone marrow, compared to aloin treatment per se. On other hand, oral iron supplementation to rats treated with doxorubicin (15 mg/kg bw) lessened the increase in the spleen iron content concomitantly with hepatic hepcidin level, rebound hepatic iron content to normal level, and by contrast augmented serum levels of iron and transferrin saturation. Also, activated Ferr-H mRNA expression and repressed TfR-1 mRNA expression were recorded, compared to doxorubicin treatment per se. Histopathological examination of the major body iron stores in rats supplemented with iron along with aloin or doxorubicin showed an increase in extramedullary hematopoiesis. In conclusion, iron supplementation restores the disturbances in iron homeostasis and erythropoiesis induced by aloin treatment.

A dual-targeting ruthenium nanodrug that inhibits primary tumor growth and lung metastasis via the PARP/ATM pathway.

BACKGROUND: Many studies have found that ruthenium complexes possess unique biochemical characteristics and inhibit tumor growth or metastasis. RESULTS: Here, we report the novel dual-targeting ruthenium candidate 2b, which has both antitumor and antimetastatic properties and targets tumor sites through the enhanced permeability and retention (EPR) effect and transferrin/transferrin receptor (TF/TFR) interaction. The candidate 2b is composed of ruthenium-complexed carboline acid and four chloride ions. In vitro, 2b triggered DNA cleavage and thus blocked cell cycle progression and induced apoptosis via the PARP/ATM pathway. In vivo, 2b inhibited not only Lewis lung cancer (LLC) tumor growth but also lung metastasis. We detected apoptosis and decreased CD31 expression in tumor tissues, and ruthenium accumulated in the primary tumor tissue of C57BL/6 mice implanted with LLC cells. CONCLUSIONS: Thus, we conclude that 2b targets tumors, inhibits tumor growth and prevents lung metastasis.

A TfR-Binding Cystine-Dense Peptide Promotes Blood-Brain Barrier Penetration of Bioactive Molecules.

The impenetrability of the blood-brain barrier (BBB) to most conventional drugs impedes the treatment of central nervous system (CNS) disorders. Interventions for diseases like brain cancer, neurodegeneration, or age-associated inflammatory processes require varied approaches to CNS drug delivery. Cystine-dense peptides (CDPs) have drawn recent interest as drugs or drug-delivery vehicles. Found throughout the phylogenetic tree, often in drug-like roles, their size, stability, and protein interaction capabilities make CDPs an attractive mid-size biologic scaffold to complement conventional antibody-based drugs. Here, we describe the identification, maturation, characterization, and utilization of a CDP that binds to the transferrin receptor (TfR), a native receptor and BBB transporter for the iron chaperone transferrin. We developed variants with varying binding affinities (KD as low as 216 pM), co-crystallized it with the receptor, and confirmed murine cross-reactivity. It accumulates in the mouse CNS at ~25% of blood levels (CNS blood content is only ~1%-6%) and delivers neurotensin, an otherwise non-BBB-penetrant neuropeptide, at levels capable of modulating CREB signaling in the mouse brain. Our work highlights the utility of CDPs as a diverse, easy-to-screen scaffold family worthy of inclusion in modern drug discovery strategies, demonstrated by the discovery of a candidate CNS drug delivery vehicle ready for further optimization and preclinical development.

Multitissue analysis of exercise and metformin on doxorubicin-induced iron dysregulation.

Doxorubicin (DOX) is an effective chemotherapeutic treatment with lasting side effects in heart and skeletal muscle. DOX is known to bind with iron, contributing to oxidative damage resulting in cardiac and skeletal muscle toxicity. However, major cellular changes to iron regulation in response to DOX are poorly understood in liver, heart, and skeletal muscle. Additionally, two cotreatments, exercise (EX) and metformin (MET), were studied for their effectiveness in reducing DOX toxicity by ameliorating iron dysregulation and preventing oxidative stress. The purposes of this study were to 1) characterize the DOX-induced changes of the major iron regulation pathway in liver, heart, and skeletal muscle and 2) to determine whether EX and MET exert their benefits by minimizing DOX-induced iron dysregulation. Mice were assigned to receive saline or DOX (15 mg/kg) treatments, paired with either EX (5 days) or MET (500 mg/kg), and were euthanized 3 days after DOX treatment. Results suggest that the cellular response to DOX is protective against oxidative stress by reducing iron availability. DOX increased iron storage capacity through elevated ferritin levels in liver, heart, and skeletal muscle. DOX reduced iron transport capacity through reduced transferrin receptor levels in heart and skeletal muscle. EX and MET cotreatments had protective effects in the liver through reduced transferrin receptor levels. At 3 days after DOX, oxidative stress was mild, as shown by normal glutathione and lipid peroxidation levels. Together these results suggest that the cellular response to reduce iron availability in response to DOX treatment is sufficient to match oxidative stress.

Characterization of hepcidin response to holotransferrin in novel recombinant TfR1 HepG2 cells.

Hepcidin is the key regulator of systemic iron homeostasis. The iron-sensing mechanisms and the role of intracellular iron in modulating hepatic hepcidin secretion are unclear. Therefore, we created a novel cell line, recombinant-TfR1 HepG2, expressing iron-response-element-independent TFRC mRNA to promote cellular iron-overload and examined the effect of excess holotransferrin (5g/L) on cell-surface TfR1, iron content, hepcidin secretion and mRNA expressions of TFRC, HAMP, SLC40A1, HFE and TFR2. Results showed that the recombinant cells exceeded levels of cell-surface TfR1 in wild-type cells under basal (2.8-fold; p<0.03) and holotransferrin-supplemented conditions for 24h and 48h (4.4- and 7.5-fold, respectively; p<0.01). Also, these cells showed higher intracellular iron content than wild-type cells under basal (3-fold; p<0.03) and holotransferrin-supplemented conditions (6.6-fold at 4h; p<0.01). However, hepcidin secretion was not higher than wild-type cells. Moreover, holotransferrin treatment to recombinant cells did not elevate HAMP responses compared to untreated or wild-type cells. In conclusion, increased intracellular iron content in recombinant cells did not increase hepcidin responses compared to wild-type cells, resembling hemochromatosis. Furthermore, TFR2 expression altered within 4h of treatment, while HFE expression altered later at 24h and 48h, suggesting that TFR2 may function prior to HFE in HAMP regulation.

[Progress on anti-tumor molecular mechanisms of dihydroartemisinin].

Artemisinin is an anti-malarial drug with poor water solubility and oral absorption; so a variety of derivatives based on the parent nucleus have been developed. Compared with artemisinin, dihydroartemisinin (DHA) has a stronger anti-malaria activity, and has the advantages of high metabolic rate and better water solubility. Recent studies have discovered that DHA has a good inhibitory effect on tumor cells, which is closely related to the peroxide bridge in its molecular structure. Since tumor cells need more Fe3+ than normal cells, there are a large number of transferrin receptors on the tumor cell membrane. DHA can break the peroxide bridge in the presence of Fe2+, and the free radicals generated can play its lethal effect on tumor cells. In addition, DHA can promote endocytosis of transferrin receptor, and thus prevent cancer cells from taking Fe3+ from microenvironment. This article reviews the anti-tumor molecular mechanism of DHA, including accelerating oxidative damage, inducing apoptosis, inhibiting the growth, proliferation and invasion of tumor cells, reversing tumor multidrug resistance.

Novel Mechanism of Cytotoxicity for the Selective Selenosemicarbazone, 2-Acetylpyridine 4,4-Dimethyl-3-selenosemicarbazone (Ap44mSe): Lysosomal Membrane Permeabilization.

Selenosemicarbazones show marked antitumor activity. However, their mechanism of action remains unknown. We examined the medicinal chemistry of the selenosemicarbazone, 2-acetylpyridine 4,4-dimethyl-3-selenosemicarbazone (Ap44mSe), and its iron and copper complexes to elucidate its mechanisms of action. Ap44mSe demonstrated a pronounced improvement in selectivity toward neoplastic relative to normal cells compared to its parent thiosemicarbazone. It also effectively depleted cellular Fe, resulting in transferrin receptor-1 up-regulation, ferritin down-regulation, and increased expression of the potent metastasis suppressor, N-myc downstream regulated gene-1. Significantly, Ap44mSe limited deleterious methemoglobin formation, highlighting its usefulness in overcoming toxicities of clinically relevant thiosemicarbazones. Furthermore, Cu-Ap44mSe mediated intracellular reactive oxygen species generation, which was attenuated by the antioxidant, N-acetyl-L-cysteine, or Cu sequestration. Notably, Ap44mSe forms redox active Cu complexes that target the lysosome to induce lysosomal membrane permeabilization. This investigation highlights novel structure-activity relationships for future chemotherapeutic design and underlines the potential of Ap44mSe as a selective anticancer/antimetastatic agent.

Transferrin receptors and glioblastoma multiforme: Current findings and potential for treatment.

The current standard treatment for glioblastoma multiforme (GBM) is surgery followed by chemotherapy and external radiation. Even with the standard treatment, the 2 year survival rate for GBM is less than 20%, making research for alternative treatments necessary. Transferrin receptor 1 (TfR1) controls the rate of cellular iron uptake by tuning the amount of iron delivered to the cells to meet metabolic needs. Kawabata et al. (J Biol Chem 1999;274:20826-32) cloned a second TfR molecule known as transferrin receptor 2 (TfR2) in 1999. Multiple experimental studies have documented increased expression of TfR1 on both proliferating cells and cells that have undergone malignant transformation. Calzolari et al. concluded that TfR2 is frequently expressed in human cell lines in 2007 (Blood Cells Mol Dis 2007;39:82-91) and in GBM in particular in 2010 (Transl Oncol 2010;3:123-34). In GBM, a highly significant correlation (p<0.0001) was found between the expression level of TfR2 and overall survival, showing that higher levels of TfR2 expression were associated with an overall longer survival. The data on which of the two transferrin receptors is the better target is also unclear and should be studied. The transferrin pathway may be a promising target, but more research should be completed on the antigenicity to discern the viability of it as an immunotherapy target.

Applying the retro-enantio approach to obtain a peptide capable of overcoming the blood-brain barrier.

The blood-brain barrier (BBB) is a formidable physical and enzymatic barrier that tightly controls the passage of molecules from the blood to the brain. In fact, less than 2 % of all potential neurotherapeutics are able to cross it. Here, by applying the retro-enantio approach to a peptide that targets the transferrin receptor, a full protease-resistant peptide with the capacity to act as a BBB shuttle was obtained and thus enabled the transport of a variety of cargos into the central nervous system.

Novel immunosuppressive agent caerulomycin A exerts its effect by depleting cellular iron content.

BACKGROUND AND PURPOSE: Recently, we have described the use of caerulomycin A (CaeA) as a potent novel immunosuppressive agent. Immunosuppressive drugs are crucial for long-term graft survival following organ transplantation and treatment of autoimmune diseases, inflammatory disorders, hypersensitivity to allergens, etc. The objective of this study was to identify cellular targets of CaeA and decipher its mechanism of action. EXPERIMENTAL APPROACH: Jurkat cells were treated with CaeA and cellular iron content, iron uptake/release, DNA content and deoxyribonucleoside triphosphate pool determined. Activation of MAPKs; expression level of transferrin receptor 1, ferritin and cell cycle control molecules; reactive oxygen species (ROS) and cell viability were measured using Western blotting, qRT-PCR or flow cytometry. KEY RESULTS: CaeA caused intracellular iron depletion by reducing its uptake and increasing its release by cells. CaeA caused cell cycle arrest by (i) inhibiting ribonucleotide reductase (RNR) enzyme, which catalyses the rate-limiting step in the synthesis of DNA; (ii) stimulating MAPKs signalling transduction pathways that play an important role in cell growth, proliferation and differentiation; and (iii) by targeting cell cycle control molecules such as cyclin D1, cyclin-dependent kinase 4 and p21(CIP1/WAF1) . The effect of CaeA on cell proliferation was reversible. CONCLUSIONS AND IMPLICATIONS: CaeA exerts its immunosuppressive effect by targeting iron. The effect is reversible, which makes CaeA an attractive candidate for development as a potent immunosuppressive drug, but also indicates that iron chelation can be used as a rationale approach to selectively suppress the immune system, because compared with normal cells, rapidly proliferating cells require a higher utilization of iron.

Receptor-based targeting of therapeutics.

Receptor-based targeting of therapeutics may be a fascinating proposition to improve the therapeutic efficacy of encapsulated drugs. The development of safe and effective nanomedicines is a prerequisite in the current nanotechnological scenario. Currently, the surface engineering of nanocarriers has attracted great attention for targeted therapeutic delivery by selective binding of targeting ligand to the specific receptors present on the surface of cells. In this review, we have discussed the current status of various receptors such as transferrin, lectoferrin, lectin, folate, human EGF receptor, scavenger, nuclear and integrin, which are over-expressed on the surface of cancer cells; along with the relevance of targeted delivery systems such as nanoparticles, polymersomes, dendrimers, liposomes and carbon nanotubes. The review also focuses on the effective utilization of receptor-based targeted delivery systems for the management of cancer in effective ways by minimizing the drug-associated side effects and improving the therapeutic efficacy of developed nano-architectures.

Immunogenicity and toxicity of transferrin receptor-targeted hybrid peptide as a potent anticancer agent.

PURPOSE: Transferrin receptor (TfR) is a cell membrane-associated glycoprotein involved in the cellular uptake of iron and the regulation of cell growth. Recent studies have shown elevated expression levels of TfR on cancer cells compared with normal cells. We previously designed a TfR-lytic hybrid peptide, which combines the TfR-binding peptide and a lytic peptide, and reported that it bound specifically to TfR and selectively killed cancer cells. Furthermore, the intravenous administration of TfR-lytic peptide in an athymic mouse model significantly inhibited tumor progression. To evaluate the immunogenicity of this peptide as a novel and potent anticancer agent, we investigated whether TfR-lytic hybrid peptide elicits cellular and humoral immune responses to produce antibodies. We also examined the toxicity of this peptide in syngeneic mice. METHODS: We performed hematologic and blood chemistry test and histological analysis and assessed hemolytic activity to check toxicity. To evaluate the immunogenicity, measurement of murine interferon-gamma and detection of TfR-lytic-specific antibody by ELISA were demonstrated. RESULTS: No T cell immune response or antibodies were detected in the group treated with TfR-lytic hybrid peptide. No hematologic toxicity, except for a decrease in leukocytes, was observed, and no remarkable influence on metabolic parameters and organs (liver, kidney, and spleen) was noted. CONCLUSIONS: Therefore, TfR-lytic hybrid peptide might provide an alternative therapeutic option for patients with cancer.

The effect of anti-TfR mouse/human chimeric antibody on anti-transplant rejection.

The expression of TfR/CD71 in T-cell surface plays a pivotal role in T-cell activation and proliferation. Anti-human-TfR monoclonal antibody could be used as an immunosuppressant in transplant therapy because of their potential to suppress T-cell responses to alloantigens. We therefore examined the feasibility of an anti-human-TfR chimeric antibody (D2C) in suppression of T-cell activation in vitro and graft-versus-host reaction (GVHR) in animals. D2C is a chimeric antibody produced by introducing the human Fc fragment. This antibody showed low antigenicity but high suppressive effect manifested by high potency to block the activation and proliferation of lymphocytes in response to alloantigens. D2C also showed capability to mediate complement-dependent cytotoxicity, which could be correlated with TfR expression in peripheral blood mononuclear cells (PBMCs). Importantly, administration of D2C significantly prolonged survival time of nude mice transplanted with human PBMCs when compared with that of control IgG-treated animals (61.2 ± 4.46 vs. 22.1 ± 5.5 days), which is associated with inhibited GVHR characterized by decreased interleukin-1 and tumor necrosis factor α production derived from transplanted PBMCs. Human-TfR chimeric antibody such as D2C could be a valuable option for the treatment of acute form of graft-versus-host disease.

Andrographolide regulates epidermal growth factor receptor and transferrin receptor trafficking in epidermoid carcinoma (A-431) cells.

BACKGROUND AND PURPOSE: Andrographolide is the active component of Andrographis paniculata, a plant used in both Indian and Chinese traditional medicine, and it has been demonstrated to induce apoptosis in different cancer cell lines. However, not much is known about how it may affect the key receptors implicated in cancer. Knowledge of how andrographolide affects receptor trafficking will allow us to better understand new mechanisms by which andrographolide may cause death in cancer cells. EXPERIMENTAL APPROACH: We utilized the well-characterized epidermal growth factor receptor (EGFR) and transferrin receptor (TfR) expressed in epidermoid carcinoma (A-431) cells as a model to study the effect of andrographolide on receptor trafficking. Receptor distribution, the total number of receptors and surface receptors were analysed by immunofluorescence, Western blot as well as flow-cytometry respectively. KEY RESULTS: Andrographolide treatment inhibited cell growth, down-regulated EGFRs on the cell surface and affected the degradation of EGFRs and TfRs. The EGFR was internalized into the cell at an increased rate, and accumulated in a compartment that co-localizes with the lysosomal-associated membrane protein in the late endosomes. CONCLUSION AND IMPLICATIONS: This study sheds light on how andrographolide may affect receptor trafficking by inhibiting receptor movement from the late endosomes to lysosomes. The down-regulation of EGFR from the cell surface also indicates a new mechanism by which andrographolide may induce cancer cell death.

Regulation of transferrin receptor 2 in human cancer cell lines.

In a recent study we have explored TfR2 expression in a panel of cancer cell lines and we observed that about 40% of these cell lines clearly express TfR2. Taking advantage of this observation and considering the frequent overexpression of c-Myc in cancer cells we have explored the existence of a possible relationship between c-Myc and TfR2 in these cell lines. Our results provided evidence that TfR2(+) cell lines express low c-Myc levels and low TfR1 levels, while TfR2(-) cell lines express high c-Myc and TfR1 levels. Using the erythroleukemic K562 TfR2(+) cells as a model, we observed that agents that enhance c-Myc expression, such as iron, determine a decrease of TfR2 expression, while molecules that induce a decreased c-Myc expression, such as the iron chelator desferoxamine or the kinase inhibitor ST 1571, induce an enhanced TfR2 expression. On the other hand, we have evaluated a possible effect of hypoxia and nitric oxide on TfR2 expression in erythroleukemia K526 and hepatoma HepG2 cells, providing evidence that: (i) agents inducing cellular hypoxia, such as CoCl(2), elicited a marked upmodulation of TfR1, but a downmodulation of TfR2 expression; (ii) NO(+) donors, such as sodium nitroprusside (SNP), induced a moderate decrease of TfR1, associated with a marked decline of TfR2 expression; (iii) NO donors, such as S-Nitroso-N-Acetylpenicillamine (SNAP), induced a clear increase of TfR1, associated with a moderate upmodulation of TfR2 expression. The ensemble of these observations suggests that in cancer cell lines TfR2 expression can be modulated through stimuli similar to those known to act on TfR1 and these findings may have important implications for our understanding of the role of TfR2 in the regulation of iron homeostasis.

Dihydroartemisinin induces apoptosis in human leukemia cells HL60 via downregulation of transferrin receptor expression.

Dihydroartemisinin (DHA), a water-soluble active metabolite of artemisinin derivatives, is the safest and most effective antimalarial analog of artemisinin. In the present investigation, we assessed the apoptotic effect of DHA on leukemia HL60 cells and its regulation of transferrin receptor (TfR). Cell growth inhibition was assessed by Trypan blue exclusive staining; the expression of caspase-3, Bcl-2, and Bax in HL60 cells was evaluated by Western blotting; DHA-induced apoptosis was determined by AO/EB double staining, DNA fragmentation assay, and flow cytometric analysis; the expression of TfR in HL60 cells was examined by real-time PCR assays, Western blotting, and flow cytometric analysis. DHA could specifically reduce the mRNA and protein expression of TfR in HL60 cells, and the flow cytometric analysis presented the unity tendency that the TfR content decreased progressively in a dose-dependent manner. Consequently, DHA exhibited high anticancer activity in HL60 cells; MTT assay and growth inhibition assay showed that DHA could specifically inhibit the growth of HL60 cells in a dose-dependent (0.25-8 micromol/l) and time-dependent (12-72 h) manner. DHA-induced DNA fragmentation also induced the activation of caspase-3 and influenced the expression of Bcl-2 and Bax. Taken together, these data from our study show that DHA can induce HL60 cell apoptosis via the effect of downregulation TfR expression resulting in an induction of apoptosis through the mitochondrial pathway, and it might be a potential antileukemia strategy for leukemia therapy.

[Transferrin conjugates in the anticancer therapy]. / Koniugaty transferyny--nadzieja w walce z nowotworami.

To enhance the therapeutic efficiacy of anticancer drugs and reducing its systemic side-effects carriers are used. Transferrin is one of the very promising protein which can be used to transport drugs, DNA and ions into the cancer cells. Because of the fact that neoplastic cells have increased number of transferrin receptors, the transferrin can deliver the drugs directly to the neoplastic cells without injury of normal cells.

Activation of transferrin receptor 1 by c-Myc enhances cellular proliferation and tumorigenesis.

Overexpression of transferrin receptor 1 (TFRC1), a major mediator of iron uptake in mammalian cells, is a common feature of human malignancies. Therapeutic strategies designed to interfere with tumor iron metabolism have targeted TFRC1. The c-Myc oncogenic transcription factor stimulates proliferation and growth by activating thousands of target genes. Here we demonstrate that TFRC1 is a critical downstream target of c-Myc. Using in vitro and in vivo models of B-cell lymphoma, we show that TFRC1 expression is activated by c-Myc. Chromatin immunoprecipitation experiments reveal that c-Myc directly binds a conserved region of TFRC1. In light of these findings, we sought to determine whether TFRC1 is required for c-Myc-mediated cellular proliferation and cell size control. TFRC1 inhibition decreases cellular proliferation and results in G1 arrest without affecting cell size. Consistent with these findings, expression profiling reveals that TFRC1 depletion alters expression of genes that regulate the cell cycle. Furthermore, enforced TFRC1 expression confers a growth advantage to cells and significantly enhances the rate of c-Myc-mediated tumor formation in vivo. These findings provide a molecular basis for increased TFRC1 expression in human tumors, illuminate the role of TFRC1 in the c-Myc target gene network, and support strategies that target TFRC1 for cancer therapy.

Temporal responses in the disruption of iron regulation by manganese.

Manganese (Mn) is an essential trace element, though at elevated exposures it is also a neurotoxicant. Several mechanisms underlying manganese toxicity have been investigated, although a consistent mechanism(s) of action at low exposures has not been fully elucidated. Here we systematically evaluated the effects of in vitro manganese exposure on intracellular iron (Fe) homeostasis and iron-regulatory protein (IRP) binding activity in undifferentiated PC12 cells over a range of manganese exposure concentrations (1, 10, 50, and 200 microM MnCl(2)) and exposure durations (12, 24, 36, and 48 hr), to test the hypothesis that moderately elevated manganese exposure disrupts cellular iron regulation. Results demonstrate that manganese exposure produces a rapid and sustained dose-dependent dysregulation of cellular iron metabolism, with effects occurring as early as 12 hr exposure and at manganese doses as low as 1 microM. Manganese exposure altered the dynamics of IRP-1 binding and the intracellular abundance of IRP-2, and altered the cellular abundance of transferrin receptor, ferritin, and mitochondrial aconitase protein levels. Cellular levels of labile iron were significantly increased with manganese exposure, although total cellular iron levels were not. The overall pattern of effects shows that manganese produced an inappropriate cellular response akin to iron deficiency, to which the cells were able to mount a compensatory response. Consistent with our previous studies, these data indicate that even low to moderate exposures to Manganese in vitro significantly disrupt cellular iron metabolism, which may be an important contributory mechanism of manganese neurotoxicity.

Desferal inhibits breast tumor growth and does not interfere with the tumoricidal activity of doxorubicin.

Desferal is a clinically approved iron chelator used to treat iron overload. Doxorubicin is an anthracycline cancer chemotherapy drug used in the treatment of breast cancer. It can undergo redox cycling in the presence of iron to produce reactive oxygen species. The oxidant-generating activity of doxorubicin is thought to be responsible for the cardiotoxic side effects of the drug, but it is unclear whether it is also required for its anti-tumor activity. To test whether an iron-chelating antioxidant would interfere with the tumor-killing activity of doxorubicin, nude mice were transplanted with xenografts of human breast cancer MDA-MB 231 cells and then treated with doxorubicin and/or desferal. Not only did desferal not interfere with the anti-tumor activity of doxorubicin, it inhibited tumor growth on its own. In vitro studies confirmed that desferal inhibits breast tumor growth. However, it did not induce apoptosis, nor did it induce cell cycle arrest. Instead, desferal caused cytostasis, apparently through iron depletion. The cytostatic activity of desferal was partially ameliorated by pretreatment with iron-saturated transferrin, and transferrin receptor expression on breast cancer cells nearly doubled after exposure to desferal. In contrast to its effect on tumor cells, desferal did not inhibit growth of normal breast epithelial cells. The data indicate that the anti-tumor activity of doxorubicin is not dependent on iron-mediated ROS production. Furthermore, desferal may have utility as an adjunctive chemotherapy due to its ability to inhibit breast tumor growth and cardiotoxic side effects without compromising the tumor-killing activity of an anthracycline chemotherapy drug.