Ferroptosis: A Regulated Cell Death Nexus Linking Metabolism, Redox Biology, and Disease.

Ferroptosis is a form of regulated cell death characterized by the iron-dependent accumulation of lipid hydroperoxides to lethal levels. Emerging evidence suggests that ferroptosis represents an ancient vulnerability caused by the incorporation of polyunsaturated fatty acids into cellular membranes, and cells have developed complex systems that exploit and defend against this vulnerability in different contexts. The sensitivity to ferroptosis is tightly linked to numerous biological processes, including amino acid, iron, and polyunsaturated fatty acid metabolism, and the biosynthesis of glutathione, phospholipids, NADPH, and coenzyme Q10. Ferroptosis has been implicated in the pathological cell death associated with degenerative diseases (i.e., Alzheimer's, Huntington's, and Parkinson's diseases), carcinogenesis, stroke, intracerebral hemorrhage, traumatic brain injury, ischemia-reperfusion injury, and kidney degeneration in mammals and is also implicated in heat stress in plants. Ferroptosis may also have a tumor-suppressor function that could be harnessed for cancer therapy. This Primer reviews the mechanisms underlying ferroptosis, highlights connections to other areas of biology and medicine, and recommends tools and guidelines for studying this emerging form of regulated cell death.

Systems biology of ferroptosis: A modeling approach.

Ferroptosis is a recently discovered form of iron-dependent regulated cell death (RCD) that occurs via peroxidation of phospholipids containing polyunsaturated fatty acid (PUFA) moieties. Activating this form of cell death is an emerging strategy in cancer treatment. Because multiple pathways and molecular species contribute to the ferroptotic process, predicting which tumors will be sensitive to ferroptosis is a challenge. We thus develop a mathematical model of several critical pathways to ferroptosis in order to perform a systems-level analysis of the process. We show that sensitivity to ferroptosis depends on the activity of multiple upstream cascades, including PUFA incorporation into the phospholipid membrane, and the balance between levels of pro-oxidant factors (reactive oxygen species, lipoxogynases) and antioxidant factors (GPX4). We perform a systems-level analysis of ferroptosis sensitivity as an outcome of five input variables (ACSL4, SCD1, ferroportin, transferrin receptor, and p53) and organize the resulting simulations into 'high' and 'low' ferroptosis sensitivity groups. We make a novel prediction corresponding to the combinatorial requirements of ferroptosis sensitivity to SCD1 and ACSL4 activity. To validate our prediction, we model the ferroptotic response of an ovarian cancer stem cell line following single- and double-knockdown of SCD1 and ACSL4. We find that the experimental outcomes are consistent with our simulated predictions. This work suggests that a systems-level approach is beneficial for understanding the complex combined effects of ferroptotic input, and in predicting cancer susceptibility to ferroptosis.

Iron and Cancer.

This review explores the multifaceted role that iron has in cancer biology. Epidemiological studies have demonstrated an association between excess iron and increased cancer incidence and risk, while experimental studies have implicated iron in cancer initiation, tumor growth, and metastasis. The roles of iron in proliferation, metabolism, and metastasis underpin the association of iron with tumor growth and progression. Cancer cells exhibit an iron-seeking phenotype achieved through dysregulation of iron metabolic proteins. These changes are mediated, at least in part, by oncogenes and tumor suppressors. The dependence of cancer cells on iron has implications in a number of cell death pathways, including ferroptosis, an iron-dependent form of cell death. Uniquely, both iron excess and iron depletion can be utilized in anticancer therapies. Investigating the efficacy of these therapeutic approaches is an area of active research that promises substantial clinical impact.

Activated Oncogenic Pathway Modifies Iron Network in Breast Epithelial Cells: A Dynamic Modeling Perspective.

Dysregulation of iron metabolism in cancer is well documented and it has been suggested that there is interdependence between excess iron and increased cancer incidence and progression. In an effort to better understand the linkages between iron metabolism and breast cancer, a predictive mathematical model of an expanded iron homeostasis pathway was constructed that includes species involved in iron utilization, oxidative stress response and oncogenic pathways. The model leads to three predictions. The first is that overexpression of iron regulatory protein 2 (IRP2) recapitulates many aspects of the alterations in free iron and iron-related proteins in cancer cells without affecting the oxidative stress response or the oncogenic pathways included in the model. This prediction was validated by experimentation. The second prediction is that iron-related proteins are dramatically affected by mitochondrial ferritin overexpression. This prediction was validated by results in the pertinent literature not used for model construction. The third prediction is that oncogenic Ras pathways contribute to altered iron homeostasis in cancer cells. This prediction was validated by a combination of simulation experiments of Ras overexpression and catalase knockout in conjunction with the literature. The model successfully captures key aspects of iron metabolism in breast cancer cells and provides a framework upon which more detailed models can be built.

DCYTB is a predictor of outcome in breast cancer that functions via iron-independent mechanisms.

BACKGROUND: Duodenal cytochrome b (DCYTB) is a ferrireductase that functions together with divalent metal transporter 1 (DMT1) to mediate dietary iron reduction and uptake in the duodenum. DCYTB is also a member of a 16-gene iron regulatory gene signature (IRGS) that predicts metastasis-free survival in breast cancer patients. To better understand the relationship between DCYTB and breast cancer, we explored in detail the prognostic significance and molecular function of DCYTB in breast cancer. METHODS: The prognostic significance of DCYTB expression was evaluated using publicly available microarray data. Signaling Pathway Impact Analysis (SPIA) of microarray data was used to identify potential novel functions of DCYTB. The role of DCYTB was assessed using immunohistochemistry and measurements of iron uptake, iron metabolism, and FAK signaling. RESULTS: High DCYTB expression was associated with prolonged survival in two large independent cohorts, together totaling 1610 patients (cohort #1, p = 1.6e-11, n = 741; cohort #2, p = 1.2e-05, n = 869; log-rank test) as well as in the Gene expression-based Outcome for Breast cancer Online (GOBO) cohort (p < 1.0e-05, n = 1379). High DCYTB expression was also associated with increased survival in homogeneously treated groups of patients who received either tamoxifen or chemotherapy. Immunohistochemistry revealed that DCYTB is localized on the plasma membrane of breast epithelial cells, and that expression is dramatically reduced in high-grade tumors. Surprisingly, neither overexpression nor knockdown of DCYTB affected levels of ferritin H, transferrin receptor, labile iron or total cellular iron in breast cancer cells. Because SPIA pathway analysis of patient microarray data revealed an association between DCYTB and the focal adhesion pathway, we examined the influence of DCYTB on FAK activation in breast cancer cells. These experiments reveal that DCYTB reduces adhesion and activation of focal adhesion kinase (FAK) and its adapter protein paxillin. CONCLUSIONS: DCYTB is an important predictor of outcome and is associated with response to therapy in breast cancer patients. DCYTB does not affect intracellular iron in breast cancer cells. Instead, DCYTB may retard cancer progression by reducing activation of FAK, a kinase that plays a central role in tumor cell adhesion and metastasis.

Yet another function of p53--the switch that determines whether radiation-induced autophagy will be cytoprotective or nonprotective: implications for autophagy inhibition as a therapeutic strategy.

The influence of autophagy inhibition on radiation sensitivity was studied in human breast, head and neck, and non-small cell lung cancer cell lines, in cell lines that were either wild type or mutant/null in p53, and in cells where p53 was inducible or silenced. Whereas ionizing radiation promoted autophagy in all tumor cell lines studied, pharmacological inhibition of autophagy and/or genetic silencing of autophagy genes failed to influence sensitivity to radiation in p53 mutant Hs578t breast tumor cells, HN6 head and neck tumor cells, and H358 non-small cell lung cancer cells. The requirement for functional p53 in the promotion of cytoprotective autophagy by radiation was confirmed by the observation that radiation-induced autophagy was nonprotective in p53 null H1299 cells but was converted to the cytoprotective form with induction of p53. Conversely, whereas p53 wild-type HN30 head and neck cancer cells did show sensitization to radiation upon autophagy inhibition, HN30 cells in which p53 was knocked down using small hairpin RNA failed to be sensitized by pharmacological autophagy inhibition. Taken together, these findings indicate that radiation-induced autophagy can be either cytoprotective or nonprotective, a functional difference related to the presence or absence of function p53. Alternatively, these findings could be interpreted to suggest that whereas radiation can induce autophagy independent of p53 status, inhibition of autophagy promotes enhanced radiation sensitivity through a mechanism that requires functional p53. These observations are likely to have direct implications with respect to clinical efforts to modulate the response of malignancies to radiation through autophagy inhibition.

Mass spectrometry imaging of metals in tissues and cells: Methods and biological applications.

BACKGROUND: Metals are pervasive throughout biological processes, where they play essential structural and catalytic roles. Metals can also exhibit deleterious effects on human health. Powerful analytical techniques, such as mass spectrometry imaging (MSI), are required to map metals due to their low concentrations within biological tissue. SCOPE OF REVIEW: This Mini Review focuses on key MSI technology that can image metal distributions in situ, describing considerations for each technique (e.g., resolution, sensitivity, etc.). We highlight recent work using MSI for mapping trace metals in tissues, detecting metal-based drugs, and simultaneously imaging metals and biomolecules. MAJOR CONCLUSIONS: MSI has enabled significant advances in locating bioactive metals at high spatial resolution and correlating their distributions with that of biomolecules. The use of metal-based immunochemistry has enabled simultaneous high-throughput protein and biomolecule imaging. GENERAL SIGNIFICANCE: The techniques and examples described herein can be applied to many biological questions concerning the important biological roles of metals, metal toxicity, and localization of metal-based drugs.

Angiotensin-(1-7) reduces proliferation and angiogenesis of human prostate cancer xenografts with a decrease in angiogenic factors and an increase in sFlt-1.

BACKGROUND: Prostate cancer is the most frequently diagnosed malignancy and the second-leading cause of cancer death in men. The purpose of this study was to determine the anti-proliferative and anti-angiogenic efficacy of angiotensin-(1-7) [Ang-(1-7)], an endogenous peptide hormone, in human prostate cancer xenografts. METHODS: Human LNCaP prostate cancer cells were injected into the flank of athymic mice and tumors were treated with Ang-(1-7) for 54 days. Tumor growth and angiogenesis were determined by immunohistochemistry and western blot hybridization. RESULTS: Ang-(1-7) markedly reduced the volume and wet weight of LNCaP xenograft tumors. Histological analysis of tumor sections from saline-treated mice showed increased Ki67 immunoreactivity and enhanced phosphorylation of the MAP kinases ERK1/2 compared to tumors from Ang-(1-7)-treated mice, suggesting that the heptapeptide reduces cell proliferation. Intratumoral vessel density was decreased in Ang-(1-7)-treated mice with an associated reduction in vascular endothelial growth factor (VEGF) and placental growth factor (PlGF), suggesting that the heptapeptide attenuates vascularization by reducing angiogenic factors. Ang-(1-7) administration markedly increased the soluble fraction of VEGF receptor 1 (sFlt-1), with a concomitant reduction in VEGF receptors 1 and 2. sFlt-1 serves as a decoy receptor that traps VEGF and PlGF, making the ligands unavailable to membrane-bound VEGF receptors and preventing activation of pro-angiogenic signaling. CONCLUSIONS: The decrease in PlGF and VEGF coupled with the increase in sFlt-1 suggests that Ang-(1-7) may serve as a novel anti-angiogenic therapy for prostate cancer. Further, the pleiotropic mechanisms of action by Ang-(1-7) may limit angiogenic resistance that occurs with VEGF inhibitors or receptor blockers.

Angiotensin-(1-7) attenuates metastatic prostate cancer and reduces osteoclastogenesis.

BACKGROUND: Angiotensin-(1-7) [Ang-(1-7)] is an endogenous, heptapeptide hormone with anti-proliferative and anti-angiogenic properties. The primary objective of this study was to determine whether Ang-(1-7) effectively reduces prostate cancer metastasis in mice. METHODS: Human PC3 prostate cancer cells were injected into the aortic arch via the carotid artery of SCID mice pre-treated with Ang-(1-7) or injected into the tibia of athymic mice, administered Ang-(1-7) for 5 weeks beginning 2 weeks post-injection. Tumor growth and volume were determined by bioluminescent and magnetic resonance imaging. The presence of tumors was confirmed by hematoxylin and eosin staining; TRAP histochemistry was used to identify osteolytic lesions. The effect of Ang-(1-7) on osteoclastogenesis was assessed in differentiated bone marrow cells. RESULTS: Pre-treatment with Ang-(1-7) prevented metastatic tumor formation following intra-aortic injection of PC3 cells, while 83% of untreated mice developed tumors in metastatic sites. Circulating VEGF was significantly higher in control mice compared to mice administered Ang-(1-7). A 5-week regimen of the heptapeptide hormone attenuated intra-tibial tumor growth; Ang-(1-7) was significantly higher in the tibia of treated mice than in control animals. Osteoclastogenesis was reduced by 50% in bone marrow cells differentiated in the presence of Ang-(1-7), suggesting that the heptapeptide hormone prevents the formation of osteolytic lesions to reduce tumor survival in the bone microenvironment. CONCLUSIONS: These findings suggest that Ang-(1-7) may serve as an anti-angiogenic and anti-metastatic agent for advanced prostate cancer. By extension, the heptapeptide hormone may provide effective therapy for bone metastasis produced from primary tumors of the lung and breast.

Ferritin H is a novel marker of early erythroid precursors and macrophages.

AIMS: Macrophages play a critical role in iron homeostasis by recycling iron from red cells and storing it in ferritin, an iron storage protein. The recycled iron is delivered to erythroid precursors for erythropoiesis. In this study, we aimed to determine whether ferritin is highly expressed in macrophages and erythroid precursors, and whether it can be used as a marker for these two cell types. METHODS AND RESULTS: A ferritin monoclonal antibody was developed, and immunohistochemistry was performed. In normal bone marrows, ferritin antibody stained early erythroid precursors and macrophages. In contrast, myeloid cells, lymphoid cells and megakaryocytes lacked ferritin expression. In leukaemic bone marrows, ferritin was selectively expressed in erythroid blasts (M6), whereas all other blasts were negative. In lymph nodes, ferritin was highly and specifically expressed in macrophages, whereas lymphocytes completely lacked ferritin expression. In non-haematopoietic tissues, ferritin antibody highlighted alveolar macrophages in the lung, as well as sinus macrophages in the liver and spleen. CONCLUSIONS: We conclude that ferritin is a novel and reliable marker for macrophages and early erythroid precursors, and may be of clinical utility in the diagnosis of diseases associated with these two cell types.

Binding and uptake of H-ferritin are mediated by human transferrin receptor-1.

Ferritin is a spherical molecule composed of 24 subunits of two types, ferritin H chain (FHC) and ferritin L chain (FLC). Ferritin stores iron within cells, but it also circulates and binds specifically and saturably to a variety of cell types. For most cell types, this binding can be mediated by ferritin composed only of FHC (HFt) but not by ferritin composed only of FLC (LFt), indicating that binding of ferritin to cells is mediated by FHC but not FLC. By using expression cloning, we identified human transferrin receptor-1 (TfR1) as an important receptor for HFt with little or no binding to LFt. In vitro, HFt can be precipitated by soluble TfR1, showing that this interaction is not dependent on other proteins. Binding of HFt to TfR1 is partially inhibited by diferric transferrin, but it is hindered little, if at all, by HFE. After binding of HFt to TfR1 on the cell surface, HFt enters both endosomes and lysosomes. TfR1 accounts for most, if not all, of the binding of HFt to mitogen-activated T and B cells, circulating reticulocytes, and all cell lines that we have studied. The demonstration that TfR1 can bind HFt as well as Tf raises the possibility that this dual receptor function may coordinate the processing and use of iron by these iron-binding molecules.

Regulatory effects of ferritin on angiogenesis.

Angiogenesis, the synthesis of new blood vessels from preexisting vessels, plays a critical role in normal wound healing and tumor growth. HKa (cleaved high molecular weight kininogen) is an endogenous inhibitor of angiogenesis formed by the cleavage of kininogen on endothelial cells. Ferritin is a protein principally known for its central role in iron storage. Here, we demonstrate that ferritin binds to HKa with high affinity (K(d) 13 nM). Further, ferritin antagonizes the antiangiogenic effects of HKa, enhancing the migration, assembly, and survival of HKa-treated endothelial cells. Effects of ferritin were independent of its iron content. Peptide mapping revealed that ferritin binds to a 22-aa subdomain of HKa that is critical to its antiangiogenic activity. In vivo, ferritin opposed HKa's antiangiogenic effects in a human prostate cancer xenograft, restoring tumor-dependent vessel growth. Ferritin-mediated regulation of angiogenesis represents a new angiogenic regulatory pathway, and identifies a new role for ferritin in cell biology.

Long-term survival following a single treatment of kidney tumors with multiwalled carbon nanotubes and near-infrared radiation.

Multiwalled carbon nanotubes (MWCNTs) exhibit physical properties that render them ideal candidates for application as noninvasive mediators of photothermal cancer ablation. Here, we demonstrate that use of MWCNTs to generate heat in response to near-infrared radiation (NIR) results in thermal destruction of kidney cancer in vitro and in vivo. We document the thermal effects of the therapy through magnetic resonance temperature-mapping and heat shock protein-reactive immunohistochemistry. Our results demonstrate that use of MWCNTs enables ablation of tumors with low laser powers (3 W/cm(2)) and very short treatment times (a single 30-sec treatment) with minimal local toxicity and no evident systemic toxicity. These treatment parameters resulted in complete ablation of tumors and a >3.5-month durable remission in 80% of mice treated with 100 microg of MWCNT. Use of MWCNTs with NIR may be effective in anticancer therapy.

Hereditary Hemochromatosis Variant Associations with Incident Nonliver Malignancies: 11-Year Follow-up in UK Biobank.

BACKGROUND: In European ancestry populations, iron overload disorder hereditary hemochromatosis is predominantly caused by HFE p.C282Y and p.H63D mutations. Male p.C282Y homozygotes have markedly increased hepatic malignancy incidence, but risks for other cancers in male and female homozygotes are unclear. METHODS: 451,143 UK Biobank European ancestry participants (aged 40-70 years; 54.3% female) were followed (mean 11.6 years) via hospital admissions and national cancer registries. We estimated risks of any incident cancer (other than nonmelanoma and liver cancer) and common incident cancers [bladder, blood (with subanalyses of leukemia and lymphoma), bone, brain, breast, colorectal, kidney, lung, melanoma, esophageal, ovarian, pancreatic, prostate and stomach] in those with p.C282Y and p.H63D genotypes, compared with participants without HFE mutations. RESULTS: Male p.C282Y homozygotes (n = 2,890, 12.1% with baseline diagnosed hereditary hemochromatosis) had increased incidence of prostate cancer [6.8% vs. 5.4% without mutations; HR = 1.32; 95% confidence interval (CI), 1.07-1.63; P = 0.01; Bonferroni adjusted P = 0.17] during follow-up. In life table estimates from ages 40 to 75 years, 14.4% of male p.C282Y homozygotes are projected to develop prostate cancer (versus 10.7% without mutations, excess 3.8%; 95% CI, 1.3-6.8). No increases in risks were found for other studied cancers in male or female p.C282Y homozygotes, or in any other p.C282Y/p.H63D genotype groups of either sex. CONCLUSIONS: In a large community sample of male p.C282Y homozygotes, there is suggestive evidence of increased prostate cancer incidence, with no evidence of excess of other studied (nonliver) cancers. IMPACT: Replication of results in other large community genotyped cohorts are needed to confirm if clinical monitoring for prostate cancer is necessary in p.C282Y homozygous males.

Complementary anti-cancer pathways triggered by inhibition of sideroflexin 4 in ovarian cancer.

DNA damaging agents are a mainstay of standard chemotherapy for ovarian cancer. Unfortunately, resistance to such DNA damaging agents frequently develops, often due to increased activity of DNA repair pathways. Sideroflexin 4 (SFXN4) is a little-studied inner mitochondrial membrane protein. Here we demonstrate that SFXN4 plays a role in synthesis of iron sulfur clusters (Fe-S) in ovarian cancer cells and ovarian cancer tumor-initiating cells, and that knockdown of SFXN4 inhibits Fe-S biogenesis in ovarian cancer cells. We demonstrate that this has two important consequences that may be useful in anti-cancer therapy. First, inhibition of Fe-S biogenesis triggers the accumulation of excess iron, leading to oxidative stress. Second, because enzymes critical to multiple DNA repair pathways require Fe-S clusters for their function, DNA repair enzymes and DNA repair itself are inhibited by reduction of SFXN4. Through this dual mechanism, SFXN4 inhibition heightens ovarian cancer cell sensitivity to DNA-damaging drugs and DNA repair inhibitors used in ovarian cancer therapy, such as cisplatin and PARP inhibitors. Sensitization is achieved even in drug resistant ovarian cancer cells. Further, knockout of SFXN4 decreases DNA repair and profoundly inhibits tumor growth in a mouse model of ovarian cancer metastasis. Collectively, these results suggest that SFXN4 may represent a new target in ovarian cancer therapy.

Serum ferritin: Past, present and future.

BACKGROUND: Serum ferritin was discovered in the 1930s, and was developed as a clinical test in the 1970s. Many diseases are associated with iron overload or iron deficiency. Serum ferritin is widely used in diagnosing and monitoring these diseases. SCOPE OF REVIEW: In this chapter, we discuss the role of serum ferritin in physiological and pathological processes and its use as a clinical tool. MAJOR CONCLUSIONS: Although many aspects of the fundamental biology of serum ferritin remain surprisingly unclear, a growing number of roles have been attributed to extracellular ferritin, including newly described roles in iron delivery, angiogenesis, inflammation, immunity, signaling and cancer. GENERAL SIGNIFICANCE: Serum ferritin remains a clinically useful tool. Further studies on the biology of this protein may provide new biological insights.

TIM-2 is expressed on B cells and in liver and kidney and is a receptor for H-ferritin endocytosis.

T cell immunoglobulin-domain and mucin-domain (TIM) proteins constitute a receptor family that was identified first on kidney and liver cells; recently it was also shown to be expressed on T cells. TIM-1 and -3 receptors denote different subsets of T cells and have distinct regulatory effects on T cell function. Ferritin is a spherical protein complex that is formed by 24 subunits of H- and L-ferritin. Ferritin stores iron atoms intracellularly, but it also circulates. H-ferritin, but not L-ferritin, shows saturable binding to subsets of human T and B cells, and its expression is increased in response to inflammation. We demonstrate that mouse TIM-2 is expressed on all splenic B cells, with increased levels on germinal center B cells. TIM-2 also is expressed in the liver, especially in bile duct epithelial cells, and in renal tubule cells. We further demonstrate that TIM-2 is a receptor for H-ferritin, but not for L-ferritin, and expression of TIM-2 permits the cellular uptake of H-ferritin into endosomes. This is the first identification of a receptor for ferritin and reveals a new role for TIM-2.

SOSTDC1 differentially modulates Smad and beta-catenin activation and is down-regulated in breast cancer.

Sclerostin domain containing 1 (SOSTDC1) protein regulates processes from development to cancer by modulating activity of bone morphogenetic protein (BMP) and wingless/int (Wnt) signaling pathways. As dysregulation of both BMP and Wnt signaling has been observed in breast cancer, we investigated whether disruption of SOSTDC1 signaling occurs in breast cancer. SOSTDC1 mRNA expression levels in breast tissue were examined using a dot blot. Affymetrix microarray data on SOSTDC1 levels were correlated with breast cancer patient survival using Kaplan-Meier plots. Correlations between SOSTDC1 protein levels and clinical parameters were assessed by immunohistochemistry of a breast cancer tissue microarray. SOSTDC1 secretion and BMP and Wnt signaling were investigated using immunoblotting. We found that SOSTDC1 is expressed in normal breast tissue and this expression is reduced in breast cancer. High levels of SOSTDC1 mRNA correlated with increased patient survival; conversely, SOSTDC1 protein levels decreased as tumor size and disease stage increased. Treatment of breast cancer cells with recombinant SOSTDC1 or Wise, a SOSTDC1 orthologue, demonstrated that SOSTDC1 selectively blocks BMP-7-induced Smad phosphorylation without diminishing BMP-2 or Wnt3a-induced signaling. In conclusion, SOSTDC1 mRNA and protein are reduced in breast cancer. High SOSTDC1 mRNA levels correlate with increased distant metastasis-free survival in breast cancer patients. SOSTDC1 differentially affects Wnt3a, BMP-2, and BMP-7 signaling in breast cancer cells. These results identify SOSTDC1 as a clinically important extracellular regulator of multiple signaling pathways in breast cancer.

Curcumin, a cancer chemopreventive and chemotherapeutic agent, is a biologically active iron chelator.

Curcumin is a natural product currently in human clinical trials for a variety of neoplastic, preneoplastic, and inflammatory conditions. We previously observed that, in cultured cells, curcumin exhibits properties of an iron chelator. To test whether the chelator activity of curcumin is sufficient to induce iron deficiency in vivo, mice were placed on diets containing graded concentrations of both iron and curcumin for 26 weeks. Mice receiving the lowest level of dietary iron exhibited borderline iron deficiency, with reductions in spleen and liver iron, but little effect on hemoglobin, hematocrit, transferrin saturation, or plasma iron. Against this backdrop of subclinical iron deficiency, curcumin exerted profound 2 effects on systemic iron, inducing a dose-dependent decline in hematocrit, hemoglobin, serum iron, and transferrin saturation, the appearance of microcytic anisocytotic red blood cells, and decreases in spleen and liver iron content. Curcumin repressed synthesis of hepcidin, a peptide that plays a central role in regulation of systemic iron balance. These results demonstrate that curcumin has the potential to affect systemic iron metabolism, particularly in a setting of subclinical iron deficiency. This may affect the use of curcumin in patients with marginal iron stores or those exhibiting the anemia of cancer and chronic disease.