Total Cellular Iron Research Articles

Magnetic Resonance Imaging of Mammalian Cells Individually Expressing Membrane-Associated Magnetosome Proteins I, L, B, and E

The magnetosome, a membrane-bound iron biomineral formed within magnetotactic bacteria, is a unique model for a magnetic resonance imaging (MRI) reporter gene contrast agent. We are translating this technology to mammalian cells by expressing essential magnetosome genes mamI, mamL, mamB, and mamE into MDA-MB-435 human melanoma cells. Currently, these genes are individually expressed in the cell although the future goal is to express all four genes together. We examined the influence of these genes on cellular MRI relaxation rates by culturing cells in the presence and absence of iron-supplemented medium and scanning them at 3 Tesla using a gelatin phantom. Total cellular iron was measured by inductively-coupled plasma mass spectrometry and correlated with relaxation rates obtained from phantom experiments. Apart from mamE, magnetosome genes that are individually expressed in mammalian cells grown in iron supplement significantly affected cellular transverse relaxation rates compared to cells grown without iron supplement. Interestingly, mamI, mamL, mamB, and mamE (even though the latter had no effect on relaxation rate) significantly affected cellular iron content. This developing gene-based contrast agent will equip MRI with improvement to imaging sensitivity and the technology to track cellular activities long term.

Hydroxysafflor Yellow A Alleviates Acute Myocardial Ischemia/Reperfusion Injury in Mice by Inhibiting Ferroptosis via the Activation of the HIF-1α/SLC7A11/GPX4 Signaling Pathway.

Ferroptosis is closely associated with the pathophysiology of myocardial ischemia. Hydroxysafflor yellow A (HSYA), the main active ingredient in the Chinese herbal medicine safflower, exerts significant protective effects against myocardial ischemia/reperfusion injury (MI/RI). The aim of this study was to investigate the protective effects of HSYA against MI/RI and identify the putative underlying mechanisms. An in vivo model of acute MI/RI was established in C57 mice. Subsequently, the effects of HSYA on myocardial tissue injury were evaluated by histology. Lipid peroxidation and myocardial injury marker contents in myocardial tissue and serum and iron contents in myocardial tissue were determined using biochemical assays. Mitochondrial damage was assessed using transmission electron microscopy. H9C2 cardiomyocytes were induced in vitro by oxygen-glucose deprivation/reoxygenation, and ferroptosis inducer erastin was administered to detect ferroptosis-related indicators, oxidative-stress-related indicators, and expressions of ferroptosis-related proteins and HIF-1α. In MI/RI model mice, HSYA reduced myocardial histopathological damage, ameliorated mitochondrial damage in myocardial cells, and decreased total cellular iron and ferrous ion contents in myocardial tissue. HSYA increased the protein levels of SLC7A11, HIF-1α, and GPX4 and mitigated erastin- or HIF-1α siRNA-induced damage in H9C2 cells. In summary, HSYA alleviated MI/RI by activating the HIF-1α/SLC7A11/GPX4 signaling pathway, thereby inhibiting ferroptosis.

PTEN-induced kinase PINK1 supports colorectal cancer growth by regulating the labile iron pool

Mitophagy is a cargo-specific autophagic process that recycles damaged mitochondria to promote mitochondrial turnover. PTEN-induced putative kinase 1 (PINK1) mediates the canonical mitophagic pathway. However, the role of PINK1 in diseases where mitophagy has been purported to play a role, such as colorectal cancer, is unclear. Our results here demonstrate that higher PINK1 expression is positively correlated with decreased colon cancer survival, and mitophagy is required for colon cancer growth. We show that doxycycline-inducible knockdown (KD) of PINK1 in a panel of colon cancer cell lines inhibited proliferation, whereas disruption of other mitophagy receptors did not impact cell growth. We observed that PINK KD led to a decrease in mitochondrial respiration, membrane hyperpolarization, accumulation of mitochondrial DNA, and depletion of antioxidant glutathione. In addition, mitochondria are important hubs for the utilization of iron and synthesizing iron-dependent cofactors such as heme and iron sulfur clusters. We observed an increase in the iron storage protein ferritin and a decreased labile iron pool in the PINK1 KD cells, but total cellular iron or markers of iron starvation/overload were not affected. Finally, cellular iron storage and the labile iron pool are maintainedviaautophagic degradation of ferritin (ferritinophagy). We found overexpressing nuclear receptor coactivator 4, a key adaptor for ferritinophagy, rescued cell growth and the labile iron pool in PINK1 KD cells. These results indicate that PINK1 integrates mitophagy and ferritinophagy to regulate intracellular iron availability and is essential for maintaining intracellular iron homeostasis to support survival and growth in colorectal cancer cells.

Monocyte MRI Relaxation Rates Are Regulated by Extracellular Iron and Hepcidin.

Many chronic inflammatory conditions are mediated by an increase in the number of monocytes in peripheral circulation, differentiation of monocytes to macrophages, and different macrophage subpopulations during pro- and anti-inflammatory stages of tissue injury. When hepcidin secretion is stimulated during inflammation, the iron export protein ferroportin is targeted for degradation on a limited number of cell types, including monocytes and macrophages. Such changes in monocyte iron metabolism raise the possibility of non-invasively tracking the activity of these immune cells using magnetic resonance imaging (MRI). We hypothesized that hepcidin-mediated changes in monocyte iron regulation influence both cellular iron content and MRI relaxation rates. In response to varying conditions of extracellular iron supplementation, ferroportin protein levels in human THP-1 monocytes decreased two- to eightfold, consistent with paracrine/autocrine regulation of iron export. Following hepcidin treatment, ferroportin protein levels further decreased two- to fourfold. This was accompanied by an approximately twofold increase in total transverse relaxation rate, R2*, compared to non-supplemented cells. A positive correlation between total cellular iron content and R2* improved from moderate to strong in the presence of hepcidin. These findings suggest that hepcidin-mediated changes detected in monocytes using MRI could be valuable for in vivo cell tracking of inflammatory responses.

NCOA4 Regulates Iron Recycling and Responds to Hepcidin Activity and Lipopolysaccharide in Macrophages.

Macrophages, via erythrophagocytosis, recycle iron from effete erythrocytes to newly developing red blood cells. Conversion of potentially cytotoxic levels of iron from its heme into nonheme form during iron recycling is safely accomplished via coordinated regulations of cellular iron transport and homeostasis. Herein, we demonstrate the roles and regulation of NCOA4 (nuclear receptor coactivator 4)-mediated ferritinophagy in macrophages after erythrophagocytosis using the mouse macrophage cell line J774 cells. Ferritin in J774 cells increased with the rise of nonheme iron by erythrocyte ingestion and declined when total cellular iron contents subsequently decreased. NCOA4, a selective autophagic cargo receptor for ferritin, was responsible for the control of cellular ferritin and total iron contents at the later stage of erythrophagocytosis. A hepcidin analog, which limits the flux of iron through iron-recycling by inhibiting iron export at the plasma membrane, repressed NCOA4 expression and led to accumulation of ferritin in the mouse macrophages. Transcriptome analyses revealed a functional association of immune response with NCOA4-dependent gene expressions, and we confirmed repression of Ncoa4 by lipopolysaccharide (LPS) in J774 cells and the spleen of mice. Collectively, our studies indicate that NCOA4 facilitates cellular ferritin turnover and the release of iron by macrophages after erythrophagocytosis and functions as a regulatory target for molecular signals of systemic iron overload and inflammation. These identify macrophage NCOA4 as a potential therapeutic target for disorders of systemic iron dysregulation, including anemia of inflammation and hemochromatosis.

The role of mitochondria in the recovery of neurons after injury.

The role of mitochondria in the recovery of neurons after injury.

Synchrotron fluorescence imaging of individual mouse beta-cells reveals changes in zinc, calcium, and iron in a model of low-grade inflammation.

Pancreatic beta-cells synthesize and secrete insulin maintaining an organism's energy homeostasis. In humans, beta-cell dysfunction and death contribute to the pathogenesis of type 2 diabetes (T2D). Although the causes of beta-cell dysfunction are complex, obesity-induced low-grade systemic inflammation plays a role. For example, obese individuals exhibiting increased levels of proinflammatory cytokines IL-6 and IL-1beta have a higher risk of beta-cell dysfunction and T2D. Interestingly, obesity-induced inflammation changes the expression of several cellular metal regulating genes, prompting this study to examine changes in the beta-cell metallome after exposure to proinflammatory-cytokines. Primary mouse beta-cells were exposed to a combination of IL-6 and IL-1beta for 48 hours, were chemically fixed and imaged by synchrotron X-ray fluorescent microscopy. Quantitative analysis showed a surprising 2.4-fold decrease in the mean total cellular content of zinc from 158± 57.7 femtograms (fg) to 65.7± 29.7 fg; calcium decreased from 216± 67.4 to 154.3± 68.7 fg (control vs. cytokines, respectively). The mean total cellular iron content slightly increased from 30.4± 12.2 to 47.2± 36.4 fg after cytokine treatment; a sub-population of cells (38%) exhibited larger increases of iron density. Changes in the subcellular distributions of zinc and calcium were observed after cytokine exposure. Beta-cells contained numerous iron puncta that accumulated still more iron after exposure to cytokines. These findings provide evidence that exposure to low levels of cytokines is sufficient to cause changes in the total cellular content and/or subcellular distribution of several metals known to be critical for normal beta-cell function.

Excessive Iron Induces Oxidative Stress Promoting Cellular Perturbations and Insulin Secretory Dysfunction in MIN6 Beta Cells.

Exposure to high levels of glucose and iron are co-related to reactive oxygen species (ROS) generation and dysregulation of insulin synthesis and secretion, although the precise mechanisms are not well clarified. The focus of this study was to examine the consequences of exposure to high iron levels on MIN6 β-cells. MIN6 pseudoislets were exposed to 20 µM (control) or 100 µM (high) iron at predefined glucose levels (5.5 mM and 11 mM) at various time points (3, 24, 48, and 72 h). Total iron content was estimated by a colourimetric FerroZine™ assay in presence or absence of transferrin-bound iron. Cell viability was assessed by a resazurin dye-based assay, and ROS-mediated cellular oxidative stress was assessed by estimating malondialdehyde levels. β-cell iron absorption was determined by a ferritin immunoassay. Cellular insulin release and content was measured by an insulin immunoassay. Expression of SNAP-25, a key protein in the core SNARE complex that modulates vesicle exocytosis, was measured by immunoblotting. Our results demonstrate that exposure to high iron levels resulted in a 15-fold (48 h) and 4-fold (72 h) increase in cellular iron accumulation. These observations were consistent with data from oxidative stress analysis which demonstrated 2.7-fold higher levels of lipid peroxidation. Furthermore, exposure to supraphysiological (11 mM) levels of glucose and high iron (100 µM) at 72 h exerted the most detrimental effect on the MIN6 β-cell viability. The effect of high iron exposure on total cellular iron content was identical in the presence or absence of transferrin. High iron exposure (100 µM) resulted in a decrease of MIN6 insulin secretion (64% reduction) as well as cellular insulin content (10% reduction). Finally, a significant reduction in MIN6 β-cell SNAP-25 protein expression was evident at 48 h upon exposure to 100 µM iron. Our data suggest that exposure to high iron and glucose concentrations results in cellular oxidative damage and may initiate insulin secretory dysfunction in pancreatic β-cells by modulation of the exocytotic machinery.

Exposure to cytokines IL‐6 and IL‐1beta changes intracellular distributions and total content of calcium, zinc and iron in mouse primary pancreatic beta‐cells

Pancreatic beta-cells secrete insulin, a hormone that maintains blood glucose homeostasis. The dysfunction of beta-cells leads to development of type2 diabetes (T2D). The causes of beta-cell dysfunction in T2D are complex. In obese individuals, increases in circulating proinflammatory cytokines IL-6 and IL-1beta are linked to a higher risk of developing diabetes, suggesting that these cytokines can be diabetes-promoting. When used at circulating concentrations found in obese individuals, these cytokines have been shown to alter calcium homeostasis in mouse beta-cells. In addition, changes in homeostasis of zinc and iron have been reported in diabetic individuals. The central hypothesis of this study: exposure to diabetes-promoting cytokines, at concentrations found in obese individuals, leads to changes in metal intracellular concentrations or distributions in beta-cells. Mouse primary beta-cells were exposed for 48 hours to cytokines: 10pg/mL IL-1beta and 20pg/mL IL-6. After cytokine exposure the cells were chemically fixed and synchrotron X-Ray fluorescence (SXRF) was employed, to investigate intracellular distributions and concentrations of zinc, calcium and iron. SXRF estimated the total cellular iron content to be 30.44 ± 12.18 (fg), and after acute exposure to cytokines the iron content was slightly increased to 47.21 ± 36.44 (fg) (mean ± S.D.). High concentrations of iron were localized in puncta structures, which were observed throughout the cytosol in all beta-cells and further analysis of the iron puncta revealed that iron was found at higher density in puncta of cells after they were exposed to cytokines, suggesting iron accumulation in these structures. The total cellular zinc content was 158.69 ± 57.68 (fg) in control cells and was significantly decreased to 65.73 ± 29.65 (fg) in cells after exposure to cytokines (mean ± S.D.) (Welch's t-test, t(28.97) = 6.655, P<0.0001). Zinc was localized to a perinuclear space of the cells in control cells and was evenly distributed after exposure to cytokines, suggesting that cytokines had a significant effect on zinc homeostasis in beta-cells. Calcium was also observed in perinuclear space of beta-cells before cytokine exposure and was localized to similar cellular compartments as zinc. Synchrotron X-ray fluorescence estimated total cellular calcium to be 216.10 ± 67.37 femtograms (fg) control cells and was significantly decreased to 154.28 ± 68.66 (fg) after the exposure to cytokines (mean ± S.D.), (Two sample t-test, t(43)=3.040, P=0.0040). In conclusion, the synchrotron X-ray fluorescence identified significant changes to zinc, calcium and iron metallomes of pancreatic beta-cells after exposure to cytokines, which warrant further investigation.

Ferrotoxicity and Its Amelioration by Calcitriol in Cultured Renal Cells.

Globally, acute kidney injury (AKI) is associated with significant mortality and an enormous economic burden. Whereas iron is essential for metabolically active renal cells, it has the potential to cause renal cytotoxicity by promoting Fenton chemistry-based oxidative stress involving lipid peroxidation. In addition, 1,25-dihydroxyvitamin D3 (calcitriol), the active form of vitamin D, is reported to have an antioxidative role. In this study, we intended to demonstrate the impact of vitamin D on iron-mediated oxidant stress and cytotoxicity of Vero cells exposed to iohexol, a low osmolar iodine-containing contrast media in vitro. Cultured Vero cells were pretreated with 1,25-dihydroxyvitamin D3 dissolved in absolute ethanol (0.05%, 2.0 mM) at a dose of 1 mM for 6 hours. Subsequently, iohexol was added at a concentration of 100 mg iodine per mL and incubated for 3 hours. Total cellular iron content was analysed by a flame atomic absorption spectrophotometer at 372 nm. Lipid peroxidation was determined by TBARS (thiobarbituric acid reactive species) assay. Antioxidants including total thiol content were assessed by Ellman's method, catalase by colorimetric method, and superoxide dismutase (SOD) by nitroblue tetrazolium assay. The cells were stained with DAPI (4′,6-diamidino-2-phenylindole), and the cytotoxicity was evaluated by viability assay (MTT assay). The results indicated that iohexol exposure caused a significant increase of the total iron content in Vero cells. A concomitant increase of lipid peroxidation and decrease of total thiol protein levels, catalase, and superoxide dismutase activity were observed along with decreased cell viability in comparison with the controls. Furthermore, these changes were significantly reversed when the cells were pretreated with vitamin D prior to incubation with iohexol. Our findings of this in vitro model of iohexol-induced renotoxicity lend further support to the nephrotoxic potential of iron and underpin the possible clinical utility of vitamin D for the treatment and prevention of AKI.

Harmful Iron-Calcium Relationship in Pantothenate kinase Associated Neurodegeneration.

Pantothenate Kinase-associated Neurodegeneration (PKAN) belongs to a wide spectrum of diseases characterized by brain iron accumulation and extrapyramidal motor signs. PKAN is caused by mutations in PANK2, encoding the mitochondrial pantothenate kinase 2, which is the first enzyme of the biosynthesis of Coenzyme A. We established and characterized glutamatergic neurons starting from previously developed PKAN Induced Pluripotent Stem Cells (iPSCs). Results obtained by inductively coupled plasma mass spectrometry indicated a higher amount of total cellular iron in PKAN glutamatergic neurons with respect to controls. PKAN glutamatergic neurons, analyzed by electron microscopy, exhibited electron dense aggregates in mitochondria that were identified as granules containing calcium phosphate. Calcium homeostasis resulted compromised in neurons, as verified by monitoring the activity of calcium-dependent enzyme calpain1, calcium imaging and voltage dependent calcium currents. Notably, the presence of calcification in the internal globus pallidus was confirmed in seven out of 15 genetically defined PKAN patients for whom brain CT scan was available. Moreover, we observed a higher prevalence of brain calcification in females. Our data prove that high amount of iron coexists with an impairment of cytosolic calcium in PKAN glutamatergic neurons, indicating both, iron and calcium dys-homeostasis, as actors in pathogenesis of the disease.

Hepcidin-mediated Iron Regulation in P19 Cells is Detectable by Magnetic Resonance Imaging

Magnetic resonance imaging can be used to track cellular activities in the body using iron-based contrast agents. However, multiple intrinsic cellular iron handling mechanisms may also influence the detection of magnetic resonance (MR) contrast: a need to differentiate among those mechanisms exists. In hepcidin-mediated inflammation, for example, downregulation of iron export in monocytes and macrophages involves post-translational degradation of ferroportin. We examined the influence of hepcidin endocrine activity on iron regulation and MR transverse relaxation rates in multi-potent P19 cells, which display high iron import and export activities, similar to alternatively-activated macrophages. Iron import and export were examined in cultured P19 cells in the presence and absence of iron-supplemented medium, respectively. Western blots indicated the levels of transferrin receptor, ferroportin and ubiquitin in the presence and absence of extracellular hepcidin. Total cellular iron was measured by inductively-coupled plasma mass spectrometry and correlated to transverse relaxation rates at 3 Tesla using a gelatin phantom. Under varying conditions of iron supplementation, the level of ferroportin in P19 cells responds to hepcidin regulation, consistent with degradation through a ubiquitin-mediated pathway. This response of P19 cells to hepcidin is similar to that of classically-activated macrophages. The correlation between total cellular iron content and MR transverse relaxation rates was different in hepcidin-treated and untreated P19 cells: slope, Pearson correlation coefficient and relaxation rate were all affected. These findings may provide a tool to non-invasively distinguish changes in endogenous iron contrast arising from hepcidin-ferroportin interactions, with potential utility in monitoring of different macrophage phenotypes involved in pro- and anti-inflammatory signaling. In addition, this work demonstrates that transverse relaxivity is not only influenced by the amount of cellular iron but also by its metabolism.

MagA expression attenuates iron export activity in undifferentiated multipotent P19 cells.

Magnetic resonance imaging (MRI) is a non-invasive imaging modality used in longitudinal cell tracking. Previous studies suggest that MagA, a putative iron transport protein from magnetotactic bacteria, is a useful gene-based magnetic resonance contrast agent. Hemagglutinin-tagged MagA was stably expressed in undifferentiated embryonic mouse teratocarcinoma, multipotent P19 cells to provide a suitable model for tracking these cells during differentiation. Western blot and immunocytochemistry confirmed the expression and membrane localization of MagA in P19 cells. Surprisingly, elemental iron analysis using inductively-coupled plasma mass spectrometry revealed significant iron uptake in both parental and MagA-expressing P19 cells, cultured in the presence of iron-supplemented medium. Withdrawal of this extracellular iron supplement revealed unexpected iron export activity in P19 cells, which MagA expression attenuated. The influence of iron supplementation on parental and MagA-expressing cells was not reflected by longitudinal relaxation rates. Measurement of transverse relaxation rates (R2* and R2) reflected changes in total cellular iron content but did not clearly distinguish MagA-expressing cells from the parental cell type, despite significant differences in the uptake and retention of total cellular iron. Unlike other cell types, the reversible component R2′ (R2* ‒ R2) provided only a moderately strong correlation to amount of cellular iron, normalized to amount of protein. This is the first report to characterize MagA expression in a previously unrecognized iron exporting cell type. The interplay between contrast gene expression and systemic iron metabolism substantiates the potential for diverting cellular iron toward the formation of a novel iron compartment, however rudimentary when using a single magnetotactic bacterial gene expression system like magA. Since relatively few mammalian cells export iron, the P19 cell line provides a tractable model of ferroportin activity, suitable for magnetic resonance analysis of key iron-handling activities and their influence on gene-based MRI contrast.

Effects of Deferoxamine on Leukemia In Vitro and Its Related Mechanism.

BackgroundThis study aimed to investigate the effect of deferoxamine (DFO) on leukemia in vitro, and to explore the underlying molecular mechanism.Material/MethodsK562 leukemia cells were treated with various concentrations of DFO (10, 50, and 100 μmol/l) with or without 10 μmol/l ferric chloride for 12 h. Then, total cellular iron was detected. CCK-8 kit and flow cytometry were used for cell viability and apoptosis detection. In addition, expression of apoptosis-related genes was determined by Western blotting and qRT-PCR, respectively.ResultsThe results suggested that DFO significantly inhibited K562 cell viability and induced cell apoptosis in a dose-dependent manner. We also found that the protein and mRNA levels of Bax, p53, and Fas dose-dependently increased in DFO-treated K562 cells, while the level of Bcl-2 markedly decreased in a dose-dependent manner. Moreover, the findings showed that ferric chloride eliminated these effects on K562 cells caused by DFO treatment.ConclusionsOur results indicate that DFO plays a protective role in leukemia via inhibiting leukemia cell viability and inducing cell apoptosis by the regulation of apoptosis-related genes expression.

Hydrogen Peroxide Causes Iron Dysregulation in C2C12 Skeletal Muscle Cells

Diabetes is becoming an increasingly prevalent issue in the United States. Diabetes and insulin resistance is associated with increased levels of circulating iron and signs of iron dysregulation. Unbound iron in cells leads to increased amounts of free radicals causing damage, including insulin resistance. Ongoing research in our lab has shown that insulin resistant rats displayed iron dysregulation in liver tissue, marked by hepatic iron accumulation, with a decreased ability to store the excess iron in ferritin storage proteins. The purpose of this study is to develop a cell model of iron dysregulation to replicate findings in previous studies in order to identify the mechanism by which this iron dysregulation occurs. Using C2C12 mouse skeletal muscle cells, we investigated the effects of hydrogen peroxide on iron regulation, and its role in the development of iron dysregulation. C2C12 cells were treated with 200 μM hydrogen peroxide for 12 hours. Following treatment with hydrogen peroxide, cells were harvested for protein and iron analysis. Protein levels of transferrin receptor (TfR) and ferritin heavy chain (FHC) were measured using western blot analysis. Total iron was measured using a total iron colorimetric assay. Hydrogen peroxide treatment decreased TfR protein levels by 24±16% (p=.03). We then developed a cellular model to reflect the stress caused by elevated circulating iron levels by treating C2C12 cells with varying concentrations (0–100 μM) of FeCl3 for 24 hours. Iron treatment resulted in an increase in total cellular iron in a dose‐dependent manner. As expected, exposure to elevated iron caused a reduction in TfR expression and an increase in FHC in a dose‐dependent manner. Using this high iron model with a 24‐hour treatment of 10 μM FeCl3, we added 200 μM hydrogen peroxide during the last 12 hours of iron treatment to impose conditions of elevated oxidative stress. The combination of iron and hydrogen peroxide resulted in an increase in TfR by 46±9% (p=.05) and a decrease in FHC by 49±13% (p=0.018), showing a complete reversal compared to hydrogen peroxide treatment alone. Total iron increased by 69±6.7% (p=0.0005). Our results show that treatment with hydrogen peroxide in the presence of iron causes an accumulation of iron and a decrease in ferritin expression, potentially leading to an increase of free iron and the production of reactive oxygen species. This represents a condition of iron dysregulation and increased oxidative stress that can contribute to insulin resistance as well as other cellular pathologies.Support or Funding InformationSupported by BYU Mentoring Enhancement GrantThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Curcumin Alters Iron Regulation in C2C12 Skeletal Muscle Cells and Prevents Iron Accumulation in a Model of Elevated Oxidative Stress

The Centers for Disease Control and Prevention have stated that as of 2017, 30.3 million people in the United States have diabetes (9.6% of total population), with an additional 84.1 million who have prediabetes (33.9% of total population). Patients with diabetes show signs of iron dysregulation, and recent studies indicate a correlation between iron dysregulation and insulin sensitivity suggesting that iron dysregulation plays a role in diabetes. Unbound cellular iron is highly reactive and can cause increased amounts of free radicals. Curcumin is a naturally occurring compound in the spice, turmeric, and has been shown to be effective in improving insulin sensitivity, although the mechanisms by which it does so are not entirely understood. Curcumin has been shown to bind iron and is very likely to have an effect on iron regulation. The purpose of this study is to understand the effects of curcumin on iron regulation and its ability to prevent iron dysregulation. Differentiated C2C12 mouse skeletal muscle cells were used in normal and high iron conditions, and protein expression and total iron content were analyzed to assess curcumin's effects on iron regulation. Cells were treated with 40 μM curcumin for 12 hours, and then harvested for analysis of protein expression and total iron content. Protein expression of transferrin receptor (TfR) and ferritin heavy chain (FHC) were measured by western blot analysis. Total iron content was measured by using a total iron colorimetric assay. C2C12 cells treated with 40 μM curcumin for 12 hours caused a reduction in TfR protein expression by 19±10% (p=0.02), as well as a reduction in FHC protein expression by 55±16% (p=0.01). The decrease in TfR expression correlated with a reduction in total cellular iron level, although the reduction in total iron was not significant. We then examined the effect of curcumin on iron regulation in high iron conditions to reflect the stress caused by elevated circulating iron levels associated with insulin resistance. In a model of high iron, pre‐treatment of 40 μM curcumin for 12 hours followed by treatment of 10 μM FeCl3 for 24 hours caused a reduction in TfR expression by 23±11% (p=0.008). Curcumin's effects on FHC expression were reversed in this high iron model, as FHC expression increased by 30±17% (p=0.04). Curcumin did not significantly alter total iron levels in this high iron model. Finally, we used a model of elevated oxidative stress to cause iron dysregulation in C2C12 cells, and analyzed curcumin's ability to prevent iron dysregulation. Cells were treated with 10 μM FeCl3 for 24 hours, with 200 μM hydrogen peroxide added during the final 12 hours of treatment. Pre‐treatment with curcumin prevented iron accumulation in cells treated with both iron and hydrogen peroxide (p=0.006). Our results show that curcumin's alteration of iron regulation is dependent upon extracellular iron levels. Results also show that curcumin has the potential to prevent iron dysregulation in an environment of elevated oxidative stress, providing a possible mechanism to its ability to improve insulin sensitivity.Support or Funding InformationThis work was supported by a BYU Mentoring Enhancement Grant.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Nitric oxide reduces oxidative stress in cancer cells by forming dinitrosyliron complexes

The chelatable iron pool (CIP) is a small but chemically significant fraction of total cellular iron. While this dynamic population of iron is limited, it is redox active and capable of generating reactive oxygen species (ROS) that can lead to oxidative stress which is associated with various pathologies. Nitric oxide (•NO), is a free radical signalling molecule that regulates numerous physiological and pathological conditions. We have previously shown that macrophages exposed to endogenously generated or exogenously administered nitric oxide (•NO) results in its interaction with CIP to form dinitrosyliron complexes with thiol containing ligands (DNICs). In this study we assessed the consequences of DNIC formation in cancer cells as •NO is known to be associated with numerous malignancies. Incubation of cancer cells with •NO led to a time and dose dependent increase in formation of DNICs. The formation of DNICs results in the sequestration of the CIP which is a major source of iron for redox reactions and reactive oxygen species (ROS) generation. Therefore, we set out to test the antioxidant effect of •NO by measuring the ability of DNICs to protect cells against oxidative stress. We observed that cancer cells treated with •NO were partially protected against H2O2 mediated cytotoxicity. This correlated to a concomitant decrease in the formation of oxidants when •NO was present during H2O2 treatment. Similar protective effects were achieved by treating cells with iron chelators in the presence of H2O2. Interestingly, •NO decreased the rate of cellular metabolism of H2O2 suggesting that a proportion of H2O2 is consumed via reactions with cellular iron. When the CIP was artificially increased by supplementation of cells with iron, a significant decrease in the cytoprotective effect of •NO was observed. Notably, •NO concentrations, at which cytoprotective and antioxidant effects were observed, correlated with concentration-dependent increases in DNIC formation. Collectively, these results demonstrate that •NO has antioxidant properties by its ability to sequester cellular iron. This could play a significant role in variety of diseases involving ROS mediated toxicity like cancer and neurodegenerative disorders where •NO has been shown to be an important etiologic factor.

Eltrombopag: a powerful chelator of cellular or extracellular iron(III) alone or combined with a second chelator

AbstractEltrombopag (ELT) is a thrombopoietin receptor agonist reported to decrease labile iron in leukemia cells. Here we examine the previously undescribed iron(III)-coordinating and cellular iron-mobilizing properties of ELT. We find a high binding constant for iron(III) (log β2=35). Clinically achievable concentrations (1 µM) progressively mobilized cellular iron from hepatocyte, cardiomyocyte, and pancreatic cell lines, rapidly decreasing intracellular reactive oxygen species (ROS) and also restoring insulin secretion in pancreatic cells. Decrements in cellular ferritin paralleled total cellular iron removal, particularly in hepatocytes. Iron mobilization from cardiomyocytes exceeded that obtained with deferiprone, desferrioxamine, or deferasirox at similar iron-binding equivalents. When combined with these chelators, ELT enhanced cellular iron mobilization more than additive (synergistic) with deferasirox. Iron-binding speciation plots are consistent with ELT donating iron to deferasirox at clinically relevant concentrations. ELT scavenges iron citrate species faster than deferasirox, but rapidly donates the chelated iron to deferasirox, consistent with a shuttling mechanism. Shuttling is also suggested by enhanced cellular iron mobilization by ELT when combined with the otherwise ineffective extracellular hydroxypyridinone chelator, CP40. We conclude that ELT is a powerful iron chelator that decreases cellular iron and further enhances iron mobilization when combined with clinically available chelators.

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

BackgroundDuodenal 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.MethodsThe 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.ResultsHigh 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.ConclusionsDCYTB 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.

Recombinant vacuolar iron transporter family homologue PfVIT from human malaria-causing Plasmodium falciparum is a Fe2+/H+exchanger

Vacuolar iron transporters (VITs) are a poorly understood family of integral membrane proteins that can function in iron homeostasis via sequestration of labile Fe2+ into vacuolar compartments. Here we report on the heterologous overexpression and purification of PfVIT, a vacuolar iron transporter homologue from the human malaria-causing parasite Plasmodium falciparum. Use of synthetic, codon-optimised DNA enabled overexpression of functional PfVIT in the inner membrane of Escherichia coli which, in turn, conferred iron tolerance to the bacterial cells. Cells that expressed PfVIT had decreased levels of total cellular iron compared with cells that did not express the protein. Qualitative transport assays performed on inverted vesicles enriched with PfVIT revealed that the transporter catalysed Fe2+/H+ exchange driven by the proton electrochemical gradient. Furthermore, the PfVIT transport function in this system did not require the presence of any Plasmodium-specific factor such as post-translational phosphorylation. PfVIT purified as a monomer and, as measured by intrinsic protein fluorescence quenching, bound Fe2+ in detergent solution with low micromolar affinity. This study of PfVIT provides material for future detailed biochemical, biophysical and structural studies to advance understanding of the vacuolar iron transporter family of membrane proteins from important human pathogens.