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.

Bioavailability of iron multi-amino acid chelate preparation in mice and human duodenal HuTu 80 cells.

Strategies for preventing Fe deficiency include Fe supplementation and Fe fortification of foods. The absorption, metabolism and chemical characteristics of Fe multi-amino acid chelate (IMAAC) are not known. Absorption of IMAAC was compared with FeSO4 in Fe-depleted mice and in vitro chemical studies of the Fe supplement was performed in HuTu 80 cells. Hb repletion study was carried out in Fe-deficient CD1 mice that were fed for 10 d a diet supplemented with ferrous IMAAC or FeSO4. A control group of Fe-replete mice was fed a diet with adequate Fe concentrations throughout the study. Tissues were collected from the mice, and the expression of Fe-related genes was determined by quantitative PCR. Ferric reductase and Fe uptake were evaluated in HuTu 80 cells. Supplementation of the diet with FeSO4 or IMAAC significantly increased Hb levels (P<0·001) in Fe-deficient mice from initial 93·9 (SD 10·8) or 116·2 (SD 9·1) to 191 (SD 0·7) or 200 (SD 0·5) g/l, respectively. Initial and final Hb for the Fe-deficient control group were 87·4 (SD 6·7) and 111 (SD 11·7) g/l, respectively. Furthermore, the liver non-haem Fe of both supplement groups increased significantly (P<0·001). IMAAC was more effective at restoring Fe in the spleen compared with FeSO4 (P<0·005). Gene expression showed the IMAAC supplement absorption is regulated by the body's Fe status as it significantly up-regulated hepcidin (P<0·001) and down-regulated duodenal cytochrome b mRNA (P<0·005), similar to the effects seen with FeSO4. A significant proportion of Fe in IMAAC is reduced by ascorbic acid. Fe absorption in mice and cells was similar for both IMAAC and FeSO4 and both compounds induce and regulate Fe metabolism genes similarly in the maintenance of homeostasis in mice.

Modulation of Dcytb (Cybrd 1) expression and function by iron, dehydroascorbate and Hif-2α in cultured cells.

BACKGROUND: Duodenal cytochrome b (Dcytb) is a mammalian plasma ferric reductase enzyme that catalyses the reduction of ferric to ferrous ion in the process of iron absorption. The current study investigates the relationship between Dcytb, iron, dehydroascorbate (DHA) and Hif-2α in cultured cell lines. METHODS: Dcytb and Hif-2α protein expression was analysed by Western blot technique while gene regulation was determined by quantitative PCR. Functional analyses were carried out by ferric reductase and (59)Fe uptake assays. RESULTS: Iron and dehydroascorbic acid treatment of cells inhibited Dcytb mRNA and protein expression. Desferrioxamine also enhanced Dcytb mRNA level after cells were treated overnight. Dcytb knockdown in HuTu cells resulted in reduced mRNA expression and lowered reductase activity. Preloading cells with DHA (to enhance intracellular ascorbate levels) did not stimulate reductase activity fully in Dcytb-silenced cells, implying a Dcytb-dependence of ascorbate-mediated ferrireduction. Moreover, Hif-2α knockdown in HuTu cells led to a reduction in reductase activity and iron uptake. CONCLUSIONS: Taken together, this study shows the functional regulation of Dcytb reductase activity by DHA and Hif-2α. GENERAL SIGNIFICANCE: Dcytb is a plasma membrane protein that accepts electrons intracellularly from DHA/ascorbic acid for ferrireduction at the apical surface of cultured cells and enterocytes.

Role of duodenal iron transporters and hepcidin in patients with alcoholic liver disease.

Patients with alcoholic liver disease (ALD) often display disturbed iron indices. Hepcidin, a key regulator of iron metabolism, has been shown to be down-regulated by alcohol in cell lines and animal models. This down-regulation led to increased duodenal iron transport and absorption in animals. In this study, we investigated gene expression of duodenal iron transport molecules and hepcidin in three groups of patients with ALD (with anaemia, with iron overload and without iron overload) and controls. Expression of DMT1, FPN1, DCYTB, HEPH, HFE and TFR1 was measured in duodenal biopsies by using real-time PCR and Western blot. Serum hepcidin levels were measured by using ELISA. Serum hepcidin was decreased in patients with ALD. At the mRNA level, expressions of DMT1, FPN1 and TFR1 genes were significantly increased in ALD. This pattern was even more pronounced in the subgroups of patients without iron overload and with anaemia. Protein expression of FPN1 paralleled the increase at the mRNA level in the group of patients with ALD. Serum ferritin was negatively correlated with DMT1 mRNA. The down-regulation of hepcidin expression leading to up-regulation of iron transporters expression in the duodenum seems to explain iron metabolism disturbances in ALD. Alcohol consumption very probably causes suppression of hepcidin expression in patients with ALD.

Increasing dietary protein acutely augments intestinal iron transporter expression and significantly increases iron absorption in rats.

Iron (Fe) deficiency is endemic worldwide. Little data are available regarding acute effects of dietary protein on intestinal Fe absorption. The current study evaluated the short-term effects of increasing dietary protein on Fe absorption and expression of genes involved in Fe homeostasis. Sprague Dawley rats (24, female) were randomly assigned to custom-formulated isocaloric diets containing 40, 20 (control), or 5% protein (as percentage of total kilocalories) for 7 d. Whole-body Fe balance studies demonstrated that Fe retention was greater in the 40% group than in the 5% group (30.8 vs. 7.3%; P<0.01). In a separate study utilizing stable iron isotopes, the 40% group absorbed 30% of ingested Fe, while the 20% group absorbed 18% (P=0.005). Whole-genome profiling revealed that increasing dietary protein from 5 to 40% increased duodenal transcript expression of divalent metal transporter 1 (DMT1) 3.2-fold, duodenal cytochrome b (Dcytb) 1.8-fold, and transferrin receptor (TfR) 1.8-fold. Consistent with these findings, DMT1 transcript expression was 4-fold higher in RNA prepared from duodenal mucosa in the 40% group compared to the 20% group (P<0.001). These data suggest that increasing dietary protein increases intestinal Fe absorption in part by up-regulating DMT1, Dcytb, and TfR.

Redox Properties of Human Erythrocytes Are Adapted for Vitamin C Recycling.

Ascorbic acid (AA; or vitamin C) is an important physiological antioxidant and radical scavenger. Some mammalian species, including homo sapiens, have lost the ability to synthetize AA and depend on its nutritional uptake. Erythrocytes from AA-auxotroph mammals express high amounts of the glucose transporter GLUT1. This isoform enables rapid uptake of glucose as well as dehydroascorbate (DHA), the fully oxidized form of AA. Here, we explored the effects of DHA uptake on the redox metabolism of human erythrocytes. DHA uptake enhanced plasma membrane electron transport (PMET) activity. This process is mediated by DCytb, a membrane bound cytochrome catalyzing extracellular reduction of Fe3+ and ascorbate free radical (AFR), the first oxidized form of AA. DHA uptake also decreased cellular radical oxygen species (ROS) levels. Both effects were massively enhanced in the presence of physiological glucose concentrations. Reduction of DHA to AA largely depleted intracellular glutathione (GSH) and induced the efflux of its oxidized form, GSSG. GSSG efflux could be inhibited by MK-571 (IC 50 = 5 µM), indicating involvement of multidrug resistance associated protein (MRP1/4). DHA-dependent GSH depletion and GSSG efflux were completely rescued in the presence of 5 mM glucose and, partially, by 2-deoxy-glucose (2-DG), respectively. These findings indicate that human erythrocytes are physiologically adapted to recycle AA both intracellularly via GLUT1-mediated DHA uptake and reduction and extracellularly via DCytb-mediated AFR reduction. We discuss the possibility that this improved erythrocyte-mediated AA recycling was a prerequisite for the emergence of AA auxotrophy which independently occurred at least twice during mammalian evolution.

Iron Supplements Containing Lactobacillus plantarum 299v Increase Ferric Iron and Up-regulate the Ferric Reductase DCYTB in Human Caco-2/HT29 MTX Co-Cultures.

Several human interventions have indicated that Lactobacillus plantarum 299v (L. plantarum 299v) increases intestinal iron absorption. The aim of the present study was to investigate possible effects of L. plantarum 299v on the mechanisms of iron absorption on the cellular level. We have previously shown that lactic fermentation of vegetables increased iron absorption in humans. It was revealed that the level of ferric iron [Fe (H2O)5]2+ was increased after fermentation. Therefore, we used voltammetry to measure the oxidation state of iron in simulated gastrointestinal digested oat and mango drinks and capsule meals containing L. plantarum 299v. We also exposed human intestinal co-cultures of enterocytes and goblet cells (Caco-2/HT29 MTX) to the supplements in order to study the effect on proteins possibly involved (MUC5AC, DCYTB, DMT1, and ferritin). We detected an increase in ferric iron in the digested meals and drinks containing L. plantarum 299v. In the intestinal cell model, we observed that the ferric reductase DCYTB increased in the presence of L. plantarum 299v, while the production of mucin (MUC5AC) decreased independently of L. plantarum 299v. In conclusion, the data suggest that the effect of L. plantarum 299v on iron metabolism is mediated through driving the Fe3+/DCYTB axis.

The active role of vitamin C in mammalian iron metabolism: much more than just enhanced iron absorption!

Ascorbate is a cofactor in numerous metabolic reactions. Humans cannot synthesize ascorbate owing to inactivation of the gene encoding the enzyme l-gulono-γ-lactone oxidase, which is essential for ascorbate synthesis. Accumulating evidence strongly suggests that in addition to the known ability of dietary ascorbate to enhance nonheme iron absorption in the gut, ascorbate within mammalian systems can regulate cellular iron uptake and metabolism. Ascorbate modulates iron metabolism by stimulating ferritin synthesis, inhibiting lysosomal ferritin degradation, and decreasing cellular iron efflux. Furthermore, ascorbate cycling across the plasma membrane is responsible for ascorbate-stimulated iron uptake from low-molecular-weight iron-citrate complexes, which are prominent in the plasma of individuals with iron-overload disorders. Importantly, this iron-uptake pathway is of particular relevance to astrocyte brain iron metabolism and tissue iron loading in disorders such as hereditary hemochromatosis and ß-thalassemia. Recent evidence also indicates that ascorbate is a novel modulator of the classical transferrin-iron uptake pathway, which provides almost all iron for cellular demands and erythropoiesis under physiological conditions. Ascorbate acts to stimulate transferrin-dependent iron uptake by an intracellular reductive mechanism, strongly suggesting that it may act to stimulate iron mobilization from the endosome. The ability of ascorbate to regulate transferrin iron uptake could help explain the metabolic defect that contributes to ascorbate-deficiency-induced anemia.

Intestinal Iron Transport and its Regulation.

In recent years, a number of components of the iron absorption pathway have been identified, greatly increasing our understanding of this important process. These include two molecules involved in brush border iron uptake, the ferric reductase DcytB and the iron transporter DMT1, and two mediating iron transfer to the body, the iron transporter Ireg1 and the ferroxidase hephaestin (Hp). Analysis of the regulation of these molecules has provided us with valuable insights into how the body responds to changes in iron requirements, and has enabled us to re-examine how iron absorption is controlled, and in particular the mucosal block phenomenon. Evidence suggests that the block to absorption that follows a priming dose of iron is the result of elevated intracellular iron levels decreasing the expression of the brush border iron transporter DMT1. Based on these observations, it is possible to propose a general model for the regulation of iron absorption whereby the basolateral transfer step involving Ireg1 and Hp controls the rate of absorption. In this model, DMT1 expression, and hence, brush border uptake, is regulated by local iron levels that are, in turn, determined by the rate of basolateral transfer.