Role of transferrin receptor and the ABC transporters ABCB6 and ABCB7 for resistance and differentiation of tumor cells towards artesunate

The anti-malarial artesunate also exerts profound anti-cancer activity. The susceptibility of tumor cells to artesunate can be enhanced by ferrous iron. The transferrin receptor (TfR) is involved in iron uptake by internalization of transferrin and is over-expressed in rapidly growing tumors. The ATP-binding cassette (ABC) transporters ABCB6 and ABCB7 are also involved in iron homeostasis. To investigate whether these proteins play a role for sensitivity towards artesunate, Oncotest's 36 cell line panel was treated with artesunate or artesunate plus iron(II) glycine sulfate (Ferrosanol). The majority of cell lines showed increased inhibition rates, for the combination of artesunate plus iron(II) glycine sulfate compared to artesunate alone. However, in 11 out of the 36 cell lines the combination treatment was not superior. Cell lines with high TfR expression significantly correlated with high degrees of modulation indicating that high TfR expressing tumor cells would be more efficiently inhibited by this combination treatment than low TfR expressing ones. Furthermore, we found a significant relationship between cellular response to artesunate and TfR expression in 55 cell lines of the National Cancer Institute (NCI), USA. A significant correlation was also found for ABCB6, but not for ABCB7 in the NCI panel. Artesunate treatment of human CCRF-CEM leukemia and MCF7 breast cancer cells induced ABCB6 expression but repressed ABCB7 expression. Finally, artesunate inhibited proliferation and differentiation of mouse erythroleukemia (MEL) cells. Down-regulation of ABCB6 by antisense oligonucleotides inhibited differentiation of MEL cells indicating that artesunate and ABCB6 may cooperate. In conclusion, our results indicate that ferrous iron improves the activity of artesunate in some but not all tumor cell lines. Several factors involved in iron homeostasis such as TfR and ABCB6 may contribute to this effect.     

Artemisinin derivatives induce iron-dependent cell death (ferroptosis) in tumor cells

BackgroundApoptosis and other forms of cell death have been intensively investigated in the past years to explain the mode of action of synthetic anticancer drugs and natural products. Recently, a new form of cell death emerged, which was termed ferroptosis, because it depends on intracellular iron. Here, the role of genes involved in iron metabolism and homeostasis for the cytotoxicity of ten artemisinin derivatives have been systematically investigated.Material and methodsLog₁₀IC₅₀ values of 10 artemisinin derivatives (artesunate, artemether, arteether, artenimol, artemisitene, arteanuin B, another monomeric artemisinin derivative and three artemisinin dimer molecules) were correlated to the microarray-based mRNA expression of 30 iron-related genes in 60 cell lines of the National Cancer Institute (NCI, USA) as determined in 218 different microarray hybridization experiments. The effect of desferoxamine and ferrostatin-1 on the cytotoxicity of artenimol of CCRF-CEM cells was determined by resazurin assays. The mRNA expression of TFRC was exemplarily validated by immunohistochemical detection of transferrin receptor protein expression.ResultsThe mRNA expression of 20 genes represented by 59 different cDNA clones significantly correlated to the log₁₀IC₅₀ values for the artemisinins, including genes encoding transferrin (TF), transferrin receptors 1 and 2 (TFRC, TFR2), cerulopasmin (CP), lactoferrin (LTF) and others. The ferroptosis inhibitor ferrostatin-1 and the iron chelator deferoxamine led to a significantly reduced cytotoxicity of artenimol, indicating ferroptosis as cell death mode.ConclusionThe numerous iron-related genes, whose expression correlated with the response to artemisinin derivatives speak in factor for the relevance of iron for the cytotoxic activity of these compounds. Treatment with ferroptosis-inducing agents such as artemisinin derivatives represents an attractive strategy for cancer therapy. Pre-therapeutic determination of iron-related genes may indicate tumor sensitivity to artemisinins. Ferroptosis induced by artemisinin-type drugs deserve further investigation for individualized tumor therapy.     

Iron-transferrin-induced increase in protein kinase C activity in CCRF-CEM cells

Iron transferrin has been found to induce a mean 10-fold increase in the activity of protein kinase C in CCRF-CEM cells. This increase was not detectable up to 45 min after treatment of cells with iron transferrin, although after 60 min, a maximal increase in enzyme activity was observed. Similarly, iron transferrin at concentrations of 0.1-0.5 microgram/ml did not alter protein kinase C activity, while concentrations of iron transferrin of 1-100 micrograms/ml induced a maximal increase in enzyme activity. Apotransferrin and iron in the form of ferric citrate, as well as complexes of transferrin with copper, nickel, zinc, manganese, and cobalt did not increase protein kinase C activity. Additionally, CCRF-CEM cells pretreated with either actinomycin D or cycloheximide and then incubated with iron transferrin did not exhibit increased enzyme activity. Treatment with iron transferrin was found to have no effect on protein kinase C activity in normal human peripheral blood lymphocytes and in HL60, Daudi, and U937 cells. However, normal lymphocytes stimulated with phytohemagglutinin for 48 hr exhibited a 2-fold increase in protein kinase C activity following treatment with iron transferrin. These results indicate a specific effect of iron transferrin on protein kinase C activity in CCRF-CEM cells and in mitogen-stimulated human lymphocytes that may occur through increased synthesis of the enzyme.     

Modulation of transferrin receptor expression by inhibitors of nucleic acid synthesis

We investigated the effects of the iron chelator desferrioxamine on the expression of transferrin receptors (TfR) by CCRF-CEM human T-cell leukaemia and B16 mouse melanoma cells growing in tissue culture. Desferrioxamine (DFOA) enhanced TfR expression when added in the dose range of 10(-5)-10(-4) to CCRF-CEM cells, but was toxic to these cells, the lower concentrations producing a slowing of cell growth with a build up in S-phase, while higher concentrations caused cell death with a block at the G1/S-phase interface. These toxic effects are compatible with its previously reported inhibition of the non-haem iron containing (M2) subunit of ribonucleotide reductase. In marked contrast, DFOA caused the growth of B16 melanoma cells to arrest in G1, without loss of cloning efficiency, and resulted in a fall in TfR expression to approximately 50% of control values. These results suggested that the effects of DFOA on TfR expression were linked to DNA synthesis rather than to a more generalised inhibition of iron-dependent cellular processes. It was subsequently found that inhibition of the M2 subunit of ribonucleotide reductase in CCRF-CEM cells with 5 X 10(-5) M hydroxyurea, which is not an iron chelator, also enhanced TfR expression, as did thymidine and cytosine arabinoside, which have different enzyme targets. By measuring cellular DNA and RNA content simultaneously it was shown that all of these agents caused unbalanced growth, i.e., inhibited DNA synthesis more than RNA synthesis. In contrast, 6-thioguanine was more inhibitory to RNA synthesis, and treatment with this drug caused a fall in TfR expression. Thus, although CCRF-CEM cells treated with DFOA show enhanced TfR expression, similar effects are also seen with other inhibitors of DNA synthesis, provided that RNA synthesis is allowed to continue. These results provide further evidence that the regulation of TfR expression by proliferating cells is specifically linked to DNA synthesis rather than to the iron requirements of other cellular processes.     

The in vitro response of human tumour cells to desferrioxamine is growth medium dependent

Abstract. Iron chelating agents have been demonstrated to inhibit tumour cell growth. However, in vitro and in vivo results using desferrioxamine a hexadentate iron chelating agent, for anti-cancer treatment are not always in agreement. Therefore, we have studied the response of three human tumour cell lines (HL-60 promyelocytic leukaemia, MCF-7 breast cancer and HepG2 hepatoma), grown in culture medium supplemented with either human pooled (HPS) or fet al bovine serum (FBS), to desferrioxamine. Desferrioxamine, at micromolar concentrations, induced severe cytotoxicity in all tumour cell lines grown in FBS medium. When grown in HPS medium, comparable desferrioxamine cytotoxicity was observed in the millimolar range. The addition of 50% saturated human transferrin to FBS medium resulted in protection against desferrioxamine cytotoxicity. HL-60 cells were further studied for iron metabolism characteristics. HL-60 cells, grown in medium with FBS, were found to have an 8.4 fold increase in surface transferrin receptor (TfR) expression ( P < 0.001) as compared with HL-60 cells grown in medium with HPS. However, iron uptake of HPS cultured HL-60 cells, after incubation with saturated human transferrin, was higher, resulting in a higher concentration of iron in HPS cultured HL-60 cells as compared with FBS cultured cells (1.72 ± 0.02 μmol/g protein v. 1.32 ± 0.14 μmol/g protein; P < 0.001). Using desferrioxamine it was shown that TfR expression is dependent on the biological availability of iron in the cell. Consistent with the lower iron content in FBS cultured cells, we conclude that the cytotoxicity of desferrioxamine is dependent on the ability of cells to replenish cellular iron stores from the culture medium. Cells grown in FBS medium lack this ability and are therefore more susceptible to desferrioxamine.     

Iron Accumulates in Huntington’s Disease Neurons: Protection by Deferoxamine

Huntington’s disease (HD) is a progressive neurodegenerative disorder caused by a polyglutamine-encoding CAG expansion in the huntingtin gene. Iron accumulates in the brains of HD patients and mouse disease models. However, the cellular and subcellular sites of iron accumulation, as well as significance to disease progression are not well understood. We used independent approaches to investigate the location of brain iron accumulation. In R6/2 HD mouse brain, synchotron x-ray fluorescence analysis revealed iron accumulation as discrete puncta in the perinuclear cytoplasm of striatal neurons. Further, perfusion Turnbull’s staining for ferrous iron (II) combined with transmission electron microscope ultra-structural analysis revealed increased staining in membrane bound peri-nuclear vesicles in R6/2 HD striatal neurons. Analysis of iron homeostatic proteins in R6/2 HD mice revealed decreased levels of the iron response proteins (IRPs 1 and 2) and accordingly decreased expression of iron uptake transferrin receptor (TfR) and increased levels of neuronal iron export protein ferroportin (FPN). Finally, we show that intra-ventricular delivery of the iron chelator deferoxamine results in an improvement of the motor phenotype in R6/2 HD mice. Our data supports accumulation of redox-active ferrous iron in the endocytic / lysosomal compartment in mouse HD neurons. Expression changes of IRPs, TfR and FPN are consistent with a compensatory response to an increased intra-neuronal labile iron pool leading to increased susceptibility to iron-associated oxidative stress. These findings, together with protection by deferoxamine, support a potentiating role of neuronal iron accumulation in HD.     

Heme activates artemisinin more efficiently than hemin, inorganic iron, or hemoglobin

Artemisinin derivatives appear to mediate their anti-malarial through an initial redox-mediated reaction. Heme, inorganic iron, and hemoglobin have all been implicated as the key molecules that activate artemisinins. The reactions of artemisinin with different redox forms of heme, ferrous iron, and deoxygenated and oxygenated hemoglobin were analyzed under similar in vitro conditions. Heme reacted with artemisinin much more efficiently than the other iron-containing molecules, supporting the role of redox active heme as the primary activator of artemisinin.     

Transferrin iron interactions with cultured hepatocellular carcinoma cells (PLC/PRF/5)

Hepatocellular carcinoma cells of the PLC/PRF/5 cell line had 1.9 x 10(5) transferrin receptors per tumor cell with a Kd of 1.5 x 10(-8) M. At high concentrations of transferrin the binding was not saturable. Transferrin internalization by hepatoma cells was shown by time and temperature-dependent binding studies and by pronase experiments. Transferrin recycling was confirmed by the demonstration of a progressive increase in the cellular molar ratios of iron to transferrin and by chase experiments. Ammonium chloride interfered with iron unloading. The vinca alkaloid vincristine inhibited iron and transferrin uptake. The hepatocarcinoma cells appeared to lack asialoglycoprotein receptors and therefore internalized partially desialated transferrin by the regular route. Iron uptake from transferrin was markedly inhibited by the hydrophobic ferrous chelator 2,2' bipyridine but was relatively unaffected by the hydrophilic ferric chelator desferroxamine. The implication that ferrous iron was involved in postendocytic transvesicular membrane iron transport was supported by a study in which hepatoma cells were shown to take up large amounts of ferrous iron suspended in 270 mM sucrose at pH 5.5. The interaction at this pH between surface labeled hepatoma cell extracts and ferrous iron on a Sephacryl S-300 column suggested that the postendocytic transvesicular transport of iron through the membrane was in part protein mediated. The endocytosed iron in hepatoma cells was found in association with ferritin (33%), transferrin (31%) and a low molecular weight fraction (21%).     

Expression of iron transport proteins divalent metal transporter-1, Ferroportin-1, HFE and transferrin receptor-1 in human monocyte-derived dendritic cells

Iron is essential for cell survival and regulates many cell functions. In the context of the immune response, iron-related metabolism is tightly controlled in activated lymphocytes as well as in cells of the innate immunity. More precisely, for dendritic cells (DCs), which are the key cell type in the development of a specific immune response, the importance of iron absorption was recently unravelled by showing that depletion of iron inhibits the maturation of DCs. On this basis, we studied in detail the expression of iron transport proteins and HFE in DCs. We found that iron uptake in this cell type is mediated by divalent-metal transporter 1 (DMT1) and transferrin receptor-1 (TfR) whereas Ferroportin-1 is very weakly expressed. HFE that regulates TfR's activity is also detected at the mRNA level. The expression of DMT1 and HFE barely varies upon endotoxin-induced maturation but TfR is up-regulated and the iron export molecule Ferroportin-1 is down-regulated. As opposed to MHC class II molecules, the intracellular localization of TfR is not changed during maturation. Our results indicate that the uptake of iron during DCs development and maturation is mediated by a strong expression of iron-uptake molecules such as DMT1 and TfR as well as a down-regulation of iron export molecules such as Ferroportin-1.     

Developmental toxicity of artesunate in the rat: comparison to other artemisinins, comparison of embryotoxicity and kinetics by oral and intravenous routes, and relationship to maternal reticulocyte count

BACKGROUND: The antimalarial, artesunate, is teratogenic and embryolethal in rats, with peak sensitivity on Days 10 and 11 postcoitum (pc). METHODS: We compared the developmental toxicity of structurally related artemisinins, dihdyroartemisinin (DHA), artemether (ARTM), and arteether (ARTE) to that of artesunate after oral administration to rats on Day 10 pc. In separate studies, embryolethality was characterized after single intravenous (IV) administration of artesunate on Day 11 pc, and toxicokinetic parameters following oral and IV administration were compared. Lastly, to determine whether maternal hematologic effects occurred at doses that affect embryonic erythroblasts, artesunate was orally administered on Day 11 pc at a dose that caused 100% embryolethality. RESULTS: All artemisinins caused the same pattern of embryolethality and fetal cardiovascular and skeletal abnormalities as previously shown for artesunate. In the IV study, marked postimplantation loss occurred at 1.5 and 3 mg/kg artesunate, but not at 0.75 mg/kg. Among the toxicokinetic parameters evaluated, only the DHA AUC_0‐t was similar at embryolethal oral and IV doses of artesunate. An embryolethal dose of artesunate caused a 15% decrease in maternal reticulocyte counts and no other hematologic effects. CONCLUSIONS: Several structurally related artemisinins cause similar developmental toxicity, suggesting an artemisinin class effect. Equally embryotoxic oral and IV treatments of one artemisinin compound (artesunate) produced similar systemic exposure to the artesunate metabolite, DHA, suggesting that DHA may be the proximate developmental toxicant. Embryolethal doses of artesunate only caused minor changes in maternal reticulocyte counts indicating that adult hematology parameters are not as sensitive as embryonic erythroblasts. Birth Defects Res (Part B) 83:397–406, 2008. © 2008 Wiley‐Liss, Inc.     

Characterization of glyceraldehyde-3-phosphate dehydrogenase as a novel transferrin receptor

A majority of cells obtain of transferrin (Tf) bound iron via transferrin receptor 1 (TfR1) or by transferrin receptor 2 (TfR2) in hepatocytes. Our study establishes that cells are capable of acquiring transferrin iron by an alternate pathway via GAPDH.These findings demonstrate that upon iron depletion, GAPDH functions as a preferred receptor for transferrin rather than TfR1 in some but not all cell types. We utilized CHO-TRVb cells that do not express TfR1 or TfR2 as a model system. A knockdown of GAPDH in these cells resulted in a decrease of not only transferrin binding but also associated iron uptake. The current study also demonstrates that, unlike TfR1 and TfR2 which are localized to a specific membrane fraction, GAPDH is located in both the detergent soluble and lipid raft fractions of the cell membrane. Further, transferrin uptake by GAPDH occurs by more than one mechanism namely clathrin mediated endocytosis, lipid raft endocytosis and macropinocytosis. By determining the kinetics of this pathway it appears that GAPDH-Tf uptake is a low affinity, high capacity, recycling pathway wherein transferrin is catabolised. Our findings provide an explanation for the detailed role of GAPDH mediated transferrin uptake as an alternate route by which cells acquire iron.     

Identification of the segments of the mouse transferrin receptor 1 required for mouse mammary tumor virus infection

Most enveloped viruses enter cells through binding of virion surface envelope proteins to receptors found on the plasma membrane of the cell. The beta retrovirus mouse mammary tumor virus (MMTV) uses transferrin receptor 1 (TfR1) to enter cells in a pH-dependent mechanism, probably co-trafficking with TfR1 to an acidic compartment where virus entry occurs. We have shown here that, although mouse and rat TfR1 function as entry receptors, cat, dog, hamster, or human TfR1s do not support MMTV infection. We also demonstrated that MMTV entry is independent of transferrin, iron, and the TfR1 cofactor hereditary hematochromatosis HFE protein. Using chimeric mouse/human hybrid TfR1 constructs, we determined the site of interaction with MMTV and found that it maps to two segments physically disparate from the TfR and HFE binding sites. Thus, MMTV has apparently evolved to enter cells independently of the iron status of the host.     

Transferrin receptor-1 suppresses neurite outgrowth in neuroblastoma Neuro2A cells

Transferrin receptor-1 (TfR1) is a cell membrane-associated glycoprotein responsible for incorporation of the iron bound to transferrin through an endocytotic process from the circulating blood. Iron is believed to play a dual role as an active center of the electron transfer system in mitochondria and as an endogenous cytotoxin through promoted generation of reactive oxygen species in different eukaryotic cells. In this study, we evaluated expression profiles of different genes related to iron mobilization across plasma membranes in neuronal cells. Marked mRNA expression was seen for various iron-related genes such as TfR1 in cultured mouse neocortical neurons, while TfR1 mRNA levels were more than doubled during culture from 3 to 6days. In mouse embryonal carcinoma P19 cells endowed to differentiate into neuronal and astroglial lineages, a transient increase was seen in both mRNA and corresponding protein for TfR1 in association with neuronal marker expression during culture with all-trans retinoic acid (ATRA). In neuronal Neuro2A cells cultured with ATRA, moreover, neurite was elongated together with increased expression of both mRNA and protein for TfR1. Overexpression of TfR1 significantly decreased the length of neurite elongated, however, while significant promotion was invariably seen in the neurite elongation in Neuro2A cells transfected with TfR1 siRNA as well as in Neuro2A cells cultured with an iron chelator. These results suggest that TfR1 would be highly expressed by neurons rather than astroglia to play a negative role in the neurite outgrowth after the incorporation of circulating transferrin in the brain.     

单核巨噬细胞铁代谢相关蛋白的表达调控

人类机体的铁代谢表现为受限制的对外界铁的吸收和有效的机体内的铁的再循环利用,单核巨噬细胞系统通过吞噬衰老的红细胞,储存和释放铁,在机体铁的循环再利用方面起到了重要的作用。因此,单核巨噬细胞系统对整个机体铁稳态的维持非常重要。近年来,随着转铁蛋白受体1(transferrin receptor1,TfR1)、铁蛋白(ferritin,Fn)、二价金属离子转运蛋白1(divalent metal transporter1,DMT1)、膜铁转运蛋白1(ferroportin1,FPN1),以及铁调素(hepcidin)等在单核巨噬细胞系统中功能和调控机制研究的不断深入,日益加深了人们对单核巨噬细胞系统的铁代谢过程和调控机制的了解。该文综述了铁水平、NO以及炎症等因素对单核巨噬细胞系统TfR1、Fn、DMT1、FPN1、hepcidin等蛋白表达的调控及其机制研究的最新进展。     

Corticosterone Induces Dysregulation of Iron Metabolism in Hippocampal Neurons In Vitro

Iron is required for neuronal function but in excess generates neurodegeneration. Although the iron homeostasis machinery in neurons has been described extensively, little is known about the influence of corticosterone on the iron homeostasis in neurons. In this study, we characterized the response of hippocampal neurons to a model of progressive corticosterone condition. We found that increasing extracellular corticosterone-induced iron accumulation killed a large proportion of neurons. Iron concentrations were significantly increased in the corticosterone-treated cells. In the hippocampal neurons, corticosterone decreased expression of ferritin and increased expression of transferrin receptor1 (TfR1), iron regulatory protein1 (IRP1), and divalent metal transporter 1. Corticosterone-induced elevation of IRP1 expression can cause increase of TfR1 and decrease of ferritin expression, which further leads to iron accumulation in hippocampal neurons and subsequently increases the oxidative damage of the neurons; it is indicated that corticosterone might be an important reason for iron deposition-caused neurodegenerative diseases.     

多孔淀粉负载青蒿素微球的抗肿瘤活性研究

探讨多孔淀粉负载青蒿素微球(ART-PS)与青蒿素原药(ART)在不同浓度下的抗肿瘤活性,以及分别联合全铁转铁蛋白后对肿瘤细胞的生长抑制作用。在体外实验中,取对数生长期的人肝癌细胞和正常肝细胞接种于96孔板中,不同浓度(0、50、100、150、200μmol·L^-1)给药处理24h后,用MTT法分别检测多孔淀粉负载青蒿素微球与青蒿素原药对细胞的生长抑制作用。MTT结果显示,同等处理浓度下,多孔淀粉负载青蒿素微球对肿瘤细胞Hep G2和SMMC-7721的抑制效果都高于青蒿素原药,但与盐酸阿霉素相比,都具有较低的细胞毒性,对正常细胞HL7702的毒副作用非常低,结果与分别联合全铁转铁蛋白后对肿瘤细胞的生长抑制作用一致。多孔淀粉负载青蒿素微球对人肝癌细胞的增殖有明显的抑制作用,效果优于青蒿素原药,并对正常肝细胞的毒副作用非常低,为青蒿素在治疗癌症的应用与研究提供了重要的参考依据。     

Erratum to: Corticosterone Induces Dysregulation of Iron Metabolism in Hippocampal Neurons In Vitro

Iron is required for neuronal function but in excess generates neurodegeneration. Although the iron homeostasis machinery in neurons has been described extensively, little is known about the influence of corticosterone on the iron homeostasis in neurons. In this study, we characterized the response of hippocampal neurons to a model of progressive corticosterone condition. We found that increasing extracellular corticosterone-induced iron accumulation killed a large proportion of neurons. Iron concentrations were significantly increased in the corticosterone-treated cells. In the hippocampal neurons, corticosterone decreased expression of ferritin and increased expression of transferrin receptor1 (TfR1), iron regulatory protein1 (IRP1), and divalent metal transporter 1. Corticosterone-induced elevation of IRP1 expression can cause increase of TfR1 and decrease of ferritin expression, which further leads to iron accumulation in hippocampal neurons and subsequently increases the oxidative damage of the neurons; it is indicated that corticosterone might be an important reason for iron deposition-caused neurodegenerative diseases.     

Release of the soluble transferrin receptor is directly regulated by binding of its ligand ferritransferrin

The human transferrin receptor (TfR) is shed by an integral metalloprotease releasing a soluble form (sTfR) into serum. The sTfR reflects the iron demand of the body and is postulated as a regulator of iron homeostasis via binding to the hereditary hemochromatosis protein HFE. To study the role of transferrin in this process, we investigated TfR shedding in HL60 cells and TfR-deficient Chinese hamster ovary cells transfected with human TfR. Independent of TfR expression, sTfR release decreases with increasing ferritransferrin concentrations, whereas apo-transferrin exhibits no inhibitory effect. To investigate the underlying mechanism, we generated several TfR mutants with different binding affinities for transferrin. Shedding of TfR mutants in transfected cells correlates exactly with their binding affinity, implying that the effect of ferritransferrin on TfR shedding is mediated by a direct molecular interaction. Analysis of sTfR release from purified microsomal membranes revealed that the regulation is independent from intracellular trafficking or cellular signaling events. Our results clearly demonstrated that sTfR does not only reflect the iron demand of the cells but also the iron availability in the bloodstream, mirrored by iron saturation of transferrin, corroborating the important potential function of sTfR as a regulator of iron homeostasis.