TfR1 Expression Research Articles

Evaluation of the expression level of apoptosis and ferroptosis cell death pathways genes in patients with osteosarcoma

BackgroundThe osteosarcoma management and failure in tumor removal remain a diagnostic challenge. ObjectivesThe expression pattern of main mediators of apoptosis and ferroptosis cell death pathways and their relevance to tumor severity and response to chemotherapy were surveyed in the current study. MethodsThe mRNA expression level of Bax, Bcl-2, caspase-8, caspase-9, TfR1, and Gpx4 were assessed in 210 bone tissue samples including benign and malignant osteosarcoma tumors and normal bone tissues via Real-time PCR. The correlation between gene expression level and patient's demographic features and the regression analysis was performed. ResultsDown-regulation of Bax, caspase-8, caspase-9, and Gpx4 was observed in bone tumors that were more prominent in high-grade, metastatic, and recurrent malignant tumors. In tumors of patients with no history of chemotherapy, lower expression of caspase-8, caspase-9 and Gpx4 and higher TfR1 expression levels were detected, indicating that ferroptosis might be selected as a substitute cell death pathway to eliminate cancer cells in osteosarcoma. The difference between malignant and benign tumors was not remarkable; while each malignant and benign tumor group compared to their adjacent normal bone tissue showed a significant reduction in the expression level of Bax, caspase-8, caspase-9, and Gpx4. The higher expression of Bcl-2, as an anti-apoptotic mediator was detected in high-grade, and metastatic osteosarcoma tumors. Also, a higher expression level of TfR1 was detected in malignant, high-grade and metastatic osteosarcoma tumors.Also, the metastatic status of malignant tumors was effective in predicting Bax, Bcl-2, Gpx4, and TfR1 expression patterns in osteosarcoma tumors. ConclusionIn osteosarcoma tumors, ferroptosis might be executed as an alternative death pathway while tumors show developing resistance to chemotherapy or when the predominant apoptotic cell death is down-regulated.

Hypoxia increases expression of selected blood\u2013brain barrier transporters GLUT-1, P-gp, SLC7A5 and TFRC, while maintaining barrier integrity, in brain capillary endothelial monolayers

BackgroundBrain capillary endothelial cells (BCECs) experience hypoxic conditions during early brain development. The newly formed capillaries are tight and functional before astrocytes and pericytes join the capillaries and establish the neurovascular unit. Brain endothelial cell phenotype markers P-gp (ABCB1), LAT-1(SLC7A5), GLUT-1(SLC2A1), and TFR(TFRC) have all been described to be hypoxia sensitive. Therefore, we hypothesized that monolayers of BCECs, cultured under hypoxic conditions, would show an increase in LAT-1, GLUT-1 and TFR expression and display tight endothelial barriers.Methods and resultsPrimary bovine BCECs were cultured under normoxic and hypoxic conditions. Chronic hypoxia induced HIF-1α stabilization and translocation to the nucleus, as judged by immunocytochemistry and confocal laser scanning imaging. Endothelial cell morphology, claudin-5 and ZO-1 localization and barrier integrity were unaffected by hypoxia, indicating that the tight junctions in the BBB model were not compromised. SLC7A5, SLC2A1, and TFRC-mRNA levels were increased in hypoxic cultures, while ABCB1 remained unchanged as shown by real-time qPCR. P-gp, TfR and GLUT-1 were found to be significantly increased at protein levels. An increase in uptake of [3H]-glucose was demonstrated, while a non-significant increase in the efflux ratio of the P-gp substrate [3H]-digoxin was observed in hypoxic cells. No changes were observed in functional LAT-1 as judged by uptake studies of [3H]-leucine. Stabilization of HIF-1α under normoxic conditions with desferrioxamine (DFO) mimicked the effects of hypoxia on endothelial cells. Furthermore, low concentrations of DFO caused an increase in transendothelial electrical resistance (TEER), suggesting that a slight activation of the HIF-1α system may actually increase brain endothelial monolayer tightness. Moreover, exposure of confluent monolayers to hypoxia resulted in markedly increase in TEER after 24 and 48 h, which corresponded to a higher transcript level of CLDN5.ConclusionsOur findings collectively suggest that hypoxic conditions increase some BBB transporters' expression via HIF-1α stabilization, without compromising monolayer integrity. This may in part explain why brain capillaries show early maturation, in terms of barrier tightness and protein expression, during embryogenesis, and provides a novel methodological tool for optimal brain endothelial culture.

Role of ferroptosis in promoting cardiotoxicity induced by Imatinib Mesylate via down-regulating Nrf2 pathways in vitro and in vivo

Imatinib Mesylate (IMA) has been widely used to treat with chronic myeloid leukemia (CML). However, cardiotoxicity associated with IMA is included among the therapeutic strategies. The present study was aimed to discover whether ferroptosis, a programmed iron-dependent cell death, is involved in IMA-induced cardiotoxicity. In vivo, mouse model was established after treated with 25 mg/kg, 50 mg/kg and 100 mg/kg IMA. Serum CK, LDH, AST activities were determined. Cardiac tissues were examined by H&E and Oil Red O staining. MDA was measured to assess production of lipid peroxide. Tissue iron and GSH content were measured. In vitro, cell viability, mitochondria membrane potential, generation of reactive oxygen species (ROS) and cellular iron levels were performed to explore the mechanism of IMA. The in vivo results revealed that IMA treatment significantly increased serum CK, LDH and AST. H&E staining showed that IMA caused cardiac structural injuries. The dose-dependent decrease of GSH and increase of tissue iron and MDA were observed in IMA-treated groups. Oil Red O staining suggested obvious cardiac lipid accumulation after treated with IMA. In H9c2 cardiomyocytes, IMA significantly inhibited cell proliferation in a dose-dependent manner. Mitochondria membrane potential assay showed that IMA destroyed the mitochondrial function. Additionally, IMA increased the cellular ROS and iron levels. Furthermore, IMA down-regulated the expression of Nrf2 and up-regulated the expression of P53 and TfR. These results provided compelling evidence that ferroptosis participates in IMA-induced cardiotoxicity. Ferroptosis could be regarded as a target to protect against cardiotoxicity in IMA-exposed patients.

E-Cadherin, Integrin Alpha2 (Cd49b), and Transferrin Receptor-1 (Tfr1) Are Promising Immunohistochemical Markers of Selected Adverse Pathological Features in Patients Treated with Radical Prostatectomy.

In patients treated for prostate cancer (PCa) with radical prostatectomy (RP), determining the risk of extraprostatic extension (EPE) and nodal involvement (NI) remains crucial for planning nerve-sparing and extended lymphadenectomy. The study aimed to determine proteins that could serve as immunohistochemical markers of locally advanced PCa. To select candidate proteins associated with adverse pathologic features (APF) reverse-phase protein array data of 498 patients was retrieved from The Cancer Genome Atlas. The analysis yielded 6 proteins which were then validated as predictors of APF utilizing immunohistochemistry in a randomly selected retrospective cohort of 53 patients. For univariate and multivariate analysis, logistic regression was used. Positive expression of TfR1 (OR 13.74; p = 0.015), reduced expression of CD49b (OR 10.15; p = 0.013), and PSA (OR 1.29; p = 0.013) constituted independent predictors of EPE, whereas reduced expression of e-cadherin (OR 10.22; p = 0.005), reduced expression of CD49b (OR 24.44; p = 0.017), and PSA (OR 1.18; p = 0.002) were independently associated with NI. Both models achieved high discrimination (AUROC 0.879 and 0.888, respectively). Immunohistochemistry constitutes a straightforward tool that might be easily utilized before RP. Expression of TfR1 and CD49b is associated with EPE, whereas expression of e-cadherin and CD49b is associated with NI. Since following immunohistochemical markers predicts respective APFs independently from PSA, in the future they might supplement existing preoperative nomograms or be implemented in novel tools.

Rebalancing Iron Homeostasis and Erythropoiesis through Tfr2 Inhibition for Correction of Anemia in CKD

BackgroundTransferrin Receptor 2 (TFR2) is a protein expressed in the liver and in the erythroid compartment. Hepatic TFR2 activates the transcription of hepcidin, the master regulator of iron homeostasis, and its inactivation causes iron overload. Erythroid TFR2 interacts with Erythropoietin (EPO) receptor and its deletion enhances erythropoiesis increasing EPO sensitivity of erythroid cells. We recently demonstrated that bone marrow (BM) Tfr2 loss improves anemia in a murine model of β-thalassemia. We hypothesized that the same approach might represent a therapeutic option also for anemias due to insufficient EPO production, as anemia of Chronic Kidney Disease (CKD).Indeed, anemia is a common complication of CKD, since EPO production is inhibited by progressive renal failure. In addition, chronic inflammation that parallels renal damage decreases EPO responsiveness of erythroid cells and enhances the production of hepcidin. Increased hepcidin levels limit iron absorption from the diet and release from the stores, reducing iron supply to erythropoiesis. All these factors contribute to anemia development.Replacement therapy with erythropoiesis stimulating agents (ESA), usually combined with iron supplementation, is effective but may lead to cardio-vascular side effects. Thus, novel and more specific strategies are needed.AimsHere, we exploit different murine models of selective Tfr2 inactivation to test whether Tfr2 targeting corrects anemia of CKD. In this context, BM Tfr2 inhibition is expected to stimulate erythropoiesis and the simultaneous downregulation of hepatic Tfr2 to correct iron deficiency.MethodsWe induced CKD using an adenine-rich diet in mice with:(1) BM-specific Tfr2 deletion (Tfr2 BMKO), generated through BM transplantation;(2) reduced Tfr2 hepatic expression, obtained treating wild-type mice with anti-Tfr2 antisense oligonucleotides (Tfr2-ASO);(3) germline Tfr2 inactivation in the whole organism (Tfr2KO).ResultsRenal damage was comparable among all the mice analyzed, excluding a differential effect of the diet in the various groups.Tfr2BMKO mice showed enhanced erythropoiesis relative to controls, due to the increased EPO responsiveness of erythroid cells lacking Tfr2, as suggested by the over-activation of the EPO-EPOR signaling pathway despite comparable EPO levels. Tfr2BMKO mice maintained higher red blood cell (RBC) count than controls for the entire protocol. Hemoglobin (Hb) levels, higher in Tfr2BMKO mice for 6 weeks, reached levels of controls at 8 weeks, concomitant with relative hypoferremia. These results indicate that BM Tfr2 deletion transiently prevents anemia until iron availability is adequate to the enhanced erythropoiesis.Then we investigated the potential beneficial effect of increasing iron availability through Tfr2-ASOs treatment, which efficiently decreased hepatic (95-97%) but not erythroid Tfr2. As expected, circulating iron levels were increased in Tfr2-ASO mice, maintaining RBC count and Hb levels in the normal range for the first 2 weeks of treatment. However, Hb and RBCs reverted to control levels at 6 weeks, before the end of the protocol. These results show that increased iron availability alone, due to hepatic Tfr2 downregulation, delays anemia development but is not sufficient to boost erythropoiesis on a long term.In agreement, Tfr2KO mice, with Tfr2 inactivation both in the liver and in the erythroid compartment, maintained higher RBC count and Hb levels compared to controls until the end of the protocol.ConclusionsThe concomitant targeting of hepatic and erythroid Tfr2, here obtained through Tfr2 germline genetic inactivation, is necessary and sufficient to ameliorate anemia of CKD. On the contrary selective BM Tfr2 deletion that enhances EPO responsiveness of erythroid cells, or hepatic Tfr2 downregulation that increases iron availability, do not correct anemia in a long term. Therefore, a specific approach able to inhibit both hepatic and erythroid Tfr2 could adjust iron availability according to the enhanced erythropoiesis, correcting both drivers of anemia development in CKD. The development of a pharmacologic tool to downregulate Tfr2 might become an alternative to the standard treatment with ESAs plus iron supplementation with limited off-target effects due to the restricted TFR2 expression. DisclosuresAghajan: Ionis Pharmaceuticals, Inc.: Current Employment. Guo: Ionis Pharmaceuticals, Inc.: Current Employment.

Atractylodin may induce ferroptosis of human hepatocellular carcinoma cells.

BackgroundIt has been reported that atractylodin has a potential antitumor effect. This study aimed to investigate the effects of atractylodin on Huh7 and Hccm hepatocellular carcinoma (HCC) cells and its molecular mechanism.MethodsHuh7 and Hccm cells were cultured in vitro, and their viability was detected by CCK-8 assay and the half inhibitory concentration (IC50) was calculated. The cells were treated with different concentrations of atractylodin, and the migration and invasion ability of cells was detected by scratch assay and Transwell assay. The cell cycle change and apoptosis rate were detected by flow cytometry. IlluminaHiSeq4000 platform was used for transcriptome sequencing, and the results were analyzed for gene differential expression, gene function, and signal pathway enrichment. Morphological changes of cells were detected by transmission electron microscopy, reactive oxygen species (ROS) levels were detected by DCFH-DA probe, and the expressions of ferroptosis related proteins GPX4, ACSL4, FTL, and TFR1 were detected by Western blot.ResultsThe results showed that atractylodin could inhibit the proliferation, migration, and invasion of Huh7 and Hccm cells, regulate the cell cycle, and induce cell apoptosis and G1 phase cell cycle arrest. In addition, it could significantly induce the increase of intracellular ROS levels, decrease the expression of GPX4 and FTL proteins, and up-regulate the expression of ACSL4 and TFR1 proteins.ConclusionsAtractylodin can inhibit the proliferation, migration, and invasion of Huh7 and Hccm liver cancer cells, and induce cell apoptosis and cell cycle arrest. In addition, our results suggest that atractylodin may induce ferroptosis in HCC cells by inhibiting the expression of GPX4 and FTL proteins, and up-regulating the expression of ACSL4 and TFR1 proteins.

Transient Reduction of Transferrin Saturation Contributes to Hepcidin Inhibition Independently of Erfe in Mice Treated with Erythropoietin

IntroductionErythropoiesis is the major consumer of iron for hemoglobin synthesis. Erythroid cells release erythroferrone (ERFE), an erythropoietin (EPO) target gene, to signal their iron needs to the liver through hepcidin inhibition (Kautz et al., 2014). Since hepcidin degrades the iron exporter ferroportin, thus reducing body iron concentration, its inhibition by ERFE increases iron availability for erythroid cells. Hepcidin is mainly controlled by the BMP-SMAD-pathway. We demonstrated that ERFE inhibits hepcidin only when the BMP-SMAD signaling is attenuated (Nai et al., 2016). However, how the BMP-SMAD pathway is reduced in a physiologic context of increased erythropoiesis remains unknown. The major source of iron for erythroid cells is the circulating diferric-transferrin (holo-TF) that is uptaken by TFR1, highly expressed by erythroid cells. Since holo-TF, measured as transferrin saturation, is involved in hepcidin activation by the BMP-SMAD pathway, we hypothesized that increase iron uptake by EPO-stimulated erythropoiesis reduces transferrin saturation, thus inhibiting hepcidin earlier than and independently of ERFE.MethodsC57BL/6 wild type and Erfe KO male mice (8-9 week-old) were challenged with a single EPO injection (200 U/mouse) and sacrificed at early time points (3-15 hrs) post-treatment. To analyze erythroid differentiation and Tfr1 levels at short time points after EPO injection (3 and 6 hrs post-treatment), Ter119+ cells were separated from bone marrow (BM) and spleen according to Forward Scatter (FSC) value and CD44 expression, as described(Liu et al., 2013). Tfr1 was measured by flow cytometry in isolated population and expressed as MFI. Expression of BM and spleen Erfe, GypA and Tfr1 was evaluated by qRT-PCR. Hamp, Id1 and Atoh8 were measured from total liver of EPO- or saline-treated mice. Serum iron, transferrin saturation (TS) and liver iron content were evaluated by standard methods (Pagani et al., 2011).ResultsWild type mice were analyzed at different time point (3, 6, 9 and 15 hrs) after EPO injection. EPO-treated mice upregulate Erfe mRNA both in BM and spleen, starting from 3 hrs post-injection. Hepcidin and the BMP-SMAD target genes Id1 and Atoh8 are significantly decreased at 9 hrs post-EPO. Although Id1 and Atoh8 start to increase 15 hrs post-EPO, hepcidin remains low due to Erfe inhibition. EPO treatment transiently (at 6 and 9 hrs) reduces both serum iron and TS that are increased thereafter (15 hrs) due to hepcidin inhibition. Since EPO stimulates Tfr1 expression in erythroid cells, we measured Tfr1mRNA and protein levels in BM and spleen. Tfr1 mRNA is slightly increased in the spleen and shows a trend towards increase in the BM at 6 hrs post-EPO, whereas it is comparable to untreated mice at 9 and 15 hrs post-EPO. However, flow cytometry analysis shows that, despite the number of erythroid precursors expressing Tfr1 is comparable in untreated and EPO treated mice, membrane Tfr1 is strongly increased at 3 and 6 hrs post-EPO, indicating that increased erythroid uptake accounts for serum iron/TS reduction shortly after EPO treatment. To demonstrate that increased Tfr1 expression by EPO in erythroid cells downregulates the liver BMP-SMAD pathway and hepcidin through a transient reduction in circulating iron/TS and independently of Erfe, we analyzed EPO-injected Erfe KO mice. Both in wild type and Erfe KO animals Id1 is strongly inhibited at 6-9 hrs post EPO. Interestingly, Erfe KO animals show a trend towards a reduction in hepcidin at 9 hrs post-EPO, and a significant decrease at 15 hrs, with low serum iron and TS.The mild but significant effect observed in Erfe KO mice suggests that downregulation of the BMP-SMAD pathway due to transient reduction of TS contributes to hepcidin inhibition in an erythroid-dependent, but Erfe-independent, manner.ConclusionsEPO stimulates erythroid expression of membrane Tfr1 at short time point after treatment, causing a transient decreased in transferrin saturation and a downregulation of the hepatic BMP-SMAD pathway that contributes to hepcidin inhibition independently of Erfe. DisclosuresFerrari:GSK: Other: B-thalassemia gene therapy was developed by Fondazione Telethon and Ospedale San Raffaele and has been inlicenced by GSK that provides funding for the clinical trial, Research Funding. Camaschella:Vifor Pharma: Membership on an entity's Board of Directors or advisory committees.

Salvia miltiorrhiza (SM) Injection Ameliorates Iron Overload-Associated Cardiac Dysfunction by Regulating the Expression of DMT1, TfR1, and FP1 in Rats.

Previous studies have found that Salvia miltiorrhiza (SM) injection have a protective effect on the iron overloaded (IO) heart. However, the mechanisms are not completely known. In the present study, we investigated the underlying mechanisms based on the iron transport-related proteins. The rats were randomly divided into five groups: control, IO group, low-dose SM group, high-dose SM group, and deferoxamine control group. Iron dextran was injected to establish the IO model. After 14 days of treatment, cardiac histological changes were observed by hematoxylin and eosin (H&E) staining. Iron uptake-related proteins divalent metal transporter-1 (DMT-1), transferrin receptor-1 (TfR-1), and iron export-related proteins ferroportin1 (FP1) in the heart were detected by Western blotting. The results showed that SM injection decreased cardiac iron deposition, ameliorated cardiac function, and inhibited cardiac oxidation. Most important of all, SM injection downregulated the expression of DMT-1 and TfR-1 and upregulated FP1 protein levels compared with the IO group. Our results indicated that reducing cardiac iron uptake and increasing iron excretion may be one of the important mechanisms of SM injection reducing cardiac iron deposition and improving cardiac function under the conditions of IO.

Transferrin receptor 1 ablation in satellite cells impedes skeletal muscle regeneration through activation of ferroptosis.

BackgroundSatellite cells (SCs) are critical to skeletal muscle regeneration. Inactivation of SCs is linked to skeletal muscle loss. Transferrin receptor 1 (Tfr1) is associated with muscular dysfunction as muscle‐specific deletion of Tfr1 results in growth retardation, metabolic disorder, and lethality, shedding light on the importance of Tfr1 in muscle physiology. However, its physiological function regarding skeletal muscle ageing and regeneration remains unexplored.MethodsRNA sequencing is applied to skeletal muscles of different ages to identify Tfr1 associated to skeletal muscle ageing. Mice with conditional SC ablation of Tfr1 were generated. Between Tfr1 SC/WT and Tfr1 SC/KO (n = 6–8 mice per group), cardiotoxin was intramuscularly injected, and transverse abdominal muscle was dissected, weighted, and cryosectioned, followed by immunostaining, haematoxylin and eosin staining, and Masson staining. These phenotypical analyses were followed with functional analysis such as flow cytometry, tread mill, Prussian blue staining, and transmission electron microscopy to identify pathological pathways that contribute to regeneration defects.ResultsBy comparing gene expression between young (2 weeks old, n = 3) and aged (80 weeks old, n = 3) mice among four types of muscles, we identified that Tfr1 expression is declined in muscles of aged mice (~80% reduction, P < 0.005), so as to its protein level in SCs of aged mice. From in vivo and ex vivo experiments, Tfr1 deletion in SCs results in an irreversible depletion of SCs (~60% reduction, P < 0.005) and cell‐autonomous defect in SC proliferation and differentiation, leading to skeletal muscle regeneration impairment, followed by labile iron accumulation, lipogenesis, and decreased Gpx4 and Nrf2 protein levels leading to reactive oxygen species scavenger defects. These abnormal phenomena including iron accumulation, activation of unsaturated fatty acid biosynthesis, and lipid peroxidation are orchestrated with the occurrence of ferroptosis in skeletal muscle. Ferroptosis further exacerbates SC proliferation and skeletal muscle regeneration. Ferrostatin‐1, a ferroptosis inhibitor, could not rescue ferroptosis. However, intramuscular administration of lentivirus‐expressing Tfr1 could partially reduce labile iron accumulation, decrease lipogenesis, and promote skeletal muscle regeneration. Most importantly, declined Tfr1 but increased Slc39a14 protein level on cellular membrane contributes to labile iron accumulation in skeletal muscle of aged rodents (~80 weeks old), leading to activation of ferroptosis in aged skeletal muscle. This is inhibited by ferrostatin‐1 to improve running time (P = 0.0257) and distance (P = 0.0248).ConclusionsSatellite cell‐specific deletion of Tfr1 impairs skeletal muscle regeneration with activation of ferroptosis. This phenomenon is recapitulated in skeletal muscle of aged rodents and human sarcopenia. Our study provides mechanistic information for developing novel therapeutic strategies against muscular ageing and diseases.

IL-6 Regulates Hepcidin Expression Via the BMP/SMAD Pathway by Altering BMP6, TMPRSS6 and TfR2 Expressions at Normal and Inflammatory Conditions in BV2 Microglia

The hormone hepcidin plays a central role in controlling iron homeostasis. Iron-mediated hepcidin synthesis is triggered via the BMP/SMAD pathway. At inflammation, mainly IL-6 pro-inflammatory cytokine mediates the regulation of hepcidin via the JAK/STAT signalling pathway. Microglial cells of the central nervous system are able to recognize a broad spectrum of pathogens via toll-like receptors and initiate inflammatory response. Although the regulation of hepcidin synthesis is well described in many tissues, little is known about the inflammation mediated hepcidin regulation in microglia. In this study, we investigated the pathways, which are involved in HAMP regulation in BV2 microglia due to inflammatory mediators and the possible relationships between the iron regulatory pathways. Our results showed that IL-6 produced by resting BV2 cells was crucial in maintaining the basal HAMP expression and hepcidin secretion. It was revealed that IL-6 neutralization decreased both STAT3 and SMAD1/5/9 phosphorylation suggesting that IL-6 proinflammatory cytokine is necessary to maintain SMAD1/5/9 activation. We revealed that IL-6 influences BMP6 and TMPRSS6 protein levels, moreover it modified TfR2 expression, as well. In this study, we revealed that BV2 microglia increased their hepcidin secretion upon IL-6 neutralization although the major regulatory pathways were inhibited. Based on our results it seems that both at inflammation and at normal condition the absence of IL-6 triggered HAMP transcription and hepcidin secretion via the NFκB pathway and possibly by the autocrine effect of TNFα cytokine on BV2 microglia.

Myotonic dystrophy kinase-related CDC42-binding kinase α, a new transferrin receptor type 2-binding partner, is a regulator of erythropoiesis.

Efficient erythropoiesis relies on the expression of the transferrin receptor type 2 (TFR2). In erythroid precursors, TFR2 facilitates the export of the erythropoietin receptor (EPOR) to cell surface, which ensures the survival and proliferation of erythroblasts. Although TFR2 has a crucial role in erythropoiesis regulation, its mechanism of action remains to be clarified. To understand its role better, we aimed at identifying its protein partners by mass-spectrometry after immunoprecipitation in erythroid cells. Here we report the kinase MRCKα (myotonic dystrophy kinase-related CDC42-binding kinase α) as a new partner of both TFR2 and EPOR in erythroblasts. We show that MRCKα is co-expressed with TFR2, and TFR1 during terminal differentiation and regulates the internalization of the two types of transferrin receptors. The knockdown of MRCKα by shRNA in human primary erythroblasts leads to a decreased cell surface expression of both TFR1 and TFR2, an increased cell-surface expression of EPOR, and a delayed differentiation. Additionally, knockout of Mrckα in the murine MEDEP cells also leads to a striking delay in erythropoiesis, showcasing the importance of this kinase in both species. Our data highlight the importance of MRCKα in the regulation of erythropoiesis.

The Protective Effect of Cyanidin-3-Glucoside on Myocardial Ischemia-Reperfusion Injury through Ferroptosis.

This study was conducted to estimate the protective effect of Cyanidin-3-glucoside (C3G) on myocardial ischemia-reperfusion (IR) injury and to explore its mechanism. The rats were subjected to left anterior descending ligation and perfusion surgery. In vitro experiments were performed on H9c2 cells using the oxygen-glucose deprivation/reoxygenation (OGD/R) model. The results showed the administration of C3G reduced the infarction area, mitigated pathological alterations, inhibited ST segment elevation, and attenuated oxidative stress and ferroptosis-related protein expression. C3G also suppressed the expressions of USP19, Beclin1, NCOA4, and LC3II/LC3I. In addition, treatment with C3G relieved oxidative stress, downregulated LC3II/LC3I, reduced autophagosome number, downregulated TfR1 expression, and upregulated the expressions of FTH1 and GPX4 in OGD/R-induced H9c2 cells. C3G could inhibit the protein levels of USP19 and LC3II. C3G promoted K11-linked ubiquitination of Beclin1. Further evidence that C3G reduced ferroptosis and ameliorated myocardial I/R injury was demonstrated with the ferroptosis promoter RSL3. Taken together, C3G could be a potential agent to protect myocardium from myocardial I/R injury.

Nitric Oxide Alters the Expression of γ-Globin, H-Ferritin, and Transferrin Receptor in Human K562 Cells at the Posttranscriptional Level

Cellular iron metabolism is altered during chronic inflammatory states, leading to reticuloendothelial iron sequestration and an associated anemia. To study the effects of nitric oxide (NO) on the expression of three genes involved in erythroid cell iron metabolism (gamma-globin, H-ferritin, and transferrin receptor [TfR]), we developed a series of human K562 erythroleukemic cell clones retrovirally transduced with inducible nitric oxide synthase (NOS-2) and producing different steady-state levels of NO. gamma-Globin and H-ferritin protein expression was reduced in NO-producing cells in relation to the amount of NO produced. Conversely, cell surface TfR expression increased in NO-producing clones. Both the inhibitory effects of NO on gamma-globin and H-ferritin expression and the stimulatory effect on TfR were reversed by the NOS inhibitor NG-methyl-L-arginine (NGMMA). gamma-Globin and H-ferritin mRNA levels were unaffected by NO production. In the case of TfR, NO appeared to stabilize mRNA in that the half life of TfR mRNA decreased from approximately 15 hours to less than 3 hours when NO production by NOS-transduced clones was inhibited. Thus, NO can regulate expression of these genes at the posttranscriptional level, an effect that is likely mediated by the known effect of NO on the RNA binding activity of iron regulatory protein-1 (Pantopoulos and Hentze, Proc Natl Acad Sci USA 92:1267, 1995). Furthermore, our findings suggest a mechanism for the observed relationship between NO production and the pathophysiology of the anemia of chronic disease.

Neuroprotective effects of kukoamine A on 6-OHDA-induced Parkinson's model through apoptosis and iron accumulation inhibition

ObjectiveParkinson's disease (PD) is characterized by the loss of dopaminergic neurons in substantia nigra (SN). Our previous study demonstrated kukoamine A (KuA) to exhibit strong neuroprotective effects through antioxidative stress, and autophagy in MPTP/MPP+-induced PD models in vivo and in vitro. It is necessary to evaluate the efficacy of the anti-PD effects under various models. MethodsIn the present study, total chemical synthesis was used to obtain KuA, which performed low content in Lycii Cortex. Then, 6-OHDA-induced PD model of PC12 cells was used to investigate the effects of KuA on PD. ResultsOur results demonstrated that KuA ameliorated cell loss and mitochondrial membrane potential (MMP) loss, and inhibited Bax/Bcl-2 ratio increase that were induced by 6-OHDA. Iron accumulation in SN is thought to participate in neuronal death in PD, which subsequently resulted in oxidative stress and overexpression of α-synuclein caused by iron metabolism protein disorder. In our study, KuA could chelate cellular iron content and decrease iron influx. Moreover, KuA could upregulate the expression of ferroportin1 and Hephaestin, downregulate the expression of DMT1, TfR, and Ferritin to maintain cellular iron homeostasis avoiding neuronal death from cellular iron deposition. Moreover, KuA could decrease the expression of a-synuclein in cells. All the results indicated that KuA protected against neurotoxin-induced PD due to the apoptosis inhibition and iron homeostasis maintaining. ConclusionKuA treatment might represent a neuroprotective treatment for PD.

Brain iron deficiency and affected contextual fear memory in mice with conditional Ferroportin1 ablation in the brain.

Fear memory is a pivotal biological function by which organisms can predict possible danger to avoid or reduce harm. However, dysregulation of fear memory processing may lead to pathological fear or anxiety and produce serious clinical symptoms, such as post-traumatic stress disorder (PTSD). Iron deficiency (ID) is reported to inhibit the initiation of fear memory. In our study, we found that ferroportin1 (FPN1), the only known cellular iron export protein in mammals, and ablation in neurons and astrocytes caused iron deficiency in the cortex and hippocampus. However, little is known about its role in the development of fear memory. Moreover, direct evidence of the role of FPN1, or the related molecular mechanisms of such a role, in balancing brain iron homeostasis, especially in neuronal cells, is lacking. Herein, we deleted Fpn1 in mouse neurons, using Nestin-cre transgenic mice, and explored the impact on neuronal iron recycling and brain iron homeostasis in the cortex and hippocampus. We investigated the response of the mice to contextual fear and found that formation of fear memory was impeded after neuronal FPN1 depletion. We also found that FPN1 ablation in neurons and astrocytes caused an atypical expression of iron metabolism-related proteins in these two regions: decreased expression of DMT1, Ft-H, and Ft-L, and increased TfR1 expression. In addition, the decreased FPN1 in brain microvascular endothelial cells (BMVECs) also shed light on the cause of the decreased iron delivery to the brain through the blood-brain barrier (BBB). Our research highlights the major role played by FPN1 in brain iron homeostasis and identifies a potential target for the treatment of PTSD.

TAMI-17. RELATIONSHIP BETWEEN IRON METABOLISM, IMMUNE CELL INFILTRATION AND SEX-BASED SURVIVAL DIFFERENCES IN GLIOMAS

Abstract Iron plays a central role in cellular metabolism, both in normal cellular functioning and in tumorigenesis. Recent evidence has shown sex-based survival differences in glioblastoma (GBM) may be related to differential expression of metabolism genes. We previously reported the iron regulating gene, HFE, was shown to have a sex-based survival impact in both low-grade gliomas and GBM. We additionally found that females with low HFE expressing tumors have significantly higher survival than males in GBM. To evaluate the relationship between iron gene expression and sex-based survival differences in GBM, we analyzed TCGA GBM gene expression and clinical data. We first analyzed the impact of iron genes on sex-based survival. In addition to HFE, FTL, TFRC, TF, and SLC39A8 (ZIP8), also showed sex-based survival differences. We then compared correlations of HFE and other iron genes to identify whether male and female GBMs differ in iron regulation and metabolism. HFE expression is significantly positively correlated with HMOX1, SLC25A28, SLC11A2, FTH1, HAMP, and TFR2 only in females. Alternatively, HFE expression is negatively correlated with ACO2 (mitochondrial aconitase) in males and ACO1 (cytoplasmic aconitase) in females. We noted that the expression of certain iron genes was highly associated with immune cell infiltration based on sex. TFR2, LRP1, and XIST expression were negatively correlated with low immune cell infiltration in females, but not males. Alternatively, in males, SLC11A2, ACO2, FOXO1, HIF1a, and HAMP genes were negatively correlated with immune infiltration. This suggests that differences in iron regulation between males and females may be contributing to differences in immune function and subsequent survival in GBM. These data suggest that the iron signature of a tumor reflects and possibly drives the metabolic and immune landscape of the tumor microenvironment thereby directly impacting survival differences between male and female GBMs.

Abstract PO-48: Cytotoxic mechanism of a novel transferrin receptor-targeting chemotherapeutic nanocarrier for use in diffuse large B-cell lymphoma

Abstract Diffuse large B-cell lymphoma (DLBCL), the most common hematologic malignancy, is an aggressive form of non-Hodgkin lymphoma. Approximately 60% of DLBCL patients achieve long-term disease-free survival from frontline R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone), but patients with relapsed or refractory disease have poor prognosis. Doxorubicin (Dox) remains the most active anti-lymphoma agent, but its use is limited by well-characterized toxicities, including potentially irreversible cardiac damage. Targeted and specific delivery of Dox to tumors to overcome these limitations has been an area of active research. Targeted delivery of Dox via the transferrin receptor (TFR1) has preclinical efficacy in solid malignancies but has yet to be explored in lymphoma. We have uncovered for the first time a significant association between TFR1 expression and poor prognosis in DLBCL. Carbon-nitride dots (CNDs) are third-generation nanocarriers with inherent photoluminescence, which are easily conjugated with a wide variety of substrates. We conjugated Dox and transferrin (TF), the ligand for TFR1, to CNDs to develop CND-Dox-TF (CDT). CDT is uniquely designed to enhance Dox delivery to TFR1-expressing DLBCL tumors while limiting effects on nonmalignant tissues. BJAB and Farage cell lines treated with CDT for 24 hours underwent apoptosis at a significantly lower concentration when compared to Dox (50 nM vs. 1000 nM). Overall, CDT was 1-2 Log10 more potent than Dox against DLBCL cell lines. Co-incubation with 250 uM of antagonist holo-transferrin significantly decreased BJAB and Farage sensitivity to CDT, confirming activity through TFR1. Confocal microscopy of BJAB cells incubated up to 24 hours with 250 nM Dox and 30 nM CDT reveled enhanced nuclear colocalization of Dox at a significantly lower concentration. Prior dose-finding experiments in non-tumor bearing mice identified a CDT safe working dose of 33.0 mg/kg containing 16% moles of Dox in comparison to Dox maximum-tolerated dose (MTD) 3.3 mg/kg. We engrafted 2 groups of 10 NSG mice with a Dox-sensitive high-TFR1 expressing patient-derived xenograft (PDX) tumor. Mice were dosed with CDT and Dox on day 0, 14, and 24. CDT demonstrated similar anti-lymphoma efficacy with reduced toxicity: CDT-treated mice displayed no significant decline in body weight, which is characteristic and was observed following each single-agent Dox treatment. Histology analyses revealed little to no obvious damage to CDT-treated nonmalignant tissues, including myocardium. With a working dose of CDT identified, we now have under way clinically relevant R-CHOP vs. R-nanoCHOP (CDT substituted for Dox) assessments in our PDX mouse model. Capitalizing on the unique design, we expect to observe improved anti-lymphoma efficacy, with decreased toxicities and an improvement in overall survival. In sum, we provide mechanistic insight for novel DLBCL nano-chemotherapy and illustrate preclinical efficacy for a promising new therapeutic approach. Citation Format: Artavazd Arumov, Piumi Y. Liyanage, Asaad Trabolsi, Evan R. Roberts, Braulio Ferreira, Daniel Bilbao, Roger M. LeBlanc, Jonathan H. Schatz. Cytotoxic mechanism of a novel transferrin receptor-targeting chemotherapeutic nanocarrier for use in diffuse large B-cell lymphoma [abstract]. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr PO-48.

Acute and Chronic Dosing of a High-Affinity Rat/Mouse Chimeric Transferrin Receptor Antibody in Mice

Non-invasive brain delivery of neurotherapeutics is challenging due to the blood-brain barrier. The revived interest in transferrin receptor antibodies (TfRMAbs) as brain drug-delivery vectors has revealed the effect of dosing regimen, valency, and affinity on brain uptake, TfR expression, and Fc-effector function side effects. These studies have primarily used monovalent TfRMAbs with a human constant region following acute intravenous dosing in mice. The effects of a high-affinity bivalent TfRMAb with a murine constant region, without a fusion partner, following extravascular dosing in mice are, however, not well characterized. Here we elucidate the plasma pharmacokinetics and safety of a high-affinity bivalent TfRMAb with a murine constant region following acute and chronic subcutaneous dosing in adult C57BL/6J male mice. Mice received a single (acute dosing) 3 mg/kg dose, or were treated for four weeks (chronic dosing). TfRMAb and control IgG1 significantly altered reticulocyte counts following acute and chronic dosing, while other hematologic parameters showed minimal change. Chronic TfRMAb dosing did not alter plasma- and brain-iron measurements, nor brain TfR levels, however, it significantly increased splenic-TfR and -iron. Plasma concentrations of TfRMAb were significantly lower in mice chronically treated with IgG1 or TfRMAb. Overall, no injection related reactions were observed in mice.

LncRNA PVT1 regulates ferroptosis through miR-214-mediated TFR1 and p53

AimThe study aims to investigate the roles of LncRNA and miRNA in ferroptosis in brain ischemia/reperfusion (I/R) in vivo and in vitro. Materials and methodsqPCR assay was used to analyze lncRNA PVT1 and miR-214 expressions in acute ischemic stroke (AIS) patients. Then, we established brain I/R mice models and OGD/R PC12 cell models to analyze the mechanism of ferroptosis. I/R mice were treated by lncRNA PVT silencing or miR-214 overexpressing lentivirus via lateral ventricles. Infarct size was analyzed by TTC staining, accompanied by the detection of ferroptosis indicators through Perls'Prussian blue staining, iron kit, MDA kit, glutathione kit, GPx activities kit and Western blotting (WB). Dual luciferase reporter assay was used to assess whether miR-214 bound to PVT1, TP53 or TFR1. Co-IP analyzed the interplay of p53 with SLC7A11. Key findingsWe found that the levels of PVT1 were upregulated and miR-214 levels were downregulated in plasma of AIS patients. NIHSS score was positively correlated with PVT1 levels but was negatively with miR-214 levels. PVT1 silencing or miR-214 overexpression significantly reduced infarct size and suppressed ferroptosis in vivo. miR-214 overexpression markedly decreased PVT1 levels. Specifically, miR-214 could bind to 3'untranslated region (3’UTR) of PVT1, TP53 or TFR1. PVT1 overexpression or miR-214 silencing markedly abolished the effects of Ferrostatin-1 on ferroptosis indicators except for TFR1 expression. Besides, miR-214 silencing counteracted the effects of PVT1 knockdown on the ferroptosis-related proteins. ConclusionPVT1 regulated ferroptosis through miR-214-mediated TFR1 and TP53 expression. There was a positive feedback loop of lncRNA PVT1/miR-214/p53 possibly.

Estrogen signaling differentially alters iron metabolism in monocytes in an Interleukin 6-dependent manner

The ability of monocytes to release or sequester iron affects their role in cancer and inflammation. Previous work has shown that while IL-6 upregulates hepcidin synthesis and enhances iron sequestration, E2 reduces hepcidin synthesis and increases iron release. Given that E2 upregulates IL-6 production in monocytes, it is likely that the exact effect of E2 on iron metabolism in monocytes is shaped by its effect on IL-6 expression. To address this issue, the expression of key iron regulatory proteins was assessed in E2-treated U937, HuT-78, THP-1 and Hep-G2 cells. Iron status was also evaluated in U937 cells treated with the ERα agonist PPT, the ER antagonist ICI-182780, dexamethasone + E2, IL-6 + E2 and in IL-6-silenced U937 cells. E2 treatment reduced hepcidin synthesis in HuT-78, THP-1 and Hep-G2 cells but increased hepcidin synthesis and reduced FPN expression in U937 cells. E2-treated U937 cells also showed reduced HIF-1α and FTH expression and increased TFR1 expression, which associated with increased labile iron content as compared with similarly treated Hep-G2 cells. While treatment of U937 cells with interleukin 6 (IL-6) resulted in increased expression of hepcidin, dexamethasone treatment resulted in reduced hepcidin synthesis relative to E2- or dexamethasone + E2-treated cells; IL-6 silencing also resulted in reduced hepcidin synthesis in U937 cells. Lastly, while iron depletion resulted in increased cell death in U937 cells, E2 treatment resulted in enhanced cell survival and reduced apoptosis. These findings suggest that E2 differentially alters iron metabolism in monocytes in an IL-6 dependent manner.