Labile Iron Pool Research Articles

Highly Selective Fluorescent Probe With an Ideal pH Profile for the Rapid and Unambiguous Determination of Subcellular Labile Iron (III) Pools in Human Cells

A convenient tool for detecting iron ions in subcellular structures is desired for better understanding its roles in biological systems. In this work, a new Fe3+ sensor, 2-(2-((1-(6-acetylpyridin-2-yl)ethylidene)amino)ethyl)-3',6'-bis(diethylamino)spiro[isoindoline-1,9'-xanthen]-3-one (RhPK), which operates across the entire cellular pH range and is capable of unambiguously detecting Fe3+ ion in live human cells at subcellular resolution, is reported. The sensor exhibits high selectivity and sensitivity toward Fe3+ with a rapid fluorescence response and a 12-fold increase in intensity upon the addition of 1 equivalent Fe3+ at pH 7.3. RhPK forms a 1:1 complex with Fe3+ with an apparent binding constant 1.54 × 107 M−1 and a detection limit of 50 nM. The sensor is stable between pH 4.2 and 9.0 and operates across the whole cellular pH range. Cell imaging demonstrates the ability of the sensor to unambiguously detect basal level Fe3+ as well as its dynamic changes in real-time in live cells at subcellular resolution, with one labile Fe3+ pool identified in mitochondria in human primary fibroblast (ws1) cells for the first time and two Fe3 + pools confirmed in mitochondria and endo/lysosomes in human SH-SY5Y neuroblastoma cells, suggesting different cell types have distinctive Fe3+ storage in subcellular compartments. The RhPK probe is powerful for rapid and sensitive bioimaging of Fe3+ at subcellular level, enabling the unambiguous detection of labile Fe3+ pools at the entire cellular pH range, which is of great significance to understand the biological chemistry of Fe3+ and its roles in physiological processes and diseases.

Iron Administration Overcomes Resistance to Erastin-Mediated Ferroptosis in Ovarian Cancer Cells.

ObjectivesDeveloping novel therapeutic approaches to defeat chemoresistance is the major goal of ovarian cancer research. Induction of ferroptosis has shown promising antitumor effects in ovarian cancer cells, but the existence of still undefined genetic and metabolic determinants of susceptibility has so far limited the application of ferroptosis inducers in vivo.MethodsErastin and/or the iron compound ferlixit were used to trigger ferroptosis in HEY, COV318, PEO4, and A2780CP ovarian cancer cell lines. Cell viability and cell death were measured by MTT and PI flow cytometry assay, respectively. The “ballooning” phenotype was tested as ferroptosis specific morphological feature. Mitochondrial dysfunction was evaluated based on ultrastructural changes, mitochondrial ROS, and mitochondrial membrane polarization. Lipid peroxidation was tested through both C11-BODIPY and malondialdehyde assays. VDAC2 and GPX4 protein levels were quantified as additional putative indicators of mitochondrial dysfunction or lipid peroxidation, respectively. The effect of erastin/ferlixit treatments on iron metabolism was analyzed by measuring intracellular labile iron pool and ROS. FtH and NCOA4 were measured as biomarkers of ferritinophagy.ResultsHere, we provide evidence that erastin is unable to induce ferroptosis in a series of ovarian cancer cell lines. In HEY cells, provided with a high intracellular labile iron pool, erastin treatment is accompanied by NCOA4-mediated ferritinophagy and mitochondrial dysfunction, thus triggering ferroptosis. In agreement, iron chelation counteracts erastin-induced ferroptosis in these cells. COV318 cells, with low baseline intracellular labile iron pool, appear resistant to erastin treatment. Notably, the use of ferlixit sensitizes COV318 cells to erastin through a NCOA4-independent intracellular iron accumulation and mitochondrial dysfunction. Ferlixit alone mimics erastin effects and promotes ferroptosis in HEY cells.ConclusionThis study proposes both the baseline and the induced intracellular free iron level as a significant determinant of ferroptosis sensitivity and discusses the potential use of ferlixit in combination with erastin to overcome ferroptosis chemoresistance in ovarian cancer.

Liraglutide attenuates hepatic iron levels and ferroptosis in db/db mice

ABSTRACT Liver pathological changes are as high as 21%-78% in diabetic patients, and treatment options are lacking. Liraglutide is a glucagon-like peptide-1 (GLP-1) receptor that is widely used in the clinic and is approved to treat obesity and diabetes. However, the specific protection mechanism needs to be clarified. In the present study, db/db mice were used to simulate Type 2 diabetes mellitus (T2DM), and they were intraperitoneally injected daily with liraglutide (200 μg/kg/d) for 5 weeks. Hepatic function, pathologic changes, oxidative stress, iron levels, and ferroptosis were evaluated. First, liraglutide decreased serum AST and ALT levels, and suppressed liver fibrosis in db/db mice. Second, liraglutide inhibited the ROS production by upregulating SOD, GSH-PX, and GSH activity as well as by downregulating MDA, 4-HNE, and NOX4 expression in db/db mice. Furthermore, liraglutide attenuated iron deposition by decreasing TfR1 expression and increasing FPN1 expression. At the same time, liraglutide decreased ferroptosis by elevating the expression of SLC7A11 and the Nrf2/HO-1/GPX4 signaling pathway in the livers of db/db mice. In addition, liraglutide decreased the high level of labile iron pools (LIPs) and intracellular lipid ROS induced by high glucose in vitro. Therefore, we speculated that liraglutide played a crucial role in reducing iron accumulation, oxidative damage and ferroptosis in db/db mice.

The Role of Ferric Nitrilotriacetate in Renal Carcinogenesis and Cell Death: From Animal Models to Clinical Implications.

Simple SummaryIron is essential for cellular growth and survival. As a consequence, iron deficiency causes pleiotropic effects on the organism, while iron overload is also deleterious by means of oxidative tissue injury, which causes hepatic cirrhosis, diabetes mellitus, and cardiomyopathy in humans. Non-heme iron comprises ferric ion (Fe(III)), which is much more prominent in the transferrin, ferritin, or labile iron pool than the ferrous ion (Fe(II)); in contrast, ferrous ion yields more reactive oxygen species (ROS) than ferric ion does. In rodents, ferric nitrilotriacetate (Fe-NTA) elicits hepatic and renal oxidized lipids via a glutathione-cycle-dependent iron reduction that eventually causes renal cell carcinoma (RCC). In addition to iron-mediated carcinogenesis, ferroptosis is triggered by the iron-dependent accumulation of lipid peroxidation to lethal levels. Here, the mechanisms of iron- and ROS-mediated RCC and the therapeutic possibility of ferroptosis are discussed.Iron is essential for cellular growth, and various ferroproteins and heme-containing proteins are involved in a myriad of cellular functions, such as DNA synthesis, oxygen transport, and catalytic reactions. As a consequence, iron deficiency causes pleiotropic effects, such as hypochromic microcytic anemia and growth disturbance, while iron overload is also deleterious by oxidative injury. To prevent the generation of iron-mediated reactive oxygen species (ROS), ferritin is synthesized to store excess iron in cells that are consistent with the clinical utility of the serum ferritin concentration to monitor the therapeutic effect of iron-chelation. Among the animal models exploring iron-induced oxidative stress, ferric nitrilotriacetate (Fe-NTA) was shown to initiate hepatic and renal lipid peroxidation and the development of renal cell carcinoma (RCC) after repeated intraperitoneal injections of Fe-NTA. Here, current understanding of Fe-NTA-induced oxidative stress mediated by glutathione-cycle-dependent iron reduction and the molecular mechanisms of renal carcinogenesis are summarized in combination with a summary of the relationship between the pathogenesis of human RCC and iron metabolism. In addition to iron-mediated carcinogenesis, the ferroptosis that is triggered by the iron-dependent accumulation of lipid peroxidation and is implicated in the carcinogenesis is discussed.

Sodium molybdate inhibits the growth of ovarian cancer cells via inducing both ferroptosis and apoptosis

Ovarian cancer has the most mortality of all gynecologic malignancies. High-grade serous ovarian carcinoma (HGSOC) is the most common and deadly type of ovarian cancer. Tumor recurrence occurs due to the emergence of chemotherapy resistance. Thus, searching for new therapeutic strategies is essential for the management of ovarian cancer. Deregulation of iron metabolism can be used by ovarian cancer cells to survive, proliferate and metastasize. Here we report that sodium molybdate, a soluble molybdenum (Mo) compound, induces the elevation of the labile iron pool (LIP) in ovarian cancer cells, correlated with the down-regulation of genes involved in extracellular matrix organization. Sodium molybdate also induces depletion of glutathione (GSH) through mediating the production of nitric oxide (NO). Elevation of LIP and depletion of GSH promote the ferroptosis of ovarian cancer cells. Meanwhile, nitric oxide induces mitochondrial damage through inhibiting mitochondrial aconitase activity, ATP production, and mitochondrial membrane potential, leading to apoptosis of ovarian cancer cells. In vivo study shows that sodium molybdate reduces tumor burden in nude mice. Xenografts treated with sodium molybdate are characterized by obvious iron accumulation, increased expression of the iron storage protein ferritin, and lipid peroxide product 4-hydroxynonenal. In addition, an elevated percentage of apoptotic cells is observed in xenografts treated with sodium molybdate. Taken together, these results demonstrate that sodium molybdate can induce both ferroptosis and apoptosis of ovarian cancer cells, making it a potential therapeutic candidate for ovarian cancer.

Endothelial cell ferroptosis mediates monocrotaline-induced pulmonary hypertension in rats by modulating NLRP3 inflammasome activation

Inflammation triggers pulmonary vascular remodelling. Ferroptosis, a nonapoptotic form of cell death that is triggered by iron-dependent lipid peroxidation and contributes to the pathogenesis of several inflammation-related diseases, but its role in pulmonary hypertension (PH) has not been studied. We examined endothelial cell ferroptosis in PH and the potential mechanisms. Pulmonary artery endothelial cells (PAECs) and lung tissues from monocrotaline (MCT)-induced PH rats were analysed for ferroptosis markers, including lipid peroxidation, the labile iron pool (LIP) and the protein expression of glutathione peroxidase 4 (GPX4), ferritin heavy chain 1 (FTH1) and NADPH oxidase-4 (NOX4). The effects of the ferroptosis inhibitor ferrostatin-1 (Fer-1) on endothelial cell ferroptosis and pulmonary vascular remodelling in MCT-induced rats were studied in vitro and in vivo. Ferroptosis was observed in PAECs from MCT-induced PH rats in vitro and in vivo and was characterized by a decline in cell viability accompanied by increases in the LIP and lipid peroxidation, the downregulation of GPX4 and FTH1 expression and the upregulation of NOX4 expression. High-mobility group box 1 (HMGB1)/Toll-like receptor 4 (TLR4)/NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome signalling was measured by western blotting. These changes were significantly blocked by Fer-1 administration in vitro and in vivo. These results suggest that Fer-1 plays a role in inhibiting ferroptosis-mediated PAEC loss during the progression of PH. The ferroptosis-induced inflammatory response depended on the activation of HMGB1/TLR4 signalling, which activated the NLRP3 inflammasome in vivo. We are the first to suggest that pulmonary artery endothelial ferroptosis triggers inflammatory responses via the HMGB1/TLR4/NLRP3 inflammasome signalling pathway in MCT-induced rats. Treating PH with a ferroptosis inhibitor and exploring new treatments based on ferroptosis regulation might be promising therapeutic strategies for PH.

The Immune-Specific E3 Ubiquitin Ligase MARCH1 Is Upregulated during Human Cytomegalovirus Infection to Regulate Iron Levels.

Human cytomegalovirus (HCMV) modulates numerous cellular pathways to facilitate infection. Iron is essential to many cellular processes and is often incorporated into proteins and enzymes involved in oxidative phosphorylation and DNA synthesis and repair, among others. Despite its prominent role in the cell, little is known about the regulation of iron metabolism during HCMV infection. Herein, we observe modulation of the transferrin receptor (TfR) during infection and a corresponding change in the cellular labile iron pool. TfR and the iron pool are increased early during infection and then return to mock levels at the late stages of infection. We identified the cellular ubiquitin ligase MARCH1 as an important regulator of TfR. MARCH1 plays a proviral role during infection, as its knockdown leads to a decrease in infectious titers. Knockdown of MARCH1 also leads to an increase in ROS, lipid peroxidation, and mitochondrial dysfunction. Inhibiting an early increase in TfR expression during infection also decreases virus production. These findings indicate the importance of tightly regulating iron metabolism during HCMV infection to facilitate efficient virus production. IMPORTANCE Iron is essential for cells, playing important roles in energy generation, DNA replication, and gene expression. During infection, HCMV alters many cellular processes to aid its replication. We found that iron levels are tightly regulated during infection and that dysregulation of iron levels alters the ability to produce infectious virions. We also found that HCMV inactivates many of the cellular safeguards put in place to deal with excess iron. Thus, infected cells become more susceptible to variations in iron levels, which could be exploited as a therapeutic strategy for dealing with HCMV infections.

Iron Withdrawal with DIBI, a Novel 3-Hydroxypyridin-4-One Chelator Iron-Binding Polymer, Attenuates Macrophage Inflammatory Responses.

Purpose: Iron is an essential trace element for the inflammatory response to infection. In this study, we determined the effect of the recently developed iron-binding polymer DIBI on the synthesis of inflammatory mediators by RAW 264.7 macrophages and bone marrow-derived macrophages (BMDMs) in response to lipopolysaccharide (LPS) stimulation. Methods: Flow cytometry was used to determine the intracellular labile iron pool, reactive oxygen species production, and cell viability. Cytokine production was measured by quantitative reverse transcription polymerase chain reaction and enzyme-linked immunosorbent assay. Nitric oxide synthesis was determined by the Griess assay. Western blotting was used to assess signal transducer and activator of transcription (STAT) phosphorylation. Results: Macrophages cultured in the presence of DIBI exhibited a rapid and significant reduction in their intracellular labile iron pool. DIBI-treated macrophages showed reduced expression of proinflammatory cytokines interferon-β, interleukin (IL)-1β, and IL-6 in response to LPS. In contrast, exposure to DIBI did not affect LPS-induced expression of tumor necrosis factor-α (TNF-α). The inhibitory effect of DIBI on IL-6 synthesis by LPS-stimulated macrophages was lost when exogenous iron in the form of ferric citrate was added to culture, confirming the selectivity of DIBI for iron. DIBI-treated macrophages showed reduced production of reactive oxygen species and nitric oxide following LPS stimulation. DIBI-treated macrophages also showed a reduction in cytokine-induced activation of STAT 1 and 3, which potentiate LPS-induced inflammatory responses. Conclusion: DIBI-mediated iron withdrawal may be able to blunt the excessive inflammatory response by macrophages in conditions such as systemic inflammatory syndrome.

Divicine induces endothelial cells injury and its potential mechanism

Divicine is an active pyrimidine aglycone, generated from vicine by the enzyme β-glucosidase upon ingestion of fava beans. In this study, we investigated the effect of divicine on cultured human umbilical vein endothelial cells (HUVECs) and explored the potential mechanisms. Incubation HUVECs with 18.5–85.1 μM divicine resulted in a concentration and time dependent decrease of cell viability, followed by decrease of cellular reduced glutathione, as well as increase of reactive oxygen species (ROS), malondialdehyde (MDA), and labile iron pool. Transmission electron microscopy confirmed that the divicine treated HUVECs’ mitochondria had shrunk. Importantly, the administration of desferrioxamine, an iron chelator, to the divicine treated HUVECs significantly reduced iron overload and cell death and decreased cellular ROS and MDA. These results demonstrated that divicine could cause damaging of endothelial cells, and ferroptosis might be involved in divicine induced HUVECs injury, reminding long term ingestion of fava beans might be harmful to vascular system, especially for those suffering from glucose 6-phosphate dehydrogenase deficiency.

Interconversion between [2Fe-2S] and [4Fe-4S] cluster glutathione complexes.

We present here how different iron-sulfide-glutathione ratios, applied in in vitro conditions comparable to those present in the mitochondrial matrix, affect the speciation of iron-sulfur cluster glutathione complexes. An excess of sulfide with respect to iron ions promotes the formation of a tetranuclear [FeII2FeIII2S4(GS)4]2- complex, while an excess of iron ions favors the formation of a dinuclear [FeIIFeIIIS2(GS)4]3- complex. These two complexes establish an interconversion equilibrium. The latter might play a role in the composition of the mitochondrial labile iron pool potentially contributing to the regulation of cellular iron homeostasis.

Iron-mediated epigenetic activation of NRF2 targets

The toxic effects of excess dietary iron within the colonic lumen are well documented, particularly in the context of Inflammatory Bowel Disease (IBD) and Colorectal Cancer (CRC). Proposed mechanisms that underpin iron-associated intestinal disease include: (1) the pro-inflammatory and ROS-promoting nature of iron, (2) gene-expression alterations, and (3) intestinal microbial dysbiosis. However, to date no studies have examined the effect of iron on the colonic epigenome. Here we demonstrate that chronic iron exposure of colonocytes leads to significant hypomethylation of the epigenome. Bioinformatic analysis highlights a significant epigenetic effect on NRF2 (nuclear factor erythroid 2-related factor 2) pathway targets (including NAD(P)H Quinone Dehydrogenase 1 [NQO1] and Glutathione peroxidase 2 [GPX2]); this demethylating effect was validated and subsequent gene and protein expression quantified. These epigenetic modifications were not observed upon the diminishment of cellular lipid peroxidation with endogenous glutathione and the subsequent removal of iron. Additionally, the induction of TET1 expression was found post-iron treatment, highlighting the possibility of an oxidative-stress induction of TET1 and subsequent hypomethylation of NRF2 targets. In addition, a strong time dependence on the establishment of iron-orchestrated hypomethylation was found which was concurrent with the increase in the intracellular labile iron pool (LIP) and lipid peroxidation levels. These epigenetic changes were further validated in murine intestinal mucosa in models administered a chronic iron diet, providing evidence for the likelihood of dietary-iron mediated epigenetic alterations in vivo. Furthermore, significant correlations were found between NQO1 and GPX2 demethylation and human intestinal tissue iron-status, thus suggesting that these iron-mediated epigenetic modifications are likely in iron-replete enterocytes. Together, these data describe a novel mechanism by which excess dietary iron is able to alter the intestinal phenotype, which could have implications in iron-mediated intestinal disease and the regulation of ferroptosis.

Inhibitory Effect of Ascorbic Acid on Myelodysplastic Syndrome Cells and Its Mechanism

To investigate the inhibitory effect of ascorbic acid single or combination of decitabine on tumor cells of myelodysplastic syndrome (MDS) and explore its related mechanism. The human MDS cell lines SKM-1 and MUTZ-1 were treated with different concentrations of ascorbic acid, and the cell proliferation activity was detected by the CCK-8 assay. The reactive oxygen species (ROS) level, labile iron pool (LIP), cell cycle, and apoptosis of SKM-1 and MUTZ-1 cells were detected by flow cytometry. The control group, ascorbic acid monotherapy group, decitabine monotherapy group, and combination group of ascorbic acid and decitabine were set up, the cell proliferation activity and apoptosis were detected in each group. High-dose ascorbic acid could reduce the cell proliferation activity of SKM-1 (R=0.886, p=0.000) and MUTZ-1 (R=0.880, p=0.000). With the increase of ascorbic acid concentration, the ROS level in SKM-1 and MUTZ-1 cells increased (r=0.816, r=0.942), the proportion of cells stagnation in G2 phase increased (r=0.970, p=0.000; r=0.962, p=0.000), the proportion of surviving cells decreased (r=-0.966, p=0.000; r=-0.952, p=0.000), and the apoptosis cells significantly increased (r=0.966, p=0.000; r=0.958, p=0.000). Nevertheless, the level of LIP showed no significant changes. After the combined application of ascorbic acid and decitabine, MDS tumor cells showed decreased proliferative activity and increased apoptosis compared with single-agent ascorbic acid and decitabine group. High-dose ascorbic acid shows a cytotoxic effect on MDS tumor cells, inhibiting cell proliferation and increasing apoptosis. Ascorbic acid combined decitabine have a synergistic effect of anti-MDS tumor cells.

JNK/Itch Axis Mediates the Lipopolysaccharide-Induced Ubiquitin-Proteasome-Dependent Degradation of Ferritin Light Chain in Murine Macrophage Cells.

Ferritin, which is composed of a heavy chain and a light chain, plays a critical role in maintaining iron homeostasis by sequestering iron. The ferritin light chain (FTL) is responsible for the stability of the ferritin complex. We have previously shown that overexpression of FTL decreases the levels of the labile iron pool (LIP) and reactive oxygen species (ROS) in lipopolysaccharide (LPS)-treated murine macrophage cells. The protein level of FTL was downregulated by LPS within a short treatment period. However, the mechanism underlying the LPS-induced changes in the FTL levels is not known. In the present study, we report that LPS induces the ubiquitin-proteasome-dependent degradation of FTL and that the mechanism of LPS-induced FTL degradation involves the JNK/Itch axis. We found that LPS downregulates the protein and mRNA levels of FTL in a time-dependent manner. The proteasome inhibitor MG-132 significantly reverses the LPS-induced decrease in FTL. Furthermore, we observed that LPS treatment cannot cause ubiquitination of the lysine site (K105 and K144) mutant of FTL. Interestingly, LPS-mediated ubiquitin-dependent degradation of FTL is significantly inhibited by the JNK-specific inhibitor SP600125. Moreover, LPS could upregulate the protein level of E3 ubiquitin ligase Itch, a substrate of JNK kinases. Immunoprecipitation analyses revealed an increase in the association of FTL with Itch, a substrate of JNK kinases, in response to LPS stimulation. SP600125 decreased LPS-induced Itch upregulation. Taken together, these results suggest that LPS stimulation leads to the degradation of FTL through the ubiquitin-proteasome proteolytic pathway, and this FTL degradation is mediated by the JNK/Itch axis in murinemacrophage cells.

Labile iron, ROS, and cell death are prominently induced by haemin, but not by non-transferrin-bound iron

BackgroundIn transfusion-related iron overload, haem-derived iron accumulation in monocytes/macrophages is the initial event. When iron loading exceeds the ferritin storage capacity, iron is released into the plasma. When iron loading exceeds transferrin binding capacity, labile, non-transferrin-bound iron (NTBI) appears and causes organ injury. Haemin-induced cell death has already been investigated; however, whether NTBI induces cell death in monocytes/macrophages remains unclear. Material and MethodsHuman monocytic THP-1 cells were treated with haemin or NTBI, particularly ferric ammonium citrate (FAC) or ferrous ammonium sulfate (FAS). The intracellular labile iron pool (LIP) was measured using an iron-sensitive fluorescent probe. Ferritin expression was measured by western blotting. ResultsLIP was elevated after haemin treatment but not after FAC or FAS treatment. Reactive oxygen species (ROS) generation and cell death induction were remarkable after haemin treatment but not after FAC or FAS treatment. Ferritin expression was not different between the FAC and haemin treatments. The combination of an iron chelator and a ferroptosis inhibitor significantly augmented the suppression of haemin cytotoxicity (p = 0.011). DiscussionThe difference in LIP suggests the different iron traffic mechanisms for haem-derived iron and NTBI. The Combination of iron chelators and antioxidants is beneficial for iron overload therapy.

Cytoplasmic Labile Iron Accumulates in Aging Stem Cells Perturbing a Key Rheostat for Identity Control

Bone marrow resident and rarely dividing hematopoietic stem cells (HSC) harbor an extensive self-renewal capacity to sustain life-long blood formation, albeit their function declines during ageing. Various molecular mechanisms confer stem cell identity, ensure long-term maintenance and are known to be deregulated in aged stem cells. How these programs are coordinated, particularly during cell division, and what triggers their ageing-associated dysfunction has been unknown.We have previously uncovered that iron chelator exposure increases the number of functional HSC ex vivo and in vivo (Kao et al., Science Transl Med 2018). While ensuring a sufficient amount of redox active, readily available iron which is required in numerous electron transfer reactions governing fundamental cellular processes, cells tightly regulate the size of the intracellular labile iron pool (LIP) to limit adverse ROS generation. Perturbations in the ability to limit intracellular iron is detrimental for cells and known to compromise HSC maintenance and function via altered redox signaling and increased macromolecule oxidation and damage. The HSC stimulatory effects of iron chelator (IC) treatment and the well characterized central roles of redox active intracellular iron in sustaining basic cell function prompted us to examine a potential regulatory role of the LIP in controlling somatic stem cell function.In this study, we quantified LIP in young and aged HSC and monitored iron homoeostasis pathway activation, hallmarked by the stabilization of transferrin receptor (Tfrc) mRNA, in stem cells for which we developed a single molecule RNA fluorescence in situ hybridization (smRNA FISH) assay enabling the quantification of Tfrc dynamics with unparalleled resolution and sensitivity. We have further used experimental LIP modulation in primary hematopoietic stem cell models to characterize the consequences of iron homeostasis pathway activation in young and aged stem cells; and employed integrated comparative quantitative transcriptomics (single cell RNA-seq) and proteomics along with genetic and pharmacological rescue models to identify the consequences and mechanisms of LIP size alterations. Our findings demonstrate that HSC, containing the lowest amount of cytoplasmic chelatable iron hematopoietic cells, activate a limited iron response during mitosis. Engagement of this iron homeostasis pathway elicits mobilization and β-oxidation of arachidonic acid and enhances stem cell-defining transcriptional programs governed by histone acetyl transferase Tip60/KAT5. We further find an age-associated expansion of the labile iron pool, along with loss of Tip60/KAT5-dependent gene regulation to contribute to the functional decline of ageing HSC, which can be mitigated by iron chelation.Together, our work reveals cytoplasmic redox active iron as a novel rheostat in adult stem cells; it demonstrates a role for the intracellular labile iron pool in coordinating a cascade of molecular events which reinforces HSC identity during cell division and to drive stem cell ageing when perturbed. As loss of iron homeostasis is commonly observed in the elderly, we anticipate these findings to trigger further studies into understanding and therapeutic mitigation of labile iron pool-dependent hematopoietic stem cell dysfunction in a wide range of degenerative and malignant hematologic pathologies. DisclosuresD'Alessandro: Omix Thecnologies: Other: Co-founder; Rubius Therapeutics: Consultancy; Forma Therapeutics: Membership on an entity's Board of Directors or advisory committees.

Intravesical Mucoadhesive Hydrogel Induces Chemoresistant Bladder Cancer Ferroptosis through Delivering Iron Oxide Nanoparticles in a Three-Tier Strategy.

Bladder cancer (BCa) is the most costly solid tumor owing to its high recurrence. Relapsed cancer is known to acquire chemoresistant features after standard intravesical chemotherapy. This cancer state is vulnerable to ferroptosis, which occurs when lipid peroxides generated by iron metabolism accumulate to lethal levels. Increasing the labile iron pool (LIP) by iron oxide nanoparticles (IONPs) promises to inhibit chemoresistant BCa (CRBCa), but systemically administered IONPs do not sufficiently accumulate at the tumor site. Therefore, their efficacy is weakened. Here, we present a three-tier delivery strategy through a mucoadhesive hydrogel platform conveying hyaluronic acid-coated IONPs (IONP-HA). When instilled, the hydrogel platform first adhered to the interface of the tumor surface, sustainably releasing IONP-HA. Subsequently, the tumor stiffness and interstitial fluid pressure were reduced by photothermal therapy, promoting IONP-HA diffusion into the deep cancer tissue. As CRBCa expressed high levels of CD44, the last delivery tier was achieved through antibody-mediated endocytosis to increase the LIP, ultimately inducing ferroptosis. This three-tiered strategy delivered the IONPs stepwise from anatomical to cellular levels and increased the iron content by up to 50-fold from that of systematic administration, which presents a potential regimen for CRBCa.

Labile iron derived from autophagy-mediated ferritin degradation in cardiomyocytes under pressure overload increases myocardial oxidative stress and develops heart failure in mice

Abstract Introduction Heart failure is the leading cause of morbidity and mortality in developed countries, and abnormal iron metabolism is common in patients with heart failure. While iron is essential for metabolic homeostasis, it can increase oxidative stress, such as lipid peroxidation resulting from the generation of harmful reactive hydroxyl radicals through the Fenton reaction. Iron is stored in ferritin, which consists of ferritin heavy (FTH1) and light chains. FTH1 has ferroxidase activity and sequestrates labile ferrous iron. Its protein expression is mediated through nuclear receptor coactivator 4 (NCOA4)-mediated autophagic degradation, known as ferritinophagy. However, the role of ferritinophagy in the stressed heart remains unclear. Methods Cardiomyocyte-specific NCOA4-deficient (KO) mice were generated and subjected to pressure overload by transverse aortic constriction to induce heart failure. Cardiac remodelling was assessed by echocardiography and histological and molecular analyses four weeks after the operation. Furthermore, lipid peroxidation was inhibited by its potent inhibitor, ferrostatin-1. Results Deletion of NCOA4 in mouse hearts did not affect cardiac phenotypes and FTH1 protein level at baseline but improved cardiac systolic function (Fractional shortening, control littermates 21.9% vs KO 45.9%) accompanied by the attenuation of ferritin degradation (The protein level of FTH1, controls 42.9% vs. KO 67.2% of sham-operated hearts) 4 weeks after pressure overload compared to the control littermates. The number of LC3B (a marker of an autophagosome)- and FTH1-positive dots (controls 2.64/103μm2 vs KO 0.30/103μm2) and that of LAMP2a (a marker of a lysosome)- and FTH1-positive dots (controls 3.68/103μm2 vs KO 1.77/103μm2) increased in TAC-operated control hearts, which was attenuated in TAC-operated NCOA4-deficient hearts. The ratio of ferrous iron to the FTH1 protein level, which represents the non-binding fraction of labile ferrous iron to FTH1, was higher in TAC-operated control hearts than in the sham-operated controls and TAC-operated NCOA4–deficient hearts. Although pressure overload increased the level of malondialdehyde, a marker for lipid peroxidation in control hearts, its level was reduced by deletion of NCOA4 (controls 1.12 nmol/mg vs KO 0.68 nmol/mg). Ferrostatin-1 significantly mitigated the development of pressure overload-induced dilated cardiomyopathy in wild-type mice (Fractional shortening, saline 21.4% vs ferrostatin-1 41.7%). Conclusions Pressure overload-induced ferritinophagy in cardiomyocytes increases myocardial labile iron pool and oxidative stress resulting in the development of heart failure in mice. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): British heart foundation

Dihydroartemisinin enhances the inhibitory effect of sorafenib on HepG2 cells by inducing ferroptosis and inhibiting energy metabolism

Although sorafenib (Sora) shows improved efficacy in clinical liver cancer therapy, its therapeutic efficacy is still greatly limited due to side effects as well as drug resistance. Thus new drug intervention strategies are imperative. Our research showed the combined application of Dihydroartemisinin (DHA) and Sora had a synergistic inhibitory effect on HepG2 and SW480 cells, and DHA enhanced Sora efficacy on xenograft tumor in nude mice. DHA and Sora significantly inhibited the cell energy metabolism by decreasing the ATP synthesis rate of oxidative phosphorylation and glycolysis rate, and induced ferroptosis by increasing the level of lipid reactive oxygen species (L-ROS), labile iron pool (LIP) as well as malondialdehyde (MDA) and decreasing the level of glutathione (GSH) in HepG2 cells. In addition, DHA and Sora significantly decreased the levels of SLC7A11 (xCT), GCLC, GPX4, and HO-1 protein in HepG2 cells. Importantly, the above-mentioned indicators changed more significantly after the combined application of DHA and Sora as compared with Sora. In conclusion, DHA and Sora had the same mechanism, and the combined application of them could have a synergistic anti-tumor effect by inducing ferroptosis and inhibiting energy metabolism in HepG2 cells.

Autophagy Regulates the Sensitivity of Acute Lymphoblastic Leukemia Cells to Ferroptosis Activator by Influencing Iron Homeostasis

To investigate the effect of autophagy to the ferroptosis in acute lymphocytic leukemia (ALL) cells and its mechanism. ALL cell lines (including Reh, Jurkat and CCRF-CEM) were selected. The cell viability was detected by MTS assay and trypan blue staining was used to evaluate the death of the cell. The expression of autophagy related protein (including p62, LC3I/II) and Ferritin in ALL cells were detected by Western blot. The alteration of labile iron pool (LIP) in ALL cells was evaluated by flow cytometry. Reh cells showed sensitivity to ferroptosis activator Erastin, while Jurkat and CCRF-CEM cells showed resistant. Autophagy activator rapamycin could promote the sensitivity of Jurkat and CCRF-CEM cells to Erastin, and the ferroptosis of the cells (P<0.001). Autophagy inhibitor chloroquine could reduce the sensitivity of Reh cells to Erastin and resist the ferroptosis of the cells (P<0.001). The expression of Ferritin could be down-regulated after autophagy was activated in Jurkat and CCRF-CEM cells (P<0.05), while the level of LIP was significantly increased (P<0.05). Inhibiting the autophgy in Reh cells could up-regulate the expression of Ferritin (P<0.01),while decrease the level of LIP (P<0.001). The iron homeostasis in cells could be regulated by autophagy through affecting Ferritin expression and LIP level. Autophagy can alter sensitivity of ALL cells to ferroptosis activator Erastin, which suggestes that combining autophagy regulator with ferroptosis activator may be a new strategy for the treatment of chemotherapy-resistant ALL.

Metastatic Colorectal Cancer: Redox Metabolism and Malignancy

Investigating the tumor microenvironment has a direct impact on the understanding of the mechanisms which regulate cancer progression and treatment. Tumor cells undergoing profound changes in their own intrinsic metabolism affect the microenvironment. Colorectal cancer (CRC) is the third most common cancer with the predominant metastases (Mts) into the liver. Mts in liver tissues (LT), adjacent to Mts (AMT) and remote tissues (RMT, taken at the distance of 5 cm from Mts) from 25 patients with metachronous liver Mts after the liver resection as well as blood and urine were studied by electron paramagnetic resonance (EPR) including the spin-trapping EPR for the detection and quantification of the activity of N2 iron-sulfur proteins, levels of NO-N2 complexes, labile iron pool (LIP), lactoferrin (Lf), superoxide and NO radicals. Activity of metalloproteinase MMP-2 and MMP-9 were determined. In adjacent and remote LT low activity of N2 in mitochondrial electron transport chain (EPR signal with g=1.94), loss of functions of detoxification system (cytochrome P450, g=2.25), appearance and growth of NO-N2 complexes (g=2.007) are obtained. Intensive EPR signals from LIP (g=2.2-2.4) and Lf (g=4.3) are registered. Superoxide generation rates were of up to 6 times higher than in the reference LT tissues and blood (p&lt;0.001). NO levels are of 1.7 times higher for the AMT compared to RMT (p&lt;0.05) while being 15 times higher for blood comparing to the reference species (p&lt;0.001). Activity of MMP-2 and MMP-9 was registered both in AMT and RMT and is in 1.7 times higher in ALT (p&lt;0.05). The obtained results can be used to estimate the functional state of organs and tissues with distant metastases, the risk of recurrence, to correct the antitumor therapeutic procedures.