Ferroportin Research Articles

Biotin-stapled mixed micelles of lapatinib: Unleashing apoptosis power in triple-negative breast cancer

The treatment options currently offered for triple-negative breast cancer (TNBC) encounter numerous obstacles, including disappointing overall survival rates, unavoidable side effects, and the development of drug resistance. Additionally, there is a growing need for nutrients, like vitamins (e.g., biotin), to sustain the unregulated growth of TNBC cells. Hence, targeted therapy (LPT B-MM) for enhancing the treatment of TNBC was explored which encompassed hydrophobized [using α-TOS (α-Tocopheryl succinate] Pluronic-based mixed micelles having inherent sodium-dependent multivitamin transporter (SMVT) targeting (biotin-stapled) ability. LPT B-MM had optimal size (210.72± 25.88 nm), PDI (0.272± 0.023), and enhanced drug loading capacity (∼13.31± 2.12%). Transmission electron microscopy validated the spherical shape of these micelles. In vitro release studies demonstrated drug release in a sustained manner without the risk of hemolysis. Importantly, LPT B-MM displayed heightened cellular uptake, increased cytotoxicity, a lower IC50 value, elevated reactive oxygen species, induced mitochondrial membrane depolarization, and a greater apoptosis index in TNBC cell lines compared to free LPT. This study highlights the considerable potential of LPT B-MM to induce apoptosis, showcasing its promise in enhancing payload capabilities and enabling SMVT specific targeting within the context of TNBC. LPT B-MM demonstrated the synergistic effects of LPT and α-TOS in inducing apoptosis in TNBC cells, leading to enhanced therapeutic efficacy. This novel combination therapy offers a promising strategy for improving treatment outcomes in TNBC patients and represents a significant advancement in the field of TNBC therapy.

Dopamine‑iron homeostasis interaction rescues mitochondrial fitness in Parkinson's disease

Imbalances of iron and dopamine metabolism along with mitochondrial dysfunction have been linked to the pathogenesis of Parkinson's disease (PD). We have previously suggested a direct link between iron homeostasis and dopamine metabolism, as dopamine can increase cellular uptake of iron into macrophages thereby promoting oxidative stress responses. In this study, we investigated the interplay between iron, dopamine, and mitochondrial activity in neuroblastoma SH-SY5Y cells and human induced pluripotent stem cell (hiPSC)-derived dopaminergic neurons differentiated from a healthy control and a PD patient with a mutation in the α-synuclein (SNCA) gene. In SH-SY5Y cells, dopamine treatment resulted in increased expression of the transmembrane iron transporters transferrin receptor 1 (TFR1), ferroportin (FPN), and mitoferrin2 (MFRN2) and intracellular iron accumulation, suggesting that dopamine may promote iron uptake. Furthermore, dopamine supplementation led to reduced mitochondrial fitness including decreased mitochondrial respiration, increased cytochrome c control efficiency, reduced mtDNA copy number and citrate synthase activity, increased oxidative stress and impaired aconitase activity. In dopaminergic neurons derived from a healthy control individual, dopamine showed comparable effects as observed in SH-SY5Y cells. The hiPSC-derived PD neurons harboring an endogenous SNCA mutation demonstrated altered mitochondrial iron homeostasis, reduced mitochondrial capacity along with increased oxidative stress and alterations of tricarboxylic acid cycle linked metabolic pathways compared with control neurons. Importantly, dopamine treatment of PD neurons promoted a rescue effect by increasing mitochondrial respiration, activating antioxidant stress response, and normalizing altered metabolite levels linked to mitochondrial function. These observations provide evidence that dopamine affects iron homeostasis, intracellular stress responses and mitochondrial function in healthy cells, while dopamine supplementation can restore the disturbed regulatory network in PD cells.

Two maize homologs of mammalian proton-coupled folate transporter, ZmMFS_1–62 and ZmMFS_1–73, are essential to salt and drought tolerance

Folates are essential to the maintenance of normal life activities in almost all organisms. Proton-coupled folate transporter (PCFT), belonging to the major facilitator superfamily, is one of the three major folate transporter types widely studied in mammals. However, information about plant PCFTs is limited. Here, a genome-wide identification of maize PCFTs was performed, and two PCFTs, ZmMFS_1-62 and ZmMFS_1-73, were functionally investigated. Both proteins contained the typical 12 transmembrane helixes with N- and C-termini located in the cytoplasm, and were localized in the plasma membrane. Molecular docking analysis indicated their binding activity with folates via hydrogen bonding. Interference with ZmMFS_1-62 and ZmMFS_1-73 in maize seedlings through virus-induced gene silencing disrupted folate homeostasis, mainly in the roots, and reduced tolerance to drought and salt stresses. Moreover, a molecular chaperone protein, ZmHSP20, was found to interact with ZmMFS_1-62 and ZmMFS_1-73, and interference with ZmHSP20 in maize seedlings also led to folate disruption and increased sensitivity to drought and salt stresses. Overall, this is the first report of functional identification of maize PCFTs, which play essential roles in salt and drought stress tolerance, thereby linking folate metabolism with abiotic stress responses in maize.

Preventive effects of lactoferrin on acute alcohol-induced liver injury via iron chelation and regulation of iron metabolism

Lactoferrin is widely found in milk and has the ability to bind iron. Previous studies have reported that lactoferrin was effective in the prevention and treatment of acute alcohol-induced liver injury (AALI). Ferroptosis is a recently discovered cell death and is involved in the development of AALI. However, the potential role of lactoferrin in acute alcohol-induced ferroptosis is still unclear. In this study, we observed that lactoferrin (10, 20 and 40 μg/mL) significantly mitigated alcohol (300 mM)-induced injury in vitro. Additionally, lactoferrin (100 and 200 mg/kg bw) significantly alleviated alcohol (4.8 g/kg bw)-induced injury in vivo. Our results showed that lactoferrin inhibited alcohol-induced upregulation of the ferroptosis marker protein ACSL4 and downregulation of GPX4. Meanwhile, lactoferrin treatment successfully reversed the elevated Malondialdehyde (MDA) levels and the reduced Glutathione (GSH) levels caused by alcohol treatment. These results can indicate that lactoferrin significantly decreased ferroptosis in vivo and in vitro. Lactoferrin has the potential to chelate iron, and our results showed that lactoferrin (20 μg/mL) significantly reduced iron ions and the expression of Ferritin Heavy Chain (FTH) under FeCl3 (100 μM) treatment. It was demonstrated that lactoferrin had a significant iron-chelating effect and reduced iron overload caused by FeCl3 in AML12 cells. Next, we examined iron content and the expression of iron metabolism marker proteins Transferrin Receptor (TFR), Divalent metal transporter 1 (DMT1), FTH, and Ferroportin (FPN). Our results showed that lactoferrin alleviated iron overload induced by acute alcohol. The expression of TFR and DMT1 was downregulated and FPN and FTH were upregulated after lactoferrin treatment in vivo and in vitro. Above all, the study suggested that lactoferrin can alleviate AALI by mitigating acute alcohol-induced ferroptosis. Lactoferrin may offer new strategies for the prevention or treatment of AALI.

Abstract 4730: Pharmacodynamic determinants of anti tumor activity of SHMT2 inhibitors

Abstract These studies investigate factors that impact the antitumor activity of SHMT2 targeting including drug accumulation, metabolism, expression of one carbon (C1) related enzymes, and tumor hypoxia. Mitochondrial C1 metabolism is upregulated in a variety of cancer types including leukemias and solid tumors. The mitochondrial C1 enzymes serine hydroxymethyltransferase (SHMT) 2 and 5,10-methylene tetrahydrofolate (THF) dehydrogenase 2 (MTHFD2) are among the top 5 overexpressed metabolic enzymes in tumors vs normal tissues in a wide range of tumor types. Thus, SHMT2 and MTHFD2 are potential therapeutic targets for cancer, sparing normal tissues that are less reliant on mitochondrial C1 metabolism. SHMT2 metabolizes serine and THF to produce glycine and 5,10-methylene THF; MTHFD2 metabolizes 5,10-methylene THF to NADH and 10-formyl THF which is converted to formate by MTHFD1L. Thus, mitochondrial C1 metabolism is the principal source of C1 units for cellular biosynthesis and provides the majority of glycine for protein, purine and glutathione synthesis. We previously discovered novel 5-substituted pyrrolo[3,2-d]pyrimidine antifolate compounds which exhibit primary inhibition at mitochondrial SHMT2 along with cytosolic SHMT1 and de novo purine biosynthesis at glycinamide ribonucleotide and 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferases. The lead compound AGF347 is taken up into cells by the facilitative transporters reduced folate carrier (SLC19A1) and the proton coupled folate transporter (SLC46A1) and like folate cofactors is metabolized to polyglutamate (PG) forms by folylpolyglutamate synthase (FPGS) which promotes retention in cytosolic and mitochondrial compartments and target enzyme engagement. We investigated the impact of folate transporter expression on metabolism to PGs for SHMT2 targeted antifolates. Folate transporter expression inversely correlated with anti-tumor efficacy of SHMT2 targeted antifolates and overexpression of FPGS resulted in dramatically increased sensitivities. However, for pyrazolopyran inhibitors of SHMT2 (e.g. SHIN1) which enter cells by diffusion and are not metabolized to polyglutamates, increased transporter and FPGS levels dramatically decreased anti-tumor activity. As SHMT2 is a target of HIF1α, we studied the impact of multitargeting SHMT2 and purine biosynthesis by pyrrolo[3,2-d]pyrimidine antifolates under hypoxic conditions. Our results establish that multi-targeting SHMT2 and purine biosynthesis preserves anti-tumor activity under hypoxia. Collectively, our results demonstrate that SHMT2 targeted pyrrolo[3,2-d]pyrimidine antifolates exhibit increased antitumor efficacies in cells expressing low folate transporter expression and is further enhanced by increased FPGS activity. Additionally, we find multitargeting of SHMT2 and de novo purine biosynthesis manifests as antitumor activity even under hypoxic conditions. Citation Format: Mathew Joseph Schneider, Carrie O'Connor, Xun Bao, Md. Junayed Nayeen, Zhanjun Hou, Jing Li, Aleem Gangjee, Larry Matherly. Pharmacodynamic determinants of anti tumor activity of SHMT2 inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4730.

Abstract 5883: Role of major facilitative folate transporters SLC19A1 and SLC46A1 in cyclic dinucleotide transport and STING signaling

Abstract Activation of stimulator of interferon genes (STING) activates the transcription factor pIRF3, promoting gene transcription encoding type I interferons including IFNβ that stimulate broader immune responses. Targeting the STING pathway for cancer is especially compelling given its importance to the malignant phenotype. Cyclic dinucleotides (CDNs) function as STING agonists. Inspired by preclinical studies, two phase I clinical trials with CDNs including ADU-S100 were initiated but concluded early due to lack of anti-tumor efficacy. The reduced folate carrier (RFC; SLC19A1) has been identified as one of the primary means to transport CDNs through the cell membrane, although a study suggested that CDN and folate binding to RFC was as least partly distinct. While depletion of the proton-coupled folate transporter (PCFT; SLC46A1) in THP-1 cells had nominal effects on CDN stimulation, overexpressing PCFT increased STING signaling. Limited clinical responses may reflect the impact of physiologic variables governing RFC and PCFT not previously considered. Understanding the mechanisms of CDN uptake by RFC and PCFT under physiologic conditions may elucidate the role of CDNs on STING signaling. To study the impact of RFC levels on CDN transport and STING signaling, we used an engineered R1-11/Tet-on-RFC HeLa cell model induced with doxycycline. In R1-11/Tet-on-RFC cells, ADU-S100 showed a low affinity for binding to RFC, as measured by direct competition for transport with [3H]methotrexate. RFC dose-dependence of ADU-S100 uptake and STING activation was confirmed by monitoring IFN-β (qPCR) and pIRF3 (Westerns). Maximum induction of pIRF3/IFNβ was seen in THP-1 and R1-11/Tet-on-RFC cells by ADU-S100 treatment at pH 7.2 (RFC pH optimum) in the presence of 25 nM leucovorin. Induction of pIRF3/IFNβ by ADU-S100 in THP-1 and R1-11/Tet-on-RFC cells were partially abolished by treatment with 200 μM of leucovorin or 10 μM of PT523 (RFC-specific inhibitor), suggesting the transport of folates and CDNs may share common albeit distinct mechanisms. ADU-S100 treatment showed no pIRF3 or IFN-β production in an engineered R1-11/Tet-on-PCFT HeLa model expressing high levels of PCFT, suggesting no direct transport by PCFT, even under optimal pH conditions. Interestingly, pIRF3/IFN-β induction was enhanced in R1-11/Tet-on-RFC/PCFT cells expressing constitutive PCFT in addition to RFC, compared to R1-11/Tet-on-RFC cells that express comparable RFC without PCFT. This may reflect previously unrecognized functional interactions between RFC and PCFT. In conclusion, RFC mediates ADU-S100 uptake and STING activation under physiologic conditions and this process is enhanced by concomitant expression of PCFT. Additional characterization of key molecular and biochemical determinants of CDN-based cancer therapeutics may lead to improved approaches for CDN therapy based on enhanced cellular uptake. Citation Format: Holly McQuithey, Madelyn Brzezinski, Carrie O’Connor, Larry Matherly, Zhanjun Hou. Role of major facilitative folate transporters SLC19A1 and SLC46A1 in cyclic dinucleotide transport and STING signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5883.

A Revised Classification of Primary Iron Overload Syndromes.

The clinical introduction of hepcidin25 (Hep25) has led to a more detailed understanding of its relationship with ferroportin (FP) and divalent metal transporter1 in primary iron overload syndromes (PIOSs). In 2012, we proposed a classification of PIOSs based on the Hep25/FP system, which consists of prehepatic aceruloplasminemia, hepatic hemochromatosis (HC), and posthepatic FP disease (FP-D). However, in consideration of accumulated evidence on PIOSs, we aimed to renew the classification. We reviewed the 2012 classification and retrospectively renewed it according to new information on PIOSs. Iron-loading anemia was included in PIOSs as a prehepatic form because of the newly discovered erythroferrone-induced suppression of Hep25, and the state of traditional FP-D was remodeled as the BIOIRON proposal. The key molecules responsible for prehepatic PIOSs are low transferrin saturation in aceruloplasminemia and increased erythroferrone production by erythroblasts in iron-loading anemia. Hepatic PIOSs comprise four genotypes of HC, in each of which the synthesis of Hep25 is inappropriately reduced in the liver. Hepatic Hep25 synthesis is adequate in posthepatic PIOSs; however, two mutant FP molecules may resist Hep25 differently, resulting in SLC40A1-HC and FP-D, respectively. PIOS phenotypes are diagnosed using laboratory tests, including circulating Hep25, followed by suitable treatments. Direct sequencing of the candidate genes may be outsourced to gene centers when needed. Laboratory kits for the prevalent mutations, such as C282Y, may be the first choice for a genetic analysis of HC in Caucasians. The revised classification may be useful worldwide.

Iron Dysregulation in Alzheimer's Disease: LA-ICP-MS Bioimaging of the Distribution of Iron and Ferroportin in the CA1 Region of the Human Hippocampus.

Alzheimer's disease (AD) is a prevalent neurodegenerative disorder characterized by cognitive decline and neuropathological hallmarks, including β-amyloid (Aβ) plaques, Tau tangles, synaptic dysfunction and neurodegeneration. Emerging evidence suggests that abnormal iron (Fe) metabolism plays a role in AD pathogenesis, but the precise spatial distribution of the Fe and its transporters, such as ferroportin (FPN), within affected brain regions remains poorly understood. This study investigates the distribution of Fe and FPN in the CA1 region of the human hippocampus in AD patients with a micrometer lateral resolution using laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). For this purpose, we visualized and quantified Fe and FPN in three separated CA1 layers: stratum molecular-radial (SMR), stratum pyramidal (SP) and stratum oriens (SO). Additionally, chromogenic immunohistochemistry was used to examine the distribution and colocalization with Tau and Aβ proteins. The results show that Fe accumulation was significantly higher in AD brains, particularly in SMR and SO. However, FPN did not present significantly changes in AD, although it showed a non-uniform distribution across CA1 layers, with elevated levels in SP and SO. Interestingly, minimal overlap was observed between Fe and FPN signals, and none between Fe and areas rich in neurofibrillary tangles (NFTs) or neuritic plaques (NP). In conclusion, the lack of correlation between Fe and FPN signals suggests complex regulatory mechanisms in AD Fe metabolism and deposition. These findings highlight the complexity of Fe dysregulation in AD and its potential role in disease progression.

Abstract A014: Role of stromal CD10 expression in progression of endometrial and endometriosis-associated cancers

Abstract Endometriosis-associated carcinomas (EACs) and endometrial malignancies have many commonalities yet their pathophysiology, particularly those with high-risk histology such as clear cell, is understudied. EACs are histologically and clinically distinct from other gynecological cancers due to their unique origin within endometriosis. The endometriotic microenvironment is uniquely hypoxic with high levels of iron due to recurrent menstrual cycling, and this iron-rich microenvironment has been linked to promoting carcinogenesis through iron-induced oxidative stress. However, the mechanism by which the endometriotic microenvironment contributes to EAC tumorigenesis and how this compares with endometrial cancer is unknown. Most of our current knowledge of EACs is built on either epidemiologic analyses or correlative studies. Additionally, most studies of the endometriotic microenvironment primarily focus on the epithelial component while the role of the stroma has been largely unexplored. Mesenchymal stem cells (MSC) are a critical component of the stromal microenvironment. Recently, we identified a key role of endometriosis derived MSCs (enMSCs) in altering ovarian clear cell carcinoma iron regulation. Interestingly, enMSCs resemble the primary endometrium derived MSCs, which suggested their role in endometrial cancer. This stromal subset is characterized by the loss of surface CD10 expression. It is known that cancer cells, particularly clear cell carcinoma (CCC), have a unique requirement for high levels of iron. We hypothesize that CD10 negative enMSCs promote growth of EAC and endometrial cancer through iron regulation. MSCs were isolated from primary human benign endometriosis, benign endometrium, or endometrial cancer. Flow cytometry was used to measure surface CD10 expression. We investigated the role of CD10 negative enMSCs on CCC iron regulation. Our results showed that CD10 negative enMSCs promote CCC growth by exporting iron through the upregulation of Hephaestin (HEPH) and Ferroportin (FPO) which then led to increase labile iron pool (LIP) in CCC cells. However, increasing the LIP does not occur with iron supplementation alone, this indicates the critical role of CD10 negative enMSCs in regulating iron handling in CCC cells. Additionally, our data showed that CD10 negative enMSCs alter the balance of Ferritin L and Ferritin H- the main iron storage proteins- in CCC cells. Mechanistically, our preliminary data showed that CD10 negative enMSCs directly donate Ferritin L into CCC cells. Further, CD10 negative enMSCs enhance Ferritin H degradation through the upregulation of ferritinophagy in CCC cells. In conclusion, our results indicate there is a sub-population of enMSCs, marked by the loss of CD10 expression, that enhances EAC and endometrial cancer cell growth through iron regulation. This highlights the existence of a tumor-promoting stromal cell within both the endometrium and endometriosis which can be capitalized on as an “Achilles' heel” to uncover novel treatment targets for EAC and endometrial cancer. Citation Format: Huda I. Atiya, Lan G. Coffman. Role of stromal CD10 expression in progression of endometrial and endometriosis-associated cancers [abstract]. In: Proceedings of the AACR Special Conference on Endometrial Cancer: Transforming Care through Science; 2023 Nov 16-18; Boston, Massachusetts. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(5_Suppl):Abstract nr A014.

Water extract of moschus alleviates erastin-induced ferroptosis by regulating the Keap1/Nrf2 pathway in HT22 cells

Ethnopharmacological relevanceMoschus, first described in the Shennong's Classic of the Materia medicine, is a scarce and precious animal medicine. Modern pharmacological researches have suggested that Moschus has neuroprotective actions, and its mechanism is related to anti-inflammatory, antioxidant, and anti-apoptosis effects. Ferroptosis is one of the major pathologies of Alzheimer's disease (AD) and is widely implicated in the pathogenesis and progression of AD. Although previous studies have suggested that Moschus possesses neuroprotective effect, whether Moschus could mitigate neuronal damages by inhibiting the onset of ferroptosis is unknown in model cells of AD. Aim of the studyThe aim of study was to explore the water extract of Moschus (WEM) on ferroptosis caused by erastin and the potential mechanism. Materials and methodsErastin was used to stimulate HT22 cells to form ferroptosis model to evaluate the anti-ferroptosis effect of WEM by cell counting kit-8 and lactic dehydrogenase (LDH) tests. The malondialdehyde (MDA) and glutathione (GSH) kits are used for detection of MDA and GSH levels, and 2′,7′-dichlorofluorescein diacetate and C11 BODIPY 581/591 fluorescence probe are used for evaluation of reactive oxygen species (ROS) and lipid peroxide (LOOH) levels. And Western blot was used to test nuclear factor erythroid 2-related factor 2 (Nrf2), Kelch-like ECH-associated protein 1 (Keap1), heme oxygenase-1 (HO-1), and ferroptosis associated proteins including glutathione peroxidase 4 (GPX4), cystine/glutamate antiporter subunit (SLC7A11), ferritin heavy chain 1 (FTH1), ferroportin1 (FPN1), transferrin receptor (TFRC). In addition, the Nrf2 inhibitor ML385 was applied to verify whether WEM prevents erastin-induced ferroptosis by activating the Keap1/Nrf2 pathway. ResultsAfter WEM treatment, erastin-induced HT22 cell survival was significantly elevated, the accumulation of intracellular MDA, ROS, and LOOH were significantly reduced, the level of GSH and expressions of ferroptosis inhibitors GPX4 and SLC7A11 were significantly increased, and iron metabolism-related proteins TFRC, FPN1, and FTH1 were regulated. These effects of WEM are implemented by activating the Keap1/Nrf2 pathway. ConclusionsThis study demonstrated that WEM could perform neuroprotective effects by alleviating ferroptosis, verified that WEM treatment of AD can be mediated by the Keap1/Nrf2 pathway, and provided theoretical support for the application of WEM in the treatment of AD.

Mitochondrial and Cytosolic One-Carbon Metabolism Is a Targetable Metabolic Vulnerability in Cisplatin-Resistant Ovarian Cancer.

One-carbon (C1) metabolism is compartmentalized between the cytosol and mitochondria with the mitochondrial C1 pathway as the major source of glycine and C1 units for cellular biosynthesis. Expression of mitochondrial C1 genes including SLC25A32, serine hydroxymethyl transferase (SHMT) 2, 5,10-methylene tetrahydrofolate dehydrogenase 2, and 5,10-methylene tetrahydrofolate dehydrogenase 1-like was significantly elevated in primary epithelial ovarian cancer (EOC) specimens compared with normal ovaries. 5-Substituted pyrrolo[3,2-d]pyrimidine antifolates (AGF347, AGF359, AGF362) inhibited proliferation of cisplatin-sensitive (A2780, CaOV3, IGROV1) and cisplatin-resistant (A2780-E80, SKOV3) EOC cells. In SKOV3 and A2780-E80 cells, colony formation was inhibited. AGF347 induced apoptosis in SKOV3 cells. In IGROV1 cells, AGF347 was transported by folate receptor (FR) α. AGF347 was also transported into IGROV1 and SKOV3 cells by the proton-coupled folate transporter (SLC46A1) and the reduced folate carrier (SLC19A1). AGF347 accumulated to high levels in the cytosol and mitochondria of SKOV3 cells. By targeted metabolomics with [2,3,3-2H]L-serine, AGF347, AGF359, and AGF362 inhibited SHMT2 in the mitochondria. In the cytosol, SHMT1 and de novo purine biosynthesis (i.e., glycinamide ribonucleotide formyltransferase, 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase) were targeted; AGF359 also inhibited thymidylate synthase. Antifolate treatments of SKOV3 cells depleted cellular glycine, mitochondrial NADH and glutathione, and showed synergistic in vitro inhibition toward SKOV3 and A2780-E80 cells when combined with cisplatin. In vivo studies with subcutaneous SKOV3 EOC xenografts in SCID mice confirmed significant antitumor efficacy of AGF347. Collectively, our studies demonstrate a unique metabolic vulnerability in EOC involving mitochondrial and cytosolic C1 metabolism, which offers a promising new platform for therapy.

Intracellular Regulation Limits the Response of Intestinal Ferroportin to Iron Status in Suckling Rats.

Iron status is regulated via iron absorption as there is no active iron excretion. Divalent metal-ion transporter-1 (DMT1) and ferroportin (FPN) are two key proteins vital for iron absorption, but the regulation of them in suckling mammals differs from that in adults. This study aims to explore regulation of iron transporters under different iron conditions during suckling. This study developed suckling rats under different iron conditions. Unexpectedly, unchanged FPN at different iron status are detected. Since FPN is the only known iron exporter for mammals, unchanged FPN limits iron exported into blood during suckling. Thus, factors regulating FPN at transcriptional, post-transcriptional, and post-translational levels are detected. Results showed that Fpn mRNA is upregulated, while micro RNA-485(miR-485) which could silence Fpn mRNA is upregulated at low iron status limiting translation of Fpn mRNA. Besides, serum hepcidin and liver Hamp mRNA are upregulated, but ring finger protein 217(Rnf217) mRNA remained unchanged at high iron status leading to FPN not downregulated as adults. Overall, this study indicates that translational regulation limits intestinal FPN protein response to iron deficiency and Rnf217 cannot effectively mediate the degradation of FPN at high iron status, which provides a reference for maintaining iron homeostasis during suckling.

Alternative splicing generates a novel ferroportin isoform with a shorter C-terminal and an intact iron- and hepcidin-binding property.

Ferroportin (FPN) is a transmembrane protein and is the only known iron exporter that helps in maintaining iron homeostasis in vertebrates. To maintain stable iron equilibrium in the body, ferroportin works in conjunction with a peptide called hepcidin. In this study, we have identified an alternatively spliced novel isoform of the human SLC40A1 gene, which encodes for the FPN protein and is found to be expressed in different tissues. The novel transcript has an alternate last exon and encodes 31-amino acid long peptide sequence that replaces 104 amino acids at C-terminal in the novel transcript. Molecular modelling and molecular dynamics (MD) simulation studies revealed key structural features of the novel isoform (FPN-N). FPN-N was predicted to have 12 transmembrane domains similar to the reported isoform (FPN), despite being much smaller in size. FPN-N was found to interact with hepcidin, a key regulator of ferroportin activity. Also, the iron-binding sites were retained in the novel isoform as revealed by the MD simulation of FPN-N in bilipid membrane. The novel isoform identified in this study may play important role in iron homeostasis. However, further studies are required to characterize the FPN-N isoform and decipher its role inside the cell.

Vitamin D promotes the folate transport and metabolism in zebrafish (Danio rerio).

Vitamin D (VD) is a fat-soluble sterol that possesses a wide range of physiological functions. The present study aimed to evaluate the effects of VD on folate metabolism in zebrafish and further investigated the underlying mechanism. Wild-type (WT) zebrafish were fed with a diet containing 0 IU/kg VD3 or 800 IU/kg VD3 for 3 wk. Meanwhile, cyp2r1 mutant zebrafish with impaired VD metabolism was used as another model of VD deficiency. Our results showed that VD deficiency in zebrafish suppressed the gene expression of folate transporters, including reduced folate carrier (RFC) and proton-coupled folate transporter (PCFT) in the intestine. Moreover, VD influenced the gene expression of several enzymes related to cellular folate metabolism in the intestine and liver of zebrafish. Importantly, VD-deficient zebrafish contained a remarkably lower level of folate content in the liver. Notably, VD was incapable of altering folate metabolism in zebrafish when gut microbiota was depleted by antibiotic treatment. Further studies proved that gut commensals from VD-deficient fish displayed a lower capacity to produce folate than those from WT fish. Our study revealed the potential correlation between VD and folate metabolism in zebrafish, and gut microbiota played a key role in VD-regulated folate metabolism in zebrafish.NEW & NOTEWORTHY Our study has identified that VD influences intestinal uptake and transport of folate in zebrafish while also altering hepatic folate metabolism and storage. Interestingly, the regulatory effects of VD on folate transport and metabolism diminished after the gut flora was interrupted by antibiotic treatment, suggesting that the regulatory effects of VD on folate metabolism in zebrafish are most likely dependent on the intestinal flora.

TFR1 knockdown alleviates iron overload and mitochondrial dysfunction during neural differentiation of Alzheimer’s disease-derived induced pluripotent stem cells by interacting with GSK3B

Iron metabolism disorders are implicated in the pathogenesis of Alzheimer’s disease (AD). It was previously reported that transferrin receptor (TFR1) expression was upregulated in AD mouse model. However, the precise biological functions of TFR1 in AD progression remains unclear. Herein, we observed a gradual increase in TFR1 protein expression during the differentiation of AD patient-derived induced pluripotent stem cells (AD-iPS). TFR1 knockdown inhibited the protein expression of ferritin and ferritin heavy chain 1 (FTH1), enhanced the expression of ferroportin 1 (FPN1), and decreased intracellular levels of total iron, labile iron, and reactive oxygen species (ROS). Moreover, TFR1 knockdown improved mitochondrial membrane potential (MMP), increased adenosine triphosphate (ATP) content, downregulated mitochondrial fission proteins, and upregulated mitochondrial fusion proteins. TFR1 knockdown alleviated iron overload and mitochondrial dysfunction in neural cells differentiated from AD-iPS, while TFR1 overexpression showed the opposite results. Additionally, TFR1interacted with glycogen synthase kinase 3 beta (GSK3B) and promoted GSK3B expression. GSK3B overexpression reversed the inhibitory effects of TFR1 knockdown on iron overload and mitochondrial dysfunction in AD-iPS differentiated neural cells. In conclusion, TFR1 knockdown alleviated iron overload and mitochondrial dysfunction in neural cells differentiated from AD-iPS by promoting GSK3B expression. Our findings provide a potential therapeutic target for the treatment of AD.Graphical

Renal-specific loss of ferroportin disrupts iron homeostasis and attenuates recovery from acute kidney injury.

Chronic kidney disease is increasing at an alarming rate and correlates with the increase in diabetes, obesity, and hypertension that disproportionately impact socioeconomically disadvantaged communities. Iron plays essential roles in many biological processes including oxygen transport, mitochondrial function, cell proliferation, and regeneration. However, excess iron induces the generation and propagation of reactive oxygen species, which lead to oxidative stress, cellular damage, and ferroptosis. Iron homeostasis is regulated in part by the kidney through iron resorption from the glomerular filtrate and exports into the plasma by ferroportin (FPN). Yet, the impact of iron overload in the kidney has not been addressed. To test more directly whether excess iron accumulation is toxic to kidneys, we generated a kidney proximal tubule-specific knockout of FPN. Despite significant intracellular iron accumulation in FPN mutant tubules, basal kidney function was not measurably different from wild type kidneys. However, upon induction of acute kidney injury (AKI), FPN mutant kidneys exhibited significantly more damage and failed recovery, evidence for ferroptosis, and increased fibrosis. Thus, disruption of iron export in proximal tubules, leading to iron overload, can significantly impair recovery from AKI and can contribute to progressive renal damage indicative of chronic kidney disease. Understanding the mechanisms that regulate iron homeostasis in the kidney may provide new therapeutic strategies for progressive kidney disease and other ferroptosis-associated disorders.NEW & NOTEWORTHY Physiological iron homeostasis depends in part on renal resorption and export into the plasma. We show that specific deletion of iron exporters in the proximal tubules sensitizes cells to injury and inhibits recovery. This can promote a chronic kidney disease phenotype. Our paper demonstrates the need for iron balance in the proximal tubules to maintain and promote healthy recovery after acute kidney injury.

Piceatannol protects against myocardial ischemia/reperfusion injury by inhibiting ferroptosis via Nrf-2 signaling-mediated iron metabolism

Myocardial tissue ischemia damages myocardial cells. Although reperfusion is an effective technique to rescue myocardial cell damage, it may also exacerbate myocardial cell damage. Ferroptosis, an iron-dependent cell death, occurs following myocardial ischemia-reperfusion (I/R). Piceatannol (PCT) is a natural stilbene compound with excellent antioxidant properties that protect against I/R injury and exerts protective effects against ferroptosis-induced cardiomyocytes following I/R injury; however, the exact mechanism remains to be elucidated. PurposeThis study aims to investigate the protective effect and mechanism of PCT on myocardial ischemia-reperfusion injury. MethodsAn ischemia-reperfusion model was established via ligation of the left anterior descending branch of mice's hearts and hypoxia-reoxygenation (H/R) of cardiomyocytes. ResultsDuring ischemia-reperfusion, Nuclear factor E2-related factor 2 (Nrf-2) expression was downregulated, the left ventricular function was impaired, intracellular iron and lipid peroxidation product levels were elevated, and cardiomyocytes underwent ferroptosis. Furthermore, ferroptosis was enhanced following treatment with an Nrf-2 inhibitor. After PCT treatment, Nrf-2 expression significantly increased, intracellular ferrous ions and lipid peroxidation products significantly reduced, Ferroportin1 (FPN1) expression increased, and transferrin receptor-1 (TfR-1) expression was inhibited. ConclusionsPCT regulates iron metabolism through Nrf-2 to protect against myocardial cell ferroptosis induced by myocardial I/R injury.

Mitochondrial ferritin upregulation reduced oxidative stress and blood-brain-barrier disruption by maintaining cellular iron homeostasis in a neonatal rat model of germinal matrix hemorrhage

Germinal matrix hemorrhage (GMH) is a devasting neurological disease in premature newborns. After GMH, brain iron overload associated with hemoglobin degradation contributed to oxidative stress, causing disruption of the already vulnerable blood-brain barrier (BBB). Mitochondrial ferritin (FTMT), a novel mitochondrial outer membrane protein, is crucial in maintaining cellular iron homeostasis. We aimed to investigate the effect of FTMT upregulation on oxidative stress and BBB disruption associated with brain iron overload in rats. A total of 222 Sprague-Dawley neonatal rat pups (7 days old) were used to establish a collagenase-induced GMH model and an iron-overload model of intracerebral FeCl2 injection. Deferiprone was administered via gastric lavage 1 h after GMH and given daily until euthanasia. FTMT CRISPR Knockout and adenovirus (Ad)-FTMT were administered intracerebroventricularly 48 h before GMH and FeCl2 injection, respectively. Neurobehavioral tests, immunofluorescence, Western blot, Malondialdehyde measurement, and brain water content were performed to evaluate neurobehavior deficits, oxidative stress, and BBB disruption, respectively. The results demonstrated that brain expressions of iron exporter Ferroportin (FPN) and antioxidant glutathione peroxidase 4 (GPX4) as well as BBB tight junction proteins including Claudin-5 and Zona Occulta (ZO)-1 were found to be decreased at 72 h after GMH. FTMT agonist Deferiprone attenuated oxidative stress and preserved BBB tight junction proteins after GMH. These effects were partially reversed by FTMT CRISPR Knockout. Iron overload by FeCl2 injection resulted in oxidative stress and BBB disruption, which were improved by Ad-FTMT mediated FTMT overexpression. Collectively, FTMT upregulation is neuroprotective against brain injury associated with iron overload. Deferiprone reduced oxidative stress and BBB disruption by maintaining cellular iron homeostasis partially by the upregulating of FTMT after GMH. Deferiprone may be an effective treatment for patients with GMH.

Engineered Bio-Heterojunction Confers Extra- and Intracellular Bacterial Ferroptosis and Hunger-Triggered Cell Protection for Diabetic Wound Repair.

Nanomaterial-mediated ferroptosis has garnered considerable interest in the antibacterial field, as it invokes the disequilibrium of ion homeostasis and boosts lipid peroxidation in extra- and intracellular bacteria. However, current ferroptosis-associated antibacterial strategies indiscriminately pose damage to healthy cells, ultimately compromising their biocompatibility. To address this daunting issue, this work has designed a precise ferroptosis bio-heterojunction (F-bio-HJ) consisting of Fe2 O3 , Ti3 C2 -MXene, and glucose oxidase (GOx) to induce extra-intracellular bacteria-targeted ferroptosis for infected diabetic cutaneous regeneration. Fe2 O3 /Ti3 C2 -MXene@GOx (FMG) catalytically generates a considerable amount of ROS which assaults the membrane of extracellular bacteria, facilitating the permeation of synchronously generated Fe2+ /Fe3+ into bacteria under near-infrared (NIR) irradiation, causing planktonic bacterial death via ferroptosis, Fe2+ overload, and lipid peroxidation. Additionally, FMG facilitates intracellular bacterial ferroptosis by transporting Fe2+ into intracellular bacteria via inward ferroportin (FPN). With GOx consuming glucose, FMG creates hunger protection which helps macrophages escape cell ferroptosis by activating the adenosine 5'-monophosphate (AMP) activated protein kinase (AMPK) pathway. In vivo results authenticate that FMG boosts diabetic infectious cutaneous regeneration without triggering ferroptosis in normal cells. As envisaged, the proposed tactic provides a promising approach to combat intractable infections by precisely terminating extra-intracellular infection via steerable ferroptosis, thereby markedly elevating the biocompatibility of therapeutic ferroptosis-mediated strategies.

Butyrate Prevents the Pathogenic Anemia-Inflammation Circuit by Facilitating Macrophage Iron Export.

Most patients with inflammatory bowel disease (IBD) develop anemia, which is attributed to the dysregulation of iron metabolism. Reciprocally, impaired iron homeostasis also aggravates inflammation. How this iron-mediated, pathogenic anemia-inflammation crosstalk is regulated in the gut remains elusive. Herein, it is for the first time revealed that anemic IBD patients exhibit impaired production of short-chain fatty acids (SCFAs), particularly butyrate. Butyrate supplementation restores iron metabolism in multiple anemia models. Mechanistically, butyrate upregulates ferroportin (FPN) expression in macrophages by reducing the enrichment of histone deacetylase (HDAC) at the Slc40a1 promoter, thereby facilitating iron export. By preventing iron sequestration, butyrate not only mitigates colitis-induced anemia but also reduces TNF-α production in macrophages. Consistently, macrophage-conditional FPN knockout mice exhibit more severe anemia and inflammation. Finally, it is revealed that macrophage iron overload impairs the therapeutic effectiveness of anti-TNF-α antibodies in colitis, which can be reversed by butyrate supplementation. Hence, this study uncovers the pivotal role of butyrate in preventing the pathogenic circuit between anemia and inflammation.