Intracellular Iron Content Research Articles

Ciclopirox ethanolamine preserves the immature state of human HSCs by mediating intracellular iron content

AbstractCulture conditions in which hematopoietic stem cells (HSCs) can be expanded for clinical benefit are highly sought after. To elucidate regulatory mechanisms governing the maintenance and propagation of human HSCs ex vivo, we screened libraries of annotated small molecules in human cord blood cells using an optimized assay for detection of functional HSCs during culture. We found that the antifungal agent ciclopirox ethanolamine (CPX) selectively supported immature CD34+CD90+ cells during culture and enhanced their long-term in vivo repopulation capacity. Purified HSCs treated with CPX showed a reduced cell division rate and an enrichment of HSC-specific gene expression patterns. Mechanistically, we found that the HSC stimulating effect of CPX was directly mediated by chelation of the intracellular iron pool, which in turn affected iron-dependent proteins and enzymes mediating cellular metabolism and respiration. Our findings unveil a significant impact of iron homeostasis in regulation of human HSCs, with important implications for both basic HSC biology and clinical hematology.

Unveiling the Therapeutic Potential of Squalene Synthase: Deciphering Its Biochemical Mechanism, Disease Implications, and Intriguing Ties to Ferroptosis.

Squalene synthase (SQS) has emerged as a promising therapeutic target for various diseases, including cancers, owing to its pivotal role in the mevalonate pathway and the antioxidant properties of squalene. Primarily, SQS orchestrates the head-to-head condensation reaction, catalyzing the fusion of two farnesyl pyrophosphate molecules, leading to the formation of squalene, which has been depicted as a highly effective oxygen-scavenging agent in in vitro studies. Recent studies have depicted this isoprenoid as a protective layer against ferroptosis due to its potential regulation of lipid peroxidation, as well as its protection against oxidative damage. Therefore, beyond its fundamental function, recent investigations have unveiled additional roles for SQS as a regulator of lipid peroxidation and programmed cell death pathways, such as ferroptosis-a type of cell death characterized by elevated levels of lipid peroxide, one of the forms of reactive oxygen species (ROS), and intracellular iron concentration. Notably, thorough explorations have shed light on the distinctive features that set SQS apart from other members within the isoprenoid synthase superfamily. Its unique biochemical structure, intricately intertwined with its reaction mechanism, has garnered significant attention. Moreover, considerable evidence substantiates the significance of SQS in various disease contexts, and its intriguing association with ferroptosis and lipid peroxidation. The objective of this report is to analyze the existing literature comprehensively, corroborating these findings, and provide an up-to-date perspective on the current understanding of SQS as a prospective therapeutic target, as well as its intricate relationship with ferroptosis. This review aims to consolidate the knowledge surrounding SQS, thereby contributing to the broader comprehension of its potential implications in disease management and therapeutic interventions.

The C-terminal domain of the ferric uptake regulator (Fur) binds a [2Fe-2S] cluster to sense the intracellular free iron content in Escherichia coli.

Escherichia coli ferric uptake regulator (Fur) binds a [2Fe-2S] cluster, not a mononuclear iron, when the intracellular free iron content is elevated in E. coli cells. Here we report that the C-terminal domain (residues 83-148) of E. coli Fur (Fur-CTD) is sufficient to bind the [2Fe-2S] cluster in response to elevation of the intracellular free iron content in E. coli cells. Deletion of gene fur in E. coli cells increases the intracellular free iron content and promotes the [2Fe-2S] cluster binding in the Fur-CTD in the cells grown in LB medium under aerobic growth conditions. When the Fur-CTD is expressed in wild type E. coli cells grown in M9 medium supplemented with increasing concentrations of iron, the Fur-CTD also progressively binds a [2Fe-2S] cluster with a maximum occupancy of about 36%. Like the E. coli Fur-CTD, the CTD of the Haemophilus influenzae Fur can also bind a [2Fe-2S] cluster in wild type E. coli cells grown in M9 medium supplemented with increasing concentrations of iron, indicating that binding of the [2Fe-2S] cluster in the C-terminal domain is highly conserved among Fur proteins. The results suggest that the Fur-CTD can be used as a physiological probe to assess the intracellular free iron content in bacteria.

Isoorientin reverses lung cancer drug resistance by promoting ferroptosis via the SIRT6/Nrf2/GPX4 signaling pathway

Cisplatin, or DDP, is a highly successful and well-known chemotherapy drug used to treat cancer. Acquired resistance to chemotherapy is a major clinical concern, yet the mechanisms of this resistance are still unknown. Ferroptosis is a type of cell death distinct from other forms, fueled by a buildup of iron-associated lipid reactive oxygen species (ROS). Gaining insight into the process of ferroptosis could lead to novel treatments for overcoming cancer resistance. In this study, the combination of isoorientin (IO) and DDP treatment resulted in a significant decrease in the viability of drug-resistant cells, a substantial increase in intracellular iron, malondialdehyde (MDA) and ROS concentrations, a notable decrease in glutathione concentration, and the occurrence of ferroptosis in cells, as revealed by in vitro and in vivo experiments. Additionally, there was a decrease in the expression of nuclear factor-erythroid factor 2-related factor 2 (Nrf2), glutathione peroxidase 4 (GPX4), and sirtuin 6 (SIRT6) proteins, and an increase in cellular ferroptosis. Isoorientin acts as a mediator to regulate cellular ferroptosis and reverse drug resistance in lung cancer cells by controlling the SIRT6/Nrf2/GPX4 signaling pathway. The findings of this study suggest that IO can promote ferroptosis and reverse drug resistance in lung cancer through the SIRT6/Nrf2/GPX4 signaling pathway, thus offering a theoretical basis for its potential clinical application.

Bacillibactin siderophore induces iron mobilisation responses inside aerobic rice variety through YSL15 transporter

Iron uptake in Oryza sativa primarily occurs with the help of phytosiderophore i.e., deoxymugineic acid in aerobic condition. Internalisation of phytosiderophore-iron complex occurs through YSL15. However, aerobic rice at high pH faces iron deficit condition due to low secretion of plant siderophore and its early diminution. There might be a possibility that microbial siderophore of predominant rhizobacteria present in soil around rice may assist in iron uptake and transport through same transporter providing survival advantage to the plant. Present study aims to explore role of siderophore of Bacillus subtilis in improving root architecture and iron uptake in aerobic rice at high pH and explore possible transporters involved in transporting siderophore-Fe3+ complex inside rice root from rhizosphere. Identification by chrome azurol sulphonate assay and characterisation of purified extract from B. subtilis culture through LC-ESI MS confirmed presence of catecholate siderophore, Bacillibactin. Aerobic rice variety viz. Swarna Shreya grown with 5 ng/ml bacillibactin showed improved morphology and anatomy than untreated sample at alkaline pH 7.5. This is possibly due to enhanced intracellular iron content as observed in atomic absorption spectroscopy. Whole root proteomic analysis of rice root by using LC-ESI MS/MS revealed presence of YSL like transporter and siderophore import proteins in treated sample. RT-qPCR analysis showed enhanced expression of gene YSL15 in siderophore treated sample than control (pH 7.5). The present study possibly suggest role of foreign siderophore (xenosiderophore) in plant growth and development through iron uptake and transport inside rice.

The Role of Fractalkine in the Regulation of Endometrial Iron Metabolism in Iron Deficiency.

Iron is a crucial element in the human body. Endometrial iron metabolism is implicated in endometrium receptivity and embryo implantation. Disturbances of the maternal as well as the endometrial iron homeostasis, such as iron deficiency, can contribute to the reduced development of the fetus and could cause an increased risk of adverse pregnancy outcomes. Fractalkine is a unique chemokine that plays a role in the communication between the mother and the fetus. It has been demonstrated that FKN is involved in the development of endometrial receptivity and embryo implantation, and it functions as a regulator of iron metabolism. In the present study, we examined the effect of FKN on the iron metabolism of HEC-1A endometrial cells in a state of iron deficiency mediated by desferrioxamine treatment. Based on the findings, FKN enhances the expression of iron metabolism-related genes in iron deficiency and modifies the iron uptake via transferrin receptor 1 and divalent metal transporter-1, and iron release via ferroportin. FKN can activate the release of iron from heme-containing proteins by elevating the level of heme oxygenase-1, contributing to the redistribution of intracellular iron content. It was revealed that the endometrium cells express both mitoferrin-1 and 2 and that their levels are not dependent on the iron availability of the cells. FKN may also contribute to maintaining mitochondrial iron homeostasis. FKN can improve the deteriorating effect of iron deficiency in HEC-1A endometrium cells, which may contribute to the development of receptivity and/or provide iron delivery towards the embryo.

Functional Characterization of FeoAB in Iron Acquisition and Pathogenicity in Riemerella anatipestifer.

The bacterium Riemerella anatipestifer requires iron for growth, but the mechanism of iron uptake is not fully understood. In this study, we disrupted the Feo system and characterized its function in iron import in R. anatipestifer ATCC 11845. Compared to the parent strain, the growth of the ΔfeoA, ΔfeoB, and ΔfeoAB strains was affected under Fe3+-limited conditions, since the absence of the feo system led to less intracellular iron than in the parent strain. In parallel, the ΔfeoAB strain was shown to be less sensitive to streptonigrin, an antibiotic that requires free iron to function. The sensitivity of the ΔfeoAB strain to hydrogen peroxide was also observed to be diminished compared with that of the parent strain, which could be related to the reduced intracellular iron content in the ΔfeoAB strain. Further research revealed that feoA and feoB were directly regulated by iron through the Fur regulator and that the transcript levels of feoA and feoB were significantly increased in medium supplemented with 1 mM MnCl2, 400 μM ZnSO4, and 200 μM CuCl2. Finally, it was shown that the ΔfeoAB strain of R. anatipestifer ATCC 11845 was significantly impaired in its ability to colonize the blood, liver, and brain of ducklings. Taken together, these results demonstrated that FeoAB supports ferrous iron acquisition in R. anatipestifer and plays an important role in R. anatipestifer colonization. IMPORTANCE In Gram-negative bacteria, the Feo system is an important ferrous iron transport system. R. anatipestifer encodes an Feo system, but its function unknown. As iron uptake may be required for oxidative stress protection and virulence, understanding the contribution of iron transporters to these processes is crucial. This study showed that the ΔfeoAB strain is debilitated in its ability to import iron and that its intracellular iron content was constitutively low, which enhanced the resistance of the deficient strain to H2O2. We were surprised to find that, in addition to responding to iron, the Feo system may play an important role in sensing manganese, zinc, and copper stress. The reduced colonization ability of the ΔfeoAB strain also sheds light on the role of iron transporters in host-pathogen interactions. This study is important for understanding the cross talk between iron and other metal transport pathways, as well as the pathogenic mechanism in R. anatipestifer.

Apolipoprotein E4 impairs iron efflux in human induced pluripotent stem cell (iPSC)‐derived cortical neurons by reducing ferroportin on cell surface

AbstractBackgroundIron homeostasis disturbance has been implicated in Alzheimer’s disease (AD), and excess iron exacerbates oxidative damage and cognitive defects. Ferroportin (Fpn) is the only known iron exporter and the expression of Fpn is decreased in the brains of AD patients. Apolipoprotein E (APOE) gene is the most important genetic risk factor for late‐onset AD. The three common proteins encoded by the gene are ApoE2, ApoE3, and ApoE4. They bind to iron with different affinities, which mediates the isoform‐specific antioxidant activity. However, whether ApoE takes part in neuronal iron homeostasis remains unclear.MethodCortical neurons were generated from human iPSCs (All from non‐demented APOE3/E3 carriers). The intracellular iron content was measured using inductively coupled plasma mass spectrometry (ICP‐MS) after co‐treatment of iron and ApoE. Western blot analysis was performed to examine the alterations of various iron storage and transport proteins. Cell surface Fpn expression levels were measured by fluorescence‐activated cell sorting (FACS).ResultIntracellular iron level in ApoE4‐treated neurons was higher than that in ApoE2‐ or ApoE3‐treated neurons. Furthermore, Fpn level on cell surface was reduced by ApoE4 treatment.ConclusionOur study demonstrates that ApoE4 impairs iron efflux and exacerbate iron accumulation via regulating surface Fpn level.

GPX4 overexpressed non-small cell lung cancer cells are sensitive to RSL3-induced ferroptosis

Ferroptosis can be induced by inhibiting antioxidant enzymes GPX4 or system Xc−, increased intracellular iron concentrations, and lipid peroxidation. Recently, it has been suggested that ferroptosis can be an effective way to induce cancer cell death, although the specific relevance and mechanism of ferroptosis have not been fully elucidated. Here, we investigated the anticancer effects of ferroptosis inducers erastin and RSL3 on non-small cell lung cancer (NSCLC) cells. RSL3 induced cell death more effectively in NSCLC cells than erastin, with limited cytotoxicity in BEAS-2B normal bronchial epithelial cells. The sensitivity of NSCLC cells to RSL3 induced death was dependent on GPX4 expression levels; the effect of RSL3 was reversed by ferrostatin-1 (a ferroptosis inhibitor) but not by Z-VAD-FMK, chloroquine, bafilomycin A1, or necrostatin-1. RSL3 induced ferroptosis by promoting lipid peroxidation, elevating intracellular LIP concentration and ROS level, and blocking GSH-to-GSSH conversion through the inhibition of GPX4 and induction of Nrf2/HO1. Furthermore, RSL3 induced autophagosomes but disrupted the formation of autolysosomes with lysosomal membrane destabilization. GPX4 knockdown had a similar effect on ferroptosis phenotypes as RSL3. Taken together, RSL3-induced ferroptosis depends on the regulation of GPX4-Nrf2/HO1 in NSCLC cells. These results may be useful in predicting the ferroptosis response in NSCLC as well as drug resistant cancer cells.

Chronic arsenic exposure induces ferroptosis via enhancing ferritinophagy in chicken livers

Arsenic (As) is a well-known pollutant in the environment, whose contamination in groundwater is a serious threat to animals and humans. Ferroptosis, a form of cell death caused by iron-dependent lipid peroxidation, is involved in various pathological processes. Ferritinophagy is the selective autophagy of ferritin and a crucial step in the induction of ferroptosis. However, the mechanism of ferritinophagy in poultry livers exposed to As remains unexplored. In this study, we investigated whether As-induced chicken liver injury is related to ferritinophagy-mediated ferroptosis at the cellular and animal levels. Our results showed that As exposure via drinking water induced hepatotoxicity in chickens, characterized by abnormal liver morphology and elevated liver function markers. Our data suggested chronic As exposure led to mitochondrial dysfunction, oxidative stress, and impaired cellular processes in chicken livers and LMH cells. Our results also showed that As exposure activated the AMPK/mTOR/ULK1 signaling pathway and significantly changed the levels of ferroptosis and autophagy-related proteins in chicken livers and LMH cells. Moreover, As exposure induced iron overload and lipid peroxidation in chicken livers and LMH cells. Interestingly, pretreatment with ferrostatin-1, chloroquine (CQ), and deferiprone alleviated these aberrant effects. Using CQ, we found that As-induced ferroptosis is autophagy-dependent. Our findings further suggested chronic As exposure induced chicken liver injury by promoting ferritinophagy-mediated ferroptosis, as evidence by activated autophagy, decreased mRNA expression of FTH1, increased intracellular iron content, and alleviation of ferroptosis through pretreatment with CQ. In conclusion, ferritinophagy-mediated ferroptosis is one of the critical mechanisms of As-induced chicken liver injury. Inhibiting ferroptosis may provide new insights for preventing and treating liver injury induced by environmental As exposure in livestock and poultry.

The molecular mechanisms of the bacterial iron sensor IdeR.

Life came to depend on iron as a cofactor for many essential enzymatic reactions. However, once the atmosphere was oxygenated, iron became both scarce and toxic. Therefore, complex mechanisms have evolved to scavenge iron from an environment in which it is poorly bioavailable, and to tightly regulate intracellular iron contents. In bacteria, this is typically accomplished with the help of one key regulator, an iron-sensing transcription factor. While Gram-negative bacteria and Gram-positive species with low guanine-cytosine (GC) content generally use Fur (ferric uptake regulator) proteins to regulate iron homeostasis, Gram-positive species with high GC content use the functional homolog IdeR (iron-dependent regulator). IdeR controls the expression of iron acquisition and storage genes, repressing the former, and activating the latter in an iron-dependent manner. In bacterial pathogens such as Corynebacterium diphtheriae and Mycobacterium tuberculosis, IdeR is also involved in virulence, whereas in non-pathogenic species such as Streptomyces, it regulates secondary metabolism as well. Although in recent years the focus of research on IdeR has shifted towards drug development, there is much left to learn about the molecular mechanisms of IdeR. Here, we summarize our current understanding of how this important bacterial transcriptional regulator represses and activates transcription, how it is allosterically activated by iron binding, and how it recognizes its DNA target sites, highlighting the open questions that remain to be addressed.

Knockdown of ABCB7 inhibits esophageal cancer progression by inhibiting the TGF-β/Smad signaling

ObjectivesEsophageal cancer accounts for the seventh in human cancers, and the sixth in the global cancer death. ATP-binding cassette, sub-family B (MDR/TAP), member 7 (ABCB7) maintains intracellular iron homeostasis and involved in the regulation of tumor progression. However, the role and mechanism of ABCB7 in esophageal cancer remained unclear. MethodsHere, we investigated its role and regulatory mechanism by knocking down ABCB7 in Eca109 and KYSE30 cells. ResultsABCB7 was significantly upregulated in esophageal cancer tissues, and was strongly associated with metastasis and poor prognosis of patients. ABCB7 knockdown inhibits the proliferation, migration and invasion of esophageal cancer cells. Importantly, ABCB7 knockdown induces apoptosis and non-apoptotic cell death in flow cytometry analysis. Higher intracellular total iron concentration was observed in ABCB7 knockdown Eca109 and KYSE30 cells. We further analyzed ABCB7 expression related genes in esophageal cancer tissues. COX7B were positively correlated with the expression of ABCB7 in 440 esophageal cancer tissues. COX7B rescued the inhibition of cell proliferation and elevated total iron concentration induced by ABCB7 knockdown. In addition, Western blot results showed that ABCB7 knockdown reversed the epithelial-mesenchymal transition (EMT) process and inhibited the TGF-β signaling pathway in Eca109 and KYSE30 cells. ConclusionIn conclusion, ABCB7 knockdown inhibits the TGF-β signaling pathway, inhibits the survival of esophageal cancer cells by inducing cell death, and reverses the EMT process. Targeting ABCB7 or COX7B could be a novel strategy for the treatment of esophageal cancer.

Effect of erythrophagocytosis-induced ferroptosis during angiogenesis in atherosclerotic plaques.

Intraplaque (IP) angiogenesis is a key feature of advanced atherosclerotic plaques. Because IP vessels are fragile and leaky, erythrocytes are released and phagocytosed by macrophages (erythrophagocytosis), which leads to high intracellular iron content, lipid peroxidation and cell death. In vitro experiments showed that erythrophagocytosis by macrophages induced non-canonical ferroptosis, an emerging type of regulated necrosis that may contribute to plaque destabilization. Erythrophagocytosis-induced ferroptosis was accompanied by increased expression of heme-oxygenase 1 and ferritin, and could be blocked by co-treatment with third generation ferroptosis inhibitor UAMC-3203. Both heme-oxygenase 1 and ferritin were also expressed in erythrocyte-rich regions of carotid plaques from ApoE-/- Fbn1C1039G+/- mice, a model of advanced atherosclerosis with IP angiogenesis. The effect of UAMC-3203 (12.35mg/kg/day) on atherosclerosis was evaluated in ApoE-/- Fbn1C1039G+/- mice fed a western-type diet (WD) for 12 weeks (n = 13 mice/group) or 20 weeks (n = 16-21 mice/group) to distinguish between plaques without and with established IP angiogenesis, respectively. A significant decrease in carotid plaque thickness was observed after 20 weeks WD (87 ± 19μm vs. 166 ± 20μm, p = 0.006), particularly in plaques with confirmed IP angiogenesis or hemorrhage (108 ± 35μm vs. 322 ± 40μm, p = 0.004). This effect was accompanied by decreased IP heme-oxygenase 1 and ferritin expression. UAMC-3203 did not affect carotid plaques after 12 weeks WD or plaques in the aorta, which typically do not develop IP angiogenesis. Altogether, erythrophagocytosis-induced ferroptosis during IP angiogenesis leads to larger atherosclerotic plaques, an effect that can be prevented by ferroptosis inhibitor UAMC-3203.

Live and inactivated Piscirickettsia salmonis activated nutritional immunity in Atlantic salmon (Salmo salar).

Nutritional immunity regulates the homeostasis of micronutrients such as iron, manganese, and zinc at the systemic and cellular levels, preventing the invading microorganisms from gaining access and thereby limiting their growth. Therefore, the objective of this study was to evaluate the activation of nutritional immunity in specimens of Atlantic salmon (Salmo salar) that are intraperitoneally stimulated with both live and inactivated Piscirickettsia salmonis. The study used liver tissue and blood/plasma samples on days 3, 7, and 14 post-injections (dpi) for the analysis. Genetic material (DNA) of P. salmonis was detected in the liver tissue of fish stimulated with both live and inactivated P. salmonis at 14 dpi. Additionally, the hematocrit percentage decreased at 3 and 7 dpi in fish stimulated with live P. salmonis, unchanged in fish challenged with inactivated P. salmonis. On the other hand, plasma iron content decreased during the experimental course in fish stimulated with both live and inactivated P. salmonis, although this decrease was statistically significant only at 3 dpi. Regarding the immune-nutritional markers such as tfr1, dmt1, and ireg1 were modulated in the two experimental conditions, compared to zip8, ft-h, and hamp, which were down-regulated in fish stimulated with live and inactivated P. salmonis during the course experimental. Finally, the intracellular iron content in the liver increased at 7 and 14 dpi in fish stimulated with live and inactivated P. salmonis, while the zinc content decreased at 14 dpi under both experimental conditions. However, stimulation with live and inactivated P. salmonis did not alter the manganese content in the fish. The results suggest that nutritional immunity does not distinguish between live and inactivated P. salmonis and elicits a similar immune response. Probably, this immune mechanism would be self-activated with the detection of PAMPs, instead of a sequestration and/or competition of micronutrients by the living microorganism.

Ferric uptake regulator (Fur) binds a [2Fe-2S] cluster to regulate intracellular iron homeostasis in Escherichia coli

Intracellular iron homeostasis in bacteria is primarily regulated by ferric uptake regulator (Fur). It has been postulated that when intracellular free iron content is elevated, Fur binds ferrous iron to downregulate the genes for iron uptake. However, the iron-bound Fur had not been identified in any bacteria until we recently found that Escherichia coli Fur binds a [2Fe-2S] cluster, but not a mononuclear iron, in E.coli mutant cells that hyperaccumulate intracellular free iron. Here, we report that E.coli Fur also binds a [2Fe-2S] cluster in wildtype E.coli cells grown in M9 medium supplemented with increasing concentrations of iron under aerobic growth conditions. Additionally, we find that binding of the [2Fe-2S] cluster in Fur turns on its binding activity for specific DNA sequences known as the Fur-box and that removal of the [2Fe-2S] cluster from Fur eliminates its Fur-box binding activity. Mutation of the conserved cysteine residues Cys-93 and Cys-96 to Ala in Fur results in the Fur mutants that fail to bind the [2Fe-2S] cluster, have a diminished binding activity for the Fur-box invitro, and are inactive to complement the function of Fur invivo. Our results suggest that Fur binds a [2Fe-2S] cluster to regulate intracellular iron homeostasis in response to elevation of intracellular free iron content in E.coli cells.

Palmitic acid-induced ferroptosis via CD36 activates ER stress to break calcium-iron balance in colon cancer cells.

Ferroptosis, featuring an iron-dependent peroxidation of lipids, is a novel form of programmed cell death that may hold great potential in cancer therapy. Our study found that palmitic acid (PA) inhibited colon cancer cell viability in vitro and in vivo, in conjunction with an accumulation of reactive oxygen species (ROS) and lipid peroxidation. The ferroptosis inhibitor Ferrostatin-1 but not Z-VAD-FMK (a pan-caspase inhibitor), Necrostatin-1 (a potent necroptosis inhibitor), or CQ (a potent inhibitor of autophagy), rescued the cell death phenotype induced by PA. Subsequently, we verified that PA induces ferroptotic cell death through excess iron as cell death was inhibited by iron chelator deferiprone (DFP), while it was exacerbated by a supplement of ferric ammonium citrate (FAC). Mechanistically, PA affects intracellular iron content by inducing endoplasmic reticulum (ER) stress leading to ER calcium release and regulating transferrin transport through increasing cytosolic calcium levels. Furthermore, we observed that cells with high expression of CD36 were more vulnerable to PA-induced ferroptosis. Altogether, our findings reveal that PA engages in anticancer properties by activating ER stress/ER calcium release/transferrin-dependent ferroptosis, and PA might serve as a compound to activate ferroptosis in colon cancer cells with high CD36 expression.

Transcriptomic analysis identified SLC40A1 as a key iron metabolism-related gene in airway macrophages in childhood allergic asthma.

Introduction: Asthma is the most common chronic condition in children, with allergic asthma being the most common phenotype, accounting for approximately 80% of cases. Growing evidence suggests that disruption of iron homeostasis and iron regulatory molecules may be associated with childhood allergic asthma. However, the underlying molecular mechanism remains unclear. Methods: Three childhood asthma gene expression datasets were analyzed to detect aberrant expression profiles of iron metabolism-related genes in the airways of children with allergic asthma. Common iron metabolism-related differentially expressed genes (DEGs) across the three datasets were identified and were subjected to functional enrichment analysis. Possible correlations between key iron metabolism-related DEGs and type 2 airway inflammatory genes were investigated. Single-cell transcriptome analysis further identified major airway cell subpopulations driving key gene expression. Key iron metabolism-related gene SLC40A1 was validated in bronchoalveolar lavage (BAL) cells from childhood asthmatics with control individuals by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and immunofluorescence. The intracellular iron content in BAL cells was assessed by Perls iron staining and the iron levels in BAL supernatant was measured by iron assay to assess airway iron metabolism status in childhood asthmatics. Results: Five common iron metabolism-related DEGs were identified, which were functionally related to iron homeostasis. Among these genes, downregulated SLC40A1 was strongly correlated with type 2 airway inflammatory markers and the gene signature of SLC40A1 could potentially be used to determine type 2-high and type 2-low subsets in childhood allergic asthmatics. Further single-cell transcriptomic analysis identified airway macrophages driving SLC40A1 expression. Immunofluorescence staining revealed colocalization of FPN (encoded by SLC40A1) and macrophage marker CD68. Down-regulation of SLC40A1 (FPN) was validated by qRT-PCR and immunofluorescence analysis. Results further indicated reduced iron levels in the BAL fluid, but increased iron accumulation in BAL cells in childhood allergic asthma patients. Furthermore, decreased expression of SLC40A1 was closely correlated with reduced iron levels in the airways of children with allergic asthma. Discussion: Overall, these findings reveal the potential role of the iron metabolism-related gene SLC40A1 in the pathogenesis of childhood allergic asthma.

Abstract 2845: PpIX synthesis increases PDT resistant cells due to the modulating effect of EGCG on the heme synthesis pathway

Abstract Introduction: Photodynamic therapy (PDT) is a treatment for non melanoma skin cancer. PDT kills cells through reactive oxygen species (ROS), produced by interaction between cellular O2 and an excited photosensitizer (PS) by a specific light. One of the most used PS in dermatology is Protoporphyrin IX (PpIX) and its precursor methyl aminolevulinate (MAL). MAL enters into cells and is converted to PpIX by enzymes of the heme group synthesis pathway. The insertion of ferrous iron into PpIX forms a heme molecule, which has no PS action. A previous study shows that efficacy of PDT, in resistant cells, can be improved by addition of epigallocatechin gallate (EGCG), a green tea polyphenol. Therefore, the aim of this work is to evaluate the rol of EGCG on PPIX synthesis. Material and methods: It was used a A-431 cell line from skin squamous cell carcinoma. The EC50 of EGCG was determined by MTT assay after 24 hours. Clonogenic assay was carried out using EGCG 10, 20 and 40 μM, for 4 and 24 hours of incubation. To evaluate PpIX and ROS, A-431 cells were exposed to EGCG (10, 20, 40 μM) with or without MAL 2 mM. After 4 hours, some plates were irradiated with red light to generate ROS. PpIX and ROS were detected by flow cytometry. Gene expression of enzymes of heme synthesis was analyzed by RT-qPCR. Intracellular iron was measured using a commercial kit.  Results: EC50 determined a concentration of 50 μM of EGCG at 24 hours. EGCG concentrations from 10 μM reduced cell clonogenic capacity, even with only 4 hours of incubation. A-431 cells show PDT resistance with MAL 2 mM and a fluence of 4 J/cm2. The addition of EGCG to MAL-PDT showed a decrement of cell viability with an increase of PpIX and ROS. Besides, incubation with EGCG for 4 hours, triggered an increment of HMBS enzyme and decrease of FECH. In addition, EGCG reduced intracellular iron concentrations. Conclusion: These findings show that EGCG has a PDT enhancing effect, decreasing intracellular iron levels and modulating gene expression of heme synthesis enzymes, which are directly related with high levels of PpIX production and ROS levels, causing the death of resistant cells.Acknowledgements: Postdoctoral FONDECYT Nº 3210618, IDeA I+D FONDEF ID21I10027 Citation Format: Daniela León, Álvaro Gutiérrez, Helga Weber, Ramón Silva, María Elena Reyes, Tamara Viscarra, Kurt Buchegger, Carmen Ili, Priscilla Brebi. PpIX synthesis increases PDT resistant cells due to the modulating effect of EGCG on the heme synthesis pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2845.

Cinobufotalin Induces Ferroptosis to Suppress Lung Cancer Cell Growth by lncRNA LINC00597/hsa-miR-367-3p/TFRC Pathway via Resibufogenin.

Lung cancer is the leading cause of cancer-associated death and the first most diagnosed cancer in the world. More than 2 million new cases are diagnosed and 1.6 million people die due to lung cancer every year. It is urgent to explore novel drugs and approaches for lung cancer treatment. Cinobufotalin is a TCM isolated from dried toad venom, which has been used to treat lung cancer. However, the precise mechanism remains unclear. This study was to investigate the mechanism of cinobufotalin treated in lung cancer. Cell growth was identified by Cell Counting Kit-8 (CCK-8) assay. Besides, ferroptosis of lung cancer cells was determined by intracellular iron content, lactate dehydrogenase (LDH) release and mitochondrial membrane potential. Moreover, RNA levels and proteins were detected by quantitative reverse transcription-PCR (qRT-PCR) and Western blot (WB), respectively. In addition, the regulatory effect of hsa-miR-367-3p on TFRC was confirmed by luciferase reporter assay. This study indicated that cinobufotalin suppressed lung cancer cell growth through resibufogenin. Besides, cinobufotalin induced ferroptosis in lung cancer cells through resibufogenin. Moreover, cinobufotalin increased lncRNA LINC00597 level, whereas it downregulated hsa-miR-367-3p expression in lung cancer cells via resibufogenin. In addition, ferroptosis inducer transferrin receptor (TFRC) was the target of hsa-miR-367-3p, and lncRNA LINC00597 upregulates TFRC expression through sponging hsa-miR-367-3p in lung cancer cells. In summary, this study indicated that cinobufotalin induced ferroptosis to suppress lung cancer cell growth by lncRNA LINC00597/hsa-miR-367-3p/TFRC pathway via resibufogenin might provide novel therapeutic targets for lung cancer therapy.

Angiotensin II-induced vascular endothelial cells ferroptosis via P53-ALOX12 signal axis

ABSTRACT Objectives The present study aimed to investigate the effect and mechanism of angiotensin II–induced ferroptosis in vascular endothelial cells. Methods In vitro, HUVECs were treated with AngII, AT1/2 R antagonist, P53 inhibitor, or their combinations. MDA and intracellular iron content were evaluated using an ELISA assay. The expression of ALOX12, P53, P21, and SLC7A11 were determined by western blotting in HUVECs and then confirmed through RT-PCR. Results As the concentration of Ang II (0, 0.1,1,10,100, and 1000uM for 48 h) increased, the level of MDA and intracellular iron content increased in HUVECs. Compared with the single AngII group, ALOX12, p53, MDA, and intracellular iron content in AT1/2R antagonist group decreased significantly. In pifithrin-α hydrobromide-treated, ALOX12, P21,MDA, and intracellular iron content decreased significantly as compared to the single AngII group. Similarly, the effect of combined use of blockers is stronger than that of blockers alone. Conclusions AngII can induce ferroptosis of vascular endothelial cells. The mechanism of AngII-induced ferroptosis may be regulated through the signal axis of p53-ALOX12.