Labile Iron Pool Levels Research Articles

Role of Ferroptosis Regulation by Nrf2/NQO1 Pathway in Alcohol-Induced Cardiotoxicity In Vitro and In Vivo.

The aim of the present study was to evaluate the cardiotoxic effects of alcohol and its potential toxic mechanism on ferroptosis in mice and H9c2 cells. Mice were intragastrically treated with three different concentrations of alcohol, 7, 14, and 28%, each day for 14 days. Body weight and electrocardiography (ECG) were recorded over the 14 day period. Serum creatine kinase (CK), lactic dehydrogenase (LDH), MDA, tissue iron, and GSH levels were measured. Cardiac tissues were examined histologically, and ferroptosis was assessed. In H9c2 cardiomyocytes, cell viability, reactive oxygen species (ROS), labile iron pool (LIP), and mitochondrial membrane potential (MMP) were measured. The proteins of ferroptosis were evaluated by the western blot technique in vivo and in vitro. The results showed that serum CK, LDH, MDA, and tissue iron levels significantly increased in the alcohol treatment group in a dose-dependent manner. The content of GSH decreased after alcohol treatment. ECG and histological examinations showed that alcohol impaired cardiac function and structure. In addition, the levels of ROS and LIP increased, and MMP levels decreased after alcohol treatment. Ferrostatin-1 (Fer-1) protected cells from lipid peroxidation. Western blotting analysis showed that alcohol downregulated the expression of Nrf2, NQO1, HO-1, and GPX4. The expressions of P53 and TfR were upregulated in vivo and in vitro. Fer-1 significantly alleviated alcohol-induced ferroptosis. In conclusion, the study showed that Nrf2/NQO1-dependent ferroptosis played a vital role in the cardiotoxicity induced by alcohol.

TRAIL predisposes non-small cell lung cancer to ferroptosis by regulating ASK-1/JNK1 pathway

ObjectiveOur current study aimed to assess the relationship between TNF-related apoptosis-inducing ligand (TRAIL) and ferroptosis in non-small cell lung cancer (NSCLC) development.MethodsThe expression of TRAIL was detected by western blot, RT-qRCR and immunohistochemistry. The viability of NSCLC cells was analyzed by CCK-8 kit. The migration and invasion of NSCLC cells were detected by wound healing assay and transwell assay, respectively. Labile iron pool (LIP) was detected based on the calcein-acetoxymethyl ester method. Ferrous iron (Fe2+) and iron levels were assessed by detection kits. The levels of superoxide dismutase (SOD), catalase (CAT), and malondialdehyde (MDA) were measured using corresponding detection kits. Mice tumor xenograft models were used for the in vivo research.ResultsThe expression of TRAIL was reduced in H1299, NCL-H1395, and A549 cells compared with BEAS-2B cells. The up-regulation of TRAIL expression significantly reduced cell viability, invasion, and migration of H1299 and A549 cells. TRAIL reduced the expression of ferroptosis-related genes (FTH1, GPX4, and SLC7A11), increased the levels of LIP, iron, and Fe2+, and promoted lipid peroxidation, thereby predisposing NSCLC cells to ferroptosis. TRAIL up-regulated the expression of phosphate modification of ASK-1 and JNK. ASKI-1 inhibitor GS-4977 attenuated the effects of TRAIL on the viability, migration, invasion, and ferroptosis of H1299 cells. Furthermore, TRAIL further suppressed tumor growth and ferroptosis in mice tumor xenograft models.ConclusionWe indicated that overexpression of TRAIL induced ferroptosis in NSCLC cells and exerted anti-tumor effects. Mechanistically, TRAIL promoted ferroptosis by the activation of the ASK-1/JNK1 pathway. Our results may provide new therapeutic strategies for NSCLC.

Erythrocyte labile iron pool indicating concealed iron overload in non-transfusion-dependent β-thalassemia

Abstract Objectives Not only do transfusion-dependent thalassemia (TDT) patients have a risk of clinical consequences arising from iron overload, non-transfusion-dependent thalassemia (NTDT) patients may encounter it also. In this study, we demonstrated the usefulness of intra-erythrocyte labile iron pool (LIP) measurement in NTDT patients with HbE/β-thalassemia in revealing a concealed iron overload and the resultant oxidative cell damage. Methods LIP and ferritin levels were assayed in 20 HbE/β-thalassemia patients, comprising 10 NTDT and 10 TDT patients and 10 healthy individuals. Erythrocyte oxidative stress parameters (intraerythrocyte reactive oxygen species (ROS) levels, numbers of erythrocyte vesicles, and cell apoptosis) were also determined and their correlations to LIP and ferritin levels were analyzed. Results LIP levels (based on different mean fluorescence intensity (MFI) values obtained by flow cytometry) were high in both the NTDT (54 [42–90]) and TDT (71 [68–77]) patients compared to the healthy individuals (43 [31–61]). The ferritin level observed in the NTDT group (2,149 [781–4,071] ng/mL) was lower than that of the TDT group (4,885 [2,343–7,826] ng/mL). The LIP level was significantly correlated with the NTDT patients’ age and all oxidative stress parameters, but the ferritin level was not. Correlation analysis predicted that NTDT patients without receiving any therapy aged 32.5 years old or over are likely at risk of iron overload status and oxidative cell damage. Conclusions The results indicate that LIP may be a helpful alternative parameter for defining the risk of iron overload.

PRDX6-mediated pulmonary artery endothelial cell ferroptosis contributes to monocrotaline-induced pulmonary hypertension

Pulmonary hypertension (PH) is a life-threatening cardiopulmonary disorder whose underlying pathogenesis is unknown. Our previous study showed that pulmonary endothelial cell (PAEC) ferroptosis is involved in the progression of PH by releasing High-mobility group box 1 (HMGB1) and activating Toll-like receptor 4/NOD-like receptor family pyrin domain containing 3 (TLR4/NLRP3) inflammasome signalling. The precise mechanisms that regulate ferroptosis in PH are unclear. This study aimed to investigate the effect of peroxiredoxin 6 (PRDX6) on PAEC ferroptosis in PH. A rat model of PH was established with monocrotaline (MCT), and the distribution and expression of PRDX6 in the pulmonary artery were examined. Lentiviral vectors carrying PRDX6 (LV-PRDX6) were transfected into PAECs and injected into MCT-induced PH rats. Cell viability, MDA levels, reactive oxygen species (ROS) levels, labile iron pool (LIP) levels and mitochondrial morphology were examined. Ferroptosis-related proteins (NADPH oxidase-4 (NOX4), glutathione peroxidase 4 (GPX4), and ferritin heavy chain 1(FTH1)), TLR4, NLRP3 inflammasome markers, HMGB1 and inflammatory cytokines were examined. Pulmonary vascular remodelling and right ventricular structure and function were measured. PRDX6 was expressed in PAECs and was significantly decreased in PH. PRDX6 overexpression significantly inhibited ferroptosis in PAECs under PH conditions in vitro and in vivo, as indicated by increased cell viability, decreased MDA, ROS and LIP levels, inhibited mitochondrial damage, upregulated GPX4 and FTH1 expression, and downregulated NOX4 expression. PRDX6 overexpression attenuated pulmonary vascular remodelling and changes in right ventricle structure and function in MCT-induced PH rats. Moreover, PRDX6 overexpression prevented HMGB1 release by PAECs and decreased TLR4 and NLRP3 inflammasome expression and inflammatory cytokine release in macrophages, while RSL3, a specific activator of ferroptosis, reversed these effects. Taken together, these findings indicate that PRDX6 regulates PAEC ferroptosis through the release of HMGB1 and activation of the TLR4/NLRP3 inflammasome signalling pathway, providing novel therapeutic targets for the treatment of PH.

FANCD2 inhibits ferroptosis by regulating the JAK2/STAT3 pathway in osteosarcoma

BackgroundThis research aimed to investigate the roles of fanconi anemia complementation group D2 (FANCD2) on the regulation of ferroptosis in osteosarcoma progression.MethodsThe function of FANCD2 on cell viability, invasion, migration, and tumor growth were explored. FANCD2 and pathway-related genes were determined by western blot. Ferroptosis-associated markers were determined, including lipid peroxidation, labile iron pool (LIP), ferrous iron (Fe2+), and ferroptosis-related genes.ResultsFANCD2 expression was increased in osteosarcoma cells. FANCD2 knockdown reduced cell viability, invasion, and migration of osteosarcoma cells. FANCD2 knockdown regulated ferroptosis-related gene expression, and distinctly increased the levels of LIP, Fe2+, and lipid peroxidation, and these effects were reversed by a ferroptosis inhibitor Fer-1. In addition, JAK2 and STAT3 expression were reduced by silencing of FANCD2, and STAT3 activator (colivelin) distinctly reversed tumor suppressor effects of FANCD2 silencing on osteosarcoma development.ConclusionThese findings suggested that FANCD2 silencing could suppress osteosarcoma cell viability, migration, invasion, and tumor growth, and induced ferroptosis by regulating the JAK2/STAT3 axis. These findings may provide novel therapeutic ideas for clinical treatment of osteosarcoma.

The Double-Edged Sword of Oleuropein in Ovarian Cancer Cells: From Antioxidant Functions to Cytotoxic Effects

Oleuropein plays a key role as a pro-oxidant as well as an antioxidant in cancer. In this study, the activity of oleuropein, in an in vitro model of ovarian (OCCs) and breast cancer cells (BCCs) was investigated. Cell viability and cell death were analyzed. Oxidative stress was measured by CM-H2DCFDA flow cytometry assay. Mitochondrial dysfunction was evaluated based on mitochondrial reactive oxygen species (ROS) and GPX4 protein levels. Further, the effects on iron metabolism were analyzed by measuring the intracellular labile iron pool (LIP). We confirmed that high doses of oleuropein show anti-proliferative and pro-apoptotic activity on HEY and MCF-7 cells. Moreover, our results indicate that low doses of oleuropein impair cell viability without affecting the mortality of cells, and also decrease the LIP and ROS levels, keeping them unchanged in MCF-7 cells. For the first time, our data show that low doses of oleuropein reduce erastin-mediated cell death. Interestingly, oleuropein decreases the levels of intracellular ROS and LIP in OCCs treated with erastin. Noteworthily, we observed an increased amount of ROS scavenging enzyme GPX4 together with a consistent reduction in mitochondrial ROS, confirming a reduction in oxidative stress in this model.

Molecular mechanism of the unusual biphasic effects of the natural compound hinokitiol on iron-induced cellular DNA damage.

Hinokitiol is a natural monoterpene compound found in the heartwood of cupressaceous plants that have anticancer and anti-inflammatory properties. However, few studies have focused on its effect on iron-mediated cellular DNA damage. Here we show that hinokitiol exhibited unusual biphasic effects on iron-induced DNA damage in a molar ratio (hinokitiol/iron) dependent manner in HeLa cells. Under low ratios (<3:1), hinokitiol markedly enhanced DNA damage induced by Fe(II) or Fe(II)-H2O2; However, when the ratios increased over 3:1, the DNA damage was progressively inhibited. We found that the total cytoplasmic and nuclear iron concentration increased as the ratios of hinokitiol/iron increased. However, the cellular level of labile iron pool (LIP) only increased at ratios lower than 3, and the ROS generation is consistent with LIP change. Hinokitiol was found to interact with iron to form lipophilic hinokitiol-iron complexes with different stoichiometry and redox-activity by complementary applications of various analytical methods. Taken together, we propose that the enhancement of iron-induced cellular DNA damage by hinokitiol at low ratios (<3:1) was due to formation of lipophilic and redox-active iron complexes which facilitated cellular iron uptake and •OH production, while the inhibition at ratios higher than 3 was due to formation of redox-inactive iron complexes. These new findings will help us to design more effective drugs for the prevention and treatment of a series of iron-related diseases via regulating the two critical physicochemical factors (lipophilicity and redox activity of iron complexes) by simple natural compounds with iron-chelating properties.

Inhibition of AEBP1 predisposes cisplatin-resistant oral cancer cells to ferroptosis

BackgroundStudies have shown that excessive iron can lead to an increased incidence of cancer. The role of adipocyte enhancer-binding protein 1 (AEBP1) on ferroptosis is unknown. Thus, we explored the effect of AEBP1 silencing in regulation of ferroptosis in cisplatin-resistant oral cancer cells.MethodsThe functions of AEBP1 silencing and sulfasalazine (SSZ) treatment were determined on oral cancer cell lines and tumor xenograft mouse models. Then we evaluated the functions of AEBP1 on cell proliferation, migration, invasion, lipid reactive oxygen species (ROS), labile iron pool (LIP) and free iron, lipid peroxidation, and expression levels of ferroptosis-related genes.ResultsAEBP1 was highly expressed in oral cancer cells and tissues. AEBP1 silencing inhibited oral cancer cell proliferation, migration, and invasion after SSZ treatment. SSZ-induced ferroptosis is due to enhanced ROS level, free iron, and lipid peroxidation, which were distinctly increased by AEBP1 silencing. Meanwhile, AEBP1 silencing enhanced the effects of SSZ on levels of LIP and Fe2+, lipid peroxidation, as well as the expression levels of ferroptosis-related genes in the tumor xenograft mouse models. Importantly, AEBP1 silencing suppressed tumor growth in vivo. Furthermore, silencing of AEBP1 might activate the JNK/ P38 /ERK pathway.ConclusionThis research suggested that silencing of AEBP1 predisposes cisplatin-resistant oral cancer cells to ferroptosis via the JNK/p38 /ERK pathway.

Genetic Engineering of Talaromyces marneffei to Enhance Siderophore Production and Preliminary Testing for Medical Application Potential.

Siderophores are compounds with low molecular weight with a high affinity and specificity for ferric iron, which is produced by bacteria and fungi. Fungal siderophores have been characterized and their feasibility for clinical applications has been investigated. Fungi may be limited in slow growth and low siderophore production; however, they have advantages of high diversity and affinity. Hence, the purpose of this study was to generate a genetically modified strain in Talaromyces marneffei that enhanced siderophore production and to identify the characteristics of siderophore to guide its medical application. SreA is a transcription factor that negatively controls iron acquisition mechanisms. Therefore, we deleted the sreA gene to enhance the siderophore production and found that the null mutant of sreA (ΔsreA) produced a high amount of extracellular siderophores. The produced siderophore was characterized using HPLC-MS, HPLC-DAD, FTIR, and 1H- and 13C-NMR techniques and identified as a coprogen B. The compound showed a powerful iron-binding activity and could reduce labile iron pool levels in iron-loaded hepatocellular carcinoma (Huh7) cells. In addition, the coprogen B showed no toxicity to the Huh7 cells, demonstrating its potential to serve as an ideal iron chelator. Moreover, it inhibits the growth of Candida albicans and Escherichia coli in a dose-dependent manner. Thus, we have generated the siderophore-enhancing strain of T. marneffei, and the coprogen B isolated from this strain could be useful in the development of a new iron-chelating agent or other medical applications.

PB1748: DIFFERENTIAL IRON HOMEOSTASIS IN CHILDHOOD ACUTE LYMPHOBLASTIC LEUKEMIA IN VITRO MODELS

Background: Iron is an essential element which plays several physiological activities in different human organs. Current studies suggest that dysregulation of iron proteins and subsequent accumulation of iron are associated with the response of leukemia to treatments. In particular, it has been reported that low levels of ferroportin-1 (FPN1), lead to an increased sensitivity to chemotherapy in acute myeloid leukemia. Aims: We aimed at analyzing the gene expression profile of iron-related genes in childhood B acute lymphoblastic leukemia (ALL) and characterize the iron metabolism in vitro models. Methods: Iron related genes (Ceruloplasmin (CP), Ferroportin-1 (SLC40A1), Transferrin Receptor 1 (TFR1) and Ferritin Light Chain (FTL)) were analyzed by qRT-PCR in BM samples of 87 childhood ALL and 8 healthy donors. Cases were selected to be representative of t(12;21), t(4;11), t(9;22) or t(1;19) subgroups, as well as MRD-based standard-risk (SR), medium-risk (MR) and high-risk (HR) response. Furthermore, we evaluated the cellular viability in REH, RS4;11, 697 and TOM-1 cell lines after exposure to iron as ferric ammonium citrate (FAC 100 to 400 µM), deferoxamine (DFO 25 µM) and FAC+DFO (100 µM + 25 µM), using MTT assay at 24-48 and 72h. TFR1 and FTL mRNA levels were assessed at 8h, 16h, 24h, 48h. Labile Iron Pool (LIP) was measured by flow-cytometry using Calcein-AM technique after the exposure to FAC (100-200-400 µM) at 48h. Results:TFR1, SLC40A1 and CP were up-regulated in HR group vs. controls. SLC40A1 and CP were down-regulated in all genetic subgroups, except for CP in t(9;22). TFR1 was slightly expressed in all subgroups. In the t(1;19) subgroup all the iron genes were downregulated, suggesting that this subgroup may not be responsive to iron, as also confirmed by our in vitro data. Iron supplementation by FAC [100 µM] resulted in a boost of viability for TOM-1, REH and RS4;11. TOM-1 and REH viability decreased with FAC 200-400 µM, while RS4;11 proliferation was stimulated by all concentrations. Together with low transcriptional levels of iron genes, 697 viability did not change with different FAC treatments. DFO reduced cell viability in a time-dependent manner. At 48h the viability started to drop for all cell lines (TOM-1 80% - RS4;11 66% - REH 71% - 697 51%) and the co-administration of iron restored the viability to levels similar or even higher than the untreated (TOM-1 100%-RS4;11 80% -REH 92% - 697 81%). FTL expression was significantly up-regulated in all cell lines, while the TFR1 was down-regulated at 8, 16 and 24h vs. untreated. Still, some differences were detected within the cellular models: RS4;11 showed highest level of FTL expression, while in 697 the transcript was slightly upregulated. In 697 cell line, the LIP increased with higher dosages of FAC, suggesting that this cell line was not able to handle high amount of free iron. TOM-1, RS4;11 and REH presented LIP levels higher than the untreated condition, still no significant differences were noticed between the concentrations. Summary/Conclusion:SLC40A1 and CP high levels of HR group potentially suggest that their refractory behavior may be correlated with ability to keep regulate iron intracellular concentration. TOM-1, REH and RS4;11 behave as resistant cell lines, whilst the 697 appear as not sensitive to iron metabolism. In conclusion, leukemia cell lines can be considered as trustable in vitro models for further studies in ALL to investigate whether iron metabolism can be determinant for survival and resistance, and consequently a target for treatment.

LncXIST Facilitates Iron Overload and Iron Overload-Induced Islet Beta Cell Injury in Type 2 Diabetes through miR-130a-3p/ALK2 Axis.

Iron overload is directly associated with diabetes mellitus, loss of islet beta cell, and insulin resistance. Likewise, long noncoding RNA (lncRNA) is associated with type 2 diabetes (T2D). Moreover, lncRNAs could be induced by iron overload. Therefore, we are going to explore the molecular mechanism of lncRNA XIST in iron overload-related T2D. Real-time quantitative PCR and Western blot were used to detect gene and protein levels, respectively. TUNEL and MTT assay were performed to examine cell survival. The glucose test strip, colorimetric analysis kit, ferritin ELISA kit, and insulin ELISA kit were performed to examine the levels of glycolic, iron, and total iron-binding capacity, ferritin, and insulin in serum. Fluorospectrophotometry assay was used to examine labile iron pool level. XIST was higher expressed in T2D and iron overload-related T2D rat tissues and cells, and iron overload-induced promoted XIST expression in T2D. Higher XIST expression was associated with iron overload in patients with T2D. Knockdown of XIST alleviated iron overload and iron overload-induced INS-1 cells injury. Further, we found that XIST can sponge miR-130a-3p to trigger receptor-like kinase 2 (ALK2) expression. Moreover, knockdown of ALK2 alleviated iron overload and iron overload-induced INS-1 cells injury by inhibiting bone morphogenetic protein 6 (BMP6)/ALK2/SMAD1/5/8 axis but reversed with XIST upregulation, which was terminally boosted by overexpression of miR-130a-3p. XIST has the capacity to promote iron overload and iron overload-related T2D initiation and development through inhibition of ALK2 expression by sponging miR-130a-3p, and that targeting this axis may be an effective strategy for treating patients with T2D.

DMT1‐mediated endosome‐mitochondria interactions regulates iron homeostasis and mitochondrial metabolism

Mitochondria‐early endosome (EE) interactions have been shown to facilitate the translocation of iron into mitochondria. Here we show that Divalent Metal Transporter 1 (DMT1) modulates iron exit from endosomes and transport into mitochondria via regulation of EE‐mitochondria interactions. In cancer cells, mitochondria are the ultimate cellular iron sink, where iron can be either stored or used for example to shift cellular metabolism towards glycolysis (Warburg effect), a key adaptive mechanism of cancer cells. Moreover, a gene signature associated with reduced intracellular iron content, including low transferrin receptor (TfR) (anti‐import) and high ferroportin (FPN) (pro‐export) expression levels, has been related to favorable breast cancer prognosis. Similarly, reduced DMT1 expression associates with improved breast cancer patient survival. We evaluated the role of DMT1 in two distinct breast cancer cell lines: estrogen receptor positive T47D and triple‐negative MDAMB231. In both cell lines, we demonstrate colocalization between EE, DMT1 and mitochondria. Interestingly, DMT1 is localized to the surface contact area between endosomes and mitochondria. To demonstrate that DMT1 plays a role in endosome‐mitochondria interactions and Mitochondrial Iron Translocation (MIT), we have generated MDAMB231 as well as T47D CRISPR/Cas9 based DMT1 knockout (KO) stable cell lines. Several lines of evidence show that DMT1 regulates MIT and labile iron pool (LIP) levels via modulation of EE‐mitochondria interactions in MDAMB231 cells. MIT decrease via DMT1 silencing was partially rescued by re‐expression of DMT1 in MDAMB231, but not in T47D cells. MDAMB231 DMT1 KO cells showed increased Ferro‐Orange staining, indicating higher LIP levels, as well as decreased TfR and increased FPN protein levels. Importantly, DMT1 silencing significantly reduced EE‐mitochondria interactions and EE speed in MDAMB231 but not in T47D. Thus, DMT1 regulates MIT and LIP levels via EE‐mitochondria interactions in MDAMB231. These results are in agreement with previous results showing that MDAMB231 display a delay in iron release in comparison to T47D, making them more sensitive to disruptions in MIT. Since mitophagy has been shown to act as a tumor suppressor in breast cancer, we tested whether it could be modulated by DMT1‐mediated MIT. We found that DMT1 silencing increases mitochondrial ferritin, global autophagy marker LC3B and PINK1/Parkin‐dependent mitophagy markers in MDAMB231; levels of all proteins evaluated were rescued to basal levels upon re‐expression of DMT1 in DMT KO cells. Moreover, DMT1 silencing decreases Tom 20 (outer mitochondrial membrane marker) with PMPCB, a known DMT1 interactor that is required for PINK1 turnover. Concurring with the role of DMT1 in mitophagy and iron metabolism, both mitochondrial metabolism and invasive cell migration are significantly impaired by DMT1 silencing and are partially rescued by re‐expression of DMT1. Overall, our results implicate DMT1 in the regulation of EE dynamics and EE‐mitochondria interactions to support higher MIT/lower LIP levels, which are necessary for sustaining mitochondrial bioenergetics and invasive cell migration. Thus, we propose DMT1 as a key player associated with aggressive phenotypes in breast cancer

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.

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.

Clioquinol Attenuates Pulmonary Fibrosis through Inactivation of Fibroblasts via Iron Chelation

Strict control of iron homeostasis is critical for the maintenance of normal lung function. Iron accumulates in the lungs of patients with idiopathic pulmonary fibrosis (IPF), but the characteristics of iron metabolism in the pathogenesis of pulmonary fibrosis (PF) and related targeting therapeutics are not well studied. In this study, we investigated the cellular and molecular characteristics of iron metabolism in fibrotic lungs and further explored the efficacy of clioquinol (CQ) for the treatment of PF as well as its functional mechanism. Iron aggregates accumulated in the lungs of patients with IPF, and ferritin light chain (FTL) transcripts were increased in their pulmonary fibroblasts. In the bleomycin (BLM)-induced pulmonary fibrosis (BLM-PF) mouse model, pulmonary iron accumulation is a very early and concomitant event of PF. Labile iron pool (LIP) levels in both fibroblasts and macrophages from the BLM-PF model were elevated, and iron metabolism was dysregulated. CQ attenuated PF induced by BLM and fluorescein isothiocyanate (FITC), and iron-saturated clioquinol (ISCQ) did not alleviate BLM-PF. Furthermore, CQ inhibited the activation of fibroblasts, including proliferation, fibrotic differentiation, proinflammatory cytokine secretion and migration. In conclusion, our study demonstrated that CQ, acting as an iron chelator, attenuates experimental PF through inactivation of fibroblasts, providing support for targeting iron metabolism as a basis for PF treatment.

Nuclear receptor coactivator 4-mediated ferritinophagy drives proliferation of dental pulp stem cells in hypoxia

Nuclear receptor coactivator 4 (NCOA4)-mediated ferritinophagy has been implicated in the ferroptosis in cancer cells and hematopoiesis in the bone marrow. However, the role of iron metabolism, especially NCOA4-mediated degradation of ferritin, has not been explored in the proliferation of mesenchymal stem cells. The present study was designed to explore the role of NCOA4-mediated ferritinophagy in hypoxia-treated dental pulp stem cells (DPSCs). Hypoxia treatment increased ROS generation, boosted cytosolic labile iron pool, increased expression of transferrin receptor 1 and NCOA4. Moreover, colocalization of LC3B with NCOA4 and ferritin was observed in hypoxia-treated DPSCs, indicating the development of ferritinophagy. Hypoxia promoted the proliferation of DPSCs, but not ferroptosis, under normal serum supplement and serum deprivation. NCOA4 knock-down reduced ferritin degradation and inhibited proliferation of DPSCs under hypoxia. Furthermore, the activation of hypoxia inducible factor 1α and p38 mitogen-activated protein kinase signaling pathway was involved in the upregulation of NCOA4 in hypoxia. Therefore, our present study suggested that NCOA4-mediated ferritinophagy promoted the level of labile iron pool, leading to enhanced iron availability and elevated cell proliferation of DPSCs. Our present study uncovered a physiological role of ferritinophagy in the proliferation and growth of mesenchymal stem cells under hypoxia.

NCOA4-mediated ferritinophagy promoted inflammatory responses in periodontitis.

Iron homeostasis plays a crucial role in the combat against pathogen invasion. Ferrous iron can trigger generous production of reactive oxygen species (ROS) by Fenton reaction. Nuclear receptor coactivator 4 (NCOA4), a selectivecargoreceptor to deliver ferritin to lysosome, may trigger release of ferritin-bound iron into the cytosol. The aim of the present study was to explore whether NCOA4-mediated ferritinophagy participated in the pathogenesis of periodontitis, and its role in promoting the periodontal inflammation. Inflamed and healthy periodontal tissues were harvested for immunobiological staining of ferritinophagy-related genes in the periodontal tissues, while real-time quantitative PCR (qPCR) was utilized to detect mRNA transcription. Periodontal ligament fibroblasts (PDLFs) were isolated and infected with Porphyromonas gingivalis. The mRNA transcription and protein expression of genes involved in the iron metabolism, including NCOA4, transferrin receptor 1 (TFR1), and ferroportin (SLC40A1) were detected by qPCR and western blot. Levels of labile iron pool and ROS production were detected by flow cytometry and confocal endoscopy. Small interference RNA was utilized to knock down NCOA4. Elevated expression of NCOA4, ferritin heavy chain, and light chain were observed in the diseased periodontal tissues. P. gingivalis infection promoted expression of TFR1, NCOA4, and microtubule-associated protein 1-light chain 3 B (LC3B), enhanced levels of intracellular labile iron pool and ROS production. NCOA4 knockdown reduced ROS generation in PDLFs in response to P. gingivalis and mitigated production of pro-inflammatory monocyte chemoattractant protein-1 and interleukin 6. P. gingivalis triggered activation of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase signaling pathway. In addition, inhibitors of JNK, SP600125, and inhibitors of p38, SB203580 blocked NCOA4 transcription. NCOA4-ferritinophagy participated in the progress of periodontitis progression. P. gingvalis-triggered ferritinophagy aggravated production of ROS and inflammatory responses in PDLFS. These findings suggest iron homeostasis plays an important role in the pathogenesis of periodontitis.

Ferronostics: Measuring Tumoral Ferrous Iron with PET to Predict Sensitivity to Iron-Targeted Cancer Therapies.

Although cancer has been known for decades to harbor an insatiable appetite for iron, only recently has the chemistry emerged to exploit this altered state therapeutically, by targeting the expanded cytosolic labile iron pool (LIP) of the cancer cell. The state of the art includes therapies that react with the LIP to produce cytotoxic radical species (in some cases also releasing drug payloads) and molecules that exacerbate LIP-induced oxidative stress to trigger ferroptosis. Effectively implementing LIP-targeted therapies in patients will require biomarkers to identify those tumors with the most elevated LIP and thus most likely to succumb to LIP-targeted interventions. Toward this goal, we tested whether tumor uptake of the novel LIP-sensing radiotracer 18F-TRX aligns with tumor sensitivity to LIP-targeted therapies. Methods:18F-TRX uptake was assessed invivo among 10 subcutaneous and orthotopic human xenograft models. Glioma and renal cell carcinoma were prioritized because these tumors have the highest relative expression levels of STEAP3, the oxidoreductase that reduces ferric iron to the ferrous oxidation state, in the Broad Institute Cancer Cell Line Encyclopedia. The antitumor effects of the LIP-activated prodrug TRX-CBI, which releases the DNA alkylator CBI, were compared in mice bearing U251 or PC3 xenografts, tumors with high and intermediate levels of 18F-TRX uptake, respectively. Results:18F-TRX showed a wide range of tumor accumulation. An antitumor assessment study showed that the growth of U251 xenografts, the model with the highest 18F-TRX uptake, was potently inhibited by TRX-CBI. Moreover, the antitumor effects against U251 were significantly greater than those observed for PC3 tumors, consistent with the relative 18F-TRX-determined LIP levels in tumors before therapy. Lastly, a dosimetry study showed that the estimated effective human doses for adult male and female mice were comparable to those of other 18F-based imaging probes. Conclusion: We report the first evidence-to our knowledge-that tumor sensitivity to an LIP-targeted therapy can be predicted with a molecular imaging tool. More generally, these data bring a new dimension to the nuclear theranostic model by showing a requirement for imaging to quantify, in situ, the concentration of a metastable bioanalyte toward predicting tumor drug sensitivity.

Sequence analysis of hepcidin in barbel steed (Hemibarbus labeo): QSHLS motif confers hepcidin iron-regulatory activity but limits its antibacterial activity

Fish hepcidin genes are generally classified into two groups: hamp1-and hamp2-type isoforms. Hamp1-type hepcidin exhibits iron regulatory and antimicrobial activity, while hamp2-type shows a unique role in the immune response against various pathogens. An iron-regulatory motif exists at the N-terminus of hamp1-type hepcidin; however, the functional effect of this motif in fish is not well understood. Here, cDNA of the barbel steed (Hemibarbus labeo) hepcidin gene was cloned and sequenced. The predicted amino acid sequence comprised a signal peptide, a prodomain, and a mature peptide. Phylogenetic tree analysis revealed that barbel steed hepcidin belongs to the fish HAMP1 cluster and is closely related to Chinese rare minnow (Gobiocypris rarus) hepcidin. Barbel steed hepcidin is constitutively expressed in healthy fish tissues, predominantly in the liver. Following iron dextran treatment or Aeromonas hydrophila infection, expression of barbel steed hepcidin increased significantly in tested tissues. In vivo administration of intact hepcidin mature peptide (hep25) significantly and dose-dependently reduced ferroportin 1 expression, while truncated hepcidin mature peptide (hep20) lacking a QSHLS motif had no such effect. In vitro treatment of barbel steed monocytes/macrophages with hep25, but not hep20, increased the labile iron pool levels. Hep25 and hep20 conferred antibacterial activity only against A. hydrophila and Vibrio vulnificus, with greater activity of the latter at low concentrations. Neither hep25 nor hep20 impaired the cell membrane integrity of A. hydrophila, but could hydrolyze its genomic DNA; lack of a QSHLS motif enables hep20 to have a better hydrolytic effect. In summary, we identified an iron-regulatory motif in a fish species and demonstrated that this motif confers hamp1-type hepcidin iron-regulatory activity, but attenuates its antibacterial activity.

Endogenous Labile Iron Pool-Mediated Free Radical Generation for Cancer Chemodynamic Therapy

Current chemodynamic therapy (CDT) primarily relies on the delivery of transition metal ions with Fenton activity to trigger hydroxyl radical production from hydrogen peroxide. However, administration of an excess amount of exogenous Fenton-type heavy metals may cause potential adverse effects to human health, including acute and chronic damages. Here, we present a new CDT strategy that uses intracellular labile iron pool (LIP) as the endogenous source of Fenton-reactive metals for eliciting free radical generation, and the discovery of hydroperoxides (R'OOH) as an optimal LIP-mediated chemodynamic agent against cancer. By simulating the metabolic fates of peroxo compounds within cells, R'OOH was found to have excellent free radical-producing ability in the presence of labile iron(II) and to suffer only moderate elimination by glutathione/glutathione peroxidase, which contributes to its superior chemodynamic efficacy. The LIP-initiated nontoxic-to-toxic transition of R'OOH, together with increased LIP levels in tumor cells, enabled efficient and specific CDT of cancer. Moreover, pH/labile iron(II) cascade-responsive nanomedicines comprising encapsulated methyl linoleate hydroperoxide and LIP-increasing agent in pH-sensitive polymer particles were fabricated to realize enhanced CDT. This work not only paves the way to using endogenous Fenton-type metals for cancer therapy but also offers a paradigm for the exploration of high-performance chemodynamic agents activated by intracellular LIP.