RESUMEN
Overexpression of ferritin heavy chain (FTH1) often associates with good prognosis in breast cancer (BCa), particularly in the triple-negative subtype (triple-negative breast cancer). However, the mechanism by which FTH1 exerts its possible tumor suppressor effects in BCa is not known. Here, we examined the bearing of FTH1 silencing or overexpression on several aspects of BCa cell growth in vitro. FTH1 silencing promoted cell growth and mammosphere formation, increased c-MYC expression, and reduced cell sensitivity to chemotherapy. In contrast, FTH1 overexpression inhibited cell growth, decreased c-MYC expression, and sensitized cancer cells to chemotherapy; silencing of c-MYC recapitulated the effects of FTH1 overexpression. These findings show for the first time that FTH1 suppresses tumor growth by inhibiting the expression of key oncogenes, such as c-MYC.
Asunto(s)
Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Ferritinas/metabolismo , Oxidorreductasas/metabolismo , Apoferritinas/biosíntesis , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , Femenino , Ferritinas/biosíntesis , Ferritinas/genética , Expresión Génica/genética , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Oxidorreductasas/genética , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/metabolismoRESUMEN
Macrophages are essential immune cells of the innate immune system. They participate in the development and regulation of inflammation. Macrophages play a fundamental role in fighting against bacterial infections by phagocytosis of bacteria, and they also have a specific role in immunomodulation by secreting pro-inflammatory cytokines. In bacterial infection, macrophages decrease the serum iron concentration by removing iron from the blood, acting as one of the most important regulatory cells of iron homeostasis. We examined whether the Gram-positive and Gram-negative cell wall components from various bacterial strains affect the cytokine production and iron transport, storage and utilization of THP-1 monocytes in different ways. We found that S. aureus lipoteichoic acid (LTA) was less effective in activating pro-inflammatory cytokine expression that may related to its effect on fractalkine production. LTA-treated cells increased iron uptake through divalent metal transporter-1, but did not elevate the expression of cytosolic and mitochondrial iron storage proteins, suggesting that the cells maintained iron efflux via the ferroportin iron exporter. E. coli and P. aeruginosa lipopolysaccharides (LPSs) acted similarly on THP-1 cells, but the rates of the alterations of the examined proteins were different. E. coli LPS was more effective in increasing the pro-inflammatory cytokine production, meanwhile it caused less dramatic alterations in iron metabolism. P. aeruginosa LPS-treated cells produced a smaller amount of pro-inflammatory cytokines, but caused remarkable elevation of both cytosolic and mitochondrial iron storage proteins and intracellular iron content compared to E. coli LPS. These results prove that LPS molecules from different bacterial sources alter diverse molecular mechanisms in macrophages that prepossess the outcome of the bacterial infection.
Asunto(s)
Pared Celular/química , Citocinas/metabolismo , Escherichia coli/química , Hierro/metabolismo , Lipopolisacáridos/farmacología , Pseudomonas aeruginosa/química , Staphylococcus aureus/química , Células THP-1/metabolismo , Ácidos Teicoicos/farmacología , Transporte Biológico , Receptor 1 de Quimiocinas CX3C/biosíntesis , Receptor 1 de Quimiocinas CX3C/genética , Quimiocina CX3CL1/metabolismo , Citocinas/biosíntesis , Citosol/metabolismo , Ferritinas/biosíntesis , Ferritinas/genética , Hemo-Oxigenasa 1/biosíntesis , Hemo-Oxigenasa 1/genética , Hepcidinas/biosíntesis , Hepcidinas/genética , Humanos , Mitocondrias/metabolismo , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Oxidorreductasas/biosíntesis , Oxidorreductasas/genética , ARN Mensajero/biosíntesis , ARN Neoplásico/genética , Células THP-1/efectos de los fármacosRESUMEN
In the present study, we demonstrate the coaction of thioredoxin and glutathione (GSH) systems in mouse liver against iron overload-induced oxidative stress (OS). Mice were injected intraperitoneally with an iron dextran solution twice a week for 3 weeks. Iron accumulation in mouse liver was demonstrated spectroscopically. To confirm the iron overload model in the liver, the increased gene expression levels of hepcidin (Hamp), ferroportin (Fpn1), and ferritin (Fth1), which regulate iron trafficking, were observed by a quantitative polymerase chain reaction. In the case of iron overload, the GSH level and the reduced glutathione/oxidized glutathione ratio, which represents a marker of OS, decreased significantly. An increase in the malondialdehyde level, one of the final products of the lipid peroxidation process, was observed. The gene expression of the thioredoxin system, including thioredoxin (Trx1) and thioredoxin reductase (TrxR1), was examined. Though TrxR1 expression decreased, no changes were observed in Trx1. The enzyme activity and semiquantitative protein expression of TRXR1 increased. The activity of GSH reductase and GSH peroxidase increased in the iron overload group. The gene and protein expressions of thioredoxininteracting protein, which is an indicator of the commitment of the cell to apoptosis, were elevated significantly. The increased protein expression of Bcl-2-related X protein and CASPASE-3, which is an indicator of apoptosis, increased significantly. In conclusion, excess iron accumulation in mouse liver tissue causes OS, which affects the redox state of the thioredoxin and GSH systems, inducing cell apoptosis and also ferroptosis due to increased lipid peroxidation and the depletion of GSH level.
Asunto(s)
Glutatión/metabolismo , Sobrecarga de Hierro/metabolismo , Hígado/metabolismo , Estrés Oxidativo , Tiorredoxinas/biosíntesis , Animales , Proteínas de Transporte de Catión/biosíntesis , Ferritinas/biosíntesis , Regulación de la Expresión Génica , Hepcidinas/biosíntesis , Sobrecarga de Hierro/patología , Hígado/patología , Masculino , Ratones , Ratones Endogámicos BALB C , Oxidorreductasas/biosíntesisRESUMEN
BACKGROUND: Renal anemia is a common complication of hemodialysis patients. Erythropoietin (EPO) hyporesponsiveness has been recognized as an important factor to poor efficacy of recombinant human erythropoietin in the treatment of renal anemia. More importantly, increased erythropoiesis resistance index (ERI) may be associated with inflammation and increased mortality. OBJECTIVE: The objective of this research was to investigate correlated factors of EPO responsiveness and to clarify the relationships between EPO hyporesponsiveness and cardiovascular mortality and all-cause mortality among maintenance hemodialysis patients. METHODS: This prospective cohort study enrolled 276 maintenance hemodialysis patients for a 55-month follow-up to investigate the factors related to ERI and its relationship to all-cause mortality and cardiovascular mortality. RESULTS: ERI was positively correlated with predialysis serum high-sensitivity C-reactive protein (r = 0.234, p < 0.001), alkaline phosphatase (r = 0.134, p = 0.028), and ferritin (r = 0.155, p = 0.010) and negatively correlated with albumin (r = -0.206, p < 0.001) and creatinine (r = -0.232, p < 0.001). As multiple linear regression showed, predialysis serum albumin, high-sensitivity C-reactive protein, ferritin, and creatinine were independent correlated factors of ERI (p < 0.05). Kaplan-Meier curves showed that the cumulative incidences of both cardiovascular mortality and all-cause mortality were significantly higher in patients with ERI > 11.04 IU/kg/w/g/dL (both p < 0.01). The high ERI group was significantly associated with higher risk for all-cause mortality (OR 1.781, 95% CI 1.091 to 2.910, p = 0.021) and cardiovascular mortality (OR 1.972, 95% CI 1.139 to 3.417, p = 0.015) after adjusting for confounders. CONCLUSIONS: Predialysis serum albumin, high-sensitivity C-reactive protein, ferritin, and creatinine were independent correlated factors of EPO responsiveness among maintenance hemodialysis patients. Patients with higher ERI values had a higher all-cause mortality rate and cardiovascular mortality rate.
Asunto(s)
Eritropoyesis , Fallo Renal Crónico/terapia , Diálisis Renal/métodos , Adulto , Anciano , Albúminas/biosíntesis , Fosfatasa Alcalina/biosíntesis , Anemia , Proteína C-Reactiva/biosíntesis , China/epidemiología , Creatinina/metabolismo , Eritropoyetina/metabolismo , Femenino , Ferritinas/biosíntesis , Humanos , Inflamación , Estimación de Kaplan-Meier , Riñón/patología , Fallo Renal Crónico/fisiopatología , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Proteínas Recombinantes/metabolismo , Análisis de Regresión , Índice de Severidad de la Enfermedad , Resultado del TratamientoRESUMEN
Parkinson's disease (PD) is a neurodegenerative disorder characterized by degeneration of dopaminergic neurons associated with dysregulation of iron homeostasis in the brain. Ferroptosis is an iron-dependent cell death process that serves as a significant regulatory mechanism in PD. However, its underlying mechanisms are not yet fully understood. By performing RNA sequencing analysis, we found that the main iron storage protein ferritin heavy chain 1 (FTH1) is differentially expressed in the rat 6-hydroyxdopamine (6-OHDA) model of PD compared with control rats. Our present work demonstrates that FTH1 is involved in iron accumulation and the ferroptosis pathway in this model. Knockdown of FTH1 in PC-12 cells significantly inhibited cell viability and caused mitochondrial dysfunction. Moreover, FTH1 was found to be involved in ferritinophagy, a selective form of autophagy involving the degradation of ferritin by ferroptosis. Overexpression of FTH1 in PC-12 cells impaired ferritinophagy and downregulated microtubule-associated protein light chain 3 and nuclear receptor coactivator 4 expression, ultimately suppressing cell death induced by ferroptosis. Consistent with these findings, the ferritinophagy inhibitors chloroquine and bafilomycin A1 inhibited ferritin degradation and ferroptosis in 6-OHDA-treated PC-12 cells. This entire process was mediated by the cyclic regulation of FTH1 and ferritinophagy. Taken together, these results suggest that FTH1 links ferritinophagy and ferroptosis in the 6-OHDA model of PD, and provide a new perspective and potential for a pharmacological target in this disease.
Asunto(s)
Ferritinas/biosíntesis , Ferroptosis/fisiología , Oxidopamina/toxicidad , Oxidorreductasas/biosíntesis , Trastornos Parkinsonianos/inducido químicamente , Trastornos Parkinsonianos/metabolismo , Animales , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Ferritinas/genética , Ferroptosis/efectos de los fármacos , Masculino , Oxidorreductasas/genética , Células PC12 , Trastornos Parkinsonianos/genética , Ratas , Ratas Sprague-DawleyRESUMEN
OBJECTIVE: Ferritin is an attractive vector for the delivery of drug molecules and antigen proteins because of its unique structural and biochemical features. In this study, recombinant ferritin from Helicobacter pylori was expressed in the soluble form employing the baculovirus expression system. RESULTS: The optimum conditions for producing recombinant ferritin comprised MOI 5 of rBV-ferritin for 96 h of infection. The recombinant ferritin was purified by Ni Sepharose™ 6 Fast Flow, with a purity and yield of 92.5% and 11.25 mg/L, respectively. In addition, the recombinant ferritin showed a multimeric structure under non-denaturing conditions, as well as self-assembled spherical cage architecture with a diameter of approximately 12 nm. Dot-ELISA results suggested that the His-tag at the N-terminus likely existed on the surface of the recombinant ferritin. CONCLUSION: Recombinant ferritin was produced by the baculovirus expression system, which has the potential to display exogenous proteins, and may aid in the delivery of drugs for disease prevention and treatment.
Asunto(s)
Proteínas Bacterianas/biosíntesis , Ferritinas/biosíntesis , Expresión Génica , Proteínas Recombinantes/biosíntesis , Células Sf9 , Animales , Proteínas Bacterianas/genética , Baculoviridae/genética , Ferritinas/genética , Vectores Genéticos , Helicobacter pylori/enzimología , Helicobacter pylori/genética , Proteínas Recombinantes/genética , SpodopteraRESUMEN
Experimental data suggest that iron sucrose (FeS) injection, used either alone or in combination with other prooxidants, can induce "renal preconditioning," in part by upregulating cytoprotective ferritin levels. However, the rapidity, degree, composition (heavy vs. light chain), and renal ferritin changes after FeS administration in humans remain to be defined. To address these issues, healthy human volunteers (n = 9) and patients with stage 3-4 chronic kidney disease(n = 9) were injected once with FeS (120, 240, or 360 mg). Plasma ferritin was measured from 0 to 8 days postinjection as an overall index of ferritin generation. Urinary ferritin served as a "biomarker" of renal ferritin production. FeS induced rapid (≤2 h), dose-dependent, plasma ferritin increases in all study participants, peaking at approximately three to five times baseline within 24-48 h. Significant urinary ferritin increases (~3 times), without dose-dependent increases in albuminuria, neutrophil gelatinase-associated lipocalin, or N-acetyl-ß-d-glucosaminidase excretion, were observed. Western blot analysis with ferritin heavy chain (Fhc)- and light chain (Flc)-specific antibodies demonstrated that FeS raised plasma Flc but not Fhc levels. Conversely, FeS increased both Fhc and Flc in urine. To assess sites of FeS-induced ferritin generation, organs from FeS-treated mice were probed for Fhc, Flc, and their mRNAs. FeS predominantly raised hepatic Flc. Conversely, marked Fhc and Flc elevations developed in the kidney and spleen. No cardiopulmonary ferritin increases occurred. Ferritin mRNAs remained unchanged throughout, implying posttranscriptional ferritin production. We conclude that FeS induces rapid, dramatic, and differential Fhc and Flc upregulation in organs. Renal Fhc and Flc increases, in the absence of nephrotoxicity, suggest potential FeS utility as a clinical renal "preconditioning" agent.
Asunto(s)
Sacarato de Óxido Férrico/farmacología , Ferritinas/biosíntesis , Precondicionamiento Isquémico , Riñón/efectos de los fármacos , Lesión Renal Aguda/prevención & control , Adulto , Anciano , Animales , Biomarcadores/orina , Femenino , Sacarato de Óxido Férrico/administración & dosificación , Sacarato de Óxido Férrico/efectos adversos , Ferritinas/sangre , Ferritinas/orina , Voluntarios Sanos , Humanos , Infusiones Parenterales , Riñón/metabolismo , Enfermedades Renales/inducido químicamente , Enfermedades Renales/patología , Masculino , Ratones , Persona de Mediana Edad , ARN Mensajero/biosíntesis , Insuficiencia Renal Crónica/metabolismo , Bazo/metabolismoRESUMEN
PM2.5 is becoming a worldwide environmental problem, which profoundly endangers public health, thus progressively capturing public attention this decade. As a fragile target of PM2.5, the underlying mechanisms of endothelial cell damage are still obscure. According to the previous microarray data and signaling pathway analysis, a new form of cell death termed ferroptosis in the current study is proposed following PM2.5 exposure. In order to verify the vital role of ferroptosis in PM2.5-induced endothelial lesion and further understand the potential mechanism involved, intracellular iron content, ROS release and lipid peroxidation, as well as biomarkers of ferroptosis were detected, respectively. As a result, uptake of particles increases cellular iron content and ROS production. Meanwhile, GSH depletion, and the decrease of GSH-Px and NADPH play significant roles in PM2.5-induced endothelial cell ferroptosis. Moreover, significantly changed expression of TFRC, FTL and FTH1 hinted that dysfunction of iron uptake and storage is a major inducer of ferroptosis. Importantly, index monitored above can be partially rescued by lipid peroxidation inhibitor ferrostatin-1 and iron chelator deferoxamine mesylate, which mediated antiferroptosis activity mainly depends on the restoration of antioxidant activity and iron metabolism. In conclusion, our data basically show that PM2.5 enhances ferroptosis sensitivity with increased ferroptotic events in endothelial cells, in which iron overload, lipid peroxidation and redox imbalance act pivotal roles.
Asunto(s)
Células Endoteliales/metabolismo , Ferroptosis/fisiología , Sobrecarga de Hierro/patología , Hierro/toxicidad , Material Particulado/toxicidad , Antígenos CD/biosíntesis , Apoferritinas/biosíntesis , Apoptosis/efectos de los fármacos , Ciclohexilaminas/farmacología , Deferoxamina/farmacología , Ferritinas/biosíntesis , Glutatión/metabolismo , Humanos , Peroxidación de Lípido/efectos de los fármacos , Oxidación-Reducción/efectos de los fármacos , Oxidorreductasas , Fenilendiaminas/farmacología , Especies Reactivas de Oxígeno/metabolismo , Receptores de Transferrina/biosíntesis , Transducción de Señal/efectos de los fármacosRESUMEN
BACKGROUND Serum ferritin is a useful tumor marker for renal cell carcinoma (RCC). However, the expression of ferritin heavy chain (FTH1), the main subunit of ferritin, is unclear in primary RCC tissues. In this study, we investigated FTH1 mRNA expression and its diagnostic and prognostic value in RCC. MATERIAL AND METHODS The mRNA expression of FTH1 was analyzed using including Oncomine, Gene Expression Omnibus, and Cancer Genome Atlas datasets, while the protein level of FTH1 was analyzed using the Human Protein Atlas database. The associations between FTH1 and clinicopathologic characteristics and survival time and Cox multivariate survival analysis were analyzed using SPSS 22.0 software. A meta-analysis was performed to assess consistency of FTH1 expression. GO, KEGG, and PPI analyses were used to predict biological functions. RESULTS According to TCGA data, overexpression of FTH1 was detected in 890 RCC tissues (15.2904±0.63157) compared to 129 normal kidney tissues (14.4502±0.51523, p<0.001). Among the clinicopathological characteristics evaluated, patients with increased pathologic T staging, lymph node metastasis, and distant metastasis were significantly associated with higher expression of FTH1. Elevated FTH1 mRNA levels were correlated with worse prognosis of RCC patients. Cox multivariate survival analysis indicated that age, stage, and M stage were predictors of poor prognosis in patients with RCC. CONCLUSIONS Our data suggest that FTH1 expression is an effective prognostic and diagnosis biomarker for RCC.
Asunto(s)
Carcinoma de Células Renales/metabolismo , Ferritinas/biosíntesis , Neoplasias Renales/metabolismo , Adulto , Anciano , Apoferritinas/genética , Apoferritinas/metabolismo , Biomarcadores de Tumor/biosíntesis , Biomarcadores de Tumor/genética , Carcinoma de Células Renales/genética , Carcinoma de Células Renales/patología , Femenino , Ferritinas/genética , Ferritinas/metabolismo , Humanos , Neoplasias Renales/genética , Neoplasias Renales/patología , Metástasis Linfática , Masculino , Persona de Mediana Edad , Oxidorreductasas , Pronóstico , ARN Mensajero/genética , ARN Mensajero/metabolismo , TranscriptomaRESUMEN
BACKGROUND: Islets of Langerhans transplantation is a promising therapy for type 1 diabetes mellitus, but this technique is compromised by transplantation stresses including inflammation. In other tissues, co-transplantation with mesenchymal stem cells has been shown to reduce damage by improving anti-inflammatory and anti-oxidant defences. Therefore, we probed the protection afforded by bone marrow mesenchymal stem cells to islets under pro-inflammatory cytokine stress. METHODS: In order to evaluate the cytoprotective potential of mesenchymal stem cells on rat islets, co-cultures were exposed to the interleukin-1, tumour necrosis factor α and interferon γ cocktail for 24 h. Islet viability and functionality tests were performed. Reactive oxygen species and malondialdehyde were measured. Expression of stress-inducible genes acting as anti-oxidants and detoxifiers, such as superoxide dismutases 1 and 2, NAD(P)H quinone oxidoreductase 1, heme oxygenase-1 and ferritin H, was compared to non-stressed cells, and the corresponding proteins were measured. Data were analysed by a two-way ANOVA followed by a Holm-Sidak post hoc analysis. RESULTS: Exposure of rat islets to cytokines induces a reduction in islet viability and functionality concomitant with an oxidative status shift with an increase of cytosolic ROS production. Mesenchymal stem cells did not significantly increase rat islet viability under exposure to cytokines but protected islets from the loss of insulin secretion. A drastic reduction of the antioxidant factors heme oxygenase-1 and ferritin H protein levels was observed in islets exposed to the cytokine cocktail with a prevention of this effect by the presence of mesenchymal stem cells. CONCLUSIONS: Our data evidenced that MSCs are able to preserve islet insulin secretion through a modulation of the oxidative imbalance mediated by heme and iron via heme oxygenase-1 and ferritin in a context of cytokine exposure.
Asunto(s)
Citocinas/farmacología , Ferritinas/biosíntesis , Hemo Oxigenasa (Desciclizante)/biosíntesis , Islotes Pancreáticos/metabolismo , Células Madre Mesenquimatosas/metabolismo , Estrés Fisiológico/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacos , Animales , Técnicas de Cocultivo , Humanos , Islotes Pancreáticos/citología , Células Madre Mesenquimatosas/citología , RatasRESUMEN
Microglial cells are brain specific professional phagocytic immune cells that play a crucial role in the inflammation- mediated neurodegeneration especially in Parkinson's disease (PD) and Alzheimer's disease. Glia maturation factor (GMF) is a neuroinflammatory protein abundantly expressed in the brain. We have previously shown that GMF expression is significantly upregulated in the substantia nigra (SN) of PD brains. However, its possible role in PD progression is still not fully understood. The Clustered-Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR Associated (Cas) protein9 (CRISPR/Cas9) system is a simple, rapid and often extremely efficient gene editing tool at desired loci, enabling complete gene knockout or homology directed repair. In this study, we examined the effect of GMF editing by using the CRISPR/Cas9 technique in BV2 microglial cells (hereafter referred to as BV2-G) on oxidative stress and nuclear factor erythroid 2-related factor 2 (NRF2)/Hemeoxygenase1 (HO-1)-dependent ferritin activation after treatment with (1-methyl-4-phenylpyridinium) MPP+. Knockout of GMF in BV2-G cells significantly attenuated oxidative stress via reduced ROS production and calcium flux. Furthermore, deficiency of GMF significantly reduced nuclear translocation of NRF2, which modulates HO-1 and ferritin activation, cyclooxygenase 2 (COX2) and nitric oxide synthase 2 (NOS2) expression in BV2 microglial cells. Lack of GMF significantly improved CD11b and CD68 positive microglial cells as compared with untreated cells. Our results also suggest that pharmacological and genetic intervention targeting GMF may represent a promising and a novel therapeutic strategy in controlling Parkinsonism by regulating microglial functions. Targeted regulation of GMF possibly mediates protein aggregation in microglial homeostasis associated with PD progression through regulation of iron metabolism by modulating NRF2-HO1 and ferritin expression.
Asunto(s)
Sistemas CRISPR-Cas/fisiología , Ferritinas/genética , Factor de Maduración de la Glia/genética , Hemo-Oxigenasa 1/genética , Proteínas de la Membrana/genética , Dinámicas Mitocondriales/fisiología , Factor 2 Relacionado con NF-E2/genética , Neuroglía/fisiología , 1-Metil-4-fenilpiridinio/toxicidad , Animales , Proteína 9 Asociada a CRISPR/biosíntesis , Proteína 9 Asociada a CRISPR/genética , Línea Celular , Ferritinas/biosíntesis , Edición Génica/métodos , Factor de Maduración de la Glia/deficiencia , Hemo-Oxigenasa 1/biosíntesis , Proteínas de la Membrana/biosíntesis , Ratones , Factor 2 Relacionado con NF-E2/biosíntesis , Neuroglía/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismoRESUMEN
BACKGROUND: Patients with thalassaemia who received regular transfusions had increased iron accumulation, leading to iron overload, which was associated with oxidative stress. Mitochondrial ferritin, encoded by the FTMT gene is an iron-storage protein in the mitochondria. The aim of this work was to investigate the expression levels of FTMT in the reticulocytes of patients with alpha-thalassaemia who were regularly transfused and rarely transfused compared with healthy controls and to evaluate the relationships of the levels of FTMT mRNA with malondialdehyde (MDA) and ferritin in these patients. METHODS: The levels of FTMT mRNA in the reticulocytes of patients (30 regularly transfused and 30 rarely transfused) and 30 healthy individuals were assessed by quantitative reverse transcription-polymerase chain reaction. The levels of ferritin and MDA were analysed by ELISA and by a thiobarbituric acid reactive substance assay, respectively. RESULTS: The levels of FTMT mRNA, ferritin and MDA in both groups of patients were significantly increased compared with those in the healthy controls. In addition, the levels of FTMT mRNA, ferritin and MDA in the regularly transfused patients were significantly higher than those in the rarely transfused patients. Furthermore, the relative expression levels of FTMT in patients correlated with those of MDA and ferritin. CONCLUSION: These results suggest that the elevation of expression levels of FTMT in the reticulocytes of patients with alpha-thalassaemia may be associated with iron loading and oxidative stress.
Asunto(s)
Ferritinas/biosíntesis , Regulación de la Expresión Génica , Proteínas Mitocondriales/biosíntesis , ARN Mensajero/biosíntesis , Reticulocitos/metabolismo , Talasemia alfa/sangre , Adolescente , Adulto , Niño , Femenino , Humanos , Masculino , Reticulocitos/patología , Talasemia alfa/patologíaRESUMEN
BACKGROUND: Although excess iron induces oxidative stress in the liver, it is unclear whether it directly activates the hepatic stellate cells (HSC). MATERIALS AND METHODS: We evaluated the effects of excess iron on fibrogenesis and transforming growth factor beta (TGF-ß) signaling in murine HSC. Cells were treated with holotransferrin (0.005-5g/L) for 24 hours, with or without the iron chelator deferoxamine (10µM). Gene expressions (α-SMA, Col1-α1, Serpine-1, TGF-ß, Hif1-α, Tfrc and Slc40a1) were analyzed by quantitative real time-polymerase chain reaction, whereas TfR1, ferroportin, ferritin, vimentin, collagen, TGF-ß RII and phospho-Smad2 proteins were evaluated by immunofluorescence, Western blot and enzyme-linked immunosorbent assay. RESULTS: HSC expressed the iron-uptake protein transferrin receptor 1 (TfR1) and the iron-export protein ferroportin. Holotransferrin upregulated TfR1 expression by 1.8-fold (P < 0.03) and ferritin accumulation (iron storage) by 2-fold (P < 0.01), and activated HSC with 2-fold elevations (P < 0.03) in α-SMA messenger RNA and collagen secretion, and a 1.6-fold increase (P < 0.01) in vimentin protein. Moreover, holotransferrin activated the TGF-ß pathway with TGF-ß messenger RNA elevated 1.6-fold (P = 0.05), and protein levels of TGF-ß RII and phospho-Smad2 increased by 1.8-fold (P < 0.01) and 1.6-fold (P < 0.01), respectively. In contrast, iron chelation decreased ferritin levels by 30% (P < 0.03), inhibited collagen secretion by 60% (P < 0.01), repressed fibrogenic genes α-SMA (0.2-fold; P < 0.05) and TGF-ß (0.4-fold; P < 0.01) and reduced levels of TGF-ß RII and phospho-Smad2 proteins. CONCLUSIONS: HSC express iron-transport proteins. Holotransferrin (iron) activates HSC fibrogenesis and the TGF-ß pathway, whereas iron depletion by chelation reverses this, suggesting that this could be a useful adjunct therapy for patients with fibrosis. Further studies in primary human HSC and animal models are necessary to confirm this.
Asunto(s)
Regulación de la Expresión Génica , Células Estrelladas Hepáticas/metabolismo , Hierro/metabolismo , Cirrosis Hepática/metabolismo , Transducción de Señal , Factor de Crecimiento Transformador beta/metabolismo , Animales , Línea Celular , Ferritinas/biosíntesis , Células Estrelladas Hepáticas/patología , Cirrosis Hepática/patología , Ratones , Proteínas Serina-Treonina Quinasas/biosíntesis , ARN Mensajero/biosíntesis , Receptor Tipo II de Factor de Crecimiento Transformador beta , Receptores de Transferrina/biosíntesis , Receptores de Factores de Crecimiento Transformadores beta/biosíntesis , Proteína Smad2/metabolismoRESUMEN
PURPOSE: Iron is essential for development and growth in young children; unfortunately, iron deficiency (ID) is a significant public health problem in this population. Young Child Formulae (YCF), milk-derived products fortified with iron and ascorbic acid (AA, an enhancer of iron absorption) may be good sources of iron to help prevent ID. Furthermore, some YCF are supplemented with prebiotics, non-digestible carbohydrates suggested to enhance iron bioavailability. The aim of our study was to evaluate iron bioavailability of YCF relative to prebiotic and AA concentrations. We hypothesised that YCF with the highest levels of prebiotics and AA would have the most bioavailable iron. METHODS: We used the in vitro digestion/Caco-2 cell model to measure iron bioavailability from 4 commercially available YCF with approximately equal amounts of iron, but varying amounts of: AA and the prebiotics fructo- and galacto-oligosaccharides. Caco-2 cell ferritin formation was used as a surrogate marker for iron bioavailability. RESULTS: The YCF with the highest concentration of prebiotics and AA had the highest iron bioavailability; conversely, the YCF with the lowest concentration of prebiotics and AA had the lowest. After the addition of exogenous prebiotics, so that all tested YCF had equivalent amounts, there was no longer a significant difference between YCF iron bioavailability. CONCLUSION: Our results suggest that ascorbic acid and prebiotics in YCF improve iron bioavailability. Ensuring that iron is delivered in a bioavailable form would improve the nutritional benefits of YCF in relation to ID/IDA amongst young children; therefore, further exploration of our findings in vivo is warranted.
Asunto(s)
Digestión , Enterocitos/metabolismo , Fórmulas Infantiles/química , Absorción Intestinal , Hierro de la Dieta/metabolismo , Prebióticos/análisis , Ácido Ascórbico/administración & dosificación , Ácido Ascórbico/análisis , Ácido Ascórbico/metabolismo , Biomarcadores/metabolismo , Células CACO-2 , Fenómenos Fisiológicos Nutricionales Infantiles , Preescolar , Ferritinas/biosíntesis , Alimentos Especializados/análisis , Calor , Humanos , Lactante , Fenómenos Fisiológicos Nutricionales del Lactante , Hierro de la Dieta/administración & dosificación , Hierro de la Dieta/análisis , Valor Nutritivo , Oligosacáridos/administración & dosificación , Oligosacáridos/análisis , Oligosacáridos/metabolismo , Prebióticos/administración & dosificación , Trisacáridos/administración & dosificación , Trisacáridos/análisis , Trisacáridos/metabolismoRESUMEN
In this work we found that the bfr gene of the rhizobial species Ensifer meliloti, encoding a bacterioferritin iron storage protein, is involved in iron homeostasis and the oxidative stress response. This gene is located downstream of and overlapping the smc03787 open reading frame (ORF). No well-predicted RirA or Irr boxes were found in the region immediately upstream of the bfr gene although two presumptive RirA boxes and one presumptive Irr box were present in the putative promoter of smc03787 We demonstrate that bfr gene expression is enhanced under iron-sufficient conditions and that Irr and RirA modulate this expression. The pattern of bfr gene expression as well as the response to Irr and RirA is inversely correlated to that of smc03787 Moreover, our results suggest that the small RNA SmelC759 participates in RirA- and Irr-mediated regulation of bfr expression and that additional unknown factors are involved in iron-dependent regulation.IMPORTANCEE. meliloti belongs to the Alphaproteobacteria, a group of bacteria that includes several species able to associate with eukaryotic hosts, from mammals to plants, in a symbiotic or pathogenic manner. Regulation of iron homeostasis in this group of bacteria differs from that found in the well-studied Gammaproteobacteria In this work we analyzed the effect of rirA and irr mutations on bfr gene expression. We demonstrate the effect of an irr mutation on iron homeostasis in this bacterial genus. Moreover, results obtained indicate a complex regulatory circuit where multiple regulators, including RirA, Irr, the small RNA SmelC759, and still unknown factors, act in concert to balance bfr gene expression.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Grupo Citocromo b/genética , Ferritinas/genética , Regulación Bacteriana de la Expresión Génica , Proteínas Reguladoras del Hierro/metabolismo , Hierro/metabolismo , ARN Bacteriano/metabolismo , Sinorhizobium meliloti/metabolismo , Factores de Transcripción/metabolismo , Proteínas Bacterianas/biosíntesis , Grupo Citocromo b/biosíntesis , Ferritinas/biosíntesis , Proteínas Reguladoras del Hierro/genética , Mutación , ARN Bacteriano/genética , Sinorhizobium meliloti/genética , Factores de Transcripción/genéticaRESUMEN
Human heavy chain ferritin (FTH1) can self-assemble into a diameter of 12-nm spherical cage with an interior cavity of 8 nm in diameter. FTH1 has great potential as a nanocage in molecular imaging and drug delivery. Different peptides have been fused with FTH1 for targeting delivery; however, the expression of FTH1 modified with peptides in soluble form is not equivalent to natural FTH1. As shown in recent study, a novel scaffold protein --thioredoxin from the archaebacterium Pyrococcus furiosus (PfTrx)--exhibits a superior solubilization capacity and thermal stability [19]. Here we report a new construct (FTH1-PfTrx-His) that can be easily expressed and purified in Escherichia coli. Of note, different peptides inserted into FTH1-PfTrx-His did not influence the expression of proteins. Finally, the doxorubicin packaging ability of FTH1-PfTrx-His is comparable to natural FTH1. Our results showed that FTH1-PfTrx-His had a potential role as a novel peptide-modified ferritin carrier for drugs or imaging probes.
Asunto(s)
Proteínas Arqueales , Ferritinas , Expresión Génica , Pyrococcus furiosus/genética , Proteínas Recombinantes de Fusión , Tiorredoxinas , Proteínas Arqueales/biosíntesis , Proteínas Arqueales/química , Proteínas Arqueales/genética , Proteínas Arqueales/aislamiento & purificación , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/metabolismo , Ferritinas/biosíntesis , Ferritinas/química , Ferritinas/genética , Ferritinas/aislamiento & purificación , Humanos , Oxidorreductasas , Pyrococcus furiosus/enzimología , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Tiorredoxinas/biosíntesis , Tiorredoxinas/química , Tiorredoxinas/genética , Tiorredoxinas/aislamiento & purificaciónRESUMEN
The complementary DNA (cDNA) of the giant panda (Ailuropoda melanoleuca) ferritin light polypeptide (FTL) gene was successfully cloned using reverse transcription-polymerase chain reaction technology. We constructed a recombinant expression vector containing FTL cDNA and overexpressed it in Escherichia coli using pET28a plasmids. The expressed protein was then purified by nickel chelate affinity chromatography. The cloned cDNA fragment was 580 bp long and contained an open reading frame of 525 bp. The deduced protein sequence was composed of 175 amino acids and had an estimated molecular weight of 19.90 kDa, with an isoelectric point of 5.53. Topology prediction revealed one N-glycosylation site, two casein kinase II phosphorylation sites, one N-myristoylation site, two protein kinase C phosphorylation sites, and one cell attachment sequence. Alignment indicated that the nucleotide and deduced amino acid sequences are highly conserved across several mammals, including Homo sapiens, Cavia porcellus, Equus caballus, and Felis catus, among others. The FTL gene was readily expressed in E. coli, which gave rise to the accumulation of a polypeptide of the expected size (25.50 kDa, including an N-terminal polyhistidine tag).
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Ferritinas/genética , Ursidae/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Secuencia Conservada , Escherichia coli , Ferritinas/biosíntesis , Ferritinas/aislamiento & purificación , Expresión Génica , Glicosilación , Punto Isoeléctrico , Peso Molecular , Procesamiento Proteico-Postraduccional , Análisis de Secuencia de ADN , Análisis de Secuencia de ProteínaRESUMEN
As a promising magnetic resonance imaging (MRI) reporter, ferritin has been used to track cells in vivo; however, its continuous overexpression can be cytotoxic, which restricts its application. In this study, we aimed to develop a switch to turn this genetic reporter "on" or "off" while monitoring cell grafts via MRI. To accomplish this, we genetically modified the ferritin heavy chain (FTH1) with a Tet-On switch and assessed the expression of FTH1 in transduced neuroblastoma cells (SK-N-SH) in vitro and in xenografted tumors in vivo. We found that FTH1 expression induced by doxycycline (Dox) in SK-N-SH-FTH1 cells depended on treatment dose and duration. We successfully detected T2-weighted MRI contrast in cell grafts after switching "on" the reporter gene using Dox, and this contrast disappeared when we switched it "off". The genetic reporter FTH1 can thus be switched "on" or "off" throughout longitudinal monitoring of cell grafts, limiting expression to when MRI contrast is needed. The controllable imaging system we have developed minimizes risks from constitutive reporter gene overexpression and facilitates tumor cell monitoring in vitro and in vivo.
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Biomarcadores de Tumor/genética , Doxiciclina/farmacología , Ferritinas/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Genes Reporteros , Imagen por Resonancia Magnética , Imagen Molecular/métodos , Neuroblastoma/diagnóstico por imagen , Animales , Biomarcadores de Tumor/metabolismo , Línea Celular Tumoral , Proliferación Celular , Relación Dosis-Respuesta a Droga , Ferritinas/biosíntesis , Xenoinjertos , Humanos , Hierro/metabolismo , Masculino , Ratones Desnudos , Trasplante de Neoplasias , Neuroblastoma/genética , Neuroblastoma/metabolismo , Oxidorreductasas , Factores de Tiempo , Transducción GenéticaRESUMEN
Metastatic renal cell carcinoma (mRCC) develops in approximately 33% of all renal cancer patients. First line treatment of mRCC includes drugs such as sunitinib, temsirolimus and pazopanib, with overall survival now reaching up to 43,6months in patients with favorable-risk metastatic disease. Several side-effects in mRCC treatment, such as hypothyroidism, can be used as positive prognostic factors and indicate good response to therapy. Hypercholesterolemia and hypertriglyceridemia independent of hypothyroidism are reported as side-effects in temsirolimus treatment and recently in sunitinib treatment, but the exact mechanism and significance of the changes remains elusive. Most likely, metabolic changes are caused by inhibition of mechanistic target of rapamycin (mTOR), a positive target of tumor growth suppression, but also a regulator of iron homeostasis. There are no clinical studies reporting changes in iron and ferritin levels during mRCC biotherapy, but we hypothesize that inhibition of mTOR will also affect iron and ferritin levels. If both lipid and iron changes correlate, there is a high possibility that both changes are primarily caused by mTOR inhibition and the level of change should correlate with the inhibition of mTOR pathway and hence the efficacy of targeted treatment. We lastly hypothesize that mRCC biotherapy causes hypercholesterolemia with a possibly improved cholesterol profile due to increase HDL/LDL ratio, so statins might not have a role as supplementary treatment, whereas a sharp rise in triglyceride levels seems to be the primary target for additional therapy.
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Biomarcadores de Tumor/metabolismo , Carcinoma de Células Renales/metabolismo , Ferritinas/sangre , Hierro/sangre , Neoplasias Renales/metabolismo , Metaboloma , Animales , Antineoplásicos/uso terapéutico , Carcinoma de Células Renales/patología , Carcinoma de Células Renales/terapia , Colesterol/metabolismo , Ferritinas/biosíntesis , Humanos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/metabolismo , Hipercolesterolemia/metabolismo , Hipertrigliceridemia/metabolismo , Hipotiroidismo/metabolismo , Indazoles , Indoles/uso terapéutico , Neoplasias Renales/patología , Neoplasias Renales/terapia , Lípidos/química , Modelos Teóricos , Metástasis de la Neoplasia , Pronóstico , Pirimidinas/uso terapéutico , Pirroles/uso terapéutico , Sirolimus/análogos & derivados , Sirolimus/metabolismo , Sulfonamidas/uso terapéutico , Sunitinib , Serina-Treonina Quinasas TOR/metabolismo , Triglicéridos/metabolismoRESUMEN
Osteogenic differentiation of multipotent mesenchymal stem cells (MSCs) plays a crucial role in bone remodeling. Numerous studies have described the deleterious effect of iron overload on bone density and microarchitecture. Excess iron decreases osteoblast activity, leading to impaired extracellular matrix (ECM) mineralization. Additionally, iron overload facilitates osteoclast differentiation and bone resorption. These processes contribute to iron overload-associated bone loss. In this study we investigated the effect of iron on osteogenic differentiation of human bone marrow MSCs (BMSCs), the third player in bone remodeling. We induced osteogenic differentiation of BMSCs in the presence or absence of iron (0-50µmol/L) and examined ECM mineralization, Ca content of the ECM, mRNA and protein expressions of the osteogenic transcription factor runt-related transcription factor 2 (Runx2), and its targets osteocalcin (OCN) and alkaline phosphatase (ALP). Iron dose-dependently attenuated ECM mineralization and decreased the expressions of Runx2 and OCN. Iron accomplished complete inhibition of osteogenic differentiation of BMSCs at 50µmol/L concentration. We demonstrated that in response to iron BMSCs upregulated the expression of ferritin. Administration of exogenous ferritin mimicked the anti-osteogenic effect of iron, and blocked the upregulation of Runx2, OCN and ALP. Iron overload in mice was associated with elevated ferritin and decreased Runx2 mRNA levels in compact bone osteoprogenitor cells. The inhibitory effect of iron is specific toward osteogenic differentiation of MSCs as neither chondrogenesis nor adipogenesis were influenced by excess iron. We concluded that iron and ferritin specifically inhibit osteogenic commitment and differentiation of BMSCs both in vitro and in vivo.