Overexpression Of Ferritin Research Articles

Quantitative MRI Evaluation of Ferritin Overexpression in Non-Small-Cell Lung Cancer.

Cancer cells frequently present elevated intracellular iron levels, which are thought to facilitate an enhanced proliferative capacity. Targeting iron metabolism within cancer cells presents an avenue to enhance therapeutic responses, necessitating the use of non-invasive models to modulate iron manipulation to predict responses. Moreover, the ubiquitous nature of iron necessitates the development of unique, non-invasive markers of metabolic disruptions to develop more personalized approaches and enhance the clinical utility of these approaches. Ferritin, an iron storage enzyme that is often upregulated as a response to iron accumulation, plays a central role in iron metabolism and has been frequently associated with unfavorable clinical outcomes in cancer. Herein, we demonstrate the successful utility, validation, and functionality of a doxycycline-inducible ferritin heavy chain (FtH) overexpression model in H1299T non-small-cell lung cancer (NSCLC) cells. Treatment with doxycycline increased the protein expression of FtH with a corresponding decrease in labile iron in vitro and in vivo, as determined by calcein-AM staining and EPR, respectively. Moreover, a subsequent increase in TfR expression was observed. Furthermore, T2* MR mapping effectively detected FtH expression in our in vivo model. These results demonstrate that T2* relaxation times can be used to monitor changes in FtH expression in tumors with bidirectional correlations depending on the model system. Overall, this study describes the development of an FtH overexpression NSCLC model and its correlation with T2* mapping for potential use in patients to interrogate iron metabolic alterations and predict clinical outcomes.

Bright ferritin for long-term MR imaging of human embryonic stem cells

BackgroundA non-invasive imaging technology that can monitor cell viability, retention, distribution, and interaction with host tissue after transplantation is needed for optimizing and translating stem cell-based therapies. Current cell imaging approaches are limited in sensitivity or specificity, or both, for in vivo cell tracking. The objective of this study was to apply a novel ferritin-based magnetic resonance imaging (MRI) platform to longitudinal tracking of human embryonic stem cells (hESCs) in vivo.MethodsHuman embryonic stem cells (hESCs) were genetically modified to stably overexpress ferritin using the CRISPR-Cas9 system. Cellular toxicity associated with ferritin overexpression and manganese (Mn) supplementation were assessed based on cell viability, proliferation, and metabolic activity. Ferritin-overexpressing hESCs were characterized based on stem cell pluripotency and cardiac-lineage differentiation capability. Cells were supplemented with Mn and imaged in vitro as cell pellets on a preclinical 3 T MR scanner. T1-weighted images and T1 relaxation times were analyzed to assess contrast. For in vivo study, three million cells were injected into the leg muscle of non-obese diabetic severe combined immunodeficiency (NOD SCID) mice. Mn was administrated subcutaneously. T1-weighted sequences and T1 mapping were used to image the animals for longitudinal in vivo cell tracking. Cell survival, proliferation, and teratoma formation were non-invasively monitored by MRI. Histological analysis was used to validate MRI results.ResultsFerritin-overexpressing hESCs labeled with 0.1 mM MnCl2 provided significant T1-induced bright contrast on in vitro MRI, with no adverse effect on cell viability, proliferation, pluripotency, and differentiation into cardiomyocytes. Transplanted hESCs displayed significant bright contrast on MRI 24 h after Mn administration, with contrast persisting for 5 days. Bright contrast was recalled at 4–6 weeks with early teratoma outgrowth.ConclusionsThe bright-ferritin platform provides the first demonstration of longitudinal cell tracking with signal recall, opening a window on the massive cell death that hESCs undergo in the weeks following transplantation before the surviving cell fraction proliferates to form teratomas.

Iron-Related Genes and Proteins in Mesenchymal Stem Cell Detection and Therapy.

Mesenchymal stem cells (MSCs) are located in various tissues of the body. These cells exhibit regenerative and reparative properties, which makes them highly valuable for cell-based therapy. Despite this, majority of MSC-related studies remain to be translated for regular clinical use. This is partly because there are methodical challenges in pre-administration MSC labelling, post-administration detection and tracking of cells, and in retention of maximal therapeutic potential in-vivo. This calls for exploration of alternative or adjunctive approaches that would enable better detection of transplanted MSCs via non-invasive methods and enhance MSC therapeutic potential in-vivo. Interestingly, these attributes have been demonstrated by some iron-related genes and proteins.Accordingly, this unique forward-looking article integrates the apparently distinct fields of iron metabolism and MSC biology, and reviews the utility of iron-related genes and iron-related proteins in facilitating MSC detection and therapy, respectively. Effects of genetic overexpression of the iron-related proteins ferritin, transferrin receptor-1 and MagA in MSCs and their utilisation as reporter genes for improving MSC detection in-vivo are critically evaluated. In addition, the beneficial effects of the iron chelator deferoxamine and the iron-related proteins haem oxygenase-1, lipocalin-2, lactoferrin, bone morphogenetic protein-2 and hepcidin in enhancing MSC therapeutics are highlighted with the consequent intracellular alterations in MSCs. This review aims to inform both regenerative and translational medicine. It can aid in formulating better methodical approaches that will improve, complement, or provide alternatives to the current pre-transplantation MSC labelling procedures, and enhance MSC detection or augment the post-transplantation MSC therapeutic potential.

The m6A Reader YTHDF1 Promotes Lung Carcinoma Progression via Regulating Ferritin Mediate Ferroptosis in an m6A-Dependent Manner.

N6-methyladenosine (m6A) plays a significant role as an epigenetic mechanism, which is involved in various cancers' progress via regulating mRNA modification. As a crucial m6A "reader", YTHDF1 is able to alter m6A-modified mRNA and promote the protein translation process in multiple cancers. However, the role of YTHDF1 in lung cancer has not been fully investigated. This study focuses on elucidating the function of YTHDF1 in the development of lung cancer and its underlying mechanism. We demonstrated that YTHDF1 was highly expressed in lung carcinoma progression; then, the loss of function experiments in lung cell lines confirmed that knockdown of YTHDF1 suppressed cell proliferation, migration and invasion and induced ferroptosis of lung cancer cells. Further functional assays showed that ferritin (FTH) was identified as the key target of YTHDF1 in lung cancer cells. Furthermore, the overexpression of ferritin in YTHDF1-depleted cells partially restored lung cancer cell suppression. Collectively, our data suggested that the upregulation of YTHDF1 promotes lung cancer carcinogenesis by accelerating ferritin translation in an m6A-dependent manner. We hope that our findings may provide a new target for lung cancer diagnosis and treatment.

Ferritin Overexpression Limits Intracellular Labile Iron and Induces Replication Stress in Lung Cancer Cells

Ferritin Overexpression Limits Intracellular Labile Iron and Induces Replication Stress in Lung Cancer Cells

Overexpression of Mitochondrial Ferritin Enhances Blood-Brain Barrier Integrity Following Ischemic Stroke in Mice by Maintaining Iron Homeostasis in Endothelial Cells.

Blood–brain barrier (BBB) breakdown, a characteristic feature of ischemic stroke, contributes to poor patient outcomes. Brain microvascular endothelial cells (BMVECs) are a key component of the BBB and dysfunction or death of these cells following cerebral ischemia reperfusion (I/R) injury can disrupt the BBB, leading to leukocyte infiltration, brain edema and intracerebral hemorrhage. We previously demonstrated that mitochondrial ferritin (FtMt) can alleviate I/R-induced neuronal ferroptosis by inhibiting inflammation-regulated iron deposition. However, whether FtMt is involved in BBB disruption during cerebral I/R is still unknown. In the present study, we found that FtMt expression in BMVECs is upregulated after I/R and overexpression of FtMt attenuates I/R-induced BBB disruption. Mechanistically, we found that FtMt prevents tight junction loss and apoptosis by inhibiting iron dysregulation and reactive oxygen species (ROS) accumulation in I/R-treated BMVECs. Chelating excess iron with deferoxamine alleviates apoptosis in the brain endothelial cell line bEnd.3 under oxygen glucose deprivation followed by reoxygenation (OGD/R) insult. In summary, our data identify a previously unexplored effect for FtMt in the BBB and provide evidence that iron-mediated oxidative stress in BMVECs is an early cause of BMVECs damage and BBB breakdown in ischemic stroke.

Formation of resting cells is accompanied with enrichment of ferritin in marine diatom Phaeodactylum tricornutum

The formation of resting cells/spores in marine diatoms helps them to survive in environmental changes, whose sedimentation characteristics also drive enhanced carbon fluxes export to the deep ocean. However, the molecular mechanism underlying the transformation remains unclear. Based on the upregulation of ferritin during dormancy in microorganism and its' crucial role in coping with environmental stress, we constructed the ferritin overexpression and silence strains in model marine diatom, Phaeodactylum tricornutum (FTN-OE and FTNSi, respectively). The morphological transformation from fusiform to ovoid were observed among the FTN-OE strains but not the wide type and FTN-Si strains. These FTN-OE oval cells exhibited typical characteristics of resting cells and benthic adaptation, such as lower growth rates, low-light adaptation, silicified valves, the excretion of exopolymeric substances, upregulation of stress-resistant proteins and increased nitrate reserves. Furthermore, FTN-OE oval cells showed increased abscisic acid synthesis and signaling pathways in the transcriptome and proteome. And the abscisic acid signal was detected in FTN-OE oval cells exclusively (2.96 ng g−1 FW), but absent in the FTNSi, FTN-OE fusiform or WT cells. Overall, our results demonstrate that ferritin overexpression in marine diatom have a close relationship with morphological transformation from fusiform to ovoid morphotype, after which they enter a resting stage, which is accompanied by abscisic acid accumulation. This improves our understanding of the molecular processes in diatom resting cells/spores' formation and suggests that ferritin accumulation might improve the sequestration of photosynthetically fixed atmospheric CO2 into the deep sea.

Silencing ferritin alleviates atherosclerosis in mice via regulating the expression levels of matrix metalloproteinases and interleukins.

This study was conducted to investigate the roles of ferritin in atherosclerosis. The mouse model of atherosclerosis was established by feeding ApoE knockout mice with a high-fat diet. The mice were then treated with ferritin-overexpressing and -silencing constructs, and assessed for interleukins (ILs) and matrix metalloproteinases (MMPs) levels using ELISA and Western blot analysis. After being fed with a high-fat diet, the ApoE knockout mice developed pro-atherogenic lipid profiles with elevated total cholesterol (TC), triglyceride (TG) and low-density lipoprotein cholesterol (LDL-C). They also showed increased atherosclerotic lesions including narrowed lumen diameter, reduced lumen area, and increased plaque size. Following injection of the overexpression and silencing constructs, mRNA levels of ferritin were increased and decreased, respectively, and at the same time the atherosclerotic lesions were aggravated and alleviated, respectively. Further analysis indicated that silencing of ferritin gene reduced IL-1β and IL-10 levels while overexpressing ferritin increased them. On other hand, the TNF-α levels showed an opposite trend. MMP8, MMP12 and MMP13 levels were increased or decreased significantly after the mice were injected with ferritin over-expression or silencing vectors, respectively. Western blot analysis showed that compared to the control, overexpressing ferritin resulted in increased expression of p-JNK while silencing ferritin decreased the expression. Meanwhile, the levels of pc-Jun remained unchanged. Our work demonstrates that ferritin can regulate the progress of atherosclerosis via regulating the expression levels of MMPs and interleukins. Silencing ferritin inhibits the development of atherosclerosis and is, therefore, worth being further investigated as a potential therapeutic approach for this disease.

Ferritin reduction is essential for cerebral ischemia-induced hippocampal neuronal death through p53/SLC7A11-mediated ferroptosis

Cerebral ischemia is the most common cause of hippocampal neuronal death and the most prevalent cause of stroke with high mortality rate. Ferroptosis has been suggested to affect the role of hippocampal neurons. This study explores the influence of lentivirus infection-induced ferritin overexpression in hippocampal neuronal injury and death through simulations in August Copenhagen Irish rat models. Twenty-four-hour cerebral ischemia–reperfusion injury was induced in the rats after 90-min middle cerebral artery occlusion (MCAO). Ferritin overexpression was induced through lentivirus infection. The Morris Water Maze (MWM) test and tau hyperphosphorylation test were performed on hippocampal neurons to establish a MCAO model. The effect of ferritin overexpression on hippocampal neuronal death was evaluated using hematoxylin–eosin staining and annexin V/propidium iodide flow cytometry. The MWM test revealed that MCAO modeling decreased the cognitive and locomotor capacity of the rats, whereas ferritin overexpression partially reversed the effect of MCAO. In addition, the hyperphosphorylation of tau caused by MCAO was reduced by ferritin. Pathogenic changes, impaired viability, increased apoptosis, and elevated caspase-9 cleavage in hippocampal neurons were clearly recovered by ferritin. Moreover, robust reactive oxygen species production and glutathione consumption, which was induced by MCAO modeling, were ameliorated by ferritin. Furthermore, two key modulators of ferroptosis, p53 and SLC7A11, were demonstrated to be upregulated by MCAO modeling and downregulated by ferritin. Ferritin reduction is essential for cerebral ischemia-induced hippocampal neuronal ferroptosis mediated via p53 and SLC7A11.

Tissue-Specific Ferritin- and GFP-Based Genetic Vectors Visualize Neurons by MRI in the Intact and Post-Ischemic Rat Brain.

(1) Background: Neurogenesis is considered to be a potential brain repair mechanism and is enhanced in stroke. It is difficult to reconstruct the neurogenesis process only from the histological sections taken from different animals at different stages of brain damage and restoration. Study of neurogenesis would greatly benefit from development of tissue-specific visualization probes. (2) Purpose: The study aimed to explore if overexpression of ferritin, a nontoxic iron-binding protein, under a doublecortin promoter can be used for non-invasive visualization of neurogenesis using magnetic resonance imaging (MRI). (3) Methods: Ferritin heavy chain (FerrH) was expressed in the adeno-associated viral backbone (AAV) under the doublecortin promoter (pDCX), specific for young neurons, in the viral construct AAV-pDCX-FerrH. Expression of the enhanced green fluorescent protein (eGFP) was used as an expression control (AAV-pDCX-eGFP). The viral vectors or phosphate-buffered saline (PBS) were injected intracerebrally into 18 adult male Sprague–Dawley rats. Three days before injection, rats underwent transient middle-cerebral-artery occlusion or sham operation. Animals were subjected to In vivo MRI study before surgery and on days 7, 14, 21, and 28 days after injection using a Bruker BioSpec 11.7 T scanner. Brain sections obtained on day 28 after injection were immunostained for ferritin, young (DCX) and mature (NeuN) neurons, and activated microglia/macrophages (CD68). Additionally, RT-PCR was performed to confirm ferritin expression. (4) Results: T2* images in post-ischemic brains of animals injected with AAV-pDCX-FerrH showed two distinct zones of MRI signal hypointensity in the ipsilesioned hemisphere starting from 14 days after viral injection—in the ischemic lesion and near the lateral ventricle and subventricular zone (SVZ). In sham-operated animals, only one zone of hypointensity near the lateral ventricle and SVZ was revealed. Immunochemistry showed that ferritin-expressing cells in ischemic lesions were macrophages (88.1%), while ferritin-expressing cells near the lateral ventricle in animals both after ischemia and sham operation were mostly mature (55.7% and 61.8%, respectively) and young (30.6% and 7.1%, respectively) neurons. RT-PCR confirmed upregulated expression of ferritin in the caudoputamen and corpus callosum. Surprisingly, in animals injected with AAV-pDCX-eGFP we similarly observed two zones of hypointensity on T2* images. Cellular studies also showed the presence of mature (81.5%) and young neurons (6.1%) near the lateral ventricle in both postischemic and sham-operated animals, while macrophages in ischemic lesions were ferritin-positive (98.2%). (5) Conclusion: Ferritin overexpression induced by injection of AAV-pDCX-FerrH was detected by MRI using T2*-weighted images, which was confirmed by immunochemistry showing ferritin in young and mature neurons. Expression of eGFP also caused a comparable reduced MR signal intensity in T2*-weighted images. Additional studies are needed to investigate the potential and tissue-specific features of the use of eGFP and ferritin expression in MRI studies.

Effect of ferritin on nitrogen fixation in Lotus japonicus nodules under various iron concentrations

In the nitrogen fixation process, iron plays a vital role by being part of many symbiotic proteins, such as nitrogenase and leghemoglobin, in an active symbiosis. Excess or insufficient iron in active nitrogen fixation negatively affects the entire process. In Lotus japonicus nodules, ferritin is expressed at the initial stages of nodule development and increases at the nodule senescence stage to mobilize iron release during that stage. In this study, we investigated the effects of overexpressing and suppressing ferritin on nitrogen fixation. Acetylene reduction activity revealed that nitrogen fixation is affected by the overexpression of ferritin at high iron concentrations, but at low iron concentrations, higher nitrogen fixation was observed in ferritin-suppressed plants. qRT-PCR data indicated that suppression of ferritin in nodules induces antioxidant genes, such as superoxide dismutase, dehydroascorbate reductase and ascorbate peroxidase, to detoxify reactive oxygen species. Our data suggest that suppressing ferritin in the nodules is effective for higher nitrogen fixation under iron deficient conditions. Overaccumulated ferritin in nodule is effective under the higher iron conditions, such as senescence state.

Bright Ferritin-a Reporter Gene Platform for On-Demand, Longitudinal Cell Tracking on MRI.

SummaryA major unresolved challenge in cell-based regenerative medicine is the absence of non-invasive technologies for tracking cell fate in deep tissue and with high spatial resolution over an extended interval. MRI is highly suited for this task, but current methods fail to provide longitudinal monitoring or high sensitivity, or both. In this study, we fill this technological gap with the first discovery and demonstration of in vivo cellular production of endogenous bright contrast via an MRI genetic reporter system that forms manganese-ferritin nanoparticles. We demonstrate this technology in human embryonic kidney cells genetically modified to stably overexpress ferritin and show that, in the presence of manganese, these cells produce far greater contrast than conventional ferritin overexpression with iron or manganese-permeable cells. In living mice, diffusely implanted bright-ferritin cells produce the highest and most sustained contrast in skeletal muscle. The bright-ferritin platform has potential for on-demand, longitudinal, and sensitive cell tracking in vivo.

Natural Saponin Formosanin C‐induced Ferroptosis in Human Hepatocellular Carcinoma Cells Involved Ferritinophagy

Formosanin C (FC) is an anti‐tumor diosgenin saponin isolated from Paris formosana Hayata (Liliaceae) which is a perennial herb grown in the humid and dark forests of Taiwan at about the level of 1000 m. The plat has been used as a folk remedy for snake‐bite inflammation and tumors. FC is also a component of “Yunnan Bai Yao”, a traditional Chinese hemostatic medicine. Ferroptosis is a new form of iron‐dependent cell death, which occurs via an increased labile iron pool and lipid reactive oxygen species (ROS). Ferritinophagy is a ferritin (an iron storage protein) degradation pathway, depending on a selective autophagic cargo receptor nuclear coactivator 4 (NCOA4). Increased labile iron pool by ferritinophagy has been recognized as an upstream regulator of ferroptosis. Thus, ferroptosis has been reported to be a form of autophagic cell death. Although iron is an essential nutrient that facilitates cell proliferation and growth, overexpression of ferritin and down‐regulation of ferroportin (an iron export protein) has recently been observed in human hepatocellular carcinoma (HCC), indicating the increased dependence of iron in the cancer cells. In the present study, FC caused a significant dosage‐related population growth inhibition in HCC HepG2 and Hep3B cells using SRB assay, which can be partially blocked by co‐administration of ferroptosis inhibitor ferrostatin‐1 (a scavenger of lipid ROS). FC induced elevation in lipid ROS formation can also be partially reversed by addition of either ferrostatin‐1, or the presence of antioxidant vitamin C and/or vitamin E using flow cytometry, confirming FC‐induced ferroptosis. Flow cytometric analysis also revealed that FC elevated the percentage of acidic vesicular organelles‐positive cells, representing the formation of autolysosomes and induction of autophagic flux in both cell lines. The FC‐induced autophagy was confirmed using Western blotting, characterized by the production of LC3‐II and a further elevation of LC3‐II expression by addition of autophagy inhibitor bafilomycin A1 to prevent the fusion of autophagosomes and lysosomes for cargo degradation. In the present of bafilomycin A1, accumulation of NCOA4 and ferritin was observed in both FC‐treated cell lines using Western blotting. Concurrently, increase in co‐localization of LC3 puncta with NCOA4 or ferritin was exhibited using confocal microscopy, suggesting FC‐induced ferritinophagy. It is noteworthy that HepG2 cells with a higher NCOA4 expression were more sensitive to FC‐induced ferroptosis and autophagy compared to Hep3B cells with a higher ferritin expression. Overall, our data suggest chemotherapeutic potential of phytochemical FC for treatment of HCC with higher expression of NCOA4 via ferritinophagy and ferroptosis.Support or Funding InformationSupported in part by the Ministry of Science and Technology, Taiwan, No. MOST 106‐2320‐B‐003‐006‐MY3 and 107‐2320‐B‐003‐002‐MY2

Minocycline attenuates neurological impairment and regulates iron metabolism in a rat model of traumatic brain injury

There is currently no effective treatment for neurological impairment caused by traumatic brain injury (TBI). It has been reported that excessive iron production in the brain may be a key factor in neurological impairment. In the present study, we investigated the effects of minocycline, a semi-synthetic tetracycline antibiotic, against TBI-induced neurological impairment and explored its underlying mechanism. Neurological impairment was assessed by foot-fault test, cylinder test, wire hang test, and Morris water maze. Nissl staining was performed to evaluate cell viability in the brain. The iron concentrations in cerebrospinal fluid (CSF), serum, and brain tissues were examined. The Fe2+- and Fe3+- chelating activity of minocycline was measured. Finally, the expression levels of important iron metabolism proteins ferritin, transferrin receptor 1 (TfR1), divalent metal transporter 1 (DMT1), ferroportin 1 (FPN1), and hepcidin in the hippocampus and cortex were measured by Western blot analysis. The results indicate that minocycline significantly attenuated the neurological impairment caused by TBI and increased neuronal viability. Minocycline showed a Fe2+- and Fe3+- chelating activity in vitro and reduced the iron concentration in CSF and brain tissues (cortex and hippocampus). Minocycline also inhibited the overexpression of ferritin and TfR1, but did not affect the expression of DMT1. Minocycline restored the expression of FPN1 by decreasing the expression of hepcidin. In conclusion, minocycline may attenuate neurological impairment caused by TBI and regulate iron metabolism.

EXTH-63. LIPOSOMAL DELIVERY OF FERRITIN HEAVY CHAIN (FTH1) siRNA RESULTS IN INCREASED RADIATION SENSITIVITY IN PATIENT DERIVED GLIOMA INITIATING CELLS

Abstract Glioblastoma (GBM) is the most lethal and incurable brain cancer. Despite maximum treatment, high recurrence rates result in median survival of less than 2 years. A subset of treatment resistant cells within GBM known as glioma initiating cells (GICs) are implicated in recurrence and exhibit ferritin overexpression. High expression of ferritin is known to be associated with poor overall survival in GBM patients. Our previous finding showed that downregulation of the ferritin heavy chain subunit (FTH1) enhanced treatment sensitivity in astrocytoma cells. Recently, functional importance of FTH1 in GICs was demonstrated by shRNA silencing leading to ablation of tumorigenic ability. We therefore posited that disrupting iron metabolism through FTH1 knockdown would radiosensitize GICs. Thus we developed multivalent cationic liposomes that enabled delivery of FTH1 siRNA to patient derived GICs isolated from pro-neural (PN) and mesenchymal (Mes) GBM. In both subtypes, FTH1 loss led to increased apoptosis and reduced cell viability. When exposed to radiation, PN GICs were unable to repair and recover from DNA damage, leading to a further decrease in cell viability and a complete loss of reproductive clonogenic potential unlike their more radioresistant Mes counterparts. Moreover, expression of stem cell marker Nestin was markedly reduced in PN but not Mes GICs. Thus, FTH1 loss is detrimental to both GIC subtypes, it seems to be critical for maintaining stemness and radiation resistance of PN GICs. These findings further support the viability of FTH1 as a therapeutic target in GBM.

MR molecular imaging of HCC employing a regulated ferritin gene carried by a modified polycation vector.

Purpose: Early diagnosis is essential for reducing liver cancer mortality, and molecular diagnosis by magnetic resonance imaging (MRI) is an emerging and promising technology. The chief aim of the present work is to use the ferritin gene, modified by the alpha-fetoprotein (AFP) promoter, carried by a highly safe vector, to produce signal contrast on T2-weighted MR imaging as an endogenous contrast agent, and to provide a highly specific target for subsequent therapy.Methods: Polyethyleneimine-β-cyclodextrin (PEI-β-CD, PC) was synthesized as a novel vector. The optimal nitrogen/phosphorus ratio (N/P) of the PC/plasmid DNA complex was determined by gel retardation, biophysical properties and transmission electron microscopy morphological analysis. The transfection efficiency was observed under a fluorescence microscope and analyzed by flow cytometry. Cellular iron accumulation caused by ferritin overexpression was verified by Prussian blue staining, and the resulting contrast imaging effect was examined by MRI.Results: The modified cationic polymer PC was much safer than high molecular weight PEI, and could condense plasmid DNA at an N/P ratio of 50 with suitable biophysical properties and a high transfection efficiency. Overexpression of ferritin enriched intracellular iron. The short-term iron imbalance initiated by AFP promoter regulation only occurred in hepatoma cells, resulting in signal contrast on MRI. The specific target TfR was also upregulated during this process.Conclusion: These results illustrate that the regulated ferritin gene carried by PC can be used as an endogenous contrast agent for MRI detection of hepatocellular carcinoma (HCC). This molecular imaging technique may promote safer early diagnosis of HCC, and provide a more highly specific target for future chemotherapy drugs.

Characteristics of the Distribution of Ferritin in Epithelial Ovarian Tumor Patients: Results of a Retrospective, Observational Study

Purpose: To investigate the ferritin distribution in epithelial ovarian cancer patients according to the FIGO stage in the prognosis of epithelial ovarian cancer. Method: All ovarian cancer patients were divided into two groups according their FIGO stage. Benign ovarian tumor patients were analyzed as the control. Serum ferritin, serum iron, and other related medical index were detected by automatic instruments for all patients. In addition, ferritin heavy chain (FHC) and ferritin light chain (FLC) proteins were detected by immunohistochemical staining in 60 epithelial ovarian cancer (EOC) patients and 30 benign ovarian tumor (BOT) patients, which were diagnosed in our department between 2011 and 2016. Results: The serum ferritin concentration was significantly higher in the EOC group than in the BOT group (172.56 ± 99.39 ng/mL vs 78.18 ± 43.06 ng/mL; p μmol/L vs 14.92 ± 6.36 μmol/L; p p p p p p p > 0.05). Conclusion: Patients showed an overexpression of ferritin and a downregulation of serum iron correlated with the prognosis of epithelial ovarian cancer, which may be a new target for diagnosis and treatment of epithelial ovarian cancer.

Adenoviral vector mediated ferritin over-expression in mesenchymal stem cells detected by 7T MRI in vitro

ObjectiveThe aim of the present work was to verify whether adenoviral vector mediated ferritin over-expression in mesenchymal stem cells could be detected by 7T MRI device, and to explore the relationship between ferritin content and MRI signal intensities.MethodsA recombined adenoviral vector (rAdV) encoding ferritin heavy chain (FTH1) subunit was specially designed for the aim of infecting bone marrow mesenchymal stem cells (BMSCs). Ferritin over-expression in BMSCs was determined by cell immunocytochemistry and the ferritin content in cells was determined by ELISA assay. BMSCs were subjected to cell viability, proliferation and multi-differentiation analyses as well as 7T MRI test using fast spin-echo pulse sequence. The R2 value andδR2 were calculated according to T2 mapping images.ResultsAs was confirmed by cell immunocytochemistry and ELISA assay, rAdV mediated ferritin was over-expressed in BMSCs. Ferritin over-expression did not interfere with stem cell viability or pluripotent differentiation but slowed cell proliferation. The R2 value of BMSCs-FTH1 vs control BMSCs from 1–4 weeks was16.65±1.28 s-1 vs 13.99±0.80 s-1, (t = 3.94, p = 0.004), 15.63±1.37 s-1 vs 13.87±0.83 s-1 (t = 2.47, p = 0.039), 15.53±0.88 s-1 vs 14.25±0.53 s-1 (t = 2.80, p = 0.023) and 14.61±1.28 s-1 vs 13.69±1.03 s-1 (t = 1.25, p = 0.24), respectively. δR2 gradually decreased from 1–4 weeks and the difference between the groups had statistical significance (F = 12.45, p<0.01).δR2 was positively correlated with OD value (r = 0.876, p<0.01) and ferritin concentration (r = 0.899, p<0.01) as determined by Pearson correlation.ConclusionsOur study confirms that ferritin could be over-expressed in BMSCs as a result of rAdV mediated infection and could be quantitatively detected by 7T MRI device. The differences in T2 signal intensities and R2 values stem from internal contrast generated by endogenous ferritin over-expression. The correlation between δR2, OD and ferritin concentration suggests that MRI can detect ferritin signal change accurately.

Activated Oncogenic Pathway Modifies Iron Network in Breast Epithelial Cells: A Dynamic Modeling Perspective

Dysregulation of iron metabolism in cancer is well documented and it has been suggested that there is interdependence between excess iron and increased cancer incidence and progression. In an effort to better understand the linkages between iron metabolism and breast cancer, a predictive mathematical model of an expanded iron homeostasis pathway was constructed that includes species involved in iron utilization, oxidative stress response and oncogenic pathways. The model leads to three predictions. The first is that overexpression of iron regulatory protein 2 (IRP2) recapitulates many aspects of the alterations in free iron and iron-related proteins in cancer cells without affecting the oxidative stress response or the oncogenic pathways included in the model. This prediction was validated by experimentation. The second prediction is that iron-related proteins are dramatically affected by mitochondrial ferritin overexpression. This prediction was validated by results in the pertinent literature not used for model construction. The third prediction is that oncogenic Ras pathways contribute to altered iron homeostasis in cancer cells. This prediction was validated by a combination of simulation experiments of Ras overexpression and catalase knockout in conjunction with the literature. The model successfully captures key aspects of iron metabolism in breast cancer cells and provides a framework upon which more detailed models can be built.

M. tuberculosis ferritin (Rv3841): Potential involvement in Amikacin (AK) & Kanamycin (KM) resistance

Tuberculosis is an infectious disease, caused by one of the most successful human pathogen, Mycobacterium tuberculosis. Aminoglycosides, Amikacin (AK) & Kanamycin (KM) are commonly used to treat drug resistant tuberculosis. They target the protein synthesis machinery by interacting with several steps of translation. Several explanations have been proposed to explain the mechanism of aminoglycoside resistance but still our information is inadequate. Iron storing/interacting proteins were found to be overexpressed in aminoglycosides resistant isolates. Iron assimilation and utilization in M. tuberculosis plays a crucial role in growth, virulence and latency. To establish the relationship of ferritin with AK & KM resistance ferritin (Rv3841/bfrB) was cloned, expressed and antimicrobial drug susceptibility testing (DST) was carried out. Rv3841/bfrB gene was cloned and expressed in E. coli BL21 using pQE2 expression vector. Etest results for DST against AK & KM showed that the minimum inhibitory concentration (MIC) of ferritin recombinant cells was changed. Recombinants showed two fold changes in MIC with AK and three fold with KM E-strips. Overexpression of ferritin reflect the MIC shift which might be playing a critical role in the survival of mycobacteria by inhibiting/modulating the effects of AK & KM. String analysis also suggests that ferritin interacted with few proteins which are directly and indirectly involved in M. tuberculosis growth, Iron assimilation, virulence, resistance, stresses and latency.