|
1
|
Modular cell-internalizing aptamer nanostructure enables targeted delivery of large functional RNAs in cancer cell lines. Nat Commun 2018; 9:2283. [PMID: 29891903 PMCID: PMC5995956 DOI: 10.1038/s41467-018-04691-x] [Citation(s) in RCA: 44] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/07/2017] [Accepted: 05/09/2018] [Indexed: 02/07/2023] Open
Abstract
Large RNAs and ribonucleoprotein complexes have powerful therapeutic potential, but effective cell-targeted delivery tools are limited. Aptamers that internalize into target cells can deliver siRNAs (<15 kDa, 19–21 nt/strand). We demonstrate a modular nanostructure for cellular delivery of large, functional RNA payloads (50–80 kDa, 175–250 nt) by aptamers that recognize multiple human B cell cancer lines and transferrin receptor-expressing cells. Fluorogenic RNA reporter payloads enable accelerated testing of platform designs and rapid evaluation of assembly and internalization. Modularity is demonstrated by swapping in different targeting and payload aptamers. Both modules internalize into leukemic B cell lines and remained colocalized within endosomes. Fluorescence from internalized RNA persists for ≥2 h, suggesting a sizable window for aptamer payloads to exert influence upon targeted cells. This demonstration of aptamer-mediated, cell-internalizing delivery of large RNAs with retention of functional structure raises the possibility of manipulating endosomes and cells by delivering large aptamers and regulatory RNAs. Large RNAs and ribonucleoprotein complexes have shown potential as novel therapeutic agents, but their targeted delivery to cells is still challenging. Here the authors present a modular aptamer nanostructure for intracellular delivery of RNAs up to 250 nucleotides to cancer cells.
Collapse
|
|
2
|
Shen Y, Li X, Dong D, Zhang B, Xue Y, Shang P. Transferrin receptor 1 in cancer: a new sight for cancer therapy. Am J Cancer Res 2018; 8:916-931. [PMID: 30034931 PMCID: PMC6048407] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/21/2018] [Accepted: 05/29/2018] [Indexed: 06/08/2023] Open
Abstract
Iron as an important element plays crucial roles in various physiological and pathological processes. Iron metabolism behaves in systemic and cellular two levels that usually are in balance conditions. The disorders of the iron metabolism balances relate with many kinds of diseases including Alzheimer's disease, osteoporosis and various cancers. In systemic iron metabolism that is regulated by hepcidin-ferroportin axis, plasma iron is bound with transferrin (TF) which has two high-affinity binding sites for ferric iron. The generic cellular iron metabolism consists of iron intake, utilization and efflux. During the iron intake process in generic cells, transferrin receptors (TFRs) act as the most important receptor mediated controls. TFR1 and TFR2 are two subtypes of TFRs those bind with iron-transferrin complex to facilitate iron into cells. TFR1 is ubiquitously expressed on the surfaces of generic cells, whereas TFR2 is specially expressed in liver cells. TFR1 has attracted more attention than TFR2 by having diverse functions in both invertebrates and vertebrates. Recently reports showed that TFR1 involved in many kinds of diseases including anemia, neurodegenerative diseases and cancers. Most importantly, TFR1 has been verified to be abnormally expressed in various cancers. Some experimental and clinical drugs and antibodies targeting TFR1 have showed strong anti-tumor effects, herein TFR1 probably become a potential molecular target for diagnosis and treatment for cancer therapy. This paper reviewed the research progresses of the roles of TFR1 in the tumorigenesis and cancer progression, the regulations of TFR1, and the therapeutic effects of targeting TFR1 on many kinds of cancers.
Collapse
Affiliation(s)
- Ying Shen
- School of Life Science, Northwestern Polytechnical UniversityXi’an 710072, Shaanxi, China
- Research and Development Institute in Shenzhen, Northwestern Polytechnical UniversityShenzhen 518057, China
| | - Xin Li
- School of Life Science, Northwestern Polytechnical UniversityXi’an 710072, Shaanxi, China
- Research and Development Institute in Shenzhen, Northwestern Polytechnical UniversityShenzhen 518057, China
| | - Dandan Dong
- School of Life Science, Northwestern Polytechnical UniversityXi’an 710072, Shaanxi, China
- Research and Development Institute in Shenzhen, Northwestern Polytechnical UniversityShenzhen 518057, China
| | - Bin Zhang
- School of Life Science, Northwestern Polytechnical UniversityXi’an 710072, Shaanxi, China
- Research and Development Institute in Shenzhen, Northwestern Polytechnical UniversityShenzhen 518057, China
| | - Yanru Xue
- School of Life Science, Northwestern Polytechnical UniversityXi’an 710072, Shaanxi, China
- Research and Development Institute in Shenzhen, Northwestern Polytechnical UniversityShenzhen 518057, China
| | - Peng Shang
- Research and Development Institute in Shenzhen, Northwestern Polytechnical UniversityShenzhen 518057, China
- Key Laboratory for Space Bioscience and Biotechnology, Institute of Special Environment Biophysics, School of Life Science, Northwestern Polytechnical UniversityXi’an 710072, Shaanxi, China
| |
Collapse
|
|
3
|
Fillebeen C, Pantopoulos K. Hepatitis C virus infection causes iron deficiency in Huh7.5.1 cells. PLoS One 2013; 8:e83307. [PMID: 24349485 PMCID: PMC3862679 DOI: 10.1371/journal.pone.0083307] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/09/2013] [Accepted: 11/05/2013] [Indexed: 02/04/2023] Open
Abstract
Patients with chronic hepatitis C virus (HCV) infection frequently develop systemic iron overload, which exacerbates morbidity. Nevertheless, iron inhibits HCV replication in cell culture models and thereby exerts antiviral activity. We hypothesized that the cellular iron status is crucial for the establishment of HCV infection. We show that HCV infection of permissive Huh7.5.1 hepatoma cells promotes an iron deficient phenotype. Thus, HCV leads to increased iron regulatory protein (IRP) activity, accumulation of IRP2 and suppression of transferrin receptor 1 (TfR1) and divalent metal transporter 1 (DMT1) in the host. These data suggest that HCV regulates cellular iron levels to bypass iron-mediated inhibition in viral replication.
Collapse
Affiliation(s)
- Carine Fillebeen
- Lady Davis Institute for Medical Research, Jewish General Hospital, Montreal, Quebec, Canada
| | - Kostas Pantopoulos
- Lady Davis Institute for Medical Research, Jewish General Hospital, Montreal, Quebec, Canada
- Department of Medicine, McGill University, Montreal, Quebec, Canada
- * E-mail:
| |
Collapse
|
|
4
|
Popovic Z, Templeton DM. Cell density-dependent shift in activity of iron regulatory protein 1 (IRP-1)/cytosolic (c-)aconitase. Metallomics 2012; 4:693-9. [PMID: 22544036 DOI: 10.1039/c2mt20027a] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/21/2022]
Abstract
Iron regulatory protein 1 (IRP-1) is a bifunctional protein involved in iron homeostasis and metabolism. In one state, it binds to specific sequences in the mRNA's of several proteins involved in iron and energy metabolism, thereby influencing their expression post-transcriptionally. In another state it contains a [4Fe-4S] iron-sulfur cofactor and displays aconitase activity in the cytosol. We have shown that this protein binds and hydrolyzes ATP, with kinetic and thermodynamic equilibrium constants that predict saturation with ATP, favouring a non-RNA-binding form at normal cellular ATP levels, and thus pointing to additional function(s) of the protein. Here we show for the first time that the RNA-binding and aconitase forms of IRP-1 can undergo interconversion dependent on the density of cells growing in culture. Thus, in high density confluent cultures, compared with low density, actively proliferating cultures, cytosolic aconitase activity is increased whereas RNA binding activity is diminished. This is accompanied by a decrease in transferrin receptor expression in confluent cells, possibly due to loss of the transcript-stabilizing activity of bound IRP-1. In high density HepG2 cultures, cytosolic glutamate and the ratio of reduced-to-oxidized glutathione were increased. We propose that increased cytosolic aconitase activity in confluent cultures may divert cytosolic citrate away from the fatty acid/membrane synthetic pathways required by dividing cells, into a glutamate-dependent maintenance of cellular macromolecular synthesis. In addition, this may confer additional protection from oxidative stress due to down-regulation of iron acquisition from transferrin and increased glutamate for glutathione synthesis.
Collapse
Affiliation(s)
- Zvezdana Popovic
- Laboratory Medicine and Pathobiology, University of Toronto, 1 King's College Circle, Toronto, Ont. M5S 1A8, Canada
| | | |
Collapse
|
|
5
|
García-Peñarrubia P, Gálvez JJ, Gálvez J. Spatio-temporal dependence of the signaling response in immune-receptor trafficking networks regulated by cell density: a theoretical model. PLoS One 2011; 6:e21786. [PMID: 21789180 PMCID: PMC3136476 DOI: 10.1371/journal.pone.0021786] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/01/2011] [Accepted: 06/09/2011] [Indexed: 12/04/2022] Open
Abstract
Cell signaling processes involve receptor trafficking through highly connected networks of interacting components. The binding of surface receptors to their specific ligands is a key factor for the control and triggering of signaling pathways. In most experimental systems, ligand concentration and cell density vary within a wide range of values. Dependence of the signal response on cell density is related with the extracellular volume available per cell. This dependence has previously been studied using non-spatial models which assume that signaling components are well mixed and uniformly distributed in a single compartment. In this paper, a mathematical model that shows the influence exerted by cell density on the spatio-temporal evolution of ligands, cell surface receptors, and intracellular signaling molecules is developed. To this end, partial differential equations were used to model ligand and receptor trafficking dynamics through the different domains of the whole system. This enabled us to analyze several interesting features involved with these systems, namely: a) how the perturbation caused by the signaling response propagates through the system; b) receptor internalization dynamics and how cell density affects the robustness of dose-response curves upon variation of the binding affinity; and c) that enhanced correlations between ligand input and system response are obtained under conditions that result in larger perturbations of the equilibrium . Finally, the results are compared with those obtained by considering that the above components are well mixed in a single compartment.
Collapse
Affiliation(s)
- Pilar García-Peñarrubia
- Department of Biochemistry and Molecular Biology and Immunology, School of Medicine, University of Murcia, Murcia, Spain
| | - Juan J. Gálvez
- Department of Information and Communications Engineering, Computer Science Faculty, University of Murcia, Murcia, Spain
| | - Jesús Gálvez
- Department of Physical Chemistry, Faculty of Chemistry, University of Murcia, Murcia, Spain
- * E-mail:
| |
Collapse
|
|
6
|
Schmidtke-Schrezenmeier G, Urban M, Musyanovych A, Mailänder V, Rojewski M, Fekete N, Menard C, Deak E, Tarte K, Rasche V, Landfester K, Schrezenmeier H. Labeling of mesenchymal stromal cells with iron oxide-poly(L-lactide) nanoparticles for magnetic resonance imaging: uptake, persistence, effects on cellular function and magnetic resonance imaging properties. Cytotherapy 2011; 13:962-75. [PMID: 21492060 PMCID: PMC3172145 DOI: 10.3109/14653249.2011.571246] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022]
Abstract
Background aims. Mesenchymal stromal cells (MSC) are the focus of research in regenerative medicine aiming at the regulatory approval of these cells for specific indications. To cope with the regulatory requirements for somatic cell therapy, novel approaches that do not interfere with the natural behavior of the cells are necessary. In this context in vivo magnetic resonance imaging (MRI) of labeled MSC could be an appropriate tool. Cell labeling for MRI with a variety of different iron oxide preparations is frequently published. However, most publications lack a comprehensive assessment of the noninterference of the contrast agent with the functionality of the labeled MSC, which is a prerequisite for the validity of cell-tracking via MRI. Methods.We studied the effects of iron oxide-poly(L-lactide) nanoparticles in MSC with flow cytom-etry, transmission electron microscopy (TEM), confocal laser scanning microscopy (CLSM), Prussian blue staining, CyQuant® proliferation testing, colony-forming unit-fibroblast (CFU-F) assays, flow chamber adhesion testing, immuno-logic tests and differentiation tests. Furthermore iron-labeled MSC were studied by MRI in agarose phantoms and Wistar rats. Results. It could be demonstrated that MSC show rapid uptake of nanoparticles and long-lasting intracellular persistence in the endosomal compartment. Labeling of the MSC with these particles has no influence on viability, differentiation, clonogenicity, proliferation, adhesion, phenotype and immunosuppressive properties. They show excellent MRI properties in agarose phantoms and after subcutaneous implantation in rats over several weeks. Conclusions. These particles qualify for studying MSC homing and trafficking via MRI.
Collapse
Affiliation(s)
- Gerlinde Schmidtke-Schrezenmeier
- DRK Blood Service Baden-Württemberg-Hessia, Institute for Clinical Transfusion Medicine and Immunogenetics Ulm and Institute of Transfusion Medicine, University of Ulm, Ulm, Germany
| | | | | | | | | | | | | | | | | | | | | | | |
Collapse
|
|
7
|
Genome-wide analysis of BEAS-2B cells exposed to trivalent arsenicals and dimethylthioarsinic acid. Toxicology 2010; 268:31-9. [DOI: 10.1016/j.tox.2009.11.018] [Citation(s) in RCA: 41] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/01/2009] [Revised: 10/30/2009] [Accepted: 11/20/2009] [Indexed: 01/22/2023]
|
|
8
|
Abstract
The liver plays a central role in iron metabolism. It is the major storage site for iron and also expresses a complex range of molecules which are involved in iron transport and regulation of iron homeostasis. An increasing number of genes associated with hepatic iron transport or regulation have been identified. These include transferrin receptors (TFR1 and 2), a ferrireductase (STEAP3), the transporters divalent metal transporter-1 (DMT1) and ferroportin (FPN) as well as the haemochromatosis protein, HFE and haemojuvelin (HJV), which are signalling molecules. Many of these genes also participate in iron regulatory pathways which focus on the hepatic peptide hepcidin. However, we are still only beginning to understand the complex interactions between liver iron transport and iron homeostasis. This review outlines our current knowledge of molecules of iron metabolism and their roles in iron transport and regulation of iron homeostasis.
Collapse
Affiliation(s)
- Ross-M Graham
- School of Medicine and Pharmacology, Fremantle Hospital, University of Western Australia, PO Box 480, Fremantle 6959, Western Australia, Australia
| | | | | | | | | |
Collapse
|
|
9
|
Schäfer R, Kehlbach R, Wiskirchen J, Bantleon R, Pintaske J, Brehm BR, Gerber A, Wolburg H, Claussen CD, Northoff H. Transferrin Receptor Upregulation: In Vitro Labeling of Rat Mesenchymal Stem Cells with Superparamagnetic Iron Oxide. Radiology 2007; 244:514-23. [PMID: 17562811 DOI: 10.1148/radiol.2442060599] [Citation(s) in RCA: 88] [Impact Index Per Article: 4.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/16/2023]
Abstract
PURPOSE To prospectively evaluate the influence of superparamagnetic iron oxide (SPIO) or ultrasmall SPIO (USPIO) particles on the surface epitope pattern of adult mesenchymal stem cells (MSCs) by regulating the expression of transferrin receptor and to prospectively evaluate the influence of transfection agents (TAs) on the uptake of SPIO or USPIO particles in MSCs. MATERIALS AND METHODS The study was approved by the institutional animal care committee of the University of Tübingen. MSCs were isolated from the bone marrow of four rats. To obtain highly homogeneous MSC populations, MSCs from one rat were single-cell cloned. One MSC clone was characterized and selected for the labeling experiments. The MSCs, which were characterized with flow cytometry and in vitro differentiation, were labeled with 200 microg/mL SPIO or USPIO or with 60 microg/mL SPIO or USPIO in combination with TAs. Aggregations of labeled cells were accommodated inside a defined volume in an agar gel matrix. Magnetic resonance (MR) imaging was performed to measure SPIO- or USPIO-induced signal voids. Quantification of cellular total iron load (TIL) (intracellular iron plus iron coating the cellular surface), determination of cellular viability, and electron microscopy were also performed. RESULTS Labeling of MSCs with SPIO or USPIO was feasible without affecting cell viability (91.1%-94.7%) or differentiation potential. For MR imaging, SPIO plus a TA was most effective, depicting 5000 cells with an average TIL of 76.5 pg per cell. SPIO or USPIO particles in combination with TAs coated the cellular surface but were not incorporated into cells. In nontransfected cells, SPIO or USPIO was taken up. MSCs labeled with SPIO or USPIO but without a TA showed enhanced expression of transferrin receptor, in contrary to both MSCs labeled with SPIO or USPIO and a TA and control cells. CONCLUSION SPIO or USPIO labeling without TAs has an influence on gene expression of MSCs upregulating transferrin receptor. Furthermore, SPIO labeling with a TA will coat the cellular surface.
Collapse
Affiliation(s)
- Richard Schäfer
- Institute of Clinical and Experimental Transfusion Medicine, University Medical Center Tübingen, Hoppe-Seyler-Str 3, D-72076 Tübingen, Germany
| | | | | | | | | | | | | | | | | | | |
Collapse
|
|
10
|
Andriopoulos B, Hegedüsch S, Mangin J, Riedel HD, Hebling U, Wang J, Pantopoulos K, Mueller S. Sustained Hydrogen Peroxide Induces Iron Uptake by Transferrin Receptor-1 Independent of the Iron Regulatory Protein/Iron-responsive Element Network. J Biol Chem 2007; 282:20301-8. [PMID: 17517884 DOI: 10.1074/jbc.m702463200] [Citation(s) in RCA: 41] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Local and systemic inflammatory conditions are characterized by the intracellular deposition of excess iron, which may promote tissue damage via Fenton chemistry. Because the Fenton reactant H(2)O(2) is continuously released by inflammatory cells, a tight regulation of iron homeostasis is required. Here, we show that exposure of cultured cells to sustained low levels of H(2)O(2) that mimic its release by inflammatory cells leads to up-regulation of transferrin receptor 1 (TfR1), the major iron uptake protein. The increase in TfR1 results in increased transferrin-mediated iron uptake and cellular accumulation of the metal. Although iron regulatory protein 1 is transiently activated by H(2)O(2), this response is not sufficient to stabilize TfR1 mRNA and to repress the synthesis of the iron storage protein ferritin. The induction of TfR1 is also independent of transcriptional activation via hypoxia-inducible factor 1alpha or significant protein stabilization. In contrast, pulse experiments with (35)S-labeled methionine/cysteine revealed an increased rate of TfR1 synthesis in cells exposed to sustained low H(2)O(2) levels. Our results suggest a novel mechanism of iron accumulation by sustained H(2)O(2), based on the translational activation of TfR1, which could provide an important (patho) physiological link between iron metabolism and inflammation.
Collapse
Affiliation(s)
- Bill Andriopoulos
- Department of Internal Medicine, Salem Medical Center, University of Heidelberg, Zeppelinstrasse 11-33, 69121 Heidelberg, Germany
| | | | | | | | | | | | | | | |
Collapse
|