ABSTRACT
The Notch pathway is dysregulated and a potential target in glioblastoma multiforme (GBM). Currently available Notch inhibitors block γ-secretase, which is necessary for Notch processing. However, Notch is first cleaved by α-secretase outside the plasma membrane, via a disintegrin and metalloproteinase-10 and -17. In this work, we used a potent α-secretase inhibitor (ASI) to test inhibition of glioblastoma growth and inhibition of Notch and of both novel and known Notch targets. Featured in this study are luciferase reporter assays and immunoblot, microarray analysis, chromatin immunoprecipitation (ChIP), quantitative real-time PCR, cell number assay, bromodeoxyuridine incorporation, plasmid rescue, orthotopic xenograft model, and local delivery of treatment with convection-enhanced delivery using nanoparticles, as well as survival, MRI, and ex vivo luciferase assay. A CBF1-luciferase reporter assay as well as an immunoblot of endogenous Notch revealed Notch inhibition by the ASI. Microarray analysis, quantitative real-time PCR, and ChIP of ASI and γ-secretase inhibitor (GSI) treatment of GBM cells identified known Notch pathway targets, as well as novel Notch targets, including YKL-40 and leukemia inhibitory factor. Finally, we found that local nanoparticle delivery of ASIs but not GSIs increased survival time significantly in a GBM stem cell xenograft treatment model, and ASI treatment resulted in decreased tumor size and Notch activity. This work indicates α-secretase as an alternative to γ-secretase for inhibition of Notch in GBM and possibly other cancers as well, and it identifies novel Notch targets with biologic relevance and potential as biomarkers.
Subject(s)
Amyloid Precursor Protein Secretases/antagonists & inhibitors , Brain Neoplasms/pathology , Cell Proliferation , Glioblastoma/pathology , Neoplastic Stem Cells/metabolism , Receptors, Notch/metabolism , Amyloid Precursor Protein Secretases/metabolism , Animals , Apoptosis , Biomarkers, Tumor/genetics , Biomarkers, Tumor/metabolism , Blotting, Western , Brain Neoplasms/drug therapy , Brain Neoplasms/metabolism , Cell Cycle , Chromatin Immunoprecipitation , Gene Expression Profiling , Glioblastoma/drug therapy , Glioblastoma/metabolism , Humans , In Vitro Techniques , Luciferases/metabolism , Magnetics , Mice , Mice, Inbred BALB C , Nanoparticles , Neoplastic Stem Cells/drug effects , Neoplastic Stem Cells/pathology , Oligonucleotide Array Sequence Analysis , Piperidines/pharmacology , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Receptors, Notch/genetics , Reverse Transcriptase Polymerase Chain Reaction , Spiro Compounds/pharmacology , Tumor Cells, Cultured , Xenograft Model Antitumor AssaysABSTRACT
Little is known of microRNA interactions with cellular pathways. Few reports have associated microRNAs with the Notch pathway, which plays key roles in nervous system development and in brain tumors. We previously implicated the Notch pathway in gliomas, the most common and aggressive brain tumors. While investigating Notch mediators, we noted microRNA-326 was upregulated following Notch-1 knockdown. This neuronally expressed microRNA was not only suppressed by Notch but also inhibited Notch proteins and activity, indicating a feedback loop. MicroRNA-326 was downregulated in gliomas via decreased expression of its host gene. Transfection of microRNA-326 into both established and stem cell-like glioma lines was cytotoxic, and rescue was obtained with Notch restoration. Furthermore, miR-326 transfection reduced glioma cell tumorigenicity in vivo. Additionally, we found microRNA-326 partially mediated the toxic effects of Notch knockdown. This work demonstrates a microRNA-326/Notch axis, shedding light on the biology of Notch and suggesting microRNA-326 delivery as a therapy.
Subject(s)
Gene Expression Regulation, Neoplastic/genetics , Glioma/pathology , MicroRNAs/metabolism , Receptor, Notch1/metabolism , Signal Transduction/physiology , Apoptosis/genetics , Arrestins/genetics , Arrestins/metabolism , Brain Neoplasms/genetics , Cell Line, Tumor , Cell Proliferation , Flow Cytometry/methods , Gene Expression Profiling , Humans , MicroRNAs/genetics , Neoplastic Stem Cells/metabolism , Neoplastic Stem Cells/pathology , Oligonucleotide Array Sequence Analysis/methods , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , RNA, Small Interfering/pharmacology , Receptor, Notch1/genetics , Signal Transduction/genetics , Transfection/methods , Tumor Stem Cell Assay/methods , beta-ArrestinsABSTRACT
Our recent findings establish a functional link between foreign nanosized bodies and autophagy. We find that nanoparticles (NP) within a certain size range act as potent autophagy activators, and that autophagic flux is an underlying physiological process of the cellular clearance of the NP. Therefore, NP may be used to study and to monitor autophagy. We provide a detailed description of laboratory protocols designed for studying NP-mediated autophagy. In addition, we review available methods of nanotechnology, which may benefit autophagy research.
Subject(s)
Autophagy/physiology , Quantum Dots , Blotting, Western , Flow Cytometry , In Situ Hybridization , Microscopy, FluorescenceABSTRACT
Nano-sized objects exist as engineered tools as well as natural or anthropogenic environmental factors. Recent progress in the field of nanotechnology allows for a deeper understanding of their impact on organisms. Recently, we showed that the size-dependent cell interaction with quantum dots is autophagy-mediated. The potential role of other endo- and exogenous nanoparticles in terms of autophagy is discussed here. Their physical properties should be taken into consideration while constructing delivery systems. Furthermore, we propose several models of targeted nanoparticles delivery. Autophagy can be considered as an additional mechanism providing intracellular selectivity for introduced nanoparticles.
Subject(s)
Autophagy , Nanoparticles , Animals , Cell Line , Drug Delivery Systems , Environment , Humans , Mice , Nanoparticles/administration & dosage , Nanoparticles/chemistry , Quantum DotsABSTRACT
Lately certain cytotoxicity of quantum dots (QDs) and some deleterious effects of labeling procedure on stem cells differentiation abilities were shown. In the present study we compared cytotoxicity and intracellular processing of two different-sized protein-conjugated QDs after labeling of the human mesenchymal stem cells (hMSC). An asymmetrical intracellular uptake of red (605 nm) and green (525 nm) quantum dots was observed. We describe for the first time a size-dependent activation of autophagy, caused by nanoparticles.
Subject(s)
Autophagy , Mesenchymal Stem Cells/physiology , Quantum Dots , Cell Differentiation , Cell Membrane/metabolism , Cell Membrane/ultrastructure , Cell Survival , Cells, Cultured , Coculture Techniques , Fluorescence , Humans , Mesenchymal Stem Cells/metabolism , Mesenchymal Stem Cells/ultrastructure , Microscopy, Electron, Transmission , Microtubule-Associated Proteins/metabolismABSTRACT
In the present review, various animal models of acute liver failure are reviewed with respect to their suitability for evaluating liver support systems (LSS) according to envisaged modes of therapy. In order to increase the value of the preclinical testing of LSS, it would be advantageous to include more than one animal model in the evaluation program. It is possible to identify appropriate sets of models, which make a suitable test system for particular clinical applications. A standardization of evaluation methods between testing groups would also be beneficial to the field of liver support.
