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      Extracellular vesicles released by fibroblasts undergoing H-Ras induced senescence show changes in lipid profile

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          Abstract

          Cells release extracellular vesicles (EVs) in their environment and cellular lipids play an important role in their formation, secretion and uptake. Besides, there is also evidence that EV transferred lipids impact on recipient’s cell signaling. Cellular senescence is characterized by a state of permanent proliferation arrest and represents a barrier towards the development of neoplastic lesions. A peculiar feature of senescence is the release of many soluble factors, the so-called Senescence-Associated Secretory Phenotype, which play a key role in triggering paracrine senescence signals. Recently, evidences have suggested that this phenotype includes not only soluble factors, but also EVs. To identify lipid signatures associated with H-Ras-induced senescence in EVs, we expressed active H-Ras (H-RasV12) in human fibroblasts and investigated how it affects EV release and lipid composition. An enrichment of hydroxylated sphingomyelin, lyso- and ether-linked phospholipids and specific H-Ras-induced senescence signatures, e.g. sphingomyelin, lysophosphatidic acid and sulfatides, were found in EVs compared to cells. Furthermore, H-RasV12 expression in fibroblasts was associated with higher levels of tetraspanins involved in vesicle formation.

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          Exosome lipidomics unravels lipid sorting at the level of multivesicular bodies.

          Exosomes are part of the family of "bioactive vesicles" and appear to be involved in distal communications between cells. They vehiculate bioactive lipids and lipolytic enzymes and their biogenesis require specific lipids and a membrane reorganisation. Their biogenesis pathway could be a way to secrete enzymes involved in lipid signalling and to generate "particulate agonists". However, this pathway seems also to be used by pathogens such as HIV. This review will consider several aspects of lipidomics studies which might help to understand the fate and role of these fascinating vesicles.
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            Senescence-associated exosome release from human prostate cancer cells.

            Males of advanced age represent a rapidly growing population at risk for prostate cancer. In the contemporary setting of earlier detection, a majority of prostate carcinomas are still clinically localized and often treated using radiation therapy. Our recent studies have shown that premature cellular senescence, rather than apoptosis, accounts for most of the clonogenic death induced by clinically relevant doses of irradiation in prostate cancer cells. We show here that this treatment-induced senescence was associated with a significantly increased release of exosome-like microvesicles. In premature senescence, this novel secretory phenotype was dependent on the activation of p53. In addition, the release of exosome-like microvesicles also increased during proliferative senescence in normal human diploid fibroblasts. These data support the hypothesis that senescence, initiated either by telomere attrition (e.g., aging) or DNA damage (e.g., radiotherapy), may induce a p53-dependent increase in the biogenesis of exosome-like vesicles. Ultrastructural analysis and RNA interference-mediated knockdown of Tsg101 provided significant evidence that the additional exosomes released by prematurely senescent prostate cancer cells were principally derived from multivesicular endosomes. Moreover, these exosomes were enriched in B7-H3 protein, a recently identified diagnostic marker for prostate cancer, and an abundance of what has recently been termed "exosomal shuttle RNA." Our findings are consistent with the proposal that exosomes can transfer cargos, with both immunoregulatory potential and genetic information, between cells through a novel mechanism that may be recruited to increase exosome release during accelerated and replicative cellular senescence.
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              Molecular lipidomics of exosomes released by PC-3 prostate cancer cells.

              The molecular lipid composition of exosomes is largely unknown. In this study, sophisticated shotgun and targeted molecular lipidomic assays were performed for in-depth analysis of the lipidomes of the metastatic prostate cancer cell line, PC-3, and their released exosomes. This study, based in the quantification of approximately 280 molecular lipid species, provides the most extensive lipid analysis of cells and exosomes to date. Interestingly, major differences were found in the lipid composition of exosomes compared to parent cells. Exosomes show a remarkable enrichment of distinct lipids, demonstrating an extraordinary discrimination of lipids sorted into these microvesicles. In particular, exosomes are highly enriched in glycosphingolipids, sphingomyelin, cholesterol, and phosphatidylserine (mol% of total lipids). Furthermore, lipid species, even of classes not enriched in exosomes, were selectively included in exosomes. Finally, it was found that there is an 8.4-fold enrichment of lipids per mg of protein in exosomes. The detailed lipid composition provided in this study may be useful to understand the mechanism of exosome formation, release and function. Several of the lipids enriched in exosomes could potentially be used as cancer biomarkers.
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                Author and article information

                Contributors
                Role: InvestigationRole: MethodologyRole: SupervisionRole: Writing – original draftRole: Writing – review & editing
                Role: InvestigationRole: MethodologyRole: SupervisionRole: Writing – original draftRole: Writing – review & editing
                Role: InvestigationRole: Methodology
                Role: InvestigationRole: MethodologyRole: Validation
                Role: InvestigationRole: MethodologyRole: Validation
                Role: MethodologyRole: Supervision
                Role: InvestigationRole: MethodologyRole: SupervisionRole: Validation
                Role: MethodologyRole: SupervisionRole: Validation
                Role: SupervisionRole: Writing – review & editing
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                28 November 2017
                2017
                : 12
                : 11
                : e0188840
                Affiliations
                [1 ] Department of Chemistry, Biology and Biotechnology, University of Perugia, Perugia, Italy
                [2 ] Department of Pharmaceutical Sciences, University of Perugia, Perugia, Italy
                [3 ] Istituto Officina dei Materiali del CNR (CNR-IOM) - Unità di Perugia, c/o Department of Physics and Geology, University of Perugia, Perugia, Italy
                [4 ] Department of Physics and Geology, University of Perugia, Perugia, Italy
                [5 ] CEMIN-Center of Excellence for Innovative Nanostructured Material, Perugia, Italy
                [6 ] Department of Pharmacological and Biomolecular Sciences, University of Milan, Milan, Italy
                Universita degli Studi di Torino, ITALY
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Author information
                http://orcid.org/0000-0003-0621-8476
                Article
                PONE-D-17-24724
                10.1371/journal.pone.0188840
                5705128
                29182668
                1ae1dc19-00bd-4310-86d8-f11490ca5cd4
                © 2017 Buratta et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 30 June 2017
                : 14 November 2017
                Page count
                Figures: 8, Tables: 0, Pages: 23
                Funding
                Funded by: funder-id http://dx.doi.org/10.13039/501100004486, Fondazione Cassa di Risparmio di Perugia;
                Award ID: 2016.0050.021
                Award Recipient :
                This work was supported by Fondazione Cassa di Risparmio di Perugia grant 2016.0050.021 to Prof. Carla Emiliani. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and Life Sciences
                Cell Biology
                Cellular Types
                Animal Cells
                Connective Tissue Cells
                Fibroblasts
                Biology and Life Sciences
                Anatomy
                Biological Tissue
                Connective Tissue
                Connective Tissue Cells
                Fibroblasts
                Medicine and Health Sciences
                Anatomy
                Biological Tissue
                Connective Tissue
                Connective Tissue Cells
                Fibroblasts
                Biology and Life Sciences
                Biochemistry
                Lipids
                Biology and Life Sciences
                Molecular Biology
                Macromolecular Structure Analysis
                Lipid Analysis
                Biology and Life Sciences
                Cell Biology
                Cellular Structures and Organelles
                Vesicles
                Biology and Life Sciences
                Biochemistry
                Lipids
                Sphingolipids
                Biology and Life Sciences
                Biochemistry
                Lipids
                Lipid Profiles
                Research and Analysis Methods
                Mathematical and Statistical Techniques
                Statistical Methods
                Multivariate Analysis
                Principal Component Analysis
                Physical Sciences
                Mathematics
                Statistics (Mathematics)
                Statistical Methods
                Multivariate Analysis
                Principal Component Analysis
                Biology and Life Sciences
                Biochemistry
                Lipids
                Phospholipids
                Custom metadata
                All relevant data are within the paper and its Supporting Information files.

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