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1.
Nature ; 579(7798): 260-264, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32132711

RESUMO

The production of pore-forming toxins that disrupt the plasma membrane of host cells is a common virulence strategy for bacterial pathogens such as methicillin-resistant Staphylococcus aureus (MRSA)1-3. It is unclear, however, whether host species possess innate immune mechanisms that can neutralize pore-forming toxins during infection. We previously showed that the autophagy protein ATG16L1 is necessary for protection against MRSA strains encoding α-toxin4-a pore-forming toxin that binds the metalloprotease ADAM10 on the surface of a broad range of target cells and tissues2,5,6. Autophagy typically involves the targeting of cytosolic material to the lysosome for degradation. Here we demonstrate that ATG16L1 and other ATG proteins mediate protection against α-toxin through the release of ADAM10 on exosomes-extracellular vesicles of endosomal origin. Bacterial DNA and CpG DNA induce the secretion of ADAM10-bearing exosomes from human cells as well as in mice. Transferred exosomes protect host cells in vitro by serving as scavengers that can bind multiple toxins, and improve the survival of mice infected with MRSA in vivo. These findings indicate that ATG proteins mediate a previously unknown form of defence in response to infection, facilitating the release of exosomes that serve as decoys for bacterially produced toxins.


Assuntos
Proteínas Relacionadas à Autofagia/metabolismo , Toxinas Bacterianas/metabolismo , Exossomos/metabolismo , Células A549 , Proteína ADAM10/metabolismo , Animais , Toxinas Bacterianas/farmacologia , Sobrevivência Celular/efeitos dos fármacos , DNA Bacteriano/farmacologia , Exossomos/efeitos dos fármacos , Exossomos/ultraestrutura , Feminino , Células HEK293 , Humanos , Masculino , Staphylococcus aureus Resistente à Meticilina/patogenicidade , Staphylococcus aureus Resistente à Meticilina/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Infecções Estafilocócicas/mortalidade
2.
Life Sci ; 246: 117401, 2020 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-32035931

RESUMO

AIMS: The management of acute liver failure (ALF) is a major challenge worldwide. The current study aimed to determine the therapeutic potential of TNF-α pretreatment of umbilical cord mesenchymal stem cell-derived exosomes (T-Exo) in ALF. MAIN METHODS: Here, we enriched T-Exo and untreated exosomes (Exo), them were measured by nanoparticle tracking analysis (NTA) for particle size detection and identified surface marker by Western blot and flow cytometry. Then the cell proliferation was detected by CCK-8 and the effect of T-Exo on the expression levels of pro-inflammatory cytokines was tested by ELISA. ALF mouse models were induced by LPS and D-GalN. H&E staining, immunohistochemistry, and Western blot were used to detect the effect of T-Exo on the levels of NLRP3 and other inflammation-related pathway proteins. qPCR was used to detect the expression level of microRNA-299-3p in T-Exo and its transfer to macrophages. Laser confocal microscopy was used to detect colocalization of exosomes,Golgi and NLRP3 in macrophages. KEY FINDINGS: Our study shows that T-Exo can reduce serum ALT, AST and proinflammatory cytokines level and inhibit activation of NLRP3 inflammation-associated pathway proteins. T-Exo treatment reduces pathological liver damage caused by ALF. Anti-inflammatory-related miRNA-299-3p is up-regulated in TNF-α-stimulated MSCs and selectively packaged into exosomes for role in exosomal treatment. And conducted preliminary exploration and hypothesis on the specific mechanism of this effect. SIGNIFICANCE: These in vitro and in vivo studies indicate that T-Exo attenuates inflammatory damage caused by ALF and promotes liver tissue repair by inhibiting the activation of the NLRP3 pathway.


Assuntos
Exossomos/efeitos dos fármacos , Falência Hepática Aguda/terapia , Macrófagos/metabolismo , Células-Tronco Mesenquimais/efeitos dos fármacos , Proteína 3 que Contém Domínio de Pirina da Família NLR/antagonistas & inibidores , Fator de Necrose Tumoral alfa/farmacologia , Animais , Western Blotting , Ensaio de Imunoadsorção Enzimática , Exossomos/fisiologia , Exossomos/transplante , Humanos , Testes de Função Hepática , Macrófagos/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Camundongos , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Reação em Cadeia da Polimerase , Células RAW 264.7 , Cordão Umbilical/citologia
3.
Int J Nanomedicine ; 14: 8121-8132, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31632022

RESUMO

Introduction: Exosomes are important mediators of intercellular communication. Previously, we characterized circulating exosomal miR-425-3p as a non-invasive prognostic marker for predicting clinical response to platinum-based chemotherapy in patients with non-small cell lung cancer (NSCLC). Methods: Circulating exosomal miR-425-3p was validated by qRT-PCR in paired serum samples from NSCLC patients during the course of platinum-based chemotherapy. Cell coculture was performed to examine the effects of exosomal miR-425-3p on the sensitivity of recipient A549 cells to cisplatin. Using bioinformatics, ChIP and luciferase reporter assays, the transcription factor essential for miR-425-3p expression was identified. Autophagic activity in the recipient cells was determined by Western blot and fluorescence microscopy. Results: Higher levels of exosomal miR-425-3p were found in serum samples from the patients in tolerance versus those at baseline. An upward trend in the expression of circulating exosomal miR-425-3p was revealed during chemotherapy. Furthermore, the expression of exosomal miR-425-3p could be induced by cisplatin in NSCLC cells. Exosomes isolated from either cisplatin-treated or cisplatin-resistant NSCLC cells conferred chemoresistance to sensitive A549 cells in a miR-425-3p-dependent manner. Cisplatin-induced c-Myc was found to directly bind the miR-425-3p promoter and transactivated its expression. Exosomal miR-425-3p facilitated autophagic activation in the recipient cells by targeting AKT1, eventually leading to chemoresistance. Discussion: Our results suggest that apart from a prognostic marker of treatment response, exosomal miR-425-3p might be a potential dynamic biomarker to tailor cisplatin resistance in NSCLC patients during the treatment and represent a promising therapeutic target for therapy-resistant NSCLC.


Assuntos
Autofagia/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/patologia , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Exossomos/metabolismo , Neoplasias Pulmonares/patologia , MicroRNAs/metabolismo , Células A549 , Apoptose/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Cisplatino/uso terapêutico , Exossomos/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , MicroRNAs/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genética , beta Catenina/metabolismo
4.
Toxicol Lett ; 316: 49-59, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31520698

RESUMO

Epidemiological studies have established the correlations between PM2.5 and a wide variety of pulmonary diseases. However, their underlying pathogeneses have not been clearly elucidated yet. In the present study, the epithelial-mesenchymal transition (EMT) phenotype with enhanced proliferation and migration activity of human pulmonary epithelial cell line BEAS-2B was observed after exposure to low dose PM2.5 exposure (50 µg/ml) for 30 passages. Then, epithelial cells derived-exosomal micro-RNA (miRNA) and intracellular total RNA were extracted, and the differentially expressed exosomal miRNAs (DE-Exo-MiRs) as well as differentially expressed protein coding genes (DEGs) were identified by RNA sequencing (RNA-seq) and transcriptome analysis. We found that chronic PM2.5 exposure stimulated the release of pulmonary epithelium derived exosomes. 45 DE-Exo-MiRs including 32 novelly predicted miRNAs and 843 DEGs between PM2.5 exposed group and the normal control were detected. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses showed that DEGs were significantly enriched in extracellular matrix organization, focal adhesion and cancer related terms. Besides, the enrichment analyses on 7774 mRNA targets of 27 DE-Exo-MiRs predicted by MiRanda software also revealed the potential regulatory role of exosomal miRNAs in pathways in cancer, Wingless/Integrated (Wnt) signaling pathway, focal adhesion related genes and other multiple pathogenic pathways. Moreover, the interactive exosomal miRNA-mRNA pair networks were constructed using Cytoscape software. Our results provided a novel basis for a better understanding of the mechanisms of chronic PM2.5 exposure induced pulmonary disorders including pulmonary fibrosis and cancer, in which exosomal miRNAs (Exo-MiRs) potentially functions by dynamically regulating gene expressions.


Assuntos
Células Epiteliais/efeitos dos fármacos , Exossomos/efeitos dos fármacos , Perfilação da Expressão Gênica/métodos , Pulmão/efeitos dos fármacos , MicroRNAs/genética , Material Particulado/toxicidade , RNA Mensageiro/genética , Transcriptoma/efeitos dos fármacos , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Biologia Computacional , Bases de Dados Genéticas , Células Epiteliais/metabolismo , Células Epiteliais/ultraestrutura , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transição Epitelial-Mesenquimal/genética , Exossomos/genética , Exossomos/metabolismo , Exossomos/patologia , Redes Reguladoras de Genes , Humanos , Pulmão/metabolismo , Pulmão/ultraestrutura , MicroRNAs/metabolismo , Tamanho da Partícula , RNA Mensageiro/metabolismo , Medição de Risco , Fatores de Tempo , Testes de Toxicidade Crônica
5.
Mol Cell Biochem ; 462(1-2): 115-122, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31473883

RESUMO

Rasal2, a Ras-GTPase-activating protein (RasGAP), is a tumor suppressor in Luminal B breast cancer, frequently metastatic and recurrent. Exosomes (Exos) are small membrane vesicles secreted by various cell types, including tumor cells, recognized as vehicles for cell-to-cell communication. Our study aimed to investigate whether Rasal2 regulates breast cancer cell growth via affecting this process. In this paper, we described that Rasal2 knockout (KO) in MCF-7 cells enhanced exosomal release and increased autophagy-related proteins in exosomal fraction, while attenuated by exosome release inhibitor GW4869. Moreover, MCF-7 cells with chloroquine (CQ) treatment boosted Rasal2 KO-induced secretory autophagy. In addition, we presented that exosomes derived from KO MCF-7 cells (KO-exo) significantly promoted breast cancer cell proliferation compared to those from MCF-7 cells transfected with an empty crispr-cas9 plasmid serving as controls (sgNT-exo); however, exosomes purified from KO MCF-7 cells co-cultured with 3-methyladenine ((3-MA + KO)-exo)/CQ ((CQ + KO)-exo) dramatically inhibited/facilitated MCF-7 cell proliferation in contrast to KO-exo group, separately. In conclusion, our findings revealed a new mechanism of Rasal2 in the regulation of breast cancer cell proliferation via autophagy-exo-mediated pathway.


Assuntos
Autofagia , Neoplasias da Mama/patologia , Proteínas Ativadoras de GTPase/metabolismo , Autofagia/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Cloroquina/farmacologia , Exossomos/efeitos dos fármacos , Exossomos/metabolismo , Exossomos/ultraestrutura , Feminino , Humanos , Células MCF-7
6.
Int J Mol Sci ; 20(15)2019 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-31370144

RESUMO

Normally ubiquitin C-terminal hydrolase L1 (UCH-L1) is expressed in the central nervous and reproductive systems of adults, but its de novo expression has been detected in many human cancers. There is a growing body of evidence that UCH-L1 de-ubiquitinating (DUB) activity plays a major pro-metastatic role in certain carcinomas. Here we tested anti-metastatic effects of the small-molecule inhibitor of UCH-L1 DUB activity, LDN-57444, in cell lines from advanced oral squamous cell carcinoma (OSCC) as well as invasive nasopharyngeal (NP) cell lines expressing the major pro-metastatic gene product of Epstein-Barr virus (EBV) tumor virus, LMP1. To overcome the limited aqueous solubility of LDN-57444 we developed a nanoparticle formulation of LDN-57444 by incorporation of the compound in polyoxazoline micellear nanoparticles (LDN-POx). LDN-POx nanoparticles were equal in effects as the native compound in vitro. Our results demonstrate that inhibition of UCH-L1 DUB activity with LDN or LDN-POx inhibits secretion of exosomes and reduces levels of the pro-metastatic factor in exosomal fractions. Both forms of UCH-L1 DUB inhibitor suppress motility of metastatic squamous carcinoma cells as well as nasopharyngeal cells expressing EBV pro-metastatic Latent membrane protein 1 (LMP1) in physiological assays. Moreover, treatment with LDN and LDN-POx resulted in reduced levels of pro-metastatic markers, a decrease of carcinoma cell adhesion, as well as inhibition of extra-cellular vesicle (ECV)-mediated transfer of viral invasive factor LMP1. We suggest that soluble inhibitors of UCH-L1 such as LDN-POx offer potential forms of treatment for invasive carcinomas including EBV-positive malignancies.


Assuntos
Antineoplásicos/farmacologia , Portadores de Fármacos , Células Epiteliais/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Indóis/farmacologia , Oximas/farmacologia , Ubiquitina Tiolesterase/genética , Proteínas da Matriz Viral/genética , Antineoplásicos/química , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Exossomos/efeitos dos fármacos , Exossomos/metabolismo , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/patogenicidade , Humanos , Indóis/química , Micelas , Boca/metabolismo , Boca/patologia , Nanopartículas/química , Nanopartículas/ultraestrutura , Nasofaringe/metabolismo , Nasofaringe/patologia , Oxazóis/química , Oximas/química , Ubiquitina Tiolesterase/antagonistas & inibidores , Ubiquitina Tiolesterase/metabolismo , Proteínas da Matriz Viral/metabolismo
7.
Int J Mol Sci ; 20(15)2019 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-31370159

RESUMO

Mesenchymal stem cells (MSCs) are present in all organs and tissues, playing a well-known function in tissue regeneration. However, there is also evidence indicating a broader role of MSCs in tissue homeostasis. In vivo studies have shown MSC paracrine mechanisms displaying proliferative, immunoregulatory, anti-oxidative, or angiogenic activity. In addition, recent studies also demonstrate that depletion and/or dysfunction of MSCs are associated with several systemic diseases, such as lupus, diabetes, psoriasis, and rheumatoid arthritis, as well as with aging and frailty syndrome. In this review, we hypothesize about the role of MSCs as keepers of tissue homeostasis as well as modulators in a variety of inflammatory and degenerative systemic diseases. This scenario opens the possibility for the use of secretome-derived products from MSCs as new therapeutic agents in order to restore tissue homeostasis, instead of the classical paradigm "one disease, one drug".


Assuntos
Anti-Inflamatórios/uso terapêutico , Artrite Reumatoide/tratamento farmacológico , Diabetes Mellitus/tratamento farmacológico , Lúpus Eritematoso Sistêmico/tratamento farmacológico , Células-Tronco Mesenquimais/efeitos dos fármacos , Psoríase/tratamento farmacológico , Idoso , Envelhecimento/genética , Animais , Artrite Reumatoide/genética , Artrite Reumatoide/metabolismo , Artrite Reumatoide/patologia , Contagem de Células , Diabetes Mellitus/genética , Diabetes Mellitus/metabolismo , Diabetes Mellitus/patologia , Modelos Animais de Doenças , Exossomos/efeitos dos fármacos , Exossomos/metabolismo , Idoso Fragilizado , Homeostase/efeitos dos fármacos , Homeostase/genética , Humanos , Lúpus Eritematoso Sistêmico/genética , Lúpus Eritematoso Sistêmico/metabolismo , Lúpus Eritematoso Sistêmico/patologia , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/patologia , Comunicação Parácrina/efeitos dos fármacos , Psoríase/genética , Psoríase/metabolismo , Psoríase/patologia
8.
Biol Pharm Bull ; 42(8): 1394-1401, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31366874

RESUMO

Autophagy-lysosome proteolysis is involved in protein quality control and classified into macroautophagy (MA), microautophagy (mA) and chaperone-mediated autophagy (CMA), by the routes of substrate delivery to lysosomes. Both autophagy-lysosome proteolysis and exosome release are strongly associated with membrane trafficking. In the present study, we investigated how chemical and small interfering RNA (siRNA)-mediated activation and inhibition of these autophagic pathways affect exosome release in AD293 cells. Activation of MA and mA by rapamycin and activation of CMA by mycophenolic acid significantly decreased exosome release. Although lysosomal inhibitors, NH4Cl and bafilomycin A1, significantly increased exosome release, a MA inhibitor, 3-methyladenine, did not affect. Exosome release was significantly increased by the siRNA-mediated knockdown of LAMP2A, which is crucial for CMA. Inversely, activity of CMA/mA was significantly increased by the prevention of exosome release, which was induced by siRNA-mediated knockdown of Rab27a. These findings indicate that CMA/mA and exosome release are reciprocally regulated. This regulation would be the molecular basis of extracellular release and propagation of misfolded proteins in various neurodegenerative diseases.


Assuntos
Exossomos , Adenina/análogos & derivados , Adenina/farmacologia , Cloreto de Amônio/farmacologia , Linhagem Celular , /genética , Exossomos/efeitos dos fármacos , Exossomos/genética , Humanos , Proteína 2 de Membrana Associada ao Lisossomo/genética , Macrolídeos/farmacologia , /genética , Ácido Micofenólico/farmacologia , RNA Interferente Pequeno/genética , Sirolimo/farmacologia
9.
Mol Cell Biochem ; 462(1-2): 1-10, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31468244

RESUMO

Diabetic cardiomyopathy is known to involve two forms of cardiac cell death: apoptosis and necrosis. However, it remains unknown whether hyperglycemia-induced apoptosis in the H9c2 cell culture system is inhibited by parasympathetic ganglionic neurons (PGN) derived exosomes (exos). We isolated PGN and sympathetic ganglionic neurons (SGN) from the right stellate ganglion in rats, and derived exos from these sources. H9c2 cells were divided into 4 groups: (1) Control, (2) H9c2 + Glucose (100 mmol/L), (3) H9c2 + Glucose + PGN-exos, and (4) H9c2 + Glucose + SGN-exos. We determined cell proliferation and viability with an MTT assay kit, and assessed apoptotic cell death with TUNEL staining and ELISA. Data were further confirmed by analyzing the presence of pro-apoptotic proteins Caspase-3 and Bax, and anti-apoptotic protein Bcl-2. Glucose exposed H9c2 cells significantly reduced cell viability, which was improved by PGN-exos, but not by SGN-exos. Furthermore, increased apoptosis in hyperglycemia in H9c2 cells was confirmed with TUNEL staining and cell death ELISA which demonstrated significantly (p < 0.05) reduction with PGN-exos treatment, but not with SGN-exos. Moreover, high expression of pro-apoptotic proteins Caspase-3 and Bax was reduced following treatment with PGN-exos; however, SGN-exos were unable to reduce the expression. Significantly reduced anti-apoptotic protein Bcl-2 following glucose treatment was improved with PGN-exos. Therefore, our data suggest that hyperglycemia induces apoptosis in H9c2 cells and decreases cell viability, and that PGN-exos are able to inhibit apoptosis, improve cell viability, and restore levels of anti-apoptotic protein Bcl-2.


Assuntos
Apoptose , Exossomos/metabolismo , Gânglios Parassimpáticos/patologia , Hiperglicemia/patologia , Miócitos Cardíacos/patologia , Neurônios/patologia , Animais , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Exossomos/efeitos dos fármacos , Glucose/toxicidade , Masculino , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Ratos Sprague-Dawley , Proteína X Associada a bcl-2/metabolismo
10.
Int J Mol Sci ; 20(13)2019 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-31284382

RESUMO

The intestinal microvasculature (iMV) plays multiple pathogenic roles during chronic inflammatory bowel disease (IBD). The iMV acts as a second line of defense and is, among other factors, crucial for the innate immunity in the gut. It is also the therapeutic location in IBD targeting aggravated leukocyte adhesion processes involving ICAM-1 and E-selectin. Specific targeting is stressed via nanoparticulate drug vehicles. Evaluating the iMV in enterocyte barrier models in vitro could shed light on inflammation and barrier-integrity processes during IBD. Therefore, we generated a barrier model by combining the enterocyte cell line Caco-2 with the microvascular endothelial cell line ISO-HAS-1 on opposite sides of a transwell filter-membrane under culture conditions which mimicked the physiological and inflamed conditions of IBD. The IBD model achieved a significant barrier-disruption, demonstrated via transepithelial-electrical resistance (TER), permeability-coefficient (Papp) and increase of sICAM sE-selectin and IL-8. In addition, the impact of a prospective model drug-vehicle (silica nanoparticles, aSNP) on ongoing inflammation was examined. A decrease of sICAM/sE-selectin was observed after aSNP-exposure to the inflamed endothelium. These findings correlated with a decreased secretion of ICAM/E-selectin bearing exosomes/microvesicles, as evaluated via ELISA. Our findings indicate that aSNP treatment of the inflamed endothelium during IBD may hamper exosomal/microvesicular systemic communication.


Assuntos
Exossomos/metabolismo , Inflamação/patologia , Nanopartículas/toxicidade , Dióxido de Silício/toxicidade , Células CACO-2 , Selectina E/metabolismo , Impedância Elétrica , Exossomos/efeitos dos fármacos , Humanos , Molécula 1 de Adesão Intercelular/metabolismo
11.
J Exp Clin Cancer Res ; 38(1): 320, 2019 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-31324203

RESUMO

BACKGROUND: Acquired resistance remains a limitation of the clinical use of 5-fluorouracil (5-FU). Because exosomes, are important vesicles participating in intercellular communication, their contribution to the development of acquired 5-FU resistance needs to be elucidated. In this study, we aimed to examine the underlying mechanisms of exosomes from 5-FU resistant cells (RKO/R) in sustaining acquired 5-FU resistance in sensitive cells (RKO/P). METHODS: Exosomes from a 5-FU-resistant cell line (RKO/R) and its parental cell line RKO/P were isolated and co-cultured with 5-FU-sensitive cells. Real-time cellular analysis (RTCA) and FACS analysis were used to examine cell viability and apoptosis. Exosomal protein profiling was performed using shotgun proteomics. Inhibitors and siRNAs were applied to study the involvement of selected proteins in 5-FU resistance. The effect of exosomal p-STAT3 (Tyr705) on the caspase cascade was examined by western blotting (WB) and high content analysis. Xenograft models were established to determine whether exosomal p-STAT3 can induce 5-FU resistance in vivo. RESULTS: Our results indicated that exosomes from RKO/R cells significantly promoted cell survival during 5-FU treatment. Proteomics and WB analysis results indicated that GSTP1 and p-STAT3 (Tyr705) were enriched in exosomes from RKO/R cells. Inhibition of p-STAT3 re-sensitized RKO/P cells to 5-FU via caspase cascade. Furthermore, p-STAT3 packaged by exosomes from RKO/R cells increased resistance of tumor cells to 5-FU in vivo. CONCLUSIONS: Our results reveal a novel mechanism by which p-STAT3-containing exosomes contribute to acquired 5-FU resistance in CRC. This study suggests a new option for potentiating the 5-FU response and finding biomarkers for chemotherapy resistance.


Assuntos
Neoplasias Colorretais/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/genética , Fluoruracila/farmacologia , Fator de Transcrição STAT3/genética , Animais , Apoptose/efeitos dos fármacos , Biomarcadores Tumorais/genética , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Exossomos/efeitos dos fármacos , Exossomos/genética , Citometria de Fluxo , Fluoruracila/efeitos adversos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glutationa S-Transferase pi/genética , Células HCT116 , Humanos , Camundongos , MicroRNAs/genética , Análise de Sequência de RNA , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
12.
PLoS One ; 14(7): e0220036, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31329632

RESUMO

Exosomes might have an unimproved potential to serve as effective delivery vehicles. However, when exosomes are developed for therapeutic applications, a method to enhance their delivery is important. This study aimed to evaluate wheather calcium chloride (CaCl2) or other chloride compounds could enhance exosome delivery to various cells without causing toxicity. Exosomes were purified from human serum by using the ExoQuick exosome precipitation kit. Isolated exosomes were mixed with CaCl2 at concentrations ranging from 100 µM to 1 mM, and then washed using Amicon filter for treating the cells. The delivery efficiency of exosomes and the viability of the cells [HEK 293 (human kidney cells) and H9C2 (rat cardiomyocytes)] were evaluated. Cellular uptake of exosomes was observed using a confocal microscope based on PKH26 labeling of exosomes. CaCl2 increased the delivery of exosomes in a dose- and treatment time-dependent manner. In HEK 293 cells, a CaCl2 concentration of 400 µM and exposure time of 12 h increased the delivery of exosomes by >20 times compared with controls. In H9C2 cells, a CaCl2 concentration of 400 µM and exposure time of >24 h increased the delivery of exosomes by >400 times compared with controls. The viability of both cell lines was maintained up to a CaCl2 concentration of 1 mM. However, cobalt chloride, cupric chloride, and magnesium chloride did not change the delivery of exosomes in both cell lines. These results suggest that the use of CaCl2 treatment might be a useful method for enhancing the delivery of exosomes.


Assuntos
Cloreto de Cálcio/farmacologia , Exossomos/metabolismo , Via Secretória , Animais , Células Cultivadas , Exocitose , Exossomos/efeitos dos fármacos , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Ratos
13.
Int J Mol Sci ; 20(11)2019 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-31146391

RESUMO

The noble gas helium (He) induces cardioprotection in vivo through unknown molecular mechanisms. He can interact with and modify cellular membranes. Caveolae are cholesterol and sphingolipid-enriched invaginations of the plasma-membrane-containing caveolin (Cav) proteins that are critical in protection of the heart. Mice (C57BL/6J) inhaled either He gas or adjusted room air. Functional measurements were performed in the isolated Langendorff perfused heart at 24 h post He inhalation. Electron paramagnetic resonance spectrometry (EPR) of samples was carried out at 24 h post He inhalation. Immunoblotting was used to detect Cav-1/3 expression in whole-heart tissue, exosomes isolated from platelet free plasma (PFP) and membrane fractions. Additionally, transmission electron microscopy analysis of cardiac tissue and serum function and metabolomic analysis were performed. In contrast to cardioprotection observed in in vivo models, the isolated Langendorff perfused heart revealed no protection after He inhalation. However, levels of Cav-1/3 were reduced 24 h after He inhalation in whole-heart tissue, and Cav-3 was increased in exosomes from PFP. Addition of serum to muscle cells in culture or naïve ventricular tissue increased mitochondrial metabolism without increasing reactive oxygen species generation. Primary and lipid metabolites determined potential changes in ceramide by He exposure. In addition to direct effects on myocardium, He likely induces the release of secreted membrane factors enriched in caveolae. Our results suggest a critical role for such circulating factors in He-induced organ protection.


Assuntos
Cardiotônicos/farmacologia , Caveolinas/metabolismo , Coração/efeitos dos fármacos , Hélio/farmacologia , Traumatismo por Reperfusão Miocárdica/tratamento farmacológico , Animais , Cardiotônicos/uso terapêutico , Cavéolas/efeitos dos fármacos , Cavéolas/metabolismo , Caveolinas/sangue , Caveolinas/genética , Células Cultivadas , Exossomos/efeitos dos fármacos , Exossomos/metabolismo , Hélio/uso terapêutico , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias Cardíacas/efeitos dos fármacos , Mitocôndrias Cardíacas/metabolismo , Traumatismo por Reperfusão Miocárdica/prevenção & controle
14.
Biomed Res Int ; 2019: 2858750, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31119161

RESUMO

Pulmonary arterial hypertension (PAH) is a serious condition. However, prevailing therapeutic strategies are not effective enough to treat PAH. Therefore, finding an effective therapy is clearly warranted. Adipose-derived mesenchymal stem cells (ASCs) and ASCs-derived exosomes (ASCs-Exos) exert protective effects in PAH, but the underlying mechanism remains unclear. Using a coculture of ASCs and monocrotaline pyrrole (MCTP)-treated human pulmonary artery endothelial cells (HPAECs), we demonstrated that ASCs increased cell proliferation in MCTP-treated HPAECs. Results showed that ASCs-Exos improved proliferation of both control HPAECs and MCTP-treated HPAECs. In addition, by transfecting ASCs with antagomir we observed that low exosomal miR-191 expression inhibited HPAECs proliferation whereas the agomir improved. Similar results were observed in vivo using a monocrotaline (MCT)-induced PAH rat model following ASCs transplantation. And ASCs transplantation attenuated MCT-induced PAH albeit less than the antagomir treated group. Finally, we found that miR-191 repressed the expression of bone morphogenetic protein receptor 2 (BMPR2) in HPAECs and PAH rats. Thus, we conjectured that miR-191, in ASCs and ASCs-Exos, plays an important role in PAH via regulation of BMPR2. These findings are expected to contribute to promising therapeutic strategies for treating PAH in the future.


Assuntos
Receptores de Proteínas Morfogenéticas Ósseas Tipo II/genética , Hipertensão Pulmonar/genética , Células-Tronco Mesenquimais/metabolismo , MicroRNAs/genética , Animais , Linhagem Celular , Proliferação de Células/genética , Células Endoteliais/metabolismo , Exossomos/efeitos dos fármacos , Exossomos/genética , Regulação da Expressão Gênica/genética , Humanos , Hipertensão Pulmonar/induzido quimicamente , Hipertensão Pulmonar/metabolismo , Hipertensão Pulmonar/patologia , Monocrotalina/toxicidade , Artéria Pulmonar/efeitos dos fármacos , Artéria Pulmonar/patologia , Ratos , Ratos Sprague-Dawley
15.
PLoS One ; 14(5): e0217394, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31136600

RESUMO

Recently, we showed that imidazole dipeptide such as carnosine contained abundantly in chicken breast meat improves brain function in a double-blind randomized controlled trial. However, the underlying molecular mechanisms remain unknown. Here, we investigated whether carnosine activates intestinal epithelial cells and induces the secretion of factors that activate brain function. We focused on exosomes derived from intestinal epithelial cells as mediators of brain-gut interaction. Results showed that exosomes derived from Caco-2 cells treated with carnosine significantly induced neurite growth in SH-SY5Y cells. To clarify the molecular basis of this finding, we performed integrated analysis of microRNAs (miRNAs) with altered expression in exosomes in response to carnosine treatment and mRNAs with altered expression in target cells in response to exosome treatment to identify related miRNAs and their target genes. The combination of miR-6769-5p and its target gene ATXN1 was found to be involved in the exosome-induced activation of neuronal cells.


Assuntos
Carnosina/farmacologia , Exossomos/efeitos dos fármacos , Exossomos/metabolismo , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Neurônios/metabolismo , Ataxina-1/genética , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Células CACO-2 , Linhagem Celular , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Exossomos/genética , Perfilação da Expressão Gênica , Humanos , Mucosa Intestinal/citologia , MicroRNAs/genética , MicroRNAs/metabolismo
16.
Mol Med Rep ; 20(1): 323-331, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31115533

RESUMO

Hypermethylation of transcription factor activating enhancer­binding protein 2e (TFAP2E) has been reported to be associated with chemoresistance to 5­fluorouracil (5­FU) in gastric cancer (GC). In the present study, the molecular mechanism governing this chemoresistance was investigated. Drug­resistant human GC MGC­803/5­FU cells were established and TFAP2E expression and methylation levels were assessed. Autocrine exosomes from GC culture medium were isolated and characterized. MicroRNA (miRNA) microarray analysis was used to determine the miRNA expression profile of GC cell­derived exosomes. Exosomes collected from MGC­803/5­FU cells were co­cultured with control cells, and 5­Aza­2'­deoxycytidine (5Aza) was added into MGC­803/5­FU cells to investigate the relationship between TFAP2E, exosomes and chemosensitivity. In the present study, it was demonstrated that hypermethylation of TFAP2E resulted in its reduced expression and 5­FU chemoresistance in GC cells. miRNAs miR­106a­5p and miR­421 were highly expressed and regulated the chemoresistance induced by TFAP2E methylation. Target gene prediction using miRBase, TargetScan and PicTar revealed that E2F1, MTOR and STAT3 may be TFAP2E target genes in GC. Collectively, our data support an important role of exosomes and exosomal miRNAs in TFAP2E methylation­induced chemoresistance to 5­FU in GC. These results highlight their potential for miRNA­based therapeutics.


Assuntos
Fluoruracila/farmacologia , MicroRNAs/genética , Neoplasias Gástricas/tratamento farmacológico , Fator de Transcrição AP-2/genética , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Metilação de DNA , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Exossomos/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Metilação/efeitos dos fármacos , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia
17.
QJM ; 112(8): 581-590, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31106370

RESUMO

BACKGROUND: Epithelial-mesenchymal transition (EMT) is an essential component of metastasis. Our previous study demonstrated that cancer-associated fibroblasts (CAFs) induce EMT in lung cancer cells. In recent years, many studies have demonstrated that CAFs induce metastasis and drug resistance in cancer cells via exosomes. AIM: We sought to discover the mechanism underlying how CAFs induce EMT in lung cancer cells, unveiling the role of exosomes in lung cancer progression. DESIGN: We cultured lung cancer cell (i) with control medium, normal fibroblasts (NFs) or CAFs; (ii) with SNAI1-transfected or NC (negative control)-transfected CAFs; (iii) with exosomes extracted from NF- or CAF-conditioned medium; (iv) with exosomes released by SNAI1 or NC-transfected CAFs; (v) with CAF-conditioned medium or exosome-depleted CAF-conditioned medium. METHODS: qRT-PCR was conducted to examine the expression of CDH1 (gene of E-cadherin) and VIM (gene of Vimentin), western blotting was conducted to examine E-cadherin and vimentin levels in lung cancer cells. RESULTS: Exosomes released by CAFs-promoted EMT in lung cancer cells. Interestingly, SNAI1 levels in exosomes secreted from CAFs were correlated with SNAI1 expression in CAFs. Furthermore, the level of SNAI1 in exosomes was crucial for inducing EMT in lung cancer cells. Finally, treatment of CAFs with GW4869, an inhibitor of exosome release, noticeably inhibited their EMT-inducing effect on recipient epithelial cells. CONCLUSIONS: The molecular mechanism underlying how CAFs induce EMT in cancer cells may be that CAFs deliver SNAI1 to recipient cancer cells via exosomes.


Assuntos
Fibroblastos Associados a Câncer/metabolismo , Exossomos/metabolismo , Neoplasias Pulmonares/patologia , Fatores de Transcrição da Família Snail/metabolismo , Compostos de Anilina/farmacologia , Compostos de Benzilideno/farmacologia , Fibroblastos Associados a Câncer/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Transição Epitelial-Mesenquimal , Exossomos/efeitos dos fármacos , Humanos , Neoplasias Pulmonares/genética , Fatores de Transcrição da Família Snail/genética
18.
Cancer Genomics Proteomics ; 16(3): 163-173, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31018947

RESUMO

BACKGROUND/AIM: We examined the gene expression changes of breast cancer cells spontaneously undergoing epithelial-mesenchymal transition (EMT) and its reverse process mesenchymal-epithelial transition (MET) and the role of exosomes in these transitions. MATERIALS AND METHODS: Highly invasive mesenchymal-like breast cancer cells, MDA-MB-231 (basal cells), EMT and MET variants, were characterized by microarray gene expression profiling, immunocytochemistry and chemo-sensitivity. RESULTS: Spontaneously disseminated cells were anoikis resistant, exhibited a dissociative, EMT-like phenotype and underwent MET when reseeded in cell-free plates. MET was inhibited by exosomes secreted by basal cells. Chemo-sensitivity to doxorubicin, vincristine and paclitaxel decreased in the order EMT

Assuntos
Antineoplásicos/farmacologia , Biomarcadores Tumorais/genética , Neoplasias da Mama/patologia , Transição Epitelial-Mesenquimal , Exossomos/patologia , Regulação Neoplásica da Expressão Gênica , Transcriptoma , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Proliferação de Células , Exossomos/efeitos dos fármacos , Exossomos/genética , Feminino , Perfilação da Expressão Gênica , Humanos , Células Tumorais Cultivadas
19.
Pathol Res Pract ; 215(6): 152379, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30878308

RESUMO

BACKGROUND: Epidemiological studies have illustrated that regular aspirin consumption may decrease the risk of non-small cell lung cancer (NSCLC). The present study aims to investigate the mechanism of aspirin-induced inhibition of NSCLC development during hypoxia. METHODS: A549 cells were pre-treated with the vehicle control or aspirin and then subjected to hypoxic culture. Cell viability was monitored by CCK-8 assay, and flow cytometry was performed to detect cell cycle distributions, apoptosis, and proportion of cancer stem cells (CSCs). Flow cytometric cell sorting was used to separate CSCs. Quantitative reverse transcription-polymerase chain reaction and Western blot were used to detect the mRNA and protein levels of stem cell markers and the related signaling molecules. The abundance of prostaglandin E2 was detected by enzyme-linked immunosorbent assay. Exosomes in the cell culture medium were isolated using ExoQuick, and the number of exosomes was quantified by the EXOCET exosome quantification assay kit. Cell migration and angiogenesis were monitored by transwell migration assay and in vitro angiogenesis experiments. RESULTS: Aspirin inhibited cell proliferation and induced G2/M cell cycle arrest in hypoxic A549 cells; it also inhibited hypoxia-enhanced stemness in both A549 and ALDH+ cells. The drug reduced hypoxia-enhanced numbers of exosomes in A549 cells and exerted negative effects on the hypoxia-mediated up-regulation of exosomal HIF-1α/COX-2 and expression of exosomal miR-135b and miR-210. While hypoxic-induced exosomes can promote the proliferation, migration, and angiogenesis of other A549 cells, aspirin can weaken this promotion by reducing the amount of exosome secreted and changing exosome contents. CONCLUSIONS: Aspirin inhibits the hypoxia-induced stemness, hypoxic-mediated exosome release, and malignant paracrine effects of A549 cells.


Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Aspirina/farmacologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Exossomos/efeitos dos fármacos , Neoplasias Pulmonares/patologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Células A549 , Hipóxia Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Humanos
20.
PLoS One ; 14(3): e0214545, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30925190

RESUMO

Doxorubicin is a chemotherapeutic agent that is commonly used to treat a broad range of cancers. However, significant cardiotoxicity, associated with prolonged exposure to doxorubicin, limits its continued therapeutic use. One strategy to prevent the uptake of doxorubicin into cardiac cells is the encapsulation of the drug to prevent non-specific uptake and also to improve the drugs' pharmacokinetic properties. Although encapsulated forms of doxorubicin limit the cardiotoxicity observed, they are not without their own liabilities as an increased amount of drug is deposited in the skin where liposomal doxorubicin can cause palmar-plantar erythrodysesthesia. Exosomes are small endogenous extracellular vesicles, that transfer bioactive material from one cell to another, and are considered attractive drug delivery vehicles due to their natural origin. In this study, we generated doxorubicin-loaded exosomes and demonstrate their rapid cellular uptake and re-distribution of doxorubicin from endosomes to the cytoplasm and nucleus resulting in enhanced potency in a number of cultured and primary cell lines when compared to free doxorubicin and liposomal formulations of doxorubicin. In contrast to other delivery methods for doxorubicin, exosomes do not accumulate in the heart, thereby providing potential for limiting the cardiac side effects and improved therapeutic index.


Assuntos
Doxorrubicina/metabolismo , Doxorrubicina/farmacologia , Exossomos/metabolismo , Apoptose/efeitos dos fármacos , Transporte Biológico , Linhagem Celular , Exossomos/efeitos dos fármacos , Humanos , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Cinética
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