RESUMEN
Epidermal growth factor receptor (EGFR) is one of the most promising molecular targets for anticancer therapy. We used boron clusters as a platform for generation of new materials. For this, functional DNA constructs conjugated with boron clusters (B-ASOs) were developed. These B-ASOs, built from 1,2-dicarba-closo-dodecaborane linked with two anti-EGFR antisense oligonucleotides (ASOs), form with their complementary congeners torus-like nanostructures, as previously shown by atomic force microscope (AFM) and transmission electron cryo-microscopy (cryo-TEM) imaging. In the present work, deepened studies were carried out on B-ASO's properties. In solution, B-ASOs formed four dominant complexes as confirmed by non-denaturing polyacrylamide gel electrophoresis (PAGE). These complexes exhibited increased stability in cell lysate comparing to the non-modified ASO. Fluorescently labeled B-ASOs localized mostly in the cytoplasm and decreased EGFR expression by activating RNase H. Moreover, the B-ASO complexes altered the cancer cell phenotype, decreased cell migration rate, and arrested the cells in the S phase of cell cycle. The 1,2-dicarba-closo-dodecaborane-containing nanostructures did not activate NLRP3 inflammasome in human macrophages. In addition, as shown by inductively coupled plasma mass spectrometry (ICP MS), these nanostructures effectively penetrated the human squamous carcinoma cells (A431), showing their potential applicability as anticancer agents.
Asunto(s)
Antineoplásicos/farmacología , Boranos/farmacología , Regulación Neoplásica de la Expresión Génica , Nanopartículas/química , Oligonucleótidos Antisentido/farmacología , Antineoplásicos/síntesis química , Antineoplásicos/metabolismo , Boranos/síntesis química , Boranos/metabolismo , Línea Celular Tumoral , Permeabilidad de la Membrana Celular , Movimiento Celular/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Células Epiteliales/patología , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/genética , Receptores ErbB/metabolismo , Células HeLa , Humanos , Cinética , Células MCF-7 , Macrófagos/citología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Oligonucleótidos Antisentido/genética , Oligonucleótidos Antisentido/metabolismo , Fase S/efectos de los fármacos , Fase S/genética , Transducción de SeñalRESUMEN
Influenza A viruses (IAVs) are aggressive pathogens that cause acute respiratory diseases and annual epidemics in humans. Host defense against IAV infection is initiated by macrophages, which are the principal effector cells of the innate immune system. We have previously shown that IAV infection of human macrophages is associated with robust secretion of proteins via conventional and unconventional protein release pathways. Here we have characterized unconventional, extracellular vesicle (EV)-mediated protein secretion in human macrophages during IAV infection using proteomics, bioinformatics, and functional studies. We demonstrate that at 9 h postinfection a robust EV-mediated protein secretion takes place. We identified 2359 human proteins from EVs of IAV-infected macrophages compared with 1448 proteins identified from EVs of control cells. Bioinformatic analysis shows that many proteins involved in translation, like components of spliceosome machinery and the ribosome, are secreted in EVs in response to IAV infection. Our data also shows that EVs derived from IAV-infected macrophages contain fatty acid-binding proteins, antiviral cytokines, copper metabolism Murr-1 domain proteins, and autophagy-related proteins. In addition, our data suggest that secretory autophagy plays a role in activating EV-mediated protein secretion during IAV infection.
Asunto(s)
Vesículas Extracelulares/genética , Interacciones Huésped-Patógeno , Subtipo H3N2 del Virus de la Influenza A/fisiología , Macrófagos/metabolismo , Proteoma/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/inmunología , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Relacionadas con la Autofagia/genética , Proteínas Relacionadas con la Autofagia/inmunología , Proteínas Relacionadas con la Autofagia/metabolismo , Biología Computacional , Citocinas/genética , Citocinas/inmunología , Citocinas/metabolismo , Vesículas Extracelulares/inmunología , Vesículas Extracelulares/virología , Proteínas de Unión a Ácidos Grasos/genética , Proteínas de Unión a Ácidos Grasos/inmunología , Proteínas de Unión a Ácidos Grasos/metabolismo , Regulación de la Expresión Génica , Ontología de Genes , Humanos , Macrófagos/inmunología , Macrófagos/virología , Anotación de Secuencia Molecular , Cultivo Primario de Células , Biosíntesis de Proteínas , Proteoma/inmunología , Proteoma/metabolismo , Transducción de SeñalRESUMEN
Extracellular ATP is an endogenous danger signal that is known to activate inflammatory responses in innate immune cells, including macrophages. Activated macrophages start to secrete proteins to induce an immune response, as well as to recruit other immune cells to the site of infection and tissue damage. In this study, we characterized the secretome (i.e., the global pattern of secreted proteins) of ATP-stimulated human macrophages. We show that ATP stimulation activates robust vesicle-mediated unconventional protein secretion, including exosome release and membrane shedding, from human macrophages. Pathway analysis of the identified secreted proteins showed that calpain-related pathways were overrepresented in the secretome of ATP-stimulated cells. In accordance with this, calpains, which are calcium-dependent nonlysosomal cysteine proteases, were activated upon ATP stimulation through a P2X purinoceptor 7 receptor-dependent pathway. Functional studies demonstrated that calpain activity is essential for the P2X purinoceptor 7 receptor-mediated activation of unconventional protein secretion. Unconventional protein secretion was followed by cell necrosis and NLRP3 inflammasome-mediated secretion of the mature form of the proinflammatory cytokine IL-1ß. Furthermore, ATP-driven NLRP3 inflammasome activation was also dependent on calpain activity. Interestingly, pro-IL-1ß and inflammasome components ASC and caspase-1 were released by ATP-activated macrophages through a vesicle-mediated secretion pathway. In conclusion, to our knowledge, we provide the first global characterization of proteins secreted by ATP-activated human macrophages and show a pivotal role for calpains in the activation of the inflammatory response during ATP exposure.