RESUMEN
Phosphatidylserine (PS) is a negatively charged phospholipid in all eukaryotic cells that is actively sequestered to the inner leaflet of the cell membrane. Exposure of PS on apoptotic cells is a normal physiological process that triggers their rapid removal by phagocytic engulfment under noninflammatory conditions via receptors primarily expressed on immune cells. PS is aberrantly exposed in the tumor microenvironment and contributes to the overall immunosuppressive signals that antagonize the development of local and systemic antitumor immune responses. PS-mediated immunosuppression in the tumor microenvironment is further exacerbated by chemotherapy and radiation treatments that result in increased levels of PS on dying cells and necrotic tissue. Antibodies targeting PS localize to tumors and block PS-mediated immunosuppression. Targeting exposed PS in the tumor microenvironment may be a novel approach to enhance immune responses to cancer.
RESUMEN
In response to cellular stress, phosphatidylserine is exposed on the outer membrane leaflet of tumor blood vessels and cancer cells, motivating the development of phosphatidylserine-specific therapies. The generation of drug-conjugated phosphatidylserine-targeting agents represents an unexplored therapeutic approach, for which antitumor effects are critically dependent on efficient internalization and lysosomal delivery of the cytotoxic drug. In the current study, we have generated phosphatidylserine-targeting agents by fusing phosphatidylserine-binding domains to a human IgG1-derived Fc fragment. The tumor localization and pharmacokinetics of several phosphatidylserine-specific Fc fusions have been analyzed in mice and demonstrate that Fc-Syt1, a fusion containing the synaptotagmin 1 C2A domain, effectively targets tumor tissue. Conjugation of Fc-Syt1 to the cytotoxic drug monomethyl auristatin E results in a protein-drug conjugate (PDC) that is internalized into target cells and, due to the Ca2+ dependence of phosphatidylserine binding, dissociates from phosphatidylserine in early endosomes. The released PDC is efficiently delivered to lysosomes and has potent antitumor effects in mouse xenograft tumor models. Interestingly, although an engineered, tetravalent Fc-Syt1 fusion shows increased binding to target cells, this higher avidity variant demonstrates reduced persistence and therapeutic effects compared with bivalent Fc-Syt1. Collectively, these studies show that finely tuned, Ca2+-switched phosphatidylserine-targeting agents can be therapeutically efficacious. Mol Cancer Ther; 17(1); 169-82. ©2017 AACR.
Asunto(s)
Calcio/metabolismo , Inmunoconjugados/metabolismo , Neoplasias/tratamiento farmacológico , Fosfatidilserinas/metabolismo , Animales , Línea Celular Tumoral , Femenino , Humanos , Masculino , Ratones , Ratones SCIDRESUMEN
BACKGROUND: There has been increasing interest in the detection of tumour exosomes in blood for cancer diagnostics. Most studies have focussed on miRNA and protein signatures that are surrogate markers for specific tumour types. Because tumour cells and tumour-derived exosomes display phosphatidylserine (PS) in their outer membrane leaflet, we developed a highly sensitive ELISA-based system that detects picogram amounts of exosomal phospholipid in plasma as a cancer biomarker. METHODS: This report describes the development of a highly specific and sensitive ELISA for the capture of PS-expressing tumour exosomes in the blood of tumour-bearing mice. To monitor the relationship between tumour burden and tumour exosome plasma concentrations, plasma from one transplantable breast cancer model (MDA-MB-231) and three genetic mouse models (MMTV-PyMT; breast and KIC and KPC; pancreatic) were screened for captured exosomal phospholipid. RESULTS: We show that quantitative assessment of PS-expressing tumour exosomes detected very early-stage malignancies before clinical evidence of disease in all four model systems. Tumour exosome levels showed significant increases by day 7 after tumour implantation in the MDA-MB-231 model while palpable tumours appeared only after day 27. For the MMTV-PyMT and KIC models, tumour exosome levels increased significantly by day 49 (P⩽0.0002) and day 21 (P⩽0.001) while tumours developed only after days 60 and 40, respectively. For the KPC model, a significant increase in blood exosome levels was detected by day 70 (P=0.023) when only preinvasive lesions are microscopically detectable. CONCLUSIONS: These data indicate that blood PS exosome levels is a specific indicator of cancer and suggest that blood PS is a biomarker for early-stage malignancies.
Asunto(s)
Biomarcadores de Tumor/sangre , Neoplasias de la Mama/sangre , Exosomas/química , Neoplasias Ováricas/sangre , Neoplasias Pancreáticas/sangre , Fosfatidilserinas/sangre , Animales , Anticuerpos/inmunología , Neoplasias de la Mama/diagnóstico , Neoplasias de la Mama/patología , Estudios de Casos y Controles , Línea Celular Tumoral , Ensayo de Inmunoadsorción Enzimática/métodos , Femenino , Humanos , Ratones , Estadificación de Neoplasias , Trasplante de Neoplasias , Neoplasias Ováricas/diagnóstico , Neoplasias Pancreáticas/diagnóstico , Neoplasias Pancreáticas/patología , Fosfatidilserinas/inmunología , Factores de Tiempo , Carga Tumoral , beta 2 Glicoproteína I/inmunologíaRESUMEN
There are no suitable screening modalities for ovarian carcinomas (OC) and repeated imaging and CA-125 levels are often needed to triage equivocal ovarian masses. Definitive diagnosis of malignancy, however, can only be established by histologic confirmation. Thus, the ability to detect OC at early stages is low, and most cases are diagnosed as advanced disease. Since tumor cells expose phosphatidylserine (PS) on their plasma membrane, we predicted that tumors might secrete PS-positive exosomes into the bloodstream that could be a surrogate biomarker for cancer. To address this, we developed a highly stringent ELISA that detects picogram quantities of PS in patient plasma. Blinded plasma from 34 suspect ovarian cancer patients and 10 healthy subjects were analyzed for the presence of PS-expressing vesicles. The nonparametric Wilcoxon rank sum test showed the malignant group had significantly higher PS values than the benign group (median 0.237 vs. -0.027, p=0.0001) and the malignant and benign groups had significantly higher PS values than the healthy group (median 0.237 vs -0.158, p<0.0001 and -0.027 vs -0.158, p=0.0002, respectively). ROC analysis of the predictive accuracy of PS-expressing exosomes/vesicles in predicting malignant against normal, benign against normal and malignant against benign revealed AUCs of 1.0, 0.95 and 0.911, respectively. This study provides proof-of-concept data that supports the high diagnostic power of PS detection in the blood of women with suspect ovarian malignancies.
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Adenocarcinoma de Células Claras/diagnóstico , Adenocarcinoma Mucinoso/diagnóstico , Biomarcadores de Tumor/sangre , Cistadenocarcinoma Seroso/diagnóstico , Neoplasias Endometriales/diagnóstico , Exosomas/metabolismo , Neoplasias Ováricas/diagnóstico , Fosfatidilserinas/sangre , Adenocarcinoma de Células Claras/sangre , Adenocarcinoma Mucinoso/sangre , Adulto , Anciano , Área Bajo la Curva , Antígeno Ca-125/sangre , Cistadenocarcinoma Seroso/sangre , Neoplasias Endometriales/sangre , Ensayo de Inmunoadsorción Enzimática , Exosomas/patología , Femenino , Estudios de Seguimiento , Humanos , Persona de Mediana Edad , Clasificación del Tumor , Estadificación de Neoplasias , Neoplasias Ováricas/sangre , Pronóstico , Curva ROC , Células Tumorales Cultivadas , Adulto JovenRESUMEN
In tumor-bearing animals, the membrane phospholipid phosphatidylserine (PS) suppresses immune responses, suggesting that PS signaling could counteract the antitumor effect of antibody-driven immune checkpoint blockade. Here, we show that treating melanoma-bearing mice with a PS-targeting antibody enhances the antitumor activity of downstream checkpoint inhibition. Combining PS-targeting antibodies with CTLA-4 or PD-1 blockade resulted in significantly greater inhibition of tumor growth than did single-agent therapy. Moreover, combination therapy enhanced CD4(+) and CD8(+) tumor-infiltrating lymphocyte numbers; elevated the fraction of cells expressing the proinflammatory cytokines IL2, IFNγ, and TNFα; and increased the ratio of CD8 T cells to myeloid-derived suppressor cells and regulatory T cells in tumors. Similar changes in immune cell profiles were observed in splenocytes. Taken together, these data show that antibody-mediated PS blockade enhances the antitumor efficacy of immune checkpoint inhibition. Cancer Immunol Res; 4(6); 531-40. ©2016 AACR.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Antígeno CTLA-4/inmunología , Melanoma Experimental/tratamiento farmacológico , Fosfatidilserinas/antagonistas & inhibidores , Receptor de Muerte Celular Programada 1/inmunología , Animales , Anticuerpos Monoclonales/administración & dosificación , Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Citocinas/biosíntesis , Femenino , Inmunofenotipificación , Linfocitos Infiltrantes de Tumor/efectos de los fármacos , Linfocitos Infiltrantes de Tumor/inmunología , Masculino , Melanoma Experimental/inmunología , Melanoma Experimental/patología , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Trasplante de Neoplasias , Fosfatidilserinas/inmunología , Bazo/inmunologíaRESUMEN
The last decade has seen an exponential growth in the number of exosome-related publications. Although many of these studies have used exosomes from biological fluids (blood, and ascites or pleural effusions) the vast majority employed vesicles isolated from large volumes of tissue culture supernatants. While several techniques are available for their isolation, all require a significant reduction in volume to obtain sufficient concentrations for study. One approach is to concentrate the medium before proceeding with their isolation, however, these procedures are very time consuming and require specialized laboratory equipment. Here we provide a new and effective method for the isolation of tumor-derived exosomes based on "charge neutralization" with acetate. We show that titration of tissue culture supernatants with 0.1M acetate to pH4.75 results in immediate precipitation of virtually all the exosomes. The precipitated exosomes can be washed to remove residual media and are readily "resolubilized" upon resuspension in acetate-free buffer at neutral pH. This simple cost effective method significantly increases the yield of exosomes from an unlimited quantity of culture supernatants. Exosomes isolated by this technique are indistinguishable from exosomes recovered by direct ultracentrifugation.
Asunto(s)
Exosomas/química , Neoplasias/química , Acetatos/química , Animales , Línea Celular Tumoral , Precipitación Química , Medios de Cultivo Condicionados/química , Humanos , Concentración de Iones de Hidrógeno , Ratones , Sales (Química)/química , Solubilidad , UltracentrifugaciónRESUMEN
The cellular and molecular pathways that regulate platelet activation, blood coagulation, and inflammation are emerging as critical players in cancer progression and metastasis. Here, we demonstrate a novel signaling mechanism whereby protease-activated receptor 1 (PAR1) mediates expression of melanoma cell adhesion molecule MCAM/MUC18 (MUC18), a critical marker of melanoma metastasis, via activation of platelet-activating factor receptor (PAFR) and cAMP-responsive element-binding protein (CREB). We found that PAR1 silencing with small hairpin RNA inhibits MUC18 expression in metastatic melanoma cells by inhibiting CREB phosphorylation, activity, and binding to the MUC18 promoter. We further demonstrate that the PAF/PAFR pathway mediates MUC18 expression downstream of PAR1. Indeed, PAR1 silencing down-regulates PAFR expression and PAF production, PAFR silencing blocks MUC18 expression, and re-expression of PAFR in PAR1-silenced cells rescues MUC18 expression. We further demonstrate that the PAR1-PAFR-MUC18 pathway mediates melanoma cell adhesion to microvascular endothelial cells, transendothelial migration, and metastatic retention in the lungs. Rescuing PAFR expression in PAR1-silenced cells fully restores metastatic phenotype of melanoma, indicating that PAFR plays critical role in the molecular mechanism of PAR1 action. Our results link the two pro-inflammatory G-protein-coupled receptors, PAR1 and PAFR, with the metastatic dissemination of melanoma and suggest that PAR1, PAFR, and MUC18 are attractive therapeutic targets for preventing melanoma metastasis.
Asunto(s)
Melanoma/metabolismo , Glicoproteínas de Membrana Plaquetaria/metabolismo , Receptor PAR-1/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Neoplasias Cutáneas/metabolismo , Antígeno CD146/metabolismo , Proteína de Unión a CREB/metabolismo , Línea Celular Tumoral , Silenciador del Gen , Humanos , Melanoma/patología , Metástasis de la Neoplasia , Regiones Promotoras Genéticas , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Neoplasias Cutáneas/patología , Factores de Transcripción/metabolismoRESUMEN
One of the hallmarks of apoptosis is the redistribution of phosphatidylserine (PS) from the inner-to-outer plasma membrane (PM) leaflet, where it functions as a ligand for phagocyte recognition and the suppression of inflammatory responses. The mechanism by which apoptotic cells externalize PS has been assumed to involve "scramblases" that randomize phospholipids across the PM bilayer. These putative activities, however, have not been unequivocally proven to be responsible for the redistribution of lipids. Because elevated cytosolic Ca(2+) is critical to this process and is also required for activation of lysosome-PM fusion during membrane repair, we hypothesized that apoptosis could activate a "pseudo"-membrane repair response that results in the fusion of lysosomes with the PM. Using a membrane-specific probe that labels endosomes and lysosomes and fluorescein-labeled annexin 5 that labels PS, we show that the appearance of PS at the cell surface during apoptosis is dependent on the fusion of lysosomes with the PM, a process that is inhibited with the lysosomotrophe, chloroquine. We demonstrate that apoptotic cells evoke a persistent pseudo-membrane repair response that likely redistributes lysosomal-derived PS to the PM outer leaflet that leads to membrane expansion and the formation of apoptotic blebs. Our data suggest that inhibition of lysosome-PM fusion-dependent redistribution of PS that occurs as a result of chemotherapy- and radiotherapy-induced apoptosis will prevent PS-dependent anti-inflammatory responses that preclude the development of tumor- and patient-specific immune responses.
Asunto(s)
Apoptosis/fisiología , Membrana Celular/metabolismo , Fosfatidilserinas/metabolismo , Animales , Células Cultivadas , Lisosomas/metabolismo , Ratones , Microscopía ConfocalRESUMEN
A hallmark of apoptotic cells is the Ca2+-dependent appearance of phosphatidylserine (PS) at the cell surface as a result of its redistribution from the inner-to-outer plasma membrane leaflet. Although endoplasmic reticulum and mitochondrial Ca2+ are known to participate in apoptosis, their role in PS externalization has not been established. In this study, several organelle-specific fluorescent markers and Ca2+-sensitive probes were used to identify the source of Ca2+ critical to PS externalization. By employing Rhod-2AM, fluorescein-labeled high molecular weight dextran, and Calcium Green 1, we provide evidence that lysosomes respond to apoptotic stimuli by releasing their luminal Ca2+ to the cytosol. Cells treated with the cytosolic phospholipase A2 inhibitor, cPLA2alpha, had no effect on caspase activation but exhibited a significant decrease in lysosomal Ca2+ release and externalization of PS in response to apoptotic stimuli. Similarly, cells depleted of lysosomal Ca2+ underwent programmed cell death yet failed to externalize PS. These data indicate that although Ca2+ release from other intracellular organelles to the cytosol is adequate for apoptosis, the release of Ca2+ from lysosomes is critical for PS externalization.
Asunto(s)
Apoptosis/fisiología , Calcio/metabolismo , Citosol/metabolismo , Lisosomas/metabolismo , Fosfatidilserinas/metabolismo , Animales , Apoptosis/efectos de los fármacos , Caspasas/metabolismo , Membrana Celular/metabolismo , Células Cultivadas , Retículo Endoplásmico/metabolismo , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Fosfolipasas A2 Grupo IV/antagonistas & inhibidores , Fosfolipasas A2 Grupo IV/metabolismo , Ratones , Sondas Moleculares/farmacologíaRESUMEN
The recognition and removal of apoptotic cells is critical to development, tissue homeostasis, and the resolution of inflammation. Many studies have shown that phagocytosis is regulated by signaling mechanisms that involve distinct ligand-receptor interactions that drive the engulfment of apoptotic cells. Studies from our laboratory have shown that the plasma protein beta-2-glycoprotein 1 (beta2GP1), a member of the short consensus repeat superfamily, binds phosphatidylserine-containing vesicles and apoptotic cells and promotes their bridging and subsequent engulfment by phagocytes. The phagocyte receptor for the protein/apoptotic cell complex, however, is unknown. Here we report that a member of the low density lipoprotein receptor-related protein family on phagocytes binds and facilitates engulfment of beta2GP1-phosphatidylserine and beta2GP1-apoptotic cell complexes. Using recombinant beta2GP1, we also show that beta2GP1-dependent uptake is mediated by bridging of the target cell to the phagocyte through the protein C- and N-terminal domains, respectively.
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Apoptosis , Macrófagos/fisiología , Receptores de LDL/metabolismo , beta 2 Glicoproteína I/fisiología , Línea Celular Tumoral , Humanos , Microscopía Fluorescente , FagocitosisRESUMEN
beta-2-Glycoprotein 1, an abundant plasma glycoprotein, binds anionic cell surfaces and functions as a regulator of thrombosis. Here, we show that cleavage of the kringle domain at Lys317/Thr318 switches its function to a regulator of angiogenesis. In vitro, the cleaved protein specifically inhibited the proliferation and migration of endothelial cells. The protein was without effect on preformed endothelial cell tubes. In vivo, the cleaved protein inhibited neovascularization into subcutaneously implanted Matrigel and Gelfoam sponge implants and the growth of orthotopically injected tumors. Collectively, these data indicate that plasmin-cleaved beta-2-glycoprotein 1 is a potent antiangiogenic and antitumor molecule of potential therapeutic significance.
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Inhibidores de la Angiogénesis/farmacología , Fibrinolisina/fisiología , Neovascularización Patológica , beta 2 Glicoproteína I/fisiología , Secuencia de Aminoácidos , Animales , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Colágeno , Combinación de Medicamentos , Células Endoteliales/efectos de los fármacos , Células Endoteliales/fisiología , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/fisiología , Esponja de Gelatina Absorbible , Kringles , Laminina , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Trasplante de Neoplasias , Neoplasias Experimentales/irrigación sanguínea , Neoplasias Experimentales/patología , Proteoglicanos , beta 2 Glicoproteína I/farmacologíaRESUMEN
Beta2-glycoprotein 1 (beta2GP1), a 50 kDa serum glycoprotein that binds anionic phospholipid-containing membranes, plays a regulatory role in physiology and pathology. The protein is a member of the short consensus repeat (SCR) superfamily containing four typical repeating domains and an aberrant fifth domain constructed into an SCR-like core at the C-terminus. To investigate the contribution of the individual domains to the binding of beta2GP1, a series of sequential domain-deleted recombinant protein fragments were generated and assessed for their interaction with PS-containing vesicles. Spectral analyses of lipid binding-dependent alterations in tryptophan emission spectra revealed that the (single) tryptophan residues of the individual domains underwent binding-dependent conformational alterations. Depending on the ionic strength, some domains moved from polar to nonpolar environments, while others moved from less polar to more polar environments. Analysis of a series of acrylamide quenching and resonance energy transfer experiments indicated that the binding of N-terminal domain 1 to PS membranes exists in two, ionic strength-dependent, conformations. At low ionic strengths, domain 1 bound to the vesicles and induced their precipitation and/or aggregation. At physiologic ionic strengths, domain 1 detached from the membrane surface while the remaining domains maintained their association with the membrane. Under these conditions, membrane-bound conformationally altered domain 1 projects away from the membrane surface, enabling it to interact with other proteins and/or cell surface ligands or receptors.
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Fosfatidilserinas/metabolismo , Liposomas Unilamelares/metabolismo , beta 2 Glicoproteína I/química , beta 2 Glicoproteína I/metabolismo , Animales , Bovinos , Precipitación Química , Electroforesis en Gel de Poliacrilamida , Transferencia de Energía , Humanos , Cinética , Ligandos , Fosfatidilcolinas/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes/metabolismo , Eliminación de Secuencia , Relación Estructura-Actividad , TriptófanoRESUMEN
The regulated loss of plasma membrane phosphatidylserine (PS) asymmetry is critical to many biological processes. In particular, the appearance of PS at the cell surface, a hallmark of apoptosis, prepares the dying cell for engulfment and elimination by phagocytes. While it is well established that PS externalization is regulated by activation of a calcium-dependent phospholipid scramblase activity in concert with inactivation of the aminophospholipid translocase, there is no evidence indicating that these processes are triggered and regulated by apoptotic regulatory mechanisms. Using a novel model system, we show that PS externalization is inducible, reversible, and independent of cytochrome c release, caspase activation, and DNA fragmentation. Additional evidence is presented indicating that the outward movement of plasma membrane PS requires sustained elevation in cytosolic Ca2+ in concert with inactivation of the aminophospholipid translocase and is inhibited by calcium channel blockers.
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Apoptosis , Membrana Celular/metabolismo , Fosfatidilserinas/química , Calcio/metabolismo , Citocromos c/metabolismo , Citosol/metabolismo , Fragmentación del ADN , Eritrocitos/metabolismo , Humanos , Células Jurkat , Células K562 , Lípidos/química , Modelos Biológicos , Fagocitos/metabolismo , Proteínas de Transferencia de Fosfolípidos/metabolismoRESUMEN
Dogma dictates that brain metastasis originate from the proliferation of extravasated tumor cells and that the blood-brain barrier (BBB) prevents the delivery of chemotherapeutic drugs to the tumors. The purpose of this study was to clarify the relationship between tumor localization and progression and the involvement of BBB function in a murine model of breast cancer brain metastasis. Green fluorescent protein expressing MDA-MB435 breast cancer cells were injected into the left ventricle of nude mice. At various time points, the entire vasculature was labeled with rhodamine-conjugated albumin. The tumors and vasculature were then imaged by laser-scanning confocal and stereo fluorescence microscopy. About 75% of the cells that reached the brain extravasated and grew perivascularly. Twenty five percent of the cells, however, proliferated within the vasculature and ultimately led to thrombosis-like infarction of the brain parenchyma. The tumorigenic "embolus" served as a sustained release source of tumor cells to downstream sites. Continuing intravascular tumor expansion led to disruption of the BBB and to overflow of cells that progressed along the vessels perivascularly to distant sites that regained protection of the BBB. Breast cancer brain metastases involve both extravascular and intravascular growth of tumor cells. These distinct pathways contribute to different pathological phenotypes that generate a heterogeneous BBB that facilitates or inhibits the delivery of chemotherapeutic drugs to the tumor.
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Barrera Hematoencefálica/patología , Neoplasias Encefálicas/irrigación sanguínea , Neoplasias Encefálicas/secundario , Neoplasias de la Mama/patología , Carcinoma/secundario , Animales , Vasos Sanguíneos/patología , Línea Celular Tumoral , Femenino , Humanos , Ratones , Ratones Desnudos , Microscopía Confocal , Trasplante de Neoplasias , Células Neoplásicas CirculantesRESUMEN
A promising target on tumor vasculature is phosphatidylserine (PS), an anionic phospholipid that resides exclusively on the inner leaflet of the plasma membrane of resting mammalian cells. We have shown previously that PS becomes exposed on the surface of endothelial cells (EC) in solid tumors. To target PS on tumor vasculature, the murine monoclonal antibody 3G4 was developed. 3G4 localizes to tumor vasculature, inhibits tumor growth, and enhances anti-tumor chemotherapies without toxicity in mice. A chimeric version of 3G4 is in clinical trials. In this study, we investigated the basis for the interaction between 3G4 and EC with surface-exposed PS. We demonstrate that antibody binding to PS is dependent on plasma protein beta-2-glycoprotein 1 (beta2GP1). beta2GP1 is a 50-kDa glycoprotein that binds weakly to anionic phospholipids under physiological conditions. We show that 3G4 enhances binding of beta2GP1 to EC induced to expose PS. We also show that divalent 3G4-beta2GP1 complexes are required for enhanced binding, since 3G4 Fab' fragments do not bind EC with exposed PS. Finally, we demonstrate that an artificial dimeric beta2GP1 construct binds to EC with exposed PS in the absence of 3G4, confirming that antibody binding is mediated by dimerization of beta2GP1. Together, these data indicate that 3G4 targets tumor EC by increasing the avidity of beta2GP1 for anionic phospholipids through formation of multivalent 3G4-beta2GP1 complexes.
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Anticuerpos Monoclonales/metabolismo , Antineoplásicos/metabolismo , Sitios de Unión de Anticuerpos , Endotelio Vascular/inmunología , Neoplasias Experimentales/inmunología , Neoplasias Experimentales/metabolismo , Fosfatidilserinas/metabolismo , beta 2 Glicoproteína I/fisiología , Animales , Anticuerpos Monoclonales/uso terapéutico , Antineoplásicos/uso terapéutico , Bovinos , Células Cultivadas , Evaluación Preclínica de Medicamentos , Endotelio Vascular/metabolismo , Endotelio Vascular/patología , Inhibidores de Crecimiento/metabolismo , Inhibidores de Crecimiento/uso terapéutico , Humanos , Ratones , Neoplasias Experimentales/irrigación sanguínea , Neoplasias Experimentales/patología , Fosfatidilserinas/inmunología , beta 2 Glicoproteína I/sangreRESUMEN
Angiogenesis, the development of new blood vessels from preexisting vessels, is crucial to tissue growth, repair, and maintenance. This process begins with the formation of endothelial cell sprouts followed by the proliferation and migration of neighboring endothelial cells along the preformed extensions. The initiating event and mechanism of sprouting is not known. We show that the phenotypic expression of negatively charged membrane surface in apoptotic cells initiates the formation of directional endothelial cell sprouts that extend toward the dying cells by a mechanism that involves endothelial cell membrane hyperpolarization and cytoskeleton reorganization but is independent of diffusible molecules.
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Apoptosis , Comunicación Celular , Movimiento Celular/fisiología , Endotelio Vascular/citología , Factor 2 de Crecimiento de Fibroblastos/fisiología , Neovascularización Fisiológica/fisiología , Electricidad Estática , Animales , Células Cultivadas , Técnicas de Cocultivo , Receptores ErbB/antagonistas & inhibidores , Ferritinas/metabolismo , Canales Iónicos/antagonistas & inhibidores , Melanoma/metabolismo , Melanoma/patología , Potenciales de la Membrana , Ratones , Fosfolípidos/metabolismo , Purinas/farmacología , Ratas , Receptores de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Sefarosa/metabolismo , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/patologíaRESUMEN
Tumors must manipulate the host vasculature to provide a blood supply adequate for their proliferation. Although tumors may arise as avascular masses, there is increasing evidence that some tumors begin to proliferate by first co-opting preexisting host blood vessels. By fluorescent vascular imaging, we provide evidence that the vasculature in orthotopically implanted melanoma arises from a preexisting red cell-deficient vascular network that remodels to patency to accommodate the requirements of the expanding tumor mass. Topical application of vascular endothelial growth factor to vascular beds generated immediate and robust vascular transitions that were morphologically similar to tumor-induced transitions. N(phi)-nitro-L-arginine, a nitric oxide inhibitor, significantly inhibited the growth of a syngeneic K1735M2 melanoma by reducing blood supply to the tumor by a mechanism independent of endothelial cell proliferation. These findings suggest that tumor-induced remodeling of red cell-deficient vessels to patency contributes to tumor vascularization and growth.
Asunto(s)
Melanoma Experimental/irrigación sanguínea , Animales , Procesos de Crecimiento Celular/efectos de los fármacos , Procesos de Crecimiento Celular/fisiología , Células Endoteliales/efectos de los fármacos , Células Endoteliales/patología , Femenino , Melanoma Experimental/patología , Ratones , Ratones Endogámicos C3H , Neovascularización Patológica/patología , Óxido Nítrico/antagonistas & inhibidores , Nitroarginina/farmacología , Piel/irrigación sanguínea , Factor A de Crecimiento Endotelial Vascular/farmacologíaRESUMEN
The plasma protein beta2GPI (beta2-glycoprotein I) has been proposed to mediate phagocytosis of apoptotic cells and to play a role in the antiphospholipid syndrome. This suggestion is based mainly on the presumption that beta2GPI has an appreciable interaction with PS (phosphatidylserine)-exposing cell membranes. However, quantitative data on the binding of beta2GPI to PS-exposing cells under physiologically relevant conditions are scarce and conflicting. Therefore we evaluated the binding of beta2GPI to PS-expressing blood platelets. Flow cytometry showed that binding of beta2GPI is negligible at physiological ionic strength, in contrast with significant binding occurring at low ionic strength. Binding parameters of beta2GPI and (for comparison) prothrombin were quantified by ellipsometric measurement of protein depletion from the supernatant following incubation with platelets. At low ionic strength (20 mM NaCl, no CaCl2), a dissociation constant (K(d)) of 0.2 microM was found for beta2GPI, with 7.4x10(5) binding sites per platelet. Under physiologically relevant conditions (120 mM NaCl and 3 mM CaCl2), binding of beta2GPI was not detectable (extrapolated K(d)>80 microM). Prothrombin binding (at 3 mM CaCl2) was much less affected by ionic strength: K(d) values of 0.5 and 1.4 muM were observed at 20 and 120 mM NaCl respectively. The low affinity and the presence of many lipid-binding proteins in plasma that can compete with the binding of beta2GPI suggest that only a small fraction (<5%) of the binding sites on PS-exposing blood cells are likely to be occupied by beta2GPI. These findings are discussed in relation to the alleged (patho-)physiological functions of beta2GPI.
Asunto(s)
Plaquetas/química , Glicoproteínas/metabolismo , Fosfatidilserinas/metabolismo , Protrombina/metabolismo , Sitios de Unión , Plaquetas/efectos de los fármacos , Plaquetas/metabolismo , Citometría de Flujo/métodos , Humanos , Ionomicina/farmacología , Concentración Osmolar , Unión Proteica , beta 2 Glicoproteína IRESUMEN
Previous studies have shown that unlike most apoptotic cells, Raji cells do not externalize phosphatidylserine (PS) upon apoptosis. Here we show that Raji cells are resistant to intrinsic apoptogenic agents, but sensitive to extrinsically triggered Fas-induced apoptosis. Treatment of intrinsic apoptosis-competent Jurkat cells with vitamin E implicated reactive oxygen species in intrinsic apoptosis because, like Raji cells, they became resistant to actinomycin D- but not Fas-triggered apoptosis. Oxidation of sulfhydryls in both cell types with N-ethylmaleimide resulted in rapid disruption of the mitochondrial membrane potential, release of cytochrome c from the mitochondria to the cytoplasm, and externalization of PS by a mechanism that was not inhibited by the pan caspase inhibiter zVAD-fmk. These results suggest that although cell death and PS externalization are both cytochrome c-dependent, they are distinct and separable processes.
Asunto(s)
Apoptosis/fisiología , Membrana Celular/metabolismo , Citocromos c/metabolismo , Potenciales de la Membrana/fisiología , Fosfatidilserinas/metabolismo , Compuestos de Sulfhidrilo/química , Antibióticos Antineoplásicos/metabolismo , Antioxidantes/metabolismo , Línea Celular Tumoral , Membrana Celular/química , Dactinomicina/metabolismo , Etilmaleimida/metabolismo , Humanos , Células Jurkat , Linfoma no Hodgkin , Oxidación-Reducción , Especies Reactivas de Oxígeno/metabolismo , Reactivos de Sulfhidrilo/metabolismo , Vitamina E/metabolismo , Receptor fas/metabolismoRESUMEN
Apoptosis and phagocytosis of apoptotic cells provide for effective and harmless clearance of unwanted or damaged cells in the body. Preferential oxidation of one particular class of phospholipids, phosphatidylserine (PS), is a typical trait of both oxidant- and nonoxidant-induced apoptosis. PS oxidation is likely to play an important role in phagocytosis either by affecting PS externalization acting as an "eat me" signal or by more effective recognition of apoptotic cells by macrophage receptors. This implies that antioxidants effective in inhibiting PS oxidation may affect PS externalization and/or effective removal of apoptotic cells. Therefore, it is essential to determine whether vitamin E, the major lipid-soluble antioxidant of membranes, inhibits PS oxidation, and hence blocks apoptosis/phagocytosis. To test this, we studied the effects of vitamin E on PS oxidation and signaling using a model of anti-Fas-triggered apoptosis in Jurkat T cells. We found that incubation of cells with vitamin E (0.25-50 micro M) resulted in its integration into cells to reach physiologically relevant concentrations. Using labeling of cell phospholipids with oxidation-sensitive and fluorescent cis-parinaric acid (PnA), we found that anti-Fas exposure caused significant and selective oxidation of PnA-PS in Jurkat T cells (22 +/- 2.1% of its content in nonexposed cells). Vitamin E protected PnA-PS against oxidation in a concentration-dependent way such that at 25 micro M and 50 micro M, a complete inhibition of anti-Fas-induced PS oxidation was achieved. At all concentrations used, vitamin E had no effect on either biomarkers of anti-Fas-induced apoptosis (PS externalization, nuclear fragmentation) or phagocytosis of anti-Fas-induced apoptotic cells by J774A.1 macrophages. We conclude that vitamin E does not significantly interfere with extrinsic (death receptor-triggered) pathways of apoptosis and does not affect phagocytosis of anti-Fas-triggered apoptotic cells.