RESUMEN
Placental growth factor (PlGF)-2 induces angio- and arteriogenesis in rodents but its therapeutic potential in a clinically representative post-infarction left ventricular (LV) dysfunction model remains unclear. We, therefore, investigated the safety and efficacy of recombinant human (rh)PlGF-2 in the infarcted porcine heart in a randomized, placebo-controlled blinded study. We induced myocardial infarction (MI) in pigs using 75 min mid-LAD balloon occlusion followed by reperfusion. After 4 w, we randomized pigs with marked LV dysfunction (LVEF < 40%) to receive continuous intravenous infusion of 5, 15, 45 µg/kg/day rhPlGF-2 or PBS (CON) for 2 w using osmotic pumps. We evaluated the treatment effect at 8 w using comprehensive MRI and immunohistochemistry and measured myocardial PlGF-2 receptor transcript levels. At 4 w after MI, infarct size was 16-18 ± 4% of LV mass, resulting in significantly impaired systolic function (LVEF 34 ± 4%). In the pilot study (3 pigs/dose), PIGF administration showed sustained dose-dependent increases in plasma concentrations for 14 days without systemic toxicity and was associated with favorable post-infarct remodeling. In the second phase (n = 42), we detected no significant differences at 8 w between CON and PlGF-treated pigs in infarct size, capillary or arteriolar density, global LV function and regional myocardial blood flow at rest or during stress. Molecular analysis showed significant downregulation of the main PlGF-2 receptor, pVEGFR-1, in dysfunctional myocardium. Chronic rhPIGF-2 infusion was safe but failed to induce therapeutic neovascularization and improve global cardiac function after myocardial infarction in pigs. Our data emphasize the critical need for properly designed trials in representative large animal models before translating presumed promising therapies to patients.
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Despite poor response rates and dose-limiting cardiotoxicity, doxorubicin (doxo) remains the standard-of-care for patients with advanced soft tissue sarcoma. We evaluated the efficacy of two tetrapeptidic doxo prodrugs (PhAc-ALGP-Dox or CBR-049 and CBR-050) that are locally activated by enzymes expressed in the tumor environment, in ten sarcoma patient-derived xenografts. Xenograft models were selected based on expression of the main activating enzyme, i.e., thimet oligopeptidase (THOP1). Mice were either randomized to vehicle, doxo, CBR-049 and CBR-050 or control, doxo, aldoxorubicin (aldoxo) and CBR-049. Treatment efficacy was assessed by tumor volume measurement and histological assessment of ex-mouse tumors. CBR-049 showed significant tumor growth delay compared to control in all xenografts investigated and was superior compared to doxo in all but one. At the same time, CBR-049 showed comparable efficacy to aldoxo but the latter was found to have a complex safety profile in mice. CBR-050 demonstrated tumor growth delay compared to control in one xenograft but was not superior to doxo. For both experimental prodrugs, strong immunostaining for THOP1 was found to predict better antitumor efficacy. The prodrugs were well tolerated without any adverse events, even though molar doses were 17-fold higher than those administered and tolerated for doxo.
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Clinical use of doxorubicin (Dox) is limited by cumulative myelo- and cardiotoxicity. This research focuses on the detailed characterization of PhAc-ALGP-Dox, a targeted tetrapeptide prodrug with a unique dual-step activation mechanism, designed to circumvent Dox-related toxicities and is ready for upcoming clinical investigation. Coupling Dox to a phosphonoacetyl (PhAc)-capped tetrapeptide forms the cell-impermeable, inactive compound, PhAc-ALGP-Dox. After extracellular cleavage by tumor-enriched thimet oligopeptidase-1 (THOP1), a cell-permeable but still biologically inactive dipeptide-conjugate is formed (GP-Dox), which is further processed intracellularly to Dox by fibroblast activation protein-alpha (FAPα) and/or dipeptidyl peptidase-4 (DPP4). In vitro, PhAc-ALGP-Dox is effective in various 2D- and 3D-cancer models, while showing improved safety toward normal epithelium, hematopoietic progenitors, and cardiomyocytes. In vivo, these results translate into a 10-fold higher tolerability and 5-fold greater retention of Dox in the tumor microenvironment compared with the parental drug. PhAc-ALGP-Dox demonstrates 63% to 96% tumor growth inhibition in preclinical models, an 8-fold improvement in efficacy in patient-derived xenograft (PDX) models, and reduced metastatic burden in a murine model of experimental lung metastasis, improving survival by 30%. The current findings highlight the potential clinical benefit of PhAc-ALGP-Dox, a targeted drug-conjugate with broad applicability, favorable tissue biodistribution, significantly improved tolerability, and tumor growth inhibition at primary and metastatic sites in numerous solid tumor models.
Asunto(s)
Antineoplásicos , Neoplasias Pulmonares , Profármacos , Animales , Antineoplásicos/química , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Doxorrubicina/química , Doxorrubicina/farmacología , Doxorrubicina/uso terapéutico , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Ratones , Profármacos/farmacología , Profármacos/uso terapéutico , Índice Terapéutico , Distribución Tisular , Microambiente TumoralRESUMEN
Given the very limited efficacy of doxorubicin (doxo) in soft tissue sarcoma, there is a clear need for more active and less toxic treatments for this family of diseases. However, due to the rarity of these malignancies and lack of reliable preclinical models, development of new therapies has lagged behind. We evaluated the efficacy of PhAc-ALGP-doxorubicin (ALGP-doxo), a prodrug metabolized to doxo by peptidases present in tumor cells and/or tumor microenvironment, in a synovial sarcoma (SynSa) and two dedifferentiated liposarcoma (DDLPS) patient-derived xenograft models. Sixty-eight mice were engrafted bilaterally with human DDLPS or SynSa and randomized to control, doxo, or ALGP-doxo treatment, which were administered using an intraperitoneal minipump. Tumor volume measurement, histopathology, and Western blotting were used to assess treatment efficacy. Tumor regrowth was evaluated in a subset of mice over a period of 2 weeks after treatment cessation. Although tumor volume in the control and doxo groups increased steadily, ALGP-doxo caused tumor volume stabilization in the DDLPS xenografts and significant tumor shrinkage in the SynSa model, continuing after treatment cessation. A significant decrease in proliferation and increase in apoptosis compared with control and doxo was observed during and after treatment with ALGP-doxo in all models. In conclusion, ALGP-doxo shows considerably higher antitumoral efficacy compared with doxo in all patient-derived xenograft models tested. Administration of a 30- to 40-fold higher dose of ALGP-doxo than doxo is tolerated without significant adverse events. These results warrant further testing of this prodrug in anthracycline-sensitive and -resistant models of soft tissue sarcoma. Mol Cancer Ther; 16(8); 1566-75. ©2017 AACR.
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Antineoplásicos/uso terapéutico , Doxorrubicina/análogos & derivados , Doxorrubicina/uso terapéutico , Profármacos/uso terapéutico , Sarcoma/tratamiento farmacológico , Animales , Antineoplásicos/química , Antineoplásicos/farmacología , Apoptosis , Diferenciación Celular , Proliferación Celular , Doxorrubicina/química , Doxorrubicina/farmacología , Humanos , Liposarcoma/tratamiento farmacológico , Liposarcoma/patología , Ratones , Proteínas de Neoplasias/metabolismo , Profármacos/química , Sarcoma/patología , Resultado del Tratamiento , Carga Tumoral , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Angiogenic growth factor therapy for ischemic cardiovascular disease carries a risk of stimulating atherosclerotic plaque growth. We evaluated risk benefit ratio of sustained administration of recombinant human placental growth factor (rhPlGF)-2 in mice with advanced atherosclerosis and chronic ischemic cardiomyopathy. We maintained apolipoprotein E-deficient mice on a high cholesterol diet and induced myocardial infarction by transient ligation at 4 weeks. At 8 weeks, we assessed left ventricular (LV) function and randomized mice to receive rhPlGF-2 or vehicle (VEH) subcutaneously for 28 days. Administration of rhPlGF-2 significantly increased PlGF plasma levels without adverse hemodynamic or systemic inflammatory effects. RhPlGF-2 did not increase plaque area, composition, or vulnerability in the aortic arch. RhPlGF-2 significantly improved contractile function and reduced LV end-systolic and end-diastolic volume indices with a concomitant increase in capillary and arteriolar density in ischemic myocardium. RhPlGF-2 may represent a promising therapeutic strategy in chronic ischemic cardiomyopathy.
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Inductores de la Angiogénesis/administración & dosificación , Aorta/efectos de los fármacos , Enfermedades de la Aorta/tratamiento farmacológico , Aterosclerosis/tratamiento farmacológico , Cardiomiopatías/tratamiento farmacológico , Infarto del Miocardio/tratamiento farmacológico , Neovascularización Fisiológica/efectos de los fármacos , Factor de Crecimiento Placentario/administración & dosificación , Función Ventricular Izquierda/efectos de los fármacos , Inductores de la Angiogénesis/toxicidad , Animales , Aorta/patología , Aorta/fisiopatología , Enfermedades de la Aorta/patología , Enfermedades de la Aorta/fisiopatología , Aterosclerosis/patología , Aterosclerosis/fisiopatología , Cardiomiopatías/diagnóstico por imagen , Cardiomiopatías/fisiopatología , Colesterol en la Dieta , Enfermedad Crónica , Modelos Animales de Enfermedad , Infusiones Subcutáneas , Masculino , Ratones Noqueados para ApoE , Contracción Miocárdica/efectos de los fármacos , Infarto del Miocardio/diagnóstico por imagen , Infarto del Miocardio/fisiopatología , Factor de Crecimiento Placentario/toxicidad , Placa Aterosclerótica , Proteínas Recombinantes/administración & dosificación , Recuperación de la Función , Volumen Sistólico/efectos de los fármacos , Factores de Tiempo , Rigidez Vascular/efectos de los fármacos , Remodelación Ventricular/efectos de los fármacosRESUMEN
The secretome of cancer and stromal cells generates a microenvironment that contributes to tumour cell invasion and angiogenesis. Here we compare the secretome of human mammary normal and cancer-associated fibroblasts (CAFs). We discover that the chloride intracellular channel protein 3 (CLIC3) is an abundant component of the CAF secretome. Secreted CLIC3 promotes invasive behaviour of endothelial cells to drive angiogenesis and increases invasiveness of cancer cells both in vivo and in 3D cell culture models, and this requires active transglutaminase-2 (TGM2). CLIC3 acts as a glutathione-dependent oxidoreductase that reduces TGM2 and regulates TGM2 binding to its cofactors. Finally, CLIC3 is also secreted by cancer cells, is abundant in the stromal and tumour compartments of aggressive ovarian cancers and its levels correlate with poor clinical outcome. This work reveals a previously undescribed invasive mechanism whereby the secretion of a glutathione-dependent oxidoreductase drives angiogenesis and cancer progression by promoting TGM2-dependent invasion.
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Canales de Cloruro/metabolismo , Progresión de la Enfermedad , Glutatión/metabolismo , Animales , Fibroblastos Asociados al Cáncer/metabolismo , Fibroblastos Asociados al Cáncer/patología , Línea Celular Tumoral , Matriz Extracelular/metabolismo , Femenino , Proteínas de Unión al GTP/metabolismo , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Ratones Endogámicos C57BL , Ratones Desnudos , Modelos Biológicos , Invasividad Neoplásica , Neoplasias/irrigación sanguínea , Neoplasias/patología , Neovascularización Patológica/metabolismo , Neovascularización Patológica/patología , Oxidorreductasas/metabolismo , Unión Proteica , Proteína Glutamina Gamma Glutamiltransferasa 2 , Proteoma/metabolismo , Proteómica , Análisis de Supervivencia , Transglutaminasas/metabolismo , Resultado del TratamientoRESUMEN
Mutations or amplification of the MET proto-oncogene are involved in the pathogenesis of several tumours, which rely on the constitutive engagement of this pathway for their growth and survival. However, MET is expressed not only by cancer cells but also by tumour-associated stromal cells, although its precise role in this compartment is not well characterized. Here we show that MET is required for neutrophil chemoattraction and cytotoxicity in response to its ligand hepatocyte growth factor (HGF). Met deletion in mouse neutrophils enhances tumour growth and metastasis. This phenotype correlates with reduced neutrophil infiltration to both the primary tumour and metastatic sites. Similarly, Met is necessary for neutrophil transudation during colitis, skin rash or peritonitis. Mechanistically, Met is induced by tumour-derived tumour necrosis factor (TNF)-α or other inflammatory stimuli in both mouse and human neutrophils. This induction is instrumental for neutrophil transmigration across an activated endothelium and for inducible nitric oxide synthase production upon HGF stimulation. Consequently, HGF/MET-dependent nitric oxide release by neutrophils promotes cancer cell killing, which abates tumour growth and metastasis. After systemic administration of a MET kinase inhibitor, we prove that the therapeutic benefit of MET targeting in cancer cells is partly countered by the pro-tumoural effect arising from MET blockade in neutrophils. Our work identifies an unprecedented role of MET in neutrophils, suggests a potential 'Achilles' heel' of MET-targeted therapies in cancer, and supports the rationale for evaluating anti-MET drugs in certain inflammatory diseases.
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Antineoplásicos/efectos adversos , Antineoplásicos/farmacología , Neoplasias/inmunología , Neoplasias/metabolismo , Neutrófilos/inmunología , Proteínas Proto-Oncogénicas c-met/metabolismo , Anciano , Animales , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Femenino , Eliminación de Gen , Factor de Crecimiento de Hepatocito , Humanos , Inflamación/inmunología , Inflamación/patología , Masculino , Ratones , Persona de Mediana Edad , Metástasis de la Neoplasia , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Óxido Nítrico/metabolismo , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas c-met/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-met/deficiencia , Proteínas Proto-Oncogénicas c-met/genética , Solubilidad , Migración Transendotelial y Transepitelial , Factor de Necrosis Tumoral alfa/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The functional diversity and molecular adaptations of reactive microglia in the chronically inflamed central nervous system (CNS) are poorly understood. We previously showed that mice lacking multifunctional protein 2 (MFP2), a pivotal enzyme in peroxisomal ß-oxidation, persistently accumulate reactive myeloid cells in the gray matter of the CNS. Here, we show that the increased numbers of myeloid cells solely derive from the proliferation of resident microglia and not from infiltrating monocytes. We defined the signature of Mfp2(-/-) microglia by gene expression profiling after acute isolation, which was validated by quantitative polymerase reaction (qPCR), immunohistochemical, and flow cytometric analysis. The features of Mfp2(-/-) microglia were compared with those from SOD1(G93A) mice, an amyotrophic lateral sclerosis model. In contrast to the neurodegenerative milieu of SOD1(G93A) spinal cord, neurons were intact in Mfp2(-/-) brain and Mfp2(-/-) microglia lacked signs of phagocytic and neurotoxic activity. The chronically reactive state of Mfp2(-/-) microglia was accompanied by the downregulation of markers that specify the unique microglial signature in homeostatic conditions. In contrast, mammalian target of rapamycin (mTOR) and downstream glycolytic and protein translation pathways were induced, indicative of metabolic adaptations. Mfp2(-/-) microglia were immunologically activated but not polarized to a pro- or anti-inflammatory phenotype. A peripheral lipopolysaccharide challenge provoked an exaggerated inflammatory response in Mfp2(-/-) brain, consistent with a primed state. Taken together, we demonstrate that chronic activation of resident microglia does not necessarily lead to phagocytosis nor overt neurotoxicity.
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Microglía/fisiología , Proteína-2 Multifuncional Peroxisomal/deficiencia , Empalme Alternativo , Esclerosis Amiotrófica Lateral/patología , Esclerosis Amiotrófica Lateral/fisiopatología , Animales , Encéfalo/patología , Encéfalo/fisiopatología , Células Cultivadas , Modelos Animales de Enfermedad , Homeostasis/fisiología , Lipopolisacáridos , Ratones Noqueados , Ratones Transgénicos , Microglía/patología , Neuroinmunomodulación/fisiología , Neuronas/patología , Neuronas/fisiología , Proteína-2 Multifuncional Peroxisomal/genética , Fagocitosis/fisiología , Médula Espinal/patología , Médula Espinal/fisiopatología , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Hypoxia confers to macrophages angiogenic and immunosuppressive properties which promote tumor growth and progression. Preventing the migration of macrophages into hypoxic tumor regions hinders angiogenesis and restores the tumor-suppressive properties of these immune cells. We have recently uncovered a neuropilin 1- and semaphorin 3A-dependent signaling pathway that defines the repositioning of macrophages to hypoxic tumor niches, a discovery that generates new options for the development of complementary anticancer treatments.
RESUMEN
Recruitment of tumor-associated macrophages (TAMs) into avascular areas sustains tumor progression; however, the underlying guidance mechanisms are unknown. Here, we report that hypoxia-induced Semaphorin 3A (Sema3A) acts as an attractant for TAMs by triggering vascular endothelial growth factor receptor 1 phosphorylation through the associated holoreceptor, composed of Neuropilin-1 (Nrp1) and PlexinA1/PlexinA4. Importantly, whereas Nrp1 levels are downregulated in the hypoxic environment, Sema3A continues to regulate TAMs in an Nrp1-independent manner by eliciting PlexinA1/PlexinA4-mediated stop signals, which retain them inside the hypoxic niche. Consistently, gene deletion of Nrp1 in macrophages favors TAMs' entrapment in normoxic tumor regions, which abates their pro-angiogenic and immunosuppressive functions, hence inhibiting tumor growth and metastasis. This study shows that TAMs' heterogeneity depends on their localization, which is tightly controlled by Sema3A/Nrp1 signaling.
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Macrófagos/fisiología , Neoplasias Experimentales/irrigación sanguínea , Neovascularización Patológica/prevención & control , Neuropilina-1/fisiología , Semaforina-3A/fisiología , Transducción de Señal/fisiología , Animales , Hipoxia de la Célula , Macrófagos/efectos de los fármacos , Ratones , Ratones Noqueados , Neoplasias Experimentales/inmunología , Neoplasias Experimentales/patología , Neuropilina-1/antagonistas & inhibidores , Neuropilina-1/genética , Semaforina-3A/antagonistas & inhibidoresRESUMEN
Expression of the mannose receptor (MRC1/CD206) identifies macrophage subtypes, such as alternatively activated macrophages (AAMs) and M2-polarized tumor-associated macrophages (TAMs), which are endowed with tissue-remodeling, proangiogenic, and protumoral activity. However, the significance of MRC1 expression for TAM's protumoral activity is unclear. Here, we describe and characterize miR-511-3p, an intronic microRNA (miRNA) encoded by both mouse and human MRC1 genes. By using sensitive miRNA reporter vectors, we demonstrate robust expression and bioactivity of miR-511-3p in MRC1(+) AAMs and TAMs. Unexpectedly, enforced expression of miR-511-3p tuned down the protumoral gene signature of MRC1(+) TAMs and inhibited tumor growth. Our findings suggest that transcriptional activation of Mrc1 in TAMs evokes a genetic program orchestrated by miR-511-3p, which limits rather than enhances their protumoral functions. Besides uncovering a role for MRC1 as gatekeeper of TAM's protumoral genetic programs, these observations suggest that endogenous miRNAs may operate to establish thresholds for inflammatory cell activation in tumors.
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Macrófagos/metabolismo , MicroARNs/metabolismo , Neoplasias/genética , Neoplasias/patología , Animales , Emparejamiento Base/genética , Secuencia de Bases , Células de la Médula Ósea/metabolismo , Línea Celular , Línea Celular Tumoral , Regulación hacia Abajo/genética , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Hematopoyesis/genética , Humanos , Inmunofenotipificación , Lectinas Tipo C/genética , Receptor de Manosa , Lectinas de Unión a Manosa/genética , Ratones , Ratones Endogámicos C57BL , MicroARNs/química , MicroARNs/genética , Datos de Secuencia Molecular , Neoplasias/irrigación sanguínea , Conformación de Ácido Nucleico , Precursores del ARN/química , Precursores del ARN/genética , Precursores del ARN/metabolismo , Receptores de Superficie Celular/genética , Quinasas Asociadas a rho/metabolismoRESUMEN
The purpose of this article is to review the suitability of the analytical and statistical techniques that have thus far been developed to assess the dissolution behavior of particles in the respirable aerodynamic size range, as generated by orally inhaled products (OIPs) such as metered-dose inhalers and dry powder inhalers. The review encompasses all analytical techniques publicized to date, namely, those using paddle-over-disk USP 2 dissolution apparatus, flow-through cell dissolution apparatus, and diffusion cell apparatus. The available techniques may have research value for both industry and academia, especially when developing modified-release formulations. The choice of a method should be guided by the question(s) that the research strives to answer, as well as by the strengths and weaknesses of the available techniques. There is still insufficient knowledge, however, for translating the dissolution data into statements about quality, performance, safety, or efficacy of OIPs in general. Any attempts to standardize a dissolution method for compendial inclusion or compendial use would therefore be premature. This review reinforces and expands on the 2008 stimulus article of the USP Inhalation Ad Hoc Advisory Panel, which "could not find compelling evidence suggesting that such dissolution testing is kinetically and/or clinically crucial for currently approved inhalation drug products."
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Preparaciones Farmacéuticas/administración & dosificación , Preparaciones Farmacéuticas/metabolismo , Administración por Inhalación , Administración Oral , Aerosoles/administración & dosificación , Aerosoles/metabolismo , SolubilidadRESUMEN
Secreted Semaphorin 3E (Sema3E) promotes cancer cell invasiveness and metastatic spreading. The pro-metastatic activity of Sema3E is due to its proteolytic fragment p61, capable of transactivating the oncogenic tyrosine kinase ErbB2 that associates with the Sema3E receptor PlexinD1 in cancer cells. Here, we show that a mutated, uncleavable variant of Sema3E (Uncl-Sema3E) binds to PlexinD1 like p61-Sema3E, but does not promote the association of PlexinD1 with ErbB2 nor activates the ensuing signalling cascade leading to metastatic spreading. Furthermore, Uncl-Sema3E competes with endogenous p61-Sema3E produced by tumour cells, thereby hampering their metastatic ability. Uncl-Sema3E also acts independently as a potent anti-angiogenic factor. It activates a PlexinD1-mediated signalling cascade in endothelial cells that leads to the inhibition of adhesion to extracellular matrix, directional migration and cell survival. The putative therapeutic potential of Uncl-Sema3E was validated in multiple orthotopic or spontaneous tumour models in vivo, where either local or systemic delivery of Uncl-Sema3E-reduced angiogenesis, growth and metastasis, even in the case of tumours refractory to treatment with a soluble vascular endothelial growth factor trap. In summary, we conclude that Uncl-Sema3E is a novel inhibitor of tumour angiogenesis and growth that concomitantly hampers metastatic spreading.
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Proliferación Celular , Furina/metabolismo , Neoplasias/metabolismo , Neoplasias/patología , Semaforinas/genética , Semaforinas/metabolismo , Animales , Moléculas de Adhesión Celular Neuronal/genética , Moléculas de Adhesión Celular Neuronal/metabolismo , Línea Celular Tumoral , Movimiento Celular , Células Endoteliales/citología , Células Endoteliales/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Péptidos y Proteínas de Señalización Intracelular , Glicoproteínas de Membrana , Ratones , Ratones Transgénicos , Mutación , Metástasis de la Neoplasia , Neoplasias/genética , Neoplasias/fisiopatología , Neovascularización Patológica , Unión Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transducción de SeñalRESUMEN
OBJECTIVE: The role of semaphorins in tumor progression is still poorly understood. In this study, we aimed at elucidating the regulatory role of semaphorin 3A (SEMA3A) in primary tumor growth and metastatic dissemination. METHODS AND RESULTS: We used 3 different experimental approaches in mouse tumor models: (1) overexpression of SEMA3A in tumor cells, (2) systemic expression of SEMA3A following liver gene transfer in mice, and (3) tumor-targeted release of SEMA3A using gene modified Tie2-expressing monocytes as delivery vehicles. In each of these experimental settings, SEMA3A efficiently inhibited tumor growth by inhibiting vessel function and increasing tumor hypoxia and necrosis, without promoting metastasis. We further show that the expression of the receptor neuropilin-1 in tumor cells is required for SEMA3A-dependent inhibition of tumor cell migration in vitro and metastatic spreading in vivo. CONCLUSIONS: In sum, both systemic and tumor-targeted delivery of SEMA3A inhibits tumor angiogenesis and tumor growth in multiple mouse models; moreover, SEMA3A inhibits the metastatic spreading from primary tumors. These data support the rationale for further investigation of SEMA3A as an anticancer molecule.
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Neoplasias de la Mama/terapia , Terapia Genética/métodos , Trasplante de Células Madre Hematopoyéticas , Neoplasias Pulmonares/prevención & control , Neovascularización Patológica/prevención & control , Semaforina-3A/metabolismo , Células Madre/metabolismo , Animales , Neoplasias de la Mama/irrigación sanguínea , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Hipoxia de la Célula , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Femenino , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundario , Ratones , Ratones Endogámicos BALB C , Necrosis , Neovascularización Patológica/genética , Neovascularización Patológica/metabolismo , Neuropilina-1/metabolismo , Comunicación Paracrina , Interferencia de ARN , Receptor TIE-2/genética , Receptor TIE-2/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Semaforina-3A/genética , Transducción de Señal , Células del Estroma/metabolismo , Factores de Tiempo , Transfección , Carga Tumoral , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The aggregation behaviour of casopitant mesylate, a new NK1 antagonist drug, was investigated by means of NMR spectroscopy and surface tension measurements. The critical micelle concentration (CMC) in glycine buffer at pH 3.5 was determined by analyzing the (1)H NMR chemical shifts variation and the surface tension in function of the concentration in a series of solutions. The temperature dependence of the CMC was also evaluated by NMR spectroscopy as well as the thermodynamic parameters contributing to the aggregation discussed. Surface tension measurements were conducted as well in the formulation conditions, e.g. in the presence of sodium chloride.
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Química Farmacéutica/métodos , Espectroscopía de Resonancia Magnética/métodos , Antagonistas del Receptor de Neuroquinina-1 , Piperazinas/farmacología , Piperidinas/farmacología , Diseño de Fármacos , Humanos , Concentración de Iones de Hidrógeno , Micelas , Modelos Químicos , Piperazina , Piperazinas/química , Propiedades de Superficie , Tecnología Farmacéutica/métodos , Temperatura , TermodinámicaRESUMEN
Semaphorin 3E (Sema3E) is a secreted molecule implicated in axonal path finding and inhibition of developmental and postischemic angiogenesis. Sema3E is also highly expressed in metastatic cancer cells, but its mechanistic role in tumor progression was not understood. Here we show that expression of Sema3E and its receptor Plexin D1 correlates with the metastatic progression of human tumors. Consistent with the clinical data, knocking down endogenous expression of either Sema3E or Plexin D1 in human metastatic carcinoma cells hampered their metastatic potential when injected into mice, while tumor growth was not markedly affected. Conversely, overexpression of exogenous Sema3E in cancer cells increased their invasiveness, transendothelial migration, and metastatic spreading, although it inhibited tumor vessel formation, resulting in reduced tumor growth in mice. The proinvasive and metastatic activity of Sema3E in tumor cells was dependent on transactivation of the Plexin D1-associated ErbB2/Neu oncogenic kinase. In sum, Sema3E-Plexin D1 signaling in cancer cells is crucially implicated in their metastatic behavior and may therefore be a promising target for strategies aimed at blocking tumor metastasis.
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Moléculas de Adhesión Celular Neuronal/fisiología , Metástasis de la Neoplasia , Semaforinas/fisiología , Transducción de Señal/fisiología , Animales , Células COS , Línea Celular Tumoral , Movimiento Celular , Chlorocebus aethiops , Femenino , Humanos , Péptidos y Proteínas de Señalización Intracelular , Glicoproteínas de Membrana , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Invasividad Neoplásica , Trasplante de Neoplasias , Receptor ErbB-2/fisiología , Semaforinas/análisis , Proteínas ras/genética , Proteínas de Unión al GTP rho/análisisRESUMEN
Semaphorins are a large family of evolutionarily conserved morphogenetic molecules originally identified for their repelling role in axonal guidance. Intriguingly, semaphorins have recently been implicated in cancer progression (Neufeld, G., T. Lange, A. Varshavsky, and O. Kessler. 2007. Adv. Exp. Med. Biol. 600:118-131). In particular, semaphorin 3B (SEMA3B) is considered a putative tumor suppressor, and yet we found that it is expressed at high levels in many invasive and metastatic human cancers. By investigating experimental tumor models, we confirmed that SEMA3B expression inhibited tumor growth, whereas metastatic dissemination was surprisingly increased. We found that SEMA3B induced the production of interleukin (IL) 8 by tumor cells by activating the p38-mitogen-activated protein kinase pathway in a neuropilin 1-dependent manner. Silencing the expression of endogenous SEMA3B in tumor cells impaired IL-8 transcription. The release of IL-8, in turn, induced the recruitment of tumor-associated macrophages and metastatic dissemination to the lung, which could be rescued by blocking IL-8 with neutralizing antibodies. In conclusion, we report that SEMA3B exerts unexpected functions in cancer progression by fostering a prometastatic environment through elevated IL-8 secretion and recruitment of macrophages coupled to the suppression of tumor growth.
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Interleucina-8/fisiología , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/fisiología , Semaforinas/genética , Semaforinas/fisiología , Neoplasias de la Mama/patología , Neoplasias de la Mama/prevención & control , Línea Celular Tumoral , Secuencia Conservada , Progresión de la Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica , Genes Supresores de Tumor , Humanos , Interleucina-8/genética , Invasividad Neoplásica , Metástasis de la Neoplasia , Neoplasias/prevención & control , Reacción en Cadena de la PolimerasaRESUMEN
Cell migration is pivotal in embryo development and in the adult. During development a wide range of progenitor cells travel over long distances before undergoing terminal differentiation. Moreover, the morphogenesis of epithelial tissues and of the cardiovascular system involves remodelling compact cell layers and sprouting of new tubular branches. In the adult, cell migration is essential for leucocytes involved in immune response. Furthermore, invasive and metastatic cancer cells have the distinctive ability to overcome normal tissue boundaries, travel in and out of blood vessels, and settle down in heterologous tissues. Cell migration normally follows strict guidance cues, either attractive, or inhibitory and repulsive. Semaphorins are a wide family of signals guiding cell migration during development and in the adult. Recent findings have established that semaphorin receptors, the plexins, govern cell migration by regulating integrin-based cell substrate adhesion and actin cytoskeleton dynamics, via specific monomeric GTPases. Plexins furthermore recruit tyrosine kinases in receptor complexes, which allows switching between multiple signaling pathways and functional outcomes. In this article, we will review the functional role of semaphorins in cell migration and the implicated molecular mechanisms controlling cell adhesion.
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Movimiento Celular/fisiología , Neuronas/citología , Neuronas/fisiología , Semaforinas/fisiología , Transducción de Señal/fisiología , Animales , Adhesión Celular/fisiología , Humanos , Sistema Nervioso/química , Sistema Nervioso/citología , Sistema Nervioso/embriología , Neuronas/química , Semaforinas/químicaRESUMEN
Plexins are transmembrane receptors for semaphorins, guiding cell migration and axon extension. Plexin activation leads to the disassembly of integrin-based focal adhesive structures and to actin cytoskeleton remodelling and inhibition of cell migration; however, the underlying molecular mechanisms are unclear. We consistently observe a transient decrease of cellular RhoA-GTP levels upon plexin activation in adherent cells. One of the main effectors of RhoA downregulation is p190, a ubiquitously expressed GTPase activating protein (GAP). We show that, in p190-deficient fibroblasts, the typical functional activities mediated by plexins (such as cell collapse and inhibition of integrin-based adhesion) are blocked or greatly impaired. Notably, the functional response can be rescued in these cells by re-expressing exogenous p190, but not a mutant form specifically lacking RhoGAP activity. We furthermore demonstrate that semaphorin function is blocked in epithelial cells, primary endothelial cells and neuroblasts upon treatment with small interfering RNAs that knockdown p190 expression. Finally, we show that p190 transiently associates with plexins, and its RhoGAP activity is increased in response to semaphorin stimulation. We conclude that p190-RhoGAP is crucially involved in semaphorin signalling to the actin cytoskeleton, via interaction with plexins.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Receptores de Superficie Celular/metabolismo , Semaforinas/metabolismo , Transducción de Señal , Animales , Antígenos CD/farmacología , Proteínas Reguladoras de la Apoptosis , Adhesión Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Quimiotaxis , Proteínas de Unión al ADN , Regulación hacia Abajo/genética , Células Endoteliales/citología , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Adhesiones Focales , Proteínas de Unión al GTP , Factores de Intercambio de Guanina Nucleótido , Humanos , Péptidos y Proteínas de Señalización Intracelular , Ratones , Células 3T3 NIH , Factor de Crecimiento Nervioso/farmacología , Neuritas , Células PC12 , Unión Proteica , ARN Interferente Pequeño/genética , Ratas , Proteínas Represoras , Semaforinas/farmacologíaRESUMEN
Vascular endothelial growth factor (VEGF) and semaphorin-3A (Sema-3A) play important roles in the transduction of promitotic and antimitotic signals, respectively. Here, we report that these conflicting signals are integrated via negative feedback between VEGF and Sema-3A pathways in several primary normal, but not malignant, mesothelial cells. Unlike malignant mesothelial (MM) cells, in which VEGF induces cell proliferation, normal mesothelial (NM) cell growth was repressed by VEGF. Although both cell-types expressed an overlapping set of VEGF tyrosine-kinase receptors, only in NM cells VEGF exposure entails a p38 mitogen-activated protein kinase (MAPK)-dependent increased of Sema-3A production. Inhibition of p38 MAPK (by SB202190 and SB203580) or a dominant-negative mutant of Sema-3A receptor plexin-A1 reversed the inhibitory effects of VEGF in NM cells, increasing cyclin D1 synthesis and cell growth. Conversely, sustained activation of p38 MAPK by the p38 MAPK-activating kinases MKK3 and MKK6 or transfection with Sema-3A inhibited VEGF-induced cyclin D1 up-regulation and MM cell proliferation. Therefore, these results delineate a new role of Sema-3A in VEGF function mediated by p38 MAPK and suggest that the abrogation of regulated Sema-3A expression is responsible for VEGF-driven growth of tumor cells.