RESUMEN
Retinal vascular diseases (RVD) have been identified as a major cause of blindness worldwide. These pathologies, including the wet form of age-related macular degeneration, retinopathy of prematurity, and diabetic retinopathy are currently treated by intravitreal delivery of anti-vascular endothelial growth factor (VEGF) agents. However, repeated intravitreal injections can lead to ocular complications and resistance to these treatments. Thus, there is a need to find new targeted therapies. Nucleolin regulates the endothelial cell (EC) activation and angiogenesis. In previous studies, we designed a pseudopeptide, N6L, that binds the nucleolin and blocks the tumor angiogenesis. In this study, the effect of N6L was investigated in two experimental models of retinopathies including oxygen-induced retinopathy (OIR) and choroidal neovascularization (CNV). We found that in mouse OIR, intraperitoneal injection of N6L is delivered to activated ECs and induced a 50% reduction of pathological neovascularization. The anti-angiogenic effect of N6L has been tested in CNV model in which the systemic injection of N6L induced a 33% reduction of angiogenesis. This effect is comparable to those obtained with VEGF-trap, a standard of care drug for RVD. Interestingly, with preventive and curative treatments, neoangiogenesis is inhibited by 59%. Our results have potential interest in the development of new therapies targeting other molecules than VEGF for RVD.
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Neovascularización Coroidal/prevención & control , Péptidos/farmacología , Fosfoproteínas/antagonistas & inhibidores , Proteínas de Unión al ARN/antagonistas & inhibidores , Enfermedades de la Retina/prevención & control , Animales , Proliferación Celular , Neovascularización Coroidal/etiología , Neovascularización Coroidal/metabolismo , Neovascularización Coroidal/patología , Modelos Animales de Enfermedad , Inyecciones Intravítreas , Ratones , Ratones Endogámicos C57BL , Oxígeno/efectos adversos , Fosforilación , Enfermedades de la Retina/etiología , Enfermedades de la Retina/metabolismo , Enfermedades de la Retina/patología , NucleolinaRESUMEN
Conventional chemotherapy used against cancer is mostly limited due to their non-targeted nature, affecting normal tissue and causing undesirable toxic effects to the affected tissue. With the aim of improving these treatments both therapeutically and in terms of their safety, numerous studies are currently being carried out using nanoparticles (NPs) as a vector combining tumor targeting and carrying therapeutic tools. In this context, it appears that nucleolin, a molecule over-expressed on the surface of tumor cells, is an interesting therapeutic target. Several ligands, antagonists of nucleolin of various origins, such as AS1411, the F3 peptide and the multivalent pseudopeptide N6L have been developed and studied as therapeutic tools against cancer. Over the last ten years or so, numerous studies have been published demonstrating that these antagonists can be used as tumor targeting agents with NPs from various origins. Focusing on nucleolin ligands, the aim of this article is to review the literature recently published or under experimentation in our research team to evaluate the efficacy and future development of these tools as anti-tumor agents.
Asunto(s)
Antineoplásicos/uso terapéutico , Aptámeros de Nucleótidos/uso terapéutico , Neoplasias/tratamiento farmacológico , Oligodesoxirribonucleótidos/uso terapéutico , Fragmentos de Péptidos/uso terapéutico , Péptidos/uso terapéutico , Fosfoproteínas/antagonistas & inhibidores , Proteínas de Unión al ARN/antagonistas & inhibidores , Antineoplásicos/química , Aptámeros de Nucleótidos/química , Portadores de Fármacos/administración & dosificación , Portadores de Fármacos/química , Expresión Génica , Proteína HMGB2/genética , Proteína HMGB2/metabolismo , Humanos , Ligandos , Terapia Molecular Dirigida/métodos , Nanopartículas/administración & dosificación , Nanopartículas/química , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patología , Oligodesoxirribonucleótidos/química , Fragmentos de Péptidos/química , Péptidos/química , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Nanomedicina Teranóstica/métodos , NucleolinaRESUMEN
Objective Weibel-Palade bodies (WPBs) are endothelial cell (EC)-specific organelles formed by vWF (von Willebrand factor) polymerization and that contain the proangiogenic factor Ang-2 (angiopoietin-2). WPB exocytosis has been shown to be implicated for vascular repair and inflammatory responses. Here, we investigate the role of WPBs during angiogenesis and vessel stabilization. Approach and Results WPB density in ECs decreased at the angiogenic front of retinal vascular network during development and neovascularization compared with stable vessels. In vitro, VEGF (vascular endothelial growth factor) induced a VEGFR-2 (vascular endothelial growth factor receptor-2)-dependent exocytosis of WPBs that contain Ang-2 and consequently the secretion of vWF and Ang-2. Blocking VEGF-dependant WPB exocytosis and Ang-2 secretion promoted pericyte migration toward ECs. Pericyte migration was inhibited by adding recombinant Ang-2 or by silencing Ang-1 (angiopoietin-1) or Tie2 (angiopoietin-1 receptor) in pericytes. Consistently, in vivo anti-VEGF treatment induced accumulation of WPBs in retinal vessels because of the inhibition of WPB exocytosis and promoted the increase of pericyte coverage of retinal vessels during angiogenesis. In tumor angiogenesis, depletion of WPBs in vWF knockout tumor-bearing mice promoted an increase of tumor angiogenesis and a decrease of pericyte coverage of tumor vessels. By another approach, normalized tumor vessels had higher WPB density. Conclusions We demonstrate that WPB exocytosis and Ang-2 secretion are regulated during angiogenesis to limit pericyte coverage of remodeling vessels by disrupting Ang-1/Tie2 autocrine signaling in pericytes.
Asunto(s)
Neovascularización Patológica/fisiopatología , Neovascularización Fisiológica/fisiología , Pericitos/fisiología , Cuerpos de Weibel-Palade/fisiología , Angiopoyetina 2/fisiología , Animales , Células Cultivadas , Células Endoteliales/fisiología , Exocitosis , Humanos , Ratones , Ratones Endogámicos C57BL , Neoplasias/irrigación sanguínea , Retina/fisiología , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Factor A de Crecimiento Endotelial Vascular/fisiologíaRESUMEN
Targeted anticancer therapies demand discovery of new cellular targets to be exploited for the delivery of toxic molecules and drugs. In this perspective, in the last few years, nucleolin has been identified as an interesting surface marker to be used for the therapy of glioblastoma. In this study, we investigated whether a synthetic antagonist of cell-surface nucleolin known as N6L, previously reported to decrease both tumor growth and tumor angiogenesis in several cancer cell lines, including glioblastoma cells, as well as endothelial cells proliferation, could be exploited to deliver a protein toxin (saporin) to glioblastoma cells. The pseudopeptide N6L cross-linked to saporin-S6 induced internalization of the toxin inside glioblastoma cancer cells. Our results in vitro demonstrated the effectiveness of this conjugate in inducing cell death, with an ID50 four orders of magnitude lower than that observed for free N6L. Furthermore, the preliminary in vivo study demonstrated efficiency in reducing the tumor mass in an orthotopic mouse model of glioblastoma.
Asunto(s)
Glioblastoma/tratamiento farmacológico , Neovascularización Patológica/tratamiento farmacológico , Péptidos/farmacología , Fosfoproteínas/farmacología , Proteínas de Unión al ARN/farmacología , Animales , Línea Celular Tumoral , Membrana Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Glioblastoma/genética , Glioblastoma/patología , Humanos , Ratones , Terapia Molecular Dirigida , Neovascularización Patológica/patología , Péptidos/química , Fosfoproteínas/química , Proteínas de Unión al ARN/química , Saporinas/química , Saporinas/farmacología , Ensayos Antitumor por Modelo de Xenoinjerto , NucleolinaRESUMEN
The synaptic protein Neuroligin 1 (NLGN1), a cell adhesion molecule, is critical for the formation and consolidation of synaptic connectivity and is involved in vascular development. The mechanism through which NLGN1 acts, especially in vascular cells, is unknown. Here, we aimed at deepening our knowledge on the cellular activities and molecular pathways exploited by endothelial NLGN1 both in vitro and in vivo. We analyzed the phenotypic consequences of NLGN1 expression modulation in endothelial cells through in vitro angiogenesis assays and the mouse postnatal retinal angiogenesis model. We demonstrate that NLGN1, whereas not affecting endothelial cell proliferation or migration, modulates cell adhesion to the vessel stabilizing protein laminin through cooperation with the α6 integrin, a specific laminin receptor. Finally, we show that in vivo, NLGN1 and α6 integrin preferentially colocalize in the mature retinal vessels, whereas NLGN1 deletion causes an aberrant VE-cadherin, laminin and α6 integrin distribution in vessels, along with significant structural defects in the vascular tree.
Asunto(s)
Moléculas de Adhesión Celular Neuronal/metabolismo , Células Endoteliales/metabolismo , Integrina alfa6/metabolismo , Neovascularización Fisiológica/fisiología , Vasos Retinianos/metabolismo , Animales , Antígenos CD/genética , Antígenos CD/metabolismo , Cadherinas/genética , Cadherinas/metabolismo , Adhesión Celular/fisiología , Moléculas de Adhesión Celular Neuronal/genética , Movimiento Celular/fisiología , Proliferación Celular , Células Endoteliales/citología , Células Endoteliales de la Vena Umbilical Humana , Humanos , Integrina alfa6/genética , Ratones , Ratones Mutantes , Vasos Retinianos/citologíaRESUMEN
Ras GTPases signal by orchestrating a balance among several effector pathways, of which those driven by the GTPases RalA and RalB are essential to Ras oncogenic functions. RalA and RalB share the same effectors but support different aspects of oncogenesis. One example is the importance of active RalA in anchorage-independent growth and membrane raft trafficking. This study has shown a new post-translational modification of Ral GTPases: nondegradative ubiquitination. RalA (but not RalB) ubiquitination increases in anchorage-independent conditions in a caveolin-dependent manner and when lipid rafts are endocytosed. Forcing RalA mono-ubiquitination (by expressing a protein fusion consisting of ubiquitin fused N-terminally to RalA) leads to RalA enrichment at the plasma membrane and increases raft exposure. This study suggests the existence of an ubiquitination/de-ubiquitination cycle superimposed on the GDP/GTP cycle of RalA, involved in the regulation of RalA activity as well as in membrane raft trafficking.
Asunto(s)
Microdominios de Membrana/metabolismo , Ubiquitina/metabolismo , Ubiquitinación/fisiología , Proteínas de Unión al GTP ral/metabolismo , Transporte Biológico/fisiología , Guanosina Difosfato/genética , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/genética , Guanosina Trifosfato/metabolismo , Células HeLa , Humanos , Microdominios de Membrana/genética , Ubiquitina/genética , Proteínas de Unión al GTP ral/genéticaRESUMEN
The Ras family G-proteins RalA and RalB make critical non-overlapping contributions to the generation of a tumorigenic regulatory network, supporting bypass of the normal restraints on both cell proliferation and survival. The Sec6/8 complex, or exocyst, has emerged as a principal direct effector complex for Ral GTPases. Here, we show that RalA and RalB support mitotic progression through mobilization of the exocyst for two spatially and kinetically distinct steps of cytokinesis. RalA is required to tether the exocyst to the cytokinetic furrow in early cytokinesis. RalB is then required for recruitment of the exocyst to the midbody of this bridge to drive abscission and completion of cytokinesis. The collaborative action of RalA and RalB is specified by discrete subcellular compartmentalization and unique pairs of RalGEF proteins that provide inputs from both Ras-family protein-dependent and protein-independent regulatory cues. This suggests that Ral GTPases integrate diverse upstream signals to choreograph multiple roles for the exocyst in mitotic progression.
Asunto(s)
Citocinesis , Regulación de la Expresión Génica , Proteínas de Unión al GTP ral/fisiología , Factor de Intercambio de Guanina Nucleótido ral/metabolismo , Núcleo Celular/metabolismo , Proliferación Celular , Supervivencia Celular , Células HeLa , Humanos , Cinética , Mitosis , Modelos Biológicos , Proteínas de Transporte Vesicular/metabolismo , Proteínas de Unión al GTP ral/metabolismo , Proteínas ras/metabolismoRESUMEN
The Pony of Esperia is an Italian autochthonous horse breed reared in the wild on the Aurunci and Ausoni Mountains. Currently, it is considered an endangered breed, as its population consists of 1623 animals. It is therefore essential to identify all aspects that can improve the management and economy of its breeding, favoring its diffusion. In this paper, the effects of intestinal strongyle infection on the chromosome stability of peripheral blood lymphocytes (PBLs) was evaluated through aneuploidy and chromosome aberration (gap, chromatid and chromosome breaks, and the number of abnormal cells) test. Statistical difference in the mean values of aneuploidy, cells with chromosome abnormalities, and chromosome and chromatid breaks were observed between ponies with high fecal egg counts (eggs per gram > 930) and those with undetectable intestinal strongylosis. The causes of this phenomenon and possible repercussions on the management of Pony of Esperia are discussed in the paper.
RESUMEN
Active drug delivery by tumor-targeting peptides is a promising approach to improve existing therapies for rhabdomyosarcoma (RMS), by increasing the therapeutic effect and decreasing the systemic toxicity, e.g., by drug-loaded peptide-targeted nanoparticles. Here, we tested 20 different tumor-targeting peptides for their ability to bind to two RMS cell lines, Rh30 and RD, using quantum dots Streptavidin and biotin-peptides conjugates as a model for nanoparticles. Four peptides revealed a very strong binding to RMS cells: NCAM-1-targeting NTP peptide, nucleolin-targeting F3 peptide, and two Furin-targeting peptides, TmR and shTmR. F3 peptide showed the strongest binding to all RMS cell lines tested, low binding to normal control myoblasts and fibroblasts, and efficient internalization into RMS cells demonstrated by the cytoplasmic delivery of the Saporin toxin. The expression of the nucleophosphoprotein nucleolin, the target of F3, on the surface of RMS cell lines was validated by competition with the natural ligand lactoferrin, by colocalization with the nucleolin-binding aptamer AS1411, and by the marked sensitivity of RMS cell lines to the growth inhibitory nucleolin-binding N6L pseudopeptide. Taken together, our results indicate that nucleolin-targeting by F3 peptide represents a potential therapeutic approach for RMS.
RESUMEN
The ocular vasculature is critically involved in many blinding diseases and is also a popular research model for the exploration of developmental and pathological angiogenesis. The development of ocular vessels is a complex, finely orchestrated sequence of events, involving spatial and temporal coordination of hyaloid, choroidal and retinal networks. Comprehensive studies of the tridimensional dynamics of microvascular remodeling are limited by the fact that preserving the spatial disposition of ocular vascular networks is cumbersome using classical histological procedures. Here, we demonstrate that light-sheet fluorescence microscopy (LFSM) of cleared mouse eyes followed by extensive virtual dissection offers a solution to this problem. To the best of our knowledge, this is the first 3D quantification of the evolution of the hyaloid vasculature and of post-occlusive venous remodeling together with the characterization of spatial distribution of various cell populations in ocular compartments, including the vitreous. These techniques will prove interesting to obtain other insights in scientific questions addressing organ-wide cell interactions.
Asunto(s)
Coroides , Vasos Retinianos , Ratones , Animales , Retina , Neovascularización Patológica , Microscopía FluorescenteRESUMEN
HYPOTHESIS: We describe the possibility of using the same block copolymer carriers prepared by PISA for in situ drug encapsulation or drug conjugation. EXPERIMENTS: Block copolymers containing poly((ethylene glycol) methacrylate)-co-poly(pentafluorophenyl methacrylate)-b-poly(hydroxypropyl methacrylate) (P((PEGMA-co-PFBMA)-b-PHPMA)) were synthesized at 10 wt% using PISA. The first approach involved in situ Doxorubicin (DOX) loading during PISA, while the second exhibited surface functionalization of PISA-made vesicles with dual drug therapies, N-acetyl cysteine (NAC) and DOX using para-fluoro-thiol reaction (PFTR) and carbodiimide chemistry, respectively. Cytotoxicity, cell uptake, and cell apoptosis were assessed on MDA-MB-231 cell lines. FINDINGS: P((PEGMA-co-PFBMA)-b-PHPMA) nanocarriers were prepared, showing size and shape transformations from spheres, cylinders to raspberry-forming vesicles. DOX was readily loaded into NPs during PISA with relatively high encapsulation efficiency of 70 %, whereas the plain PISA-made vesicles could be functionalized with NAC and DOX at high yields. DOX-free NPs showed biocompatibility, whilst DOX-conjugated NPs imparted a concentration-dependent cytotoxicity, as well as an enhanced cell uptake compared to free DOX. The results demonstrated that the same PISA-derived self-assemblies enabled either in situ drug encapsulation, or post-polymerization surface engineering with useful functionalities upon tuning the macro-CTA block, thus holding promises for future drug delivery and biomedical applications.
Asunto(s)
Nanopartículas , Neoplasias , Doxorrubicina/metabolismo , Doxorrubicina/farmacología , Portadores de Fármacos , Humanos , Metacrilatos , Micelas , Polimerizacion , PolímerosRESUMEN
In the original publication [...].
RESUMEN
Background: The pancreatic ductal adenocarcinoma (PDAC) microenvironment is highly fibrotic and hypoxic, with poor immune cell infiltration. Recently, we showed that nucleolin (NCL) inhibition normalizes tumour vessels and impairs PDAC growth. Methods: Immunocompetent mouse models of PDAC were treated by the pseudopeptide N6L, which selectively inhibits NCL. Tumour-infiltrating immune cells and changes in the tumour microenvironment were analysed. Results: N6L reduced the proportion of regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs) and increased tumour-infiltrated T lymphocytes (TILs) with an activated phenotype. Low-dose anti-VEGFR2 treatment normalized PDAC vessels but did not modulate the immune suppressive microenvironment. RNAseq analysis of N6L-treated PDAC tumours revealed a reduction of cancer-associated fibroblast (CAF) expansion in vivo and in vitro. Notably, N6L treatment decreased IL-6 levels both in tumour tissues and in serum. Treating mPDAC by an antibody blocking IL-6 reduced the proportion of Tregs and MDSCs and increased the amount of TILs, thus mimicking the effects of N6L. Conclusions: These results demonstrate that NCL inhibition blocks the amplification of lymphoid and myeloid immunosuppressive cells and promotes T cell activation in PDAC through a new mechanism of action dependent on the direct inhibition of the tumoral stroma.
RESUMEN
We previously showed that N6L, a pseudopeptide that targets nucleolin, impairs pancreatic ductal adenocarcinoma (PDAC) growth and normalizes tumor vessels in animal models. In this study, we analyzed the translatome of PDAC cells treated with N6L to identify the pathways that were either repressed or activated. We observed a strong decrease in global protein synthesis. However, about 6% of the mRNAs were enriched in the polysomes. We identified a 5'TOP motif in many of these mRNAs and demonstrated that a chimeric RNA bearing a 5'TOP motif was up-regulated by N6L. We demonstrated that N6L activates the mTOR pathway, which is required for the translation of these mRNAs. An inhibitory synergistic effect in PDAC cell lines, including patient-derived xenografts and tumor-derived organoids, was observed when N6L was combined with mTOR inhibitors. In conclusion, N6L reduces pancreatic cells proliferation, which then undergoes translational reprogramming through activation of the mTOR pathway. N6L and mTOR inhibitors act synergistically to inhibit the proliferation of PDAC and human PDX cell lines. This combotherapy of N6L and mTOR inhibitors could constitute a promising alternative to treat pancreatic cancer.
RESUMEN
Only a fraction of cancer patients benefits from immune checkpoint inhibitors. This may be partly due to the dense extracellular matrix (ECM) that forms a barrier for T cells. Comparing five preclinical mouse tumor models with heterogeneous tumor microenvironments, we aimed to relate the rate of tumor stiffening with the remodeling of ECM architecture and to determine how these features affect intratumoral T cell migration. An ECM-targeted strategy, based on the inhibition of lysyl oxidase, was used. In vivo stiffness measurements were found to be strongly correlated with tumor growth and ECM crosslinking but negatively correlated with T cell migration. Interfering with collagen stabilization reduces ECM content and tumor stiffness leading to improved T cell migration and increased efficacy of anti-PD-1 blockade. This study highlights the rationale of mechanical characterizations in solid tumors to understand resistance to immunotherapy and of combining treatment strategies targeting the ECM with anti-PD-1 therapy.
Asunto(s)
Fenómenos Fisiológicos Celulares/efectos de los fármacos , Colágeno/metabolismo , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Linfocitos T/metabolismo , Microambiente Tumoral/fisiología , Animales , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Matriz Extracelular/metabolismo , Femenino , Inhibidores de Puntos de Control Inmunológico/farmacología , Ratones , Ratones Endogámicos C57BL , Neoplasias Experimentales , Proteína-Lisina 6-Oxidasa/metabolismoRESUMEN
Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive and resistant cancer with no available effective therapy. We have previously demonstrated that nucleolin targeting by N6L impairs tumor growth and normalizes tumor vessels in PDAC mouse models. Here, we investigated new pathways that are regulated by nucleolin in PDAC. We found that N6L and nucleolin interact with ß-catenin. We found that the Wnt/ß-catenin pathway is activated in PDAC and is necessary for tumor-derived 3D growth. N6L and nucleolin loss of function induced by siRNA inhibited Wnt pathway activation by preventing ß-catenin stabilization in PDAC cells. N6L also inhibited the growth and the activation of the Wnt/ß-catenin pathway in vivo in mice and in 3D cultures derived from MIA PaCa2 tumors. On the other hand, nucleolin overexpression increased ß-catenin stabilization. In conclusion, in this study, we identified ß-catenin as a new nucleolin interactor and suggest that the Wnt/ß-catenin pathway could be a new target of the nucleolin antagonist N6L in PDAC.
RESUMEN
BACKGROUND & AIMS: Cancer-associated fibroblasts (CAFs) from pancreatic adenocarcinoma (PDA) present high protein synthesis rates. CAFs express the G-protein-coupled somatostatin receptor sst1. The sst1 agonist SOM230 blocks CAF protumoral features in vitro and in immunocompromised mice. We have explored here the therapeutic potential of SOM230, and underlying mechanisms, in immunocompetent models of murine PDA mimicking the heavy fibrotic and immunosuppressive stroma observed in patient tumors. METHODS: Large-scale mass spectrometry analyses were performed on media conditioned from 9 patient PDA-derived CAF primary cultures. Spontaneous transgenic and experimental (orthotopic co-graft of tumor cells plus CAFs) PDA-bearing mice were longitudinally ultrasound-monitored for tumor and metastatic progression. Histopathology and flow cytometry analyses were performed on primary tumors and metastases. Stromal signatures were functionally validated through bioinformatics using several published, and 1 original, PDA database. RESULTS: Proteomics on the CAF secretome showed that SOM230 controls stromal activities including inflammatory responses. Among the identified secreted proteins, we validated that colony-stimulating factor 1 (CSF-1) (a macrophage growth factor) was reduced by SOM230 in the tumor and plasma of PDA-harboring mice, alongside intratumor stromal normalization (reduced CAF and macrophage activities), and dramatic metastasis reduction. In transgenic mice, these SOM230 benefits alleviate the chemotherapy-induced (gemcitabine) immunosuppressive stroma reshaping. Mechanistically, SOM230 acts in vivo on CAFs through sst1 to disrupt prometastatic CAF production of CSF-1 and cross-talk with macrophages. We found that in patients, stromal CSF-1 was associated with aggressive PDA forms. CONCLUSIONS: We propose SOM230 as an antimetastatic therapy in PDA for its capacity to remodel the fibrotic and immunosuppressive myeloid stroma. This pharmacotherapy should benefit PDA patients treated with chemotherapies.
Asunto(s)
Fibroblastos Asociados al Cáncer/efectos de los fármacos , Carcinoma Ductal Pancreático/tratamiento farmacológico , Macrófagos/efectos de los fármacos , Neoplasias Pancreáticas/tratamiento farmacológico , Secretoma/efectos de los fármacos , Somatostatina/análogos & derivados , Anciano , Anciano de 80 o más Años , Animales , Fibroblastos Asociados al Cáncer/metabolismo , Fibroblastos Asociados al Cáncer/patología , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/secundario , Femenino , Hormonas/farmacología , Humanos , Macrófagos/metabolismo , Macrófagos/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Persona de Mediana Edad , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Somatostatina/farmacologíaRESUMEN
During angiogenic remodeling, Ang-1, the ligand of Tie2 tyrosine kinase, is involved in vessel sprouting and stabilization through unclear effects on nascent capillaries and mural cells. In our study, we hypothesized that the Ang-1/Tie2 system could cross-talk with integrins, and be influenced by the dynamic interactions between extracellular matrix and endothelial cells (ECs). Here, we show that alpha5beta1 specifically sensitizes and modulates Tie2 receptor activation and signaling, allowing EC survival at low concentrations of Ang-1 and inducing persistent EC motility. Tie2 and alpha5beta1 interact constitutively; alpha5beta1 binding to fibronectin increases this association, whereas Ang-1 stimulation recruits p85 and FAK to this complex. Furthermore, we demonstrate that Ang-1 is able to mediate selectively alpha5beta1 outside-in FAK phosphorylation. Thus, Ang-1 triggers signaling pathways through Tie2 and alpha5beta1 receptors that could cross-talk when Tie2/alpha5beta1 interaction occurs in ECs plated on fibronectin. By using blocking antibodies, we consistently found that alpha5beta1, but not alphavbeta3 activation, is essential to Ang-1-dependent angiogenesis in vivo.
Asunto(s)
Angiopoyetina 1/farmacología , Endotelio Vascular/efectos de los fármacos , Integrina alfa5beta1/metabolismo , Receptor TIE-2/metabolismo , Animales , Células CHO , Adhesión Celular/efectos de los fármacos , Técnicas de Cultivo de Célula , Línea Celular , Movimiento Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Embrión de Pollo , Membrana Corioalantoides/irrigación sanguínea , Cromonas/farmacología , Colágeno Tipo I/metabolismo , Cricetinae , Cricetulus , Relación Dosis-Respuesta a Droga , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Fibrinógeno/metabolismo , Fibronectinas/metabolismo , Humanos , Cinética , Morfolinas/farmacología , Neovascularización Fisiológica/efectos de los fármacos , Pruebas de Precipitina , Venas Umbilicales/citologíaRESUMEN
Large-pore mesoporous silica (LPMS) microspheres with tunable pore size have received intensive interest in the field of drug delivery due to their high storage capacity and fast delivery rate of drugs. In this work, a facile salt-assisted spray-drying method has been developed to fabricate LPMS microspheres using continuous spray-drying of simple inorganic salts as pore templates and colloidal SiO2 nanoparticles as building blocks, followed by washing with water to remove the templates. More importantly, the porosity of the LPMS microspheres can be finely tuned by adjusting the furnace temperature and relative concentration of the salt to SiO2, which could lead to optimal pharmaceutical outcomes. Then, the biological roles of these LPMS microspheres were evaluated in antibacterial and cancer therapy. In this regard, rhodamine b as a probe was initially loaded inside the LPMS microspheres. The obtained particles not only showed high entrapment efficiency (up to 30%) and a pH-responsive drug release but also presented pore-size-controlled drug release performance. Then, in vitro antibacterial activities of multiple antibiotics, namely nalidixic acid, chloramphenicol, and ciprofloxacin, loaded in the LPMS particles were investigated against two pathogenic bacteria, Escherichia coli (Gram-negative) and Staphylococcus aureus (Gram-positive). The results indicated bacterial inhibition up to 70% and 20% in less than 2 h for Escherichia coli and Staphylococcus aureus, respectively. This inhibition of bacterial growth was accompanied by no bacterial regrowth within 30 h. Finally, the versatility of LPMS microspheres as drug carriers in pancreatic cancer treatment was explored. In this regard, a pro-apoptotic NCL antagonist agent (N6L) as an antitumor agent was successfully loaded onto LPMS microspheres. Interestingly, the resulting particles showed pore-size-dependent anticancer activity with inhibition of cancer cell growth up to 60%.
Asunto(s)
Neoplasias Pancreáticas , Dióxido de Silicio , Antibacterianos/farmacología , Composición de Medicamentos , Humanos , Microesferas , Neoplasias Pancreáticas/tratamiento farmacológico , Tamaño de la Partícula , PorosidadRESUMEN
Lung cancer is the most common cause of cancer death in the United States. The genome of non-small cell lung cancer (NSCLC), the most frequent lung cancer type, is strongly affected by Long Interspersed Nuclear Element (LINE1) insertions. Active LINE1s are repetitive DNA sequences that can amplify themselves in the genome utilizing a retrotransposition mechanism whereby LINE1 is copied via reverse transcription and inserted at target sites. ORF1p and ORF2p are LINE1 encoded proteins essential for LINE1 retrotransposition. LINE1s are silenced epigenetically in somatic tissues, and their reactivation has been associated with cancer pathogenesis. Here, we present evidence that nucleolin (NCL) regulates expression of LINE1-ORF1p (L1-ORF1p) in NSCLC cells. Genetic knockdown of NCL significantly inhibited expression of L1-ORF1p in various NSCLC cell lines. Treatment with the investigational NCL antagonist N6L ablated L1-ORF1p expression in all cell lines constitutively expressing L1-ORFp. N6L displayed a stronger antiproliferative activity in NSCLC tumor cell lines expressing the highest L1-ORF1p protein levels. Moreover, N6L treatment of nude mice bearing NSCLC tumor xenografts blocked L1-ORF1p expression and effectively inhibited tumor growth. These data indicate that L1-ORF1p expression is regulated by NCL and identify NCL as a novel promising target for pharmacological inhibition of LINE1.