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BACKGROUND: In the past decades studies on anti-tumoral drugs inhibiting matrix metalloproteinase (MMPs) were disappointing. Recently, we demonstrated that mature endothelial cells (ECs) and endothelial colony forming cells (ECFCs) can switch between invasion modes to cope with challenging environments, performing the "amoeboid angiogenesis" in the absence of proteases activity. METHODS: We first set out to investigate by ELISA if the inhibitors of the main protease family involved in angiogenesis were differently expressed during breast cancer progression. We used Marimastat, a broad-spectrum MMP inhibitor, as a means of inducing amoeboid characteristics and studied VEGF role in amoeboid angiogenesis. Thus, we performed invasion and capillary morphogenesis assay, morphological, cell signaling and in vivo mouse studies. RESULTS: Our data showed that TIMP1, TIMP2, alpha2-antiplasmin, PAI-1 and cystatin increase in breast cancer serum of patients with primary cancer and lymph node positive compared to healthy women. In vitro results revealed that the most high-powered protease inhibitors able to induce amoeboid invasion of ECFCs were TIMP1, 2 and 3. Surprisingly, Marimastat promotes ECFC invasion and tubular formation in vitro and in vivo, inducing amoeboid characteristics. We observed that the combination of Marimastat plus VEGF doesn't boost neither cell invasion nor vessel formation capacity. Moreover, inhibition of VEGF activity with Bevacizumab in the presence of Marimastat confirmed that amoeboid angiogenesis is independent from the stimulus of the main vascular growth factor, VEGF. CONCLUSIONS: We underline the importance to consider the amoeboid mechanism of endothelial and cancer cell invasion, probably responsible for the failure of synthetic metalloproteinase inhibitors as cancer therapy and tumor resistance to VEGF-targeted therapies, to set-up new drugs to be used in cancer therapy.
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Amoeba , Neoplasias , Animales , Femenino , Ratones , Amoeba/metabolismo , Inhibidores de la Angiogénesis/farmacología , Inhibidores de la Angiogénesis/uso terapéutico , Células Endoteliales/metabolismo , Metaloproteinasas de la Matriz/metabolismo , Morfogénesis , Neoplasias/tratamiento farmacológico , Neovascularización Patológica/tratamiento farmacológico , Neovascularización Patológica/metabolismo , Transducción de Señal , Factor A de Crecimiento Endotelial Vascular/metabolismo , Sistema de Señalización de MAP QuinasasRESUMEN
Senescence occurs upon critical telomere shortening, or following DNA damage, oncogenic activation, hypoxia and oxidative stress, overall referred to stress-induced premature senescence (SIPS). In response to DNA damage, senescent cells release cytoplasmic chromatin fragments (CCFs), and express an altered secretome, the senescence-associated secretory phenotype (SASP), which contributes to generate a pro-inflammatory and pro-tumoral extracellular milieu. Polyphenols have gained significant attention owing to their anti-inflammatory and anti-tumour activities. Here, we studied the effect of oleuropein aglycone (OLE) and hydroxytyrosol (HT) on DNA damage, CCF appearance and SASP in a model of irradiation-induced senescence. Neonatal human dermal fibroblasts (NHDFs) were γ-irradiated and incubated with OLE, 5 µM and HT, 1 µM. Cell growth and senescence-associated (SA)-ß-Gal-staining were used as senescence markers. DNA damage was evaluated by Comet assay, lamin B1 expression, release of CCFs, cyclic GMP-AMP Synthase (cGAS) activation. IL-6, IL-8, MCP-1 and RANTES were measured by ELISA assay. Our results showed that OLE and HT exerted a protective effect on 8 Gy irradiation-induced senescence, preserving lamin B1 expression and reducing cGAS/STING/NFκB-mediated SASP. The ability of OLE and HT to mitigate DNA damage, senescence status and the related SASP in normal cells can be exploited to improve the efficacy and safety of cancer radiotherapy.
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Neoplasias , Olea , Senescencia Celular , Daño del ADN , Humanos , Lamina Tipo B , FN-kappa B/genética , Neoplasias/metabolismo , Nucleotidiltransferasas/genética , Olea/metabolismo , Fenoles/farmacología , Radiación IonizanteRESUMEN
How T-helper (Th) lymphocyte subpopulations identified in synovial fluid from patients with juvenile idiopathic arthritis (JIA) (Th17, classic Th1, or nonclassic Th1) drive joint damage is of great interest for the possible use of biological drugs that inhibit the specific cytokines. Our objective was to clarify the role of such Th subpopulations in the pathogenesis of articular cartilage destruction by synovial fibroblasts (SFbs), and the effect of Th17 blockage in an animal model. SFbs were isolated from healthy subjects and patients with JIA, and peripheral blood Th lymphocytes subsets were obtained from healthy subjects. Fragments of human cartilage from healthy subjects in a collagen matrix containing JIA or normal SFbs grafted underskin in SCID mice were used to measure cartilage degradation under the effects of Th supernatants. JIA SFbs overexpress MMP9 and MMP2 and Th17 induce both MMPs in normal SFbs, while nonclassic Th1 upregulate urokinase plasminogen activator (uPA) activity. In vitro invasive phenotype of normal SFbs is stimulated with conditioned medium of Th17 and nonclassic-Th1. In the in vivo "inverse wrap" model, normal SFbs stimulated with supernatants of Th17-lymphocytes and nonclassic Th1 produced a cartilage invasion and degradation similar to JIA SFbs. Secukinumab inhibits the cartilage damage triggered by factors produced by Th17.
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Anticuerpos Monoclonales Humanizados/uso terapéutico , Artritis Juvenil/inmunología , Artritis Juvenil/terapia , Cartílago Articular/inmunología , Cartílago Articular/patología , Células Th17/inmunología , Células Th17/patología , Adolescente , Animales , Artritis Experimental/inmunología , Artritis Experimental/patología , Artritis Experimental/terapia , Artritis Juvenil/patología , Cartílago Articular/metabolismo , Estudios de Casos y Controles , Niño , Preescolar , Citocinas/inmunología , Modelos Animales de Enfermedad , Fibroblastos/inmunología , Fibroblastos/metabolismo , Fibroblastos/patología , Humanos , Técnicas In Vitro , Interleucina-17/antagonistas & inhibidores , Ratones , Ratones SCID , Proteolisis , Membrana Sinovial/inmunología , Membrana Sinovial/metabolismo , Membrana Sinovial/patologíaRESUMEN
Exosomes (Exos) have been reported to promote pre-metastatic niche formation, proliferation, angiogenesis and metastasis. We have investigated the role of uPAR in melanoma cell lines-derived Exos and their pro-angiogenic effects on human microvascular endothelial cells (HMVECs) and endothelial colony-forming cells (ECFCs). Melanoma Exos were isolated from conditioned media of A375 and M6 cells by differential centrifugation and filtration. Tunable Resistive Pulse Sensing (TRPS) and Nanoparticle tracking analysis were performed to analyze dimension and concentration of Exos. The CRISPR-Cas 9 technology was exploited to obtain a robust uPAR knockout. uPAR is expressed in melanoma Exos that are internalized by HMVECs and ECFCs, enhancing VE-Cadherin, EGFR and uPAR expression in endothelial cells that undergo a complete angiogenic program, including proliferation, migration and tube formation. uPAR loss reduced the pro-angiogenic effects of melanoma Exos in vitro and in vivo by inhibition of VE-Cadherin, EGFR and uPAR expression and of ERK1,2 signaling in endothelial cells. A similar effect was obtained with a peptide that inhibits uPAR-EGFR interaction and with the EGFR inhibitor Gefitinib, which also inhibited melanoma Exos-dependent EGFR phosphorylation. This study suggests that uPAR is required for the pro-angiogenic activity of melanoma Exos. We propose the identification of uPAR-expressing Exos as a potentially useful biomarker for assessing pro-angiogenic propensity and eventually monitoring the response to treatment in metastatic melanoma patients.
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Antígenos CD/metabolismo , Cadherinas/metabolismo , Exosomas/metabolismo , Receptores del Activador de Plasminógeno Tipo Uroquinasa/metabolismo , Transducción de Señal , Animales , Antígenos CD/genética , Cadherinas/genética , Línea Celular , Células Endoteliales/citología , Células Endoteliales/metabolismo , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/genética , Receptores ErbB/metabolismo , Gefitinib/farmacología , Edición Génica , Humanos , Melanoma/metabolismo , Melanoma/patología , Ratones , Ratones SCID , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Neovascularización Fisiológica , Fosforilación/efectos de los fármacos , Interferencia de ARN , ARN Interferente Pequeño , Receptores del Activador de Plasminógeno Tipo Uroquinasa/antagonistas & inhibidores , Receptores del Activador de Plasminógeno Tipo Uroquinasa/genéticaRESUMEN
In this manuscript, we show the involvement of the uPA/uPAR system in the regulation of aerobic glycolysis of melanoma cells. uPAR over-expression in human melanoma cells controls an invasive and glycolytic phenotype in normoxic conditions. uPAR down-regulation by siRNA or its uncoupling from integrins, and hence from integrin-linked tyrosine kinase receptors (IL-TKRs), by an antagonist peptide induced a striking inhibition of the PI3K/AKT/mTOR/HIF1α pathway, resulting into impairment of glucose uptake, decrease of several glycolytic enzymes and of PKM2, a checkpoint that controls metabolism of cancer cells. Further, binding of uPA to uPAR regulates expression of molecules that govern cell invasion, including extracellular matrix metallo-proteinases inducer (EMPPRIN) and enolase, a glycolytyc enzyme that also serves as a plasminogen receptor, thus providing a common denominator between tumor metabolism and phenotypic invasive features. Such effects depend on the α5ß1-integrin-mediated uPAR connection with EGFR in melanoma cells with engagement of the PI3K-mTOR-HIFα pathway. HIF-1α trans-activates genes whose products mediate tumor invasion and glycolysis, thus providing the common denominator between melanoma metabolism and its invasive features. These findings unveil a unrecognized interaction between the invasion-related uPAR and IL-TKRs in the control of glycolysis and disclose a new pharmacological target (i.e., uPAR/IL-TKRs axis) for the therapy of melanoma.
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Melanoma/metabolismo , Receptores del Activador de Plasminógeno Tipo Uroquinasa/metabolismo , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo , Animales , Línea Celular Tumoral , Regulación hacia Abajo , Femenino , Glucólisis , Células HEK293 , Xenoinjertos , Humanos , Melanoma/patología , Ratones , Ratones Desnudos , Ratones SCID , Invasividad Neoplásica , FenotipoRESUMEN
Bone metastasis is a common burden in many types of cancer and has a severe impact on the quality of life in patients. Hence, specific therapeutic strategies inhibiting tumor induced osteolysis are urgently needed. In this study, we aimed to interfere with integrin adhesion receptors, which are central players of the bone resorption process. For this purpose, we used cilengitide, a cyclic RGD peptide, which blocks integrin αVß3 and αVß5-ligand binding. Our results revealed that cilengitide blocked osteoclast maturation in a dose-dependent manner. In detail, pre-osteoclasts treated with cilengitide exhibited reduced cell spreading, cell migration and cell adhesion on RGD-containing matrix proteins, which are ligands of integrin αV. The activation of the most upstream signal transduction molecules of the integrin receptor-initiated pathway, FAK and c-Src, were consistently blocked by cilengitide. First evidence suggests that cilengitide might interfere with metastatic bone disease in vivo and this study describes a potential underlying mechanism of the inhibitory effect of cilengitide on αV-integrin expressing pre-osteoclasts by blocking integrin ligand binding and interfering with osteoclast maturation and cell behavior. In conclusion, our findings suggest that cilengitide, which interferes with αV-integrins on osteoclasts, may represent a novel therapeutic strategy in the treatment of malignant bone disease.
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Antineoplásicos/farmacología , Neoplasias Óseas/prevención & control , Neoplasias de la Mama/tratamiento farmacológico , Osteoclastos/efectos de los fármacos , Venenos de Serpiente/farmacología , Animales , Neoplasias Óseas/secundario , Neoplasias de la Mama/patología , Adhesión Celular , Línea Celular Tumoral , Movimiento Celular , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Humanos , Cadenas alfa de Integrinas/antagonistas & inhibidores , Cadenas alfa de Integrinas/metabolismo , Ratones , Osteoclastos/fisiología , Transducción de Señal/efectos de los fármacosRESUMEN
Research on the nanoscale membrane structures known as lipid rafts is relevant to the fields of cancer biology, inflammation and ischaemia. Lipid rafts recruit molecules critical to signalling and regulation of the invasion process in malignant cells, the leukocytes that provide immunity in inflammation and the endothelial cells that build blood and lymphatic vessels, as well as the patterning of neural networks. As angiogenesis is a common denominator, regulation of receptors and signalling molecules critical to angiogenesis is central to the design of new approaches aimed at reducing, promoting or normalizing the angiogenic process. The goal of this review is to highlight some of the key issues that indicate the involvement of endothelial cell lipid rafts at each step of so-called 'sprouting angiogenesis', from stimulation of the vascular endothelial growth factor to the choice of tip cells, activation of migratory and invasion pathways, recruitment of molecules that guide axons in vascular patterning and maturation of blood vessels. Finally, the review addresses opportunities for future studies to define how these lipid domains (and their constituents) may be manipulated to stimulate the so-called 'normalization' of vascular networks within tumors, and be identified as the main target, enabling the development of more efficient chemotherapeutics and cancer immunotherapies.
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Vasos Sanguíneos/metabolismo , Microdominios de Membrana/metabolismo , Axones/metabolismo , Caveolinas/metabolismo , Células Endoteliales/metabolismo , Humanos , Neoplasias/metabolismo , Neoplasias/patología , Neovascularización Patológica , Transducción de Señal , Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Gangliosides and the urokinase plasminogen activator receptor (uPAR) tipically partition in specialized membrane microdomains called lipid-rafts. uPAR becomes functionally important in fostering angiogenesis in endothelial progenitor cells (EPCs) upon recruitment in caveolar-lipid rafts. Moreover, cell membrane enrichment with exogenous GM1 ganglioside is pro-angiogenic and opposite to the activity of GM3 ganglioside. On these basis, we first checked the interaction of uPAR with membrane models enriched with GM1 or GM3, relying on the adoption of solid-supported mobile bilayer lipid membranes with raft-like composition formed onto solid hydrophilic surfaces, and evaluated by surface plasmon resonance (SPR) the extent of uPAR recruitment. We estimated the apparent dissociation constants of uPAR-GM1/GM3 complexes. These preliminary observations, indicating that uPAR binds preferentially to GM1-enriched biomimetic membranes, were validated by identifying a pro-angiogenic activity of GM1-enriched EPCs, based on GM1-dependent uPAR recruitment in caveolar rafts. We have observed that addition of GM1 to EPCs culture medium promotes matrigel invasion and capillary morphogenesis, as opposed to the anti-angiogenesis activity of GM3. Moreover, GM1 also stimulates MAPKinases signalling pathways, typically associated with an angiogenesis program. Caveolar-raft isolation and Western blotting of uPAR showed that GM1 promotes caveolar-raft partitioning of uPAR, as opposed to control and GM3-challenged EPCs. By confocal microscopy, we have shown that in EPCs uPAR is present on the surface in at least three compartments, respectively, associated to GM1, GM3 and caveolar rafts. Following GM1 exogenous addition, the GM3 compartment is depleted of uPAR which is recruited within caveolar rafts thereby triggering angiogenesis.
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Caveolas/metabolismo , Células Progenitoras Endoteliales/metabolismo , Gangliósido G(M1)/farmacología , Gangliósido G(M3)/farmacología , Microdominios de Membrana/metabolismo , Neovascularización Fisiológica/efectos de los fármacos , Receptores del Activador de Plasminógeno Tipo Uroquinasa/metabolismo , Caveolas/efectos de los fármacos , Caveolina 1/metabolismo , Ensayo de Unidades Formadoras de Colonias , Células Progenitoras Endoteliales/efectos de los fármacos , Humanos , Recién Nacido , Cinética , Microdominios de Membrana/efectos de los fármacos , Fenotipo , Transducción de SeñalRESUMEN
Endothelial cell caveolar-rafts are considered functional platforms that recruit several pro-angiogenic molecules to realize an efficient angiogenic program. Here we studied the differential caveolar-raft protein composition of endothelial colony-forming cells following stimulation with VEGF, which localizes in caveolae on interaction with its type-2 receptor. Endothelial colony-forming cells are a cell population identified in human umbilical blood that show all the properties of an endothelial progenitor cell and a high proliferative rate. Two-dimensional gel electrophoresis analysis was coupled with mass spectrometry to identify candidate proteins. The twenty-eight differentially expressed protein spots were grouped according to their function using Gene Ontology classification. In particular, functional categories relative to cell death inhibition and hydrogen peroxide metabolic processes resulted enriched. In these categories, Peroxiredoxin-2 and 6, that control hydrogen peroxide metabolic processes, are the main enriched molecules together with the anti-apoptotic 78 kDa glucose regulated protein. Some of the proteins we identified had never before identified as caveolar-raft components. Other identified proteins include calpain small subunit-1, known to mediates angiogenic response to VEGF, gelsolin, which regulates stress fiber assembly, and annexin A3, an angiogenic mediator that induces VEGF production. We validated the functional activity of the above proteins, showing that the siRNA silencing of these resulted in the inhibition of capillary morphogenesis. Overall, our data show that VEGF stimulation triggers the caveolar-raft recruitment of proteins that warrant a physiological amount of reactive oxygen species to maintain a proper angiogenic function of endothelial colony-forming cells and preserve the integrity of the actin cytoskeleton.
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Caveolas/metabolismo , Células Endoteliales/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Sangre Fetal/citología , Humanos , Neovascularización Fisiológica/fisiología , Proteómica , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
OBJECTIVE: Clinical evidence suggests that the vascular abnormalities of systemic sclerosis (SSc) precede the onset of fibrosis, but molecular cues accounting for a possible vascular connection of SSc fibrosis have been elusive, although they have been searched for intensively. Since we had previously shown that connective tissue growth factor (CCN2), endowed with fibroblast-oriented activities, was overexpressed by endothelial cells (ECs) from SSc patients, we undertook this study to investigate its role and mechanisms in fibroblast activation. METHODS: Normal fibroblasts were challenged with conditioned medium of normal microvascular ECs (MVECs) and MVECs obtained from SSc patients with the diffuse form of the disease. Fibroblast invasion was studied using the Boyden chamber Matrigel assay. Fibroblast activation was evaluated by Western blotting and immunofluorescence of α-smooth muscle actin (α-SMA), vimentin, and type I collagen. Matrix metalloproteinase (MMP) production was evaluated by zymography and reverse transcription-polymerase chain reaction. Signal transduction and activation of the small GTPases RhoA and Rac1 were studied by Western blotting. Inhibition of SSc MVEC conditioned medium-dependent fibroblast activation was obtained by anti-CCN2 antibodies and the transforming growth factor ß (TGFß) antagonist peptide p17. RESULTS: SSc MVEC CCN2 stimulated fibroblast activation and invasion. Such activities depended on CCN2-induced overexpression of TGFß and its type I, II, and III receptors combined with overproduction of MMP-2 and MMP-9 gelatinases. All of these effects were reversed by the TGFß antagonist peptide p17. Motility increase required Rac1 activation and RhoA inhibition and was inhibited by an MMP inhibitor. These features connoted a mesenchymal style of cell invasion. Since fibroblast activation also fostered overexpression of α-SMA, vimentin, and type I collagen, the CCN2-dependent increase in fibroblast activities recapitulated the characteristics of a mesenchymal-to-mesenchymal transition. CONCLUSION: SSc MVECs recruit and activate dermal fibroblasts by induction of a CCN2/TGFß-dependent mesenchymal-to-mesenchymal transition.
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Factor de Crecimiento del Tejido Conjuntivo/metabolismo , Células Endoteliales/metabolismo , Fibroblastos/efectos de los fármacos , Esclerodermia Sistémica/metabolismo , Piel/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Western Blotting , Colágeno , Combinación de Medicamentos , Femenino , Fibroblastos/metabolismo , Fibroblastos/patología , Técnica del Anticuerpo Fluorescente , Humanos , Laminina , Masculino , Mesodermo/patología , Proteoglicanos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Esclerodermia Sistémica/patología , Transducción de Señal , Piel/patología , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
This study investigates the distinctive characteristics of iron oxide magnetic nanoparticles (mNPs) and their potential application in cancer therapy, focusing on melanoma. Three types of mNPs, pre-validated for safety, underwent molecular analysis to uncover the activated signaling pathways in melanoma cells. Using the Western blot technique, the study revealed that mNPs induce cytotoxicity, hinder proliferation through ERK1/2 dephosphorylation, and prompt proapoptotic effects, including DNA damage by inducing H2AX phosphorylation. Additionally, in vitro magnetic hyperthermia notably enhanced cellular damage in melanoma cells. Moreover, the quantification of intracellular iron levels through Inductively Coupled Plasma Mass Spectrometry (ICP-MS) analysis unveils the precise dosage required to induce cellular damage effectively. These compelling findings not only shed light on the therapeutic potential of mNPs in melanoma treatment but also open exciting avenues for future research, heralding a new era in the development of targeted and effective cancer therapies. Indeed, by discerning the effective dose, our approach becomes instrumental in optimizing the therapeutic utilization of iron oxide magnetic nanoparticles, enabling the induction of precisely targeted and controlled cellular responses.
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Endothelial urokinase-type plasminogen activator receptor (uPAR) is thought to provide a regulatory mechanism in angiogenesis. Here we studied the proangiogenic role of uPAR in endothelial colony-forming cells (ECFCs), a cell population identified in human umbilical blood that embodies all of the properties of an endothelial progenitor cell matched with a high proliferative rate. By using caveolae-disrupting agents and by caveolin-1 silencing, we have shown that the angiogenic properties of ECFCs depend on caveolae integrity and on the presence of full-length uPAR in such specialized membrane invaginations. Inhibition of uPAR expression by antisense oligonucleotides promoted caveolae disruption, suggesting that uPAR is an inducer of caveolae organization. Vascular endothelial growth factor (VEGF) promoted accumulation of uPAR in ECFC caveolae in its undegraded form. We also demonstrated that VEGF-dependent ERK phosphorylation required integrity of caveolae as well as caveolar uPAR expression. VEGF activity depends on inhibition of ECFC MMP12 production, which results in impairment of MMP12-dependent uPAR truncation. Further, MMP12 overexpression in ECFC inhibited vascularization in vitro and in vivo. Our data suggest that intratumor homing of ECFCs suitably engineered to overexpress MMP12 could have the chance to control uPAR-dependent activities required for tumor angiogenesis and malignant cells spreading.
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Caveolas/metabolismo , Células Endoteliales/fisiología , Neovascularización Fisiológica/fisiología , Receptores del Activador de Plasminógeno Tipo Uroquinasa/metabolismo , Células Madre/fisiología , Animales , Células Cultivadas , Células Endoteliales/metabolismo , Humanos , Recién Nacido , Masculino , Metaloproteinasa 12 de la Matriz/genética , Metaloproteinasa 12 de la Matriz/metabolismo , Ratones , Ratones Endogámicos C57BL , Neovascularización Fisiológica/genética , Isoformas de Proteínas/metabolismo , Transporte de Proteínas , Células Madre/metabolismo , Distribución TisularRESUMEN
OBJECTIVE: Urokinase plasminogen activator (uPA), uPA receptor (uPAR), and PA inhibitor 1 (PAI-1) have pivotal roles in the proliferation and invasion of several cell types, including synovial fibroblasts (SFs). The aim of this study was to investigate the possibility of controlling the invasion of rheumatoid arthritis (RA) SFs in vitro and in vivo by inhibiting uPA and uPAR. METHODS: Normal SFs, SFs from patients with RA, and SFs from patients with psoriatic arthritis (PsA) were used. The levels of uPA, uPAR, and PAI-1 were measured by enzyme-linked immunosorbent assay and reverse transcription-polymerase chain reaction analysis of messenger RNA. The activity of uPA was studied by zymography. Proliferation was measured by cell counting, and cell invasion was measured with a Boyden chamber assembled with Matrigel-coated porous filters. Human cartilage and RA SF implantation in the SCID mouse model of RA were used to study cartilage invasion in vivo. RESULTS: RA SFs and PsA SFs overexpressed uPAR and as a result were more active than their normal counterparts in terms of both Matrigel invasion and proliferation. This effect was counteracted by a specific inhibitor of uPA enzymatic activity (WX-340) and by uPAR antisense treatment. The use of both WX-340 and uPAR antisense treatment in vitro showed cooperative effects in RA SFs that were more intense than the effects of either treatment alone. Significant inhibition of cartilage invasion was obtained in vivo with uPAR antisense treatment, while uPA inhibition was inefficient, either alone or in combination with antisense treatment. CONCLUSION: The decrease in uPAR expression in RA SFs reduced invasion of human cartilage in vitro and in the SCID mouse model.
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Artritis Reumatoide/metabolismo , Cartílago Articular/metabolismo , Fibroblastos/metabolismo , Membrana Sinovial/metabolismo , Animales , Recuento de Células , Proliferación Celular , Modelos Animales de Enfermedad , Humanos , Ratones , Oligodesoxirribonucleótidos Antisentido , Inhibidor 1 de Activador Plasminogénico/genética , Inhibidor 1 de Activador Plasminogénico/metabolismo , Receptores del Activador de Plasminógeno Tipo Uroquinasa/genética , Receptores del Activador de Plasminógeno Tipo Uroquinasa/metabolismo , Activador de Plasminógeno de Tipo Uroquinasa/genética , Activador de Plasminógeno de Tipo Uroquinasa/metabolismoRESUMEN
OBJECTIVE: Studies have shown that in systemic sclerosis (SSc) endothelial cells, overproduction of matrix metalloproteinase 12 (MMP-12) and pentraxin 3 (PTX3) is associated with defective angiogenesis. This study was undertaken to examine whether overexpression of the relevant molecules could inhibit angiogenesis of normal microvascular endothelial cells (MVECs), and whether silencing of these molecules in SSc MVECs could restore the lost angiogenic properties of the cells in vitro and in vivo. METHODS: Transient transfection of MVECs with human MMP12 and PTX3 was performed by electroporation. Silencing of MMP12 and PTX3 was obtained by treatment with small interfering RNA, and treatment effects were validated by Western blotting with specific antibodies and a fluorimetric assay. In vitro cell migration and capillary morphogenesis were studied on Matrigel substrates. In vivo angiogenesis was studied using a Matrigel sponge assay in mice. RESULTS: Transfection of MMP12 and PTX3 in normal MVECs resulted in loss of proliferation, invasion, and capillary morphogenesis in vitro, attributed to truncation of the urokinase-type plasminogen activator receptor by MMP12 and to the anti-fibroblast growth factor 2/anti-vascular endothelial growth factor activity of PTX3. These effects were particularly evident in mixed populations of transfected normal MVECs (50% transfected with MMP12 and 50% with PTX3). Silencing of the same molecules in SSc MVECs increased their invasion in Matrigel. Single-gene silencing did not increase the capillary morphogenesis of SSc MVECs, whereas double-gene-silenced cells showed a burst of capillary tube formation. Culture medium of silenced SSc MVECs stimulated angiogenesis in assays of Matrigel sponge invasion in mice. CONCLUSION: Overexpression of either MMP12 or PTX3 in normal MVECs blunts their angiogenic properties. Loss of function of MMP12 and PTX3 in SSc MVECs restores the ability of the cells to produce capillaries in vitro and induces vascularization in vivo on a Matrigel sponge.
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Proteína C-Reactiva/metabolismo , Células Endoteliales/metabolismo , Endotelio Vascular/metabolismo , Metaloproteinasa 12 de la Matriz/metabolismo , Neovascularización Fisiológica/fisiología , Esclerodermia Sistémica/metabolismo , Componente Amiloide P Sérico/metabolismo , Western Blotting , Proteína C-Reactiva/genética , Movimiento Celular/fisiología , Proliferación Celular , Endotelio Vascular/citología , Humanos , Metaloproteinasa 12 de la Matriz/genética , Neovascularización Patológica/metabolismo , Componente Amiloide P Sérico/genética , TransfecciónRESUMEN
In their new article in the Journal of Investigative Dermatology, Tseng et al. (2021) confirm that the sensitivity of melanoma cells to antiâPD-L1 checkpoint inhibitor therapy is correlated with high PD-L1 surface expression. By blocking PD-L1 membrane clearing, controlled by LRP1 and PAI-1, the expression of high-cell-surface levels of PD-L1 was maintained.
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Melanoma , Inhibidor 1 de Activador Plasminogénico , Humanos , Factores Inmunológicos , Inmunoterapia , Melanoma/tratamiento farmacológicoRESUMEN
uPAR is a globular protein, tethered to the cell membrane by a GPI-anchor involved in several cancer-related properties and its overexpression commonly correlates with poor prognosis and metastasis. We investigated the consequences of uPAR irreversible loss in human melanoma and colon cancer cell lines, knocking out its expression by CRISPR/Cas9. We analyzed through flow cytometry, western blotting and qPCR, the modulation of the most known cancer stem cells-associated genes and the EGFR while we observed the proliferation rate exploiting 2D and 3D cellular models. We also generated uPAR "rescue" expression cell lines as well as we promoted the expression of only its 3'UTR to demonstrate the involvement of uPAR mRNA in tumor progression. Knocking out PLAUR, uPAR-encoding gene, we observed an inhibited growth ratio unexpectedly coupled with a significant percentage of cells acquiring a stem-like phenotype. In vivo experiments demonstrated that uPAR loss completely abrogates tumorigenesis despite the gained stem-like profile. Nonetheless, we proved that the reintroduction of the 3'UTR of PLAUR gene was sufficient to restore the wild-type status validating the hypothesis that such a region may act as a "molecular sponge". In particular miR146a, by binding PLAUR 3' UTR region might be responsible for uPAR-dependent inhibition of EGFR expression.
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Near infrared (NIR)-resonant gold nanoparticles (AuNPs) hold great promise in cancer diagnostics and treatment. However, translating the theranostic potential of AuNPs into clinical applications still remains a challenge due to the difficulty to improve the efficiency and specificity of tumor delivery in vivo as well as the clearance from liver and spleen to avoid off target toxicity. In this study, endothelial colony forming cells (ECFCs) are exploited as vehicles to deliver AuNPs to tumors. It is first demonstrated that ECFCs display a great capability to intake AuNPs without losing viability, and exert antitumor activity per se. Using a human melanoma xenograft mouse model, it is next demonstrated that AuNP-loaded ECFCs retain their capacity to migrate to tumor sites in vivo 1 day after injection and stay in the tumor mass for more than 1 week. In addition, it is demonstrated that ECFC-loaded AuNPs are efficiently cleared by the liver over time and do not elicit any sign of damage to healthy tissue.
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OBJECTIVE: Proteases are considered critical in peri-cystic tissue degradation required in jaw cyst expansion. We studied the expression of the plasminogen activation system (plasminogen activators; their inhibitor type-1, PAI-1; the receptor for the urokinase-type plasminogen activator, uPAR) in follicular and inflammatory cysts of the jaw, to identify a possible role of this system in jaw cyst enlargement. MATERIALS AND METHODS: Jaw cysts were collected by therapeutic enucleation. ELISA and casein zymography were used to measure and characterize plasminogen activators in cyst fluid. By immunohistochemistry we examined the presence of uPAR in cyst walls and inflammatory cells, and by Western blotting the molecular forms of uPAR within the cyst fluid. RESULTS: Inflammatory cysts fluid contained higher amounts of plasminogen activators of the urinary-type (uPA), and lower amounts of PAI-1, when compared to follicular cysts fluid. Epithelial layers of both types of cysts and inflammatory cells expressed uPAR. Native 3-domain uPAR was scarcely detectable within cysts, where its cleavage was accounted for by uPA. CONCLUSION: These data suggest a plasminogen activation-dependent mechanism of cyst enlargement, where only the outward uPAR expressed on epithelial cells and on inflammatory cells direct the peri-cystic protease cascade, in a way similar to tumor enlargement within tissues.
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Quistes Maxilomandibulares/patología , Quistes Odontogénicos/patología , Receptores del Activador de Plasminógeno Tipo Uroquinasa/análisis , Activador de Plasminógeno de Tipo Uroquinasa/análisis , Adulto , Anciano , Anciano de 80 o más Años , Western Blotting , Líquido Quístico/química , Células Epiteliales/enzimología , Células Epiteliales/patología , Femenino , Humanos , Inmunohistoquímica , Inflamación , Quistes Maxilomandibulares/enzimología , Antígenos Comunes de Leucocito/análisis , Masculino , Persona de Mediana Edad , Quistes Odontogénicos/enzimología , Inhibidor 1 de Activador Plasminogénico/análisis , Activadores Plasminogénicos/análisisRESUMEN
Targeted and immune therapies have unquestionably improved the prognosis of melanoma patients. However the treatment of this neoplasm still requires approaches with a higher therapeutic index, in order to reduce shortcomings related to toxic effects and aspecific targeting. This means developing therapeutic tools derived with high affinity molecules for tumor components differentially expressed in melanoma cells with respect to their normal counterpart. Nanomedicine has sought to address this problem owing to the high modulability of nanoparticles. This approach exploits not only the enhanced permeability and retention effect typical of the tumor microenvironment (passive targeting), but also the use of specific "molecular antennas" that recognize some tumor-overexpressed molecules (active targeting). This line of research has given rise to the so-called "smart nanoparticles," some of which have already passed the preclinical phase and are under clinical trials in melanoma patients. To further improve nanoparticles partition within tumors, for some years now a line of thought is exploiting the molecular systems that regulate the innate tumor-homing activity of platelets, granulocytes, monocytes/macrophages, stem cells, endothelial-colony-forming cells, and red blood cells loaded with nanoparticles. This new vision springs from the results obtained with some of these cells in regenerative medicine, an approach called "cell therapy." This review takes into consideration the advantages of cell therapy as the only one capable of overcoming the limits of targeting imposed by the increased interstitial pressure of tumors.
RESUMEN
Urokinase Plasminogen Activator (uPA) Receptor (uPAR) is a well-known GPI-anchored three-domain membrane protein with pro-tumor roles largely shown in all the malignant tumors where it is over-expressed. Here we have exploited the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 gene knock out approach to investigate its role in the oxidative metabolism in human melanoma and colon cancer as the consequences of its irreversible loss. Knocking out PLAUR, a uPAR-encoding gene, in A375p, A375M6 and HCT116, which are two human melanoma and a colon carcinoma, respectively, we have observed an increased number of mitochondria in the two melanoma cell lines, while we evidenced an immature biogenesis of mitochondria in the colon carcinoma culture. Such biological diversity is, however, reflected in a significant enhancement of the mitochondrial spare respiratory capacity, fueled by an increased expression of GLS2, and in a decreased glycolysis paired with an increased secretion of lactate by all uPAR KO cells. We speculated that this discrepancy might be explained by an impaired ratio between LDHA and LDHB.