RESUMO
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.
Assuntos
Antígenos CD/metabolismo , Caderinas/metabolismo , Exossomos/metabolismo , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Transdução de Sinais , Animais , Antígenos CD/genética , Caderinas/genética , Linhagem Celular , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Receptores ErbB/metabolismo , Gefitinibe/farmacologia , Edição de Genes , Humanos , Melanoma/metabolismo , Melanoma/patologia , Camundongos , Camundongos SCID , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Neovascularização Fisiológica , Fosforilação/efeitos dos fármacos , Interferência de RNA , RNA Interferente Pequeno , Receptores de Ativador de Plasminogênio Tipo Uroquinase/antagonistas & inibidores , Receptores de Ativador de Plasminogênio Tipo Uroquinase/genéticaRESUMO
Vitronectin, extracellular matrix protein, plays an important role in embryonic development and in organ and tissue reparation. A unique characteristic of vitronectin is specific binding of various biological molecules, including urokinase receptor (uPAR), extracellular matrix components, adhesion receptors, growth factors, thus supporting the modulation of cell behavior. Vitronectin is in fact not found in intact myocardium, while after infarction its level increases significantly, which correlates with accumulation of uPAR+ progenitor cardiac cells in the focus. The cells isolated from the heart of wild type mice are characterized by higher adhesion to vitronectin than progenitor cardiac cells from the myocardium of uPAR knockout mice. In addition, inhibition of urokinase receptor with specific antibodies on the surface of the progenitor cardiac cells of wild type mice leads to attenuation of their adhesive activity and flattening on vitronectin matrix, which can be important for their distribution in the postinfarction myocardium and realization of the reparative functions.
Assuntos
Adesão Celular/fisiologia , Miocárdio/metabolismo , Receptores de Ativador de Plasminogênio Tipo Uroquinase/fisiologia , Células-Tronco/fisiologia , Vitronectina/metabolismo , Animais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infarto do Miocárdio/patologia , Miocárdio/citologia , Receptores de Ativador de Plasminogênio Tipo Uroquinase/antagonistas & inibidores , Receptores de Ativador de Plasminogênio Tipo Uroquinase/genéticaRESUMO
Protein-protein interactions drive every aspect of cell signaling, yet only a few small-molecule inhibitors of these interactions exist. Despite our ability to identify critical residues known as hot spots, little is known about how to effectively engage them to disrupt protein-protein interactions. Here, we take advantage of the ease of preparation and stability of pyrrolinone 1, a small-molecule inhibitor of the tight interaction between the urokinase receptor (uPAR) and its binding partner, the urokinase-type plasminogen activator uPA, to synthesize more than 40 derivatives and explore their effect on the protein-protein interaction. We report the crystal structure of uPAR bound to previously discovered pyrazole 3 and to pyrrolinone 12. While both 3 and 12 bind to uPAR and compete with a fluorescently labeled peptide probe, only 12 and its derivatives inhibit the full uPAR·uPA interaction. Compounds 3 and 12 mimic and engage different hot-spot residues on uPA and uPAR, respectively. Interestingly, 12 is involved in a π-cation interaction with Arg-53, which is not considered a hot spot. Explicit-solvent molecular dynamics simulations reveal that 3 and 12 exhibit dramatically different correlations of motion with residues on uPAR. Free energy calculations for the wild-type and mutant uPAR bound to uPA or 12 show that Arg-53 interacts with uPA or with 12 in a highly cooperative manner, thereby altering the contributions of hot spots to uPAR binding. The direct engagement of peripheral residues not considered hot spots through π-cation or salt-bridge interactions could provide new opportunities for enhanced small-molecule engagement of hot spots to disrupt challenging protein-protein interactions.
Assuntos
Pirazóis/síntese química , Pirróis/síntese química , Receptores de Ativador de Plasminogênio Tipo Uroquinase/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/síntese química , Ativador de Plasminogênio Tipo Uroquinase/antagonistas & inibidores , Cristalografia por Raios X , Corantes Fluorescentes/química , Expressão Gênica , Humanos , Cinética , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Ligação Proteica/efeitos dos fármacos , Pirazóis/farmacologia , Pirróis/farmacologia , Receptores de Ativador de Plasminogênio Tipo Uroquinase/química , Receptores de Ativador de Plasminogênio Tipo Uroquinase/genética , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Coloração e Rotulagem/métodos , Relação Estrutura-Atividade , Termodinâmica , Ativador de Plasminogênio Tipo Uroquinase/química , Ativador de Plasminogênio Tipo Uroquinase/genética , Ativador de Plasminogênio Tipo Uroquinase/metabolismoRESUMO
The urokinase receptor (uPAR) plays a critical role in breast cancer (BC) progression and metastases and is a validated target for novel therapies. The current study investigates the effects of MV-uPA, an oncolytic measles virus fully retargeted against uPAR in syngeneic and xenograft BC metastases models. In vitro replication and cytotoxicity of MVs retargeted against human (MV-h-uPA) or mouse (MV-m-uPA) uPAR were assessed in human and murine cancer and non-cancer mammary epithelial cells. The in vivo effects of species-specific uPAR retargeted MVs were assessed in syngeneic and xenograft models of experimental metastases, established by intravenous administration of luciferase expressing 4T1 or MDA-MD-231 cells. Metastases progression was assessed by in vivo bioluminescence imaging. Tumor targeting was evaluated by qRT-PCR of MV-N, rescue of viable viral particles, and immunostaining of MV particles in lungs from tumor bearing mice. In vitro, MV-h-uPA and MV-m-uPA selectively infected, replicated, and induced cytotoxicity in cancer compared to non-cancer cells in a species-specific manner. In vivo, MV-m-uPA delayed 4T1 lung metastases progression and prolonged survival. These effects were associated with identification of viable viral particles, viral RNA, and detection of MV-N by immunostaining from lung tissues in treated mice. In the human MDA-MB-231 metastases model, intravenous administration of MV-h-uPA markedly inhibited metastases progression and significantly improved survival, compared to controls. No significant treatment-related toxicity was observed in treated mice. The above preclinical findings strongly suggest that uPAR retargeted measles virotherapy is a novel and feasible systemic therapy strategy against metastatic breast cancer.
Assuntos
Neoplasias da Mama/genética , Neoplasias Mamárias Experimentais/genética , Vírus do Sarampo/genética , Terapia Viral Oncolítica , Receptores de Ativador de Plasminogênio Tipo Uroquinase/biossíntese , Administração Intravenosa , Animais , Neoplasias da Mama/terapia , Neoplasias da Mama/virologia , Linhagem Celular Tumoral , Feminino , Humanos , Neoplasias Mamárias Experimentais/terapia , Neoplasias Mamárias Experimentais/virologia , Camundongos , Metástase Neoplásica , Vírus Oncolíticos/genética , Receptores de Ativador de Plasminogênio Tipo Uroquinase/antagonistas & inibidores , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The relationship between the positioning of ligands on the surface of nanoparticles and the structural features of nanoconjugates has been underestimated for a long time, albeit of primary importance to promote specific biological recognition at the nanoscale. In particular, it has been formerly observed that a proper molecular orientation can play a crucial role, first optimizing ligand immobilization onto the nanoparticles and, second, improving the targeting efficiency of the nanoconjugates. In this work, we present a novel strategy to afford peptide-oriented ligation using genetically modified cutinase fusion proteins, which combines the presence of a site-directed "capture" module based on an enzymatic unit and a "targeting" moiety consisting of the ligand terminal end of a genetically encoded polypeptide chain. As an example, the oriented presentation of U11 peptide, a sequence specific for the recognition of urokinase plasminogen activator receptor (uPAR), was achieved by enzyme-mediated conjugation with an irreversible inhibitor of cutinase, an alkylphosphonate p-nitrophenol ester linker, covalently bound to the surface of iron oxide nanoparticles. The targeting efficiency of the resulting protein-nanoparticle conjugates was assessed using uPAR-positive breast cancer cells exploiting confocal laser scanning microscopy and quantitative fluorescence analysis of confocal images. Ultrastructural analysis of transmission electron micrographs provided evidence of a receptor-mediated pathway of endocytosis. Our results showed that, despite the small average number of targeting peptides presented on the nanoparticles, our ligand-oriented nanoconjugates proved to be very effective in selectively binding to uPAR and in promoting the uptake in uPAR-positive cancer cells.
Assuntos
Hidrolases de Éster Carboxílico/química , Sistemas de Liberação de Medicamentos/métodos , Nanoconjugados/química , Peptídeos/química , Proteínas Recombinantes de Fusão/química , Antineoplásicos/química , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Hidrolases de Éster Carboxílico/genética , Hidrolases de Éster Carboxílico/metabolismo , Linhagem Celular Tumoral , Endocitose , Compostos Férricos/química , Humanos , Modelos Moleculares , Nanoconjugados/ultraestrutura , Nanopartículas/química , Nanopartículas/ultraestrutura , Nitrofenóis/química , Peptídeos/genética , Peptídeos/metabolismo , Peptídeos/farmacologia , Receptores de Ativador de Plasminogênio Tipo Uroquinase/antagonistas & inibidores , Receptores de Ativador de Plasminogênio Tipo Uroquinase/genética , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Relação Estrutura-AtividadeRESUMO
Effective removal of apoptotic cells, particularly apoptotic neutrophils, is essential for the successful resolution of acute inflammatory conditions. In these experiments, we found that whereas interaction between vitronectin and integrins diminished the ability of macrophages to ingest apoptotic cells, interaction between vitronectin with urokinase-type plasminogen activator receptor (uPAR) on the surface of apoptotic cells also had equally important inhibitory effects on efferocytosis. Preincubation of vitronectin with plasminogen activator inhibitor-1 eliminated its ability to inhibit phagocytosis of apoptotic cells. Similarly, incubation of apoptotic cells with soluble uPAR or Abs to uPAR significantly diminished efferocytosis. In the setting of LPS-induced ALI, enhanced efferocytosis and decreased numbers of neutrophils were found in bronchoalveolar lavage obtained from vitronectin-deficient (vtn(-/-)) mice compared with wild type (vtn(+/+)) mice. Furthermore, there was increased clearance of apoptotic vtn(-/-) as compared with vtn(+/+) neutrophils after introduction into the lungs of vtn(-/-) mice. Incubation of apoptotic vtn(-/-) neutrophils with purified vitronectin before intratracheal instillation decreased efferocytosis in vivo. These findings demonstrate that the inhibitory effects of vitronectin on efferocytosis involve interactions with both the engulfing phagocyte and the apoptotic target cell.
Assuntos
Lesão Pulmonar Aguda/imunologia , Apoptose/efeitos dos fármacos , Macrófagos Peritoneais/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Fagocitose/efeitos dos fármacos , Timócitos/efeitos dos fármacos , Vitronectina/imunologia , Lesão Pulmonar Aguda/induzido quimicamente , Lesão Pulmonar Aguda/metabolismo , Lesão Pulmonar Aguda/patologia , Animais , Anticorpos/farmacologia , Apoptose/imunologia , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/imunologia , Contagem de Células , Técnicas de Cocultura , Feminino , Lipopolissacarídeos , Macrófagos Peritoneais/imunologia , Macrófagos Peritoneais/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neutrófilos/imunologia , Neutrófilos/patologia , Fagocitose/imunologia , Inibidor 1 de Ativador de Plasminogênio/farmacologia , Receptores de Ativador de Plasminogênio Tipo Uroquinase/antagonistas & inibidores , Receptores de Ativador de Plasminogênio Tipo Uroquinase/genética , Receptores de Ativador de Plasminogênio Tipo Uroquinase/imunologia , Timócitos/imunologia , Timócitos/patologia , Vitronectina/deficiência , Vitronectina/genéticaRESUMO
This study examined the mechanism of action of amiloride, a urokinase-type plasminogen activator receptor inhibitor, in lowering proteinuria. Podocytes were resuscitated to allow for their proliferation and were observed for morphological changes. In the in vitro experiment, control, lipopolysaccharide, and lipopolysaccharide + amiloride groups were established. The expression of urokinase-type plasminogen activator receptor (uPAR) in podocytes was detected with a flow cytometer and cell motility was detected with the transwell migration assay. In the in vivo test, the urine protein volume of the model was detected at 24 h using Coomassie brilliant blue staining and the morphological changes of the podocytes were detected with immunofluorescence. The protein expression rate of uPAR in the lipopolysaccharide group was significantly higher than those in the control and lipopolysaccharide + amiloride groups (P < 0.05). The viability of cells in the lipopolysaccharide group was significantly higher than those in the control and lipopolysaccharide + amiloride groups (P < 0.05). Compared with the urine protein level in the control group at 24 h, the level in the lipopolysaccharide group increased significantly (P < 0.05), whereas compared with the urine protein level in the lipopolysaccharide group, the level in the lipopolysaccharide + amiloride group decreased (P < 0.05). uPAR expression was significantly downregulated, and the fusion of the podocyte-specific skelemin synaptopodin on the glomerulus podocytes was significantly decreased in the lipopolysaccharide + amiloride group. These results suggest that amiloride is able to reduce cell motility and thus lower proteinuria by inhibiting the expression of uPAR in podocytes.
Assuntos
Amilorida/farmacologia , Podócitos/efeitos dos fármacos , Podócitos/metabolismo , Proteinúria/metabolismo , Receptores de Ativador de Plasminogênio Tipo Uroquinase/antagonistas & inibidores , Amilorida/administração & dosagem , Animais , Linhagem Celular Transformada , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Modelos Animais de Doenças , Expressão Gênica , Camundongos , Podócitos/patologia , Proteinúria/tratamento farmacológico , Proteinúria/urina , Receptores de Ativador de Plasminogênio Tipo Uroquinase/genética , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismoRESUMO
Glioma is a highly complex brain tumor characterized by the dysregulation of proteins and genes that leads to tumor metastasis. Cathepsin B and uPAR are overexpressed in gliomas and they are postulated to play central roles in glioma metastasis. In this study, efficient downregulation of cathepsin B and uPAR by siRNA treatments significantly reduced glioma cell adhesion to laminin as compared to vitronectin, fibronectin, or collagen I in U251 and 4910 glioma cell lines. Brain glioma tissue array analysis showed high expression of CD151 in clinical samples when compared with normal brain tissue. Cathepsin B and uPAR siRNA treatment led to the downregulation of CD151 and laminin-binding integrins α3 and ß1. Co-immunoprecipitation experiments revealed that downregulation of cathepsin B and uPAR decreased the interaction of CD151 with uPAR cathepsin B, and α3ß1 integrin. Studies on the downstream signaling cascade of uPAR/CD151/α3ß1 integrin have shown that phosphorylation of FAK, SRC, paxillin, and expression of adaptor cytoskeletal proteins talin and vinculin were reduced with knockdown of cathepsin B, uPAR, and CD151. Treatment with the bicistronic construct reduced interactions between uPAR and CD151 as well as lowering α3ß1 integrin, talin, and vinculin expression levels in pre-established glioma tumors of nude mice. In conclusion, our results show that downregulation of cathepsin B and uPAR alone and in combination inhibit glioma cell adhesion by downregulating CD151 and its associated signaling molecules in vitro and in vivo. Taken together, the results of the present study show that targeting the uPAR-cathepsin B system has possible therapeutic potential.
Assuntos
Catepsina B/metabolismo , Adesão Celular , Movimento Celular , Glioma/patologia , Integrina alfa3beta1/metabolismo , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Tetraspanina 24/metabolismo , Animais , Apoptose , Western Blotting , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Catepsina B/antagonistas & inibidores , Catepsina B/genética , Moléculas de Adesão Celular/metabolismo , Proliferação de Células , Regulação para Baixo , Citometria de Fluxo , Imunofluorescência , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Glioma/genética , Glioma/metabolismo , Humanos , Técnicas Imunoenzimáticas , Imunoprecipitação , Integrina alfa3beta1/antagonistas & inibidores , Integrina alfa3beta1/genética , Camundongos , Camundongos Nus , Fosforilação , RNA Interferente Pequeno/genética , Receptores de Ativador de Plasminogênio Tipo Uroquinase/antagonistas & inibidores , Receptores de Ativador de Plasminogênio Tipo Uroquinase/genética , Tetraspanina 24/antagonistas & inibidores , Tetraspanina 24/genética , Células Tumorais Cultivadas , CalininaRESUMO
The uPAR·uPA protein-protein interaction (PPI) is involved in signaling and proteolytic events that promote tumor invasion and metastasis. A previous study had identified 4 (IPR-803) from computational screening of a commercial chemical library and shown that the compound inhibited uPAR·uPA PPI in competition biochemical assays and invasion cellular studies. Here, we synthesize 4 to evaluate in vivo pharmacokinetic (PK) and efficacy studies in a murine breast cancer metastasis model. First, we show, using fluorescence polarization and saturation transfer difference (STD) NMR, that 4 binds directly to uPAR with sub-micromolar affinity of 0.2 µM. We show that 4 blocks invasion of breast MDA-MB-231, and inhibits matrix metalloproteinase (MMP) breakdown of the extracellular matrix (ECM). Derivatives of 4 also inhibited MMP activity and blocked invasion in a concentration-dependent manner. Compound 4 also impaired MDA-MB-231 cell adhesion and migration. Extensive in vivo PK studies in NOD-SCID mice revealed a half-life of nearly 5h and peak concentration of 5 µM. Similar levels of the inhibitor were detected in tumor tissue up to 10h. Female NSG mice inoculated with highly malignant TMD-MDA-MB-231 in their mammary fat pads showed that 4 impaired metastasis to the lungs with only four of the treated mice showing severe or marked metastasis compared to ten for the untreated mice. Compound 4 is a promising template for the development of compounds with enhanced PK parameters and greater efficacy.
Assuntos
Antineoplásicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Metástase Neoplásica/tratamento farmacológico , Mapas de Interação de Proteínas/efeitos dos fármacos , Receptores de Ativador de Plasminogênio Tipo Uroquinase/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/uso terapêutico , Ativador de Plasminogênio Tipo Uroquinase/antagonistas & inibidores , Animais , Antineoplásicos/química , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Mama/efeitos dos fármacos , Mama/metabolismo , Mama/patologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Feminino , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Invasividade Neoplásica/patologia , Metástase Neoplásica/patologia , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacocinética , Bibliotecas de Moléculas Pequenas/farmacologia , Ativador de Plasminogênio Tipo Uroquinase/metabolismoRESUMO
Head and neck cancer is the sixth most common cancer in the world and most of them are squamous cell carcinomas. High frequency of cisplatin resistance in head and neck squamous cell carcinoma (HNSCC) leads to tumor relapse and irresponsiveness to cisplatin-based chemotherapy. However, the mechanisms underlying cisplatin resistance is still largely unrevealed. In this study, we found that CD147 was overexpressed in cisplatin-resistant HNSCC cell lines. Based on the result, CD147 expression was down-regulated in the cisplatin-resistant cell line and we observed that the sensitivity to cisplatin increased, as showed in the results of MTT assay and PI-staining apoptotic detection. Meanwhile, transfection of CD147 expression vector promoted the occurrence of cisplatin resistance in the cisplatin-sensitive cell line. Simultaneously blocking of uPAR with neutralizing antibody would significantly prevent the occurrence of cisplatin resistance induced by CD147 overexpression. In conclusion, our study finds that CD147 is also involved in mediating cisplatin resistance in HNSCC and uPAR serves as a possible candidate that collaborates with CD147 in this process.
Assuntos
Basigina/genética , Carcinoma de Células Escamosas/genética , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias de Cabeça e Pescoço/genética , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cisplatino/farmacologia , Regulação Neoplásica da Expressão Gênica/genética , Vetores Genéticos/genética , Humanos , RNA Interferente Pequeno/genética , Receptores de Ativador de Plasminogênio Tipo Uroquinase/antagonistas & inibidores , TransfecçãoRESUMO
RATIONALE: Binding of maternal anti-Ro/La antibodies to cognate antigen expressed on apoptotic cardiocytes decreases clearance by healthy cardiocytes, which may contribute to the development of autoimmune associated congenital heart block and fatal cardiomyopathy. OBJECTIVE: Given recent evidence implicating the urokinase plasminogen activator receptor (uPAR) as a "don't eat me" signal during efferocytosis, experiments addressed whether surface bound anti-Ro antibodies inhibit apoptotic cell removal via an effect on the expression/function of the urokinase-type plasminogen activator protease uPA/uPAR system. METHODS AND RESULTS: As assessed by flow cytometry and confocal microscopy, uPAR colocalizes and interacts with Ro60 on the surface of apoptotic human fetal cardiocytes. Blocking of uPAR enhances phagocytosis of apoptotic cardiocytes by healthy cardiocytes and reverses the anti-Ro60-dependent impaired clearance of apoptotic cardiocytes. Binding of anti-Ro60 antibodies to apoptotic cardiocytes results in increased uPAR expression, as well as enhanced uPA activity. The binding of anti-Ro60 did not alter other surface molecules involved in cell recognition (calreticulin, CD31, or CD47). CONCLUSIONS: These data suggest that increased uPAR expression and uPA activity induced by anti-Ro60 binding to the apoptotic fetal cardiocyte provide a molecular basis by which these antibodies inhibit efferocytosis and ultimately lead to scar of the fetal conduction system and working myocardium.
Assuntos
Bloqueio Cardíaco/congênito , Receptores de Ativador de Plasminogênio Tipo Uroquinase/genética , Animais , Anticorpos Monoclonais , Apoptose , Antígeno CD47/metabolismo , Calreticulina/metabolismo , Cardiomiopatias/imunologia , Galinhas/imunologia , Feminino , Doenças Fetais/genética , Feto/fisiologia , Citometria de Fluxo , Bloqueio Cardíaco/etiologia , Bloqueio Cardíaco/genética , Sistema de Condução Cardíaco/patologia , Humanos , Imunoglobulina G , Camundongos , Microscopia Confocal , Miócitos Cardíacos/imunologia , Miócitos Cardíacos/patologia , Miócitos Cardíacos/fisiologia , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Gravidez , Receptores de Ativador de Plasminogênio Tipo Uroquinase/antagonistas & inibidores , Ativador de Plasminogênio Tipo Uroquinase/metabolismoRESUMO
Cathepsin B and urokinase plasminogen activator receptor (uPAR) are postulated to play key roles in glioma invasion. Calcineurin is one of the key regulators of mitochondrial-dependent apoptosis, but its mechanism is poorly understood. Hence, we studied subcellular localization of calcineurin after transcriptional downregulation of uPAR and cathepsin B in glioma. In the present study, efficient downregulation of uPAR and cathepsin B increased the translocation of calcineurin A from the mitochondria to the cytosol, decreased pBAD (S136) expression and its interaction with 14-3-3ζ and increased the interaction of BAD with Bcl-xl. Co-depletion of uPAR and cathepsin B induced mitochondrial translocation of BAD, activation of caspase 3 as well as PARP and cytochrome c and SMAC release. These effects were inhibited by FK506 (10 µM), a specific inhibitor of calcineurin. Calcineurin A was co-localized and also co-immunoprecipitated with Bcl-2. This interaction decreased with co-depletion of uPAR and cathepsin B and also with Bcl-2 inhibitor, HA 14-1 (20 µg/ml). Altered localization and interaction of calcineurin A with Bcl-2 was also observed in vivo when uPAR and cathepsin B were downregulated. In conclusion, downregulation of uPAR and cathepsin B induced apoptosis by targeting calcineurin A to BAD via Bcl-2 in glioma.
Assuntos
Apoptose , Calcineurina/metabolismo , Catepsina B/metabolismo , Glioma/metabolismo , Glioma/patologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Proteína de Morte Celular Associada a bcl/metabolismo , Western Blotting , Caspase 3/metabolismo , Catepsina B/antagonistas & inibidores , Linhagem Celular Tumoral , Citocromos c/metabolismo , Regulação para Baixo , Imunofluorescência , Humanos , Técnicas Imunoenzimáticas , Imunoprecipitação , Potencial da Membrana Mitocondrial , Receptores de Ativador de Plasminogênio Tipo Uroquinase/antagonistas & inibidores , Transdução de Sinais , Frações SubcelularesRESUMO
The diffuse-type gastric cancer (DGC) is a subtype of gastric cancer (GC) associated with low HER2 positivity rate and insensitivity to chemotherapy and immune checkpoint inhibitors. Here, we identify urokinase-type plasminogen activator receptor (uPAR) as a potential therapeutic target for DGC. We have developed a novel anti-uPAR monoclonal antibody, which targets the domains II and III of uPAR and blocks the binding of urokinase-type plasminogen activator to uPAR. We show that the combination of anti-uPAR and anti-Programmed cell death protein 1 (PD-1) remarkably inhibits tumor growth and prolongs survival via multiple mechanisms, using cell line-derived xenograft and patient-derived xenograft mouse models. Furthermore, uPAR chimeric antigen receptor-expressing T cells based on the novel anti-uPAR effectively kill DGC patient-derived organoids and exhibit impressive survival benefit in the established mouse models, especially when combined with PD-1 blockade therapy. Our study provides a new possibility of DGC treatment by targeting uPAR in a unique manner.
Assuntos
Receptor de Morte Celular Programada 1 , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Neoplasias Gástricas , Animais , Antineoplásicos Imunológicos/imunologia , Antineoplásicos Imunológicos/farmacologia , Humanos , Camundongos , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptor de Morte Celular Programada 1/imunologia , Receptores de Ativador de Plasminogênio Tipo Uroquinase/antagonistas & inibidores , Receptores de Ativador de Plasminogênio Tipo Uroquinase/imunologia , Transdução de Sinais , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/imunologia , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Ativador de Plasminogênio Tipo Uroquinase/metabolismoRESUMO
Interactions between urokinase plasminogen activator receptor (uPAR) and its various ligands regulate tumor growth, invasion, and metastasis. Antibodies that bind specific uPAR epitopes may disrupt these interactions, thereby inhibiting these processes. Using a highly diverse and naïve human fragment of the antigen binding (Fab) phage display library, we identified 12 unique human Fabs that bind uPAR. Two of these antibodies compete against urokinase plasminogen activator (uPA) for uPAR binding, whereas a third competes with beta1 integrins for uPAR binding. These competitive antibodies inhibit uPAR-dependent cell signaling and invasion in the non-small cell lung cancer cell line, H1299. Additionally, the integrin-blocking antibody abrogates uPAR/beta1 integrin-mediated H1299 cell adhesion to fibronectin and vitronectin. This antibody and one of the uPAR/uPA antagonist antibodies shows a significant combined effect in inhibiting cell invasion through Matrigel/Collagen I or Collagen I matrices. Our results indicate that these antagonistic antibodies have potential for the detection and treatment of uPAR-expressing tumors.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Movimento Celular/efeitos dos fármacos , Fragmentos Fab das Imunoglobulinas/farmacologia , Proteínas de Neoplasias/antagonistas & inibidores , Receptores de Ativador de Plasminogênio Tipo Uroquinase/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Animais , Anticorpos Monoclonais , Carcinoma Pulmonar de Células não Pequenas/imunologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Movimento Celular/imunologia , Humanos , Fragmentos Fab das Imunoglobulinas/genética , Fragmentos Fab das Imunoglobulinas/imunologia , Invasividade Neoplásica , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/imunologia , Proteínas de Neoplasias/metabolismo , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , Ligação Proteica/imunologia , Receptores de Ativador de Plasminogênio Tipo Uroquinase/genética , Receptores de Ativador de Plasminogênio Tipo Uroquinase/imunologia , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Transdução de Sinais/imunologia , Spodoptera , Ativador de Plasminogênio Tipo Uroquinase/genética , Ativador de Plasminogênio Tipo Uroquinase/imunologia , Ativador de Plasminogênio Tipo Uroquinase/metabolismoRESUMO
Urokinase-type plasminogen activator receptor (uPAR) mediates a multitude of biological activities, has key roles in several clinical indications, including malignancies and inflammation, and, thus, has attracted intensive research over the past few decades. The pleiotropic functions of uPAR can be attributed to its interaction with an array of partners. Many inhibitors have been developed to intervene with the interaction of uPAR with these partners. Here, we review the development of these classes of uPAR inhibitor and their inhibitory mechanisms to promote the translation of these inhibitors to clinical applications.
Assuntos
Descoberta de Drogas/métodos , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Transdução de Sinais/efeitos dos fármacos , Humanos , Receptores de Ativador de Plasminogênio Tipo Uroquinase/antagonistas & inibidores , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismoRESUMO
With a 5-year recurrence rate of 30-78%, urothelial cell carcinoma (UCC) rates amongst the highest of all solid malignancies. Consequently, after transurethral resection, patients are subjugated to life-long endoscopic surveillance. A multimodal near-infrared (NIR) fluorescence-based imaging strategy can improve diagnosis, resection and surveillance, hence increasing quality of life. METHODS: Expression of urokinase plasminogen activator receptor (uPAR) and epithelial cell adhesion molecule (EpCAM) are determined on paraffin-embedded human UCC using immunohistochemistry and on UCC cell lines by flow cytometry. MNPR-101, a humanised monoclonal antibody targeting uPAR is conjugated to IRDye800CW and binding is validated in vitro using surface plasmon resonance and cell-based binding assays. In vivo NIR fluorescence and photoacoustic three-dimensional (3D) imaging are performed with subcutaneously growing human UM-UC-3luc2 cells in BALB/c-nude mice. The translational potential is confirmed in a metastasising UM-UC-3luc2 orthotopic mouse model. Infliximab-IRDye800CW and rituximab-IRDye800CW are used as controls. RESULTS: UCCs show prominent uPAR expression at the tumour-stroma interface and EpCAM on epithelial cells. uPAR and EpCAM are expressed by 6/7 and 4/7 UCC cell lines, respectively. In vitro, MNPR-101-IRDye800CW has a picomolar affinity for domain 2-3 of uPAR. In vivo fluorescence imaging with MNPR-101-IRDye800CW, specifically delineates both subcutaneous and orthotopic tumours with tumour-to-background ratios reaching as high as 6.8, differing significantly from controls (p < 0.0001). Photoacoustic 3D in depth imaging confirms the homogenous distribution of MNPR-101-IRDye800CW through the tumour. CONCLUSIONS: MNPR-101-IRDye800CW is suitable for multimodal imaging of UCC, awaiting clinical translation.
Assuntos
Anticorpos Monoclonais/farmacologia , Imagem Molecular/métodos , Imagem Óptica/métodos , Receptores de Ativador de Plasminogênio Tipo Uroquinase/antagonistas & inibidores , Cirurgia Assistida por Computador/métodos , Neoplasias da Bexiga Urinária/diagnóstico , Animais , Apoptose , Proliferação de Células , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Prognóstico , Receptores de Ativador de Plasminogênio Tipo Uroquinase/imunologia , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Células Tumorais Cultivadas , Neoplasias da Bexiga Urinária/imunologia , Neoplasias da Bexiga Urinária/cirurgia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The urokinase receptor (uPAR) is a cell surface receptor that binds to the serine protease urokinase-type plasminogen activator (uPA) with high affinity. This interaction is beneficial for extravascular fibrin clearance, but it has also been associated with a broad range of pathological conditions including cancer, atherosclerosis, and kidney disease. Here, starting with a small molecule that we previously discovered by virtual screening and cheminformatics analysis, we design and synthesize several derivatives that were tested for binding and inhibition of the uPAR â uPA interaction. To confirm the binding site and establish a binding mode of the compounds, we carried out biophysical studies using uPAR mutants, among them uPARH47C-N259C , a mutant previously developed to mimic the structure of uPA-bound uPAR. Remarkably, a substantial increase in potency is observed for inhibition of uPARH47C-N259C binding to uPA compared to wild-type uPAR, consistent with our use of the structure of uPAR in its uPA-bound state to design small-molecule uPAR â uPA antagonists. Combined with the biophysical studies, molecular docking followed by extensive explicit-solvent molecular dynamics simulations and MM-GBSA free energy calculations yielded the most favorable binding pose of the compound. Collectively, these results suggest that potent inhibition of uPAR binding to uPA with small molecules will likely only be achieved by developing small molecules that exhibit high-affinity to solution apo structures of uPAR, rather than uPA-bound structures of the receptor.
Assuntos
Receptores de Ativador de Plasminogênio Tipo Uroquinase/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Ativador de Plasminogênio Tipo Uroquinase/antagonistas & inibidores , Sítios de Ligação/efeitos dos fármacos , Quimioinformática , Relação Dose-Resposta a Droga , Humanos , Modelos Moleculares , Conformação Molecular , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Bibliotecas de Moléculas Pequenas/química , Ativador de Plasminogênio Tipo Uroquinase/metabolismoRESUMO
Research is currently underway worldwide into the development of receptor-specific radiopharmaceuticals for the imaging and treatment of cancer. The successful clinical development of radiolabeled somatostatin analogs for imaging and treatment of cancers overexpressing somatostatin receptors has catalyzed further preclinical investigation of other radiolabeled peptides for molecular imaging and peptide-receptor radiotherapy, including such well-studied peptide vectors as cholecystokinin, neurotensin, bombesin and RGD peptides. Within this larger context, this article will focus on the current status of two more recent additions to the list of molecular imaging targets - guanylate cyclase C, a specific marker for colorectal cancer, and the urokinase plasminogen activator receptor, a cell-surface receptor overexpressed in diverse cancer types.
Assuntos
Guanilato Ciclase/antagonistas & inibidores , Neoplasias/diagnóstico por imagem , Neoplasias/radioterapia , Fragmentos de Peptídeos/uso terapêutico , Compostos Radiofarmacêuticos/uso terapêutico , Receptores de Ativador de Plasminogênio Tipo Uroquinase/antagonistas & inibidores , Animais , Ensaios Clínicos como Assunto , Humanos , Prognóstico , RadiografiaRESUMO
In the present work, an iridium (Ir) complex loaded theranostic nanoplatform was designed for high-efficiency triple-negative breast cancer (TNBC) therapy. For this purpose, the Ir complex was firstly loaded on a photothermal agent of gold nanostars (GNS) by simply mixing followed by functionalization using a urokinase-type plasminogen activator receptor (uPAR) targeted polyetherimide-AE105 peptide conjugate (P-AE105) with the formation of GNS@Ir@P-AE105. It was demonstrated that the resultant GNS@Ir@P-AE105 was a multifunctional nanoplatform with advantages of (1) NIR laser controlled release of the Ir complex; (2) precise delivery of the Ir complex to TNBC cells; (3) excellent photothermal (PT)/photoacoustic (PA)/X-ray computed tomography (CT) tri-modal imaging ability; and (4) a synergistic photothermal-chemotherapeutic effect. An in-depth investigation of the mechanism revealed that binding forces of the Ir complex-GNS and P-AE105-GNS were significantly diminished upon NIR laser irradiation, which conferred an NIR laser-responsive Ir complex release property to the nanoplatform. Moreover, the nanoplatform exerted high efficiency anti-TNBC effects via a ROS-induced p53 apoptotic pathway. Specifically, combinational photothermal-chemotherapeutic treatments stimulated intracellular ROS generation, which significantly up-regulated apoptotic-relative p53 gene expression either by causing severe DNA damage or inducing an arrest effect on the sub-G1 phase of the cell cycle. Taken together, our work provides a novel theranostic nanoplatform for efficient and simultaneous diagnosis and therapy of TNBC.
Assuntos
Antineoplásicos/farmacologia , Lasers , Imagem Multimodal , Fototerapia , Receptores de Ativador de Plasminogênio Tipo Uroquinase/antagonistas & inibidores , Neoplasias de Mama Triplo Negativas/terapia , Animais , Proliferação de Células/efeitos dos fármacos , Terapia Combinada , Liberação Controlada de Fármacos , Humanos , Raios Infravermelhos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Imagem Óptica , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Células Tumorais CultivadasRESUMO
Ligand-targeted toxins (LTTs) are bioengineered molecules which are composed of a targeting component linked to a toxin that induces cell death once the LTT binds its target. Bispecific targeting allows for the simultaneous targeting of two receptors. In this review, we mostly focus on the epidermal growth factor receptor (EGFR) as a target. We discuss the development and testing of a bispecific LTT targeting EGFR and urokinase-type plasminogen activator receptor (uPAR) as two attractive targets implicated in tumor growth and in the regulation of the tumor microvasculature in solid tumors. In vitro and mouse xenograft studies have shown that EGFR-targeted bispecific angiotoxin (eBAT) is effective against human solid tumors. Canine studies have shown that eBAT is both safe and effective against canine hemangiosarcoma, which is physiologically similar to human angiosarcoma. Finding the appropriate dosing strategy and sequencing of eBAT administration, in combination with other therapeutics, are among important factors for future directions. Together, the data indicate that eBAT targets cancer stem cells, it may have a role in inhibiting human tumor vasculature, and its bispecific conformation may have a role in reducing toxicity in comparative oncologic trials in dogs.