Assuntos
Corantes Fluorescentes , Meningioma , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Humanos , Neoplasias Meníngeas/diagnóstico por imagem , Neoplasias Meníngeas/cirurgia , Meningioma/diagnóstico por imagem , Meningioma/cirurgia , Receptores de Ativador de Plasminogênio Tipo Uroquinase/químicaRESUMO
Proteolysis is a key event in several biological processes; proteolysis must be tightly controlled because its improper activation leads to dramatic consequences. Deregulation of proteolytic activity characterizes many pathological conditions, including cancer. The plasminogen activation (PA) system plays a key role in cancer; it includes the serine-protease urokinase-type plasminogen activator (uPA). uPA binds to a specific cellular receptor (uPAR), which concentrates proteolytic activity at the cell surface, thus supporting cell migration. However, a large body of evidence clearly showed uPAR involvement in the biology of cancer cell independently of the proteolytic activity of its ligand. In this review we will first describe this multifunctional molecule and then we will discuss how uPAR can sustain most of cancer hallmarks, which represent the biological capabilities acquired during the multistep cancer development. Finally, we will illustrate the main data available in the literature on uPAR as a cancer biomarker and a molecular target in anti-cancer therapy.
Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias/metabolismo , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Animais , Biomarcadores Tumorais/genética , Humanos , Neoplasias/diagnóstico , Neoplasias/genética , Neoplasias/terapia , Receptores de Ativador de Plasminogênio Tipo Uroquinase/química , Receptores de Ativador de Plasminogênio Tipo Uroquinase/genéticaRESUMO
Despite being recognized as a therapeutic target in the processes of cancer cell proliferation and metastasis for over 50 years, the interaction of the urokinase plasminogen activator uPA with its receptor uPAR still needs an improved understanding. High resolution crystallographic data (PDB ) of the uPA-uPAR binding geometry was used to perform quantum biochemistry computations within the density functional theory (DFT) framework. A divide to conquer methodology considering a mixed homogeneous/inhomogeneous dielectric model and explicitly taking water molecules into account was employed to obtain a large set of uPA-uPAR residue-residue interaction energies. In order of importance, not only were Phe25 > Tyr24 > Trp30 > Ile28 shown to be the most relevant uPA residues binding it to uPAR, but the residues Lys98 > His87 > Gln40 > Asn22 > Lys23 > Val20 also had significant interaction energies, which helps to explain published experimental mutational data. Furthermore, the results obtained with the uPA-uPAR in/homogeneous dielectric function show that a high dielectric constant value ε = 40 is adequate to take into account the electrostatic environment at the interface between the proteins, while using a smaller value of ε (<10) leads to an overestimation of the uPA-uPAR binding energy. Hot spots of the uPA-uPAR binding domain were identified and a quantum biochemistry description of the uPAR blockers uPA21-30 and cyclo21,29uPA21-29[(S21C;H29C)] was performed, demonstrating that cyclization improves the stability of mimetic peptides without compromising their binding energies to uPAR.
Assuntos
Receptores de Ativador de Plasminogênio Tipo Uroquinase/química , Ativador de Plasminogênio Tipo Uroquinase/química , Sequência de Aminoácidos , Aminoácidos/química , Teoria da Densidade Funcional , Peptídeos Cíclicos/química , Ligação Proteica , Conformação Proteica , Eletricidade Estática , Relação Estrutura-Atividade , TermodinâmicaRESUMO
The interaction between the short 88Ser-Arg-Ser-Arg-Tyr92 sequence of the urokinase receptor (uPAR) and the formyl peptide receptor type 1 (FPR1) elicits cell migration. We generated the Ac-(D)-Tyr-(D)-Arg-Aib-(D)-Arg-NH2 (RI-3) peptide which inhibits the uPAR/FPR1 interaction, reducing migration of FPR1 expressing cells toward N-formyl-methionyl-leucyl-phenylalanine (fMLF) and Ser-Arg-Ser-Arg-Tyr (SRSRY) peptides. To understand the structural basis of the RI-3 inhibitory effects, the FPR1/fMLF, FPR1/SRSRY and FPR1/RI-3 complexes were modeled and analyzed, focusing on the binding pocket of FPR1 and the interaction between the amino acids that signal to the FPR1 C-terminal loop. We found that RI-3 shares the same binding site of fMLF and SRSRY on FPR1. However, while fMLF and SRSRY display the same agonist activation signature (i.e. the series of contacts that transmit the conformational transition throughout the complex), translating binding into signaling, RI-3 does not interact with the activation region of FPR1 and hence does not activate signaling. Indeed, fluorescein-conjugated RI-3 prevents either fMLF and SRSRY uptake on FPR1 without triggering FPR1 internalization and cell motility in the absence of any stimulus. Collectively, our data show that RI-3 is a true FPR1 antagonist and suggest a pharmacophore model useful for development of compounds that selectively inhibit the uPAR-triggered, FPR1-mediated cell migration.
Assuntos
Peptídeos/metabolismo , Receptores de Formil Peptídeo/metabolismo , Receptores de Ativador de Plasminogênio Tipo Uroquinase/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Células HEK293 , Humanos , Simulação de Dinâmica Molecular , Peptídeos/química , Peptídeos/farmacologia , Ligação Proteica , Mapas de Interação de Proteínas , Estrutura Terciária de Proteína , Ratos , Receptores de Formil Peptídeo/química , Receptores de Formil Peptídeo/genética , Relação Estrutura-AtividadeRESUMO
Soluble urokinase plasminogen activator receptor (suPAR) is an immune-derived circulating signaling molecule that has been implicated in chronic kidney disease, such as focal segmental glomerulosclerosis (FSGS). Typically, native uPAR (isoform 1) translates to a 3-domain protein capable of binding and activating integrins, yet the function of additional isoforms generated by alternative splicing is unknown. Here, we characterized mouse uPAR isoform 2 (msuPAR2), encoding domain I and nearly one-half of domain II, as a dimer in solution, as revealed by 3D electron microscopy structural analysis. In vivo, msuPAR2 transgenic mice exhibited signs of severe renal disease characteristic of FSGS with proteinuria, loss of kidney function, and glomerulosclerosis. Sequencing of the glomerular RNAs from msuPAR2-Tg mice revealed a differentially expressed gene signature that includes upregulation of the suPAR receptor Itgb3, encoding ß3 integrin. Crossing msuPAR2-transgenic mice with 3 different integrin ß3 deficiency models rescued msuPAR2-mediated kidney function. Further analyses indicated a central role for ß3 integrin and c-Src in msuPAR2 signaling and in human FSGS kidney biopsies. Administration of Src inhibitors reduced proteinuria in msuPAR2-transgenic mice. In conclusion, msuPAR2 may play an important role in certain forms of scarring kidney disease.
Assuntos
Nefropatias/genética , Receptores de Ativador de Plasminogênio Tipo Uroquinase/química , Receptores de Ativador de Plasminogênio Tipo Uroquinase/genética , Adipócitos/citologia , Animais , Biópsia , Modelos Animais de Doenças , Células HEK293 , Humanos , Camundongos , Camundongos Transgênicos , Microscopia Eletrônica , Podócitos/citologia , Domínios Proteicos , Isoformas de Proteínas , Multimerização Proteica , Receptor PAR-2/genética , Estudos Retrospectivos , Transdução de SinaisRESUMO
We report a ratiometric strategy for detection of different types of breast cancer cells by surface-enhanced Raman spectroscopy (SERS), which simultaneously quantifies the levels of dual biomarkers distinctly expressed on cancer cells to consequently achieve their expression ratio. Two SERS nanoprobes that are encoded with distinct SERS signatures are conjugated with urokinase plasminogen activation receptor (uPAR)- and epidermal growth factor receptor (EGFR)-targeting peptides. The SERS imaging of single live cells can accurately quantify the cellular biomarker expression difference from the SERS intensity ratio and is further employed for cancer cell screening. The results show that MDA-MB-231 and MCF-7 exhibit distinct expression of the uPAR and EGFR and they can be respectively discriminated by the intensity ratio of SERS signals from uPAR- and EGFR-targeting SERS nanoprobes. The ratiometric strategy permits background-free SERS detection of cancer cells and dramatically improves the signal-to-noise ratio of targeted cellular SERS imaging, thus enabling accurate cancer cell screening without the need for additional references. It is believed that the present ratiometric method should be a promising avenue for breast cancer diagnostics and screening, which can be easily extended for detection of other cancer cell types.
Assuntos
Nanopartículas Metálicas/química , Neoplasias/diagnóstico , Análise Espectral Raman/métodos , Neoplasias da Mama/diagnóstico , Linhagem Celular Tumoral , Receptores ErbB/química , Receptores ErbB/uso terapêutico , Humanos , Receptores de Ativador de Plasminogênio Tipo Uroquinase/química , Receptores de Ativador de Plasminogênio Tipo Uroquinase/uso terapêuticoRESUMO
BACKGROUND: TEM8 is a cell membrane protein predominantly expressed in tumor endothelium, which serves as a receptor for the protective antigen (PA) of anthrax toxin. However, the physiological ligands for TEM8 remain unknown. RESULTS: Here we identified uPA as an interacting partner of TEM8. Binding of uPA stimulated the phosphorylation of TEM8 and augmented phosphorylation of EGFR and ERK1/2. Finally, TEM8-Fc, a recombinant fusion protein comprising the extracellular domain of human TEM8 linked to the Fc portion of human IgG1, efficiently abrogated the interaction between uPA and TEM8, blocked uPA-induced migration of HepG2 cells in vitro and inhibited the growth and metastasis of human MCF-7 xenografts in vivo. uPA, TEM8 and EGFR overexpression and ERK1/2 phosphorylation were found co-located on frozen cancer tissue sections. CONCLUSIONS: Taken together, our data provide evidence that TEM8 is a novel receptor for uPA, which may play a significant role in the regulation of tumor growth and metastasis.
Assuntos
Receptores ErbB/metabolismo , Proteínas de Neoplasias/metabolismo , Receptores de Superfície Celular/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Proliferação de Células , Humanos , Cinética , Proteínas dos Microfilamentos , Metástase Neoplásica , Fosforilação , Domínios Proteicos , Receptores de Ativador de Plasminogênio Tipo Uroquinase/química , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/químicaRESUMO
The receptor for the urokinase-type plasminogen activator (uPAR) is a widely recognized master regulator of cell migration. We and others have previously documented that the uPAR(84-95) sequence, interacts with the formyl peptide receptors (FPR)s, henceforth inducing cell migration of several cell lines, including leukocytes, and the synthetic shorter peptide (Ser88-Arg-Ser-Arg-Tyr92, SRSRY) retains chemotactic activity in vitro and in vivo. Recently, we have developed the head-to-tail cyclic analog [SRSRY], a new potent and stable inhibitor of monocyte trafficking. This prompted us to develop novel cyclic and linear analogs of [SRSRY] with the aim to broaden the knowledge about structure-activity relationships of peptide [SRSRY]. Herein we report their synthesis, effects on cell migration, conformational and docking analyses which served to envisage a new pharmacophore model for inhibitors of FPR1-triggered cell migration.
Assuntos
Peptídeos/farmacologia , Receptores de Formil Peptídeo/antagonistas & inibidores , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Animais , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Estrutura Molecular , Peptídeos/síntese química , Peptídeos/química , Ratos , Receptores de Ativador de Plasminogênio Tipo Uroquinase/química , Relação Estrutura-AtividadeRESUMO
The urokinase receptor (uPAR) stimulates cell proliferation by forming a macromolecular complex with αvß3 integrin and the epidermal growth factor receptor (EGFR, ErbB1 or HER1) that we name the uPAR proliferasome. uPAR transactivates EGFR, which in turn mediates uPAR-initiated mitogenic signal to the cell. EGFR activation and EGFR-dependent cell growth are blocked in the absence of uPAR expression or when uPAR activity is inhibited by antibodies against either uPAR or EGFR. The mitogenic sequence of uPAR corresponds to the D2A motif present in domain 2. NMR analysis revealed that D2A synthetic peptide has a particular three-dimensional structure, which is atypical for short peptides. D2A peptide is as effective as EGF in promoting EGFR phosphorylation and cell proliferation that were inhibited by AG1478, a specific inhibitor of the tyrosine kinase activity of EGFR. Both D2A and EGF failed to induce proliferation of NR6-EGFR-K721A cells expressing a kinase-defective mutant of EGFR. Moreover, D2A peptide and EGF phosphorylate ERK demonstrating the involvement of the MAP kinase signalling pathway. Altogether, this study reveals the importance of sequence D2A of uPAR, and the interdependence of uPAR and EGFR.
Assuntos
Proliferação de Células , Receptores ErbB/metabolismo , Integrina alfaVbeta3/metabolismo , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Sequência de Aminoácidos , Linhagem Celular Tumoral , Células HEK293 , Humanos , Sistema de Sinalização das MAP Quinases , Modelos Moleculares , Fosforilação , Receptores de Ativador de Plasminogênio Tipo Uroquinase/químicaRESUMO
D2A-Ala is a synthetic peptide that has been created by introducing mutations in the original D2A sequence, 130IQEGEEGRPKDDR142 of human urokinase receptor (uPAR). In vitro, D2A-Ala peptide displays strong anti-tumoural properties inhibiting EGF-induced chemotaxis, invasion and proliferation of a human fibrosarcoma cell line, HT 1080, and a human colorectal adenocarcinoma cell line, HT 29. D2A-Ala exerts its effects by preventing EGF receptor (EGFR) phosphorylation. To test D2A-Ala in vivo, this peptide was PEGylated generating polyethyleneglycol (PEG)-D2A-Ala peptide. PEGylation did not alter the inhibitory properties of D2A-Ala. Human tumour xenografts in the immunodeficient nude mice using HT 1080 and HT 29 cell lines showed that PEG-D2A-Ala significantly prevents tumour growth decreasing size, weight and density of tumours. The most efficient doses of the peptide were 5 and 10â¯mg/kg, thereby relevant for possible development of the peptide into a drug against cancer in particular tumours expressing EGFR.
Assuntos
Adenossarcoma/tratamento farmacológico , Antineoplásicos/farmacologia , Quimiotaxia/efeitos dos fármacos , Neoplasias Colorretais/tratamento farmacológico , Fibrossarcoma/tratamento farmacológico , Oligopeptídeos/farmacologia , Receptores de Ativador de Plasminogênio Tipo Uroquinase/química , Adenossarcoma/metabolismo , Adenossarcoma/patologia , Animais , Antineoplásicos/química , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Feminino , Fibrossarcoma/metabolismo , Fibrossarcoma/patologia , Células HT29 , Humanos , Camundongos , Camundongos Nus , Invasividade Neoplásica , Oligopeptídeos/química , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
C4.4A is a glycosylphosphatidylinositol-anchored membrane protein comprised of two LU domains (Ly6/uPAR-like domains) and an extensively O-glycosylated C-terminal Ser/Thr/Pro-rich region. C4.4A is a novel biomarker for squamous epithelial differentiation. Its expression is dysregulated under various pathological conditions and it is a robust biomarker for poor prognosis in various malignant conditions such as pulmonary adenocarcinoma. To facilitate crystallization, the two LU domains were excised from intact C4.4A by limited proteolysis, purified and crystallized by the sitting-drop vapour-diffusion method. The crystals diffracted to 2.7â Å resolution and belonged to space group C2221, with unit-cell parameters a = 55.49, b = 119.63, c = 168.54â Å. The statistics indicated good quality of the data, which form a solid basis for the determination of the C4.4A structure.
Assuntos
Sequência de Aminoácidos , Biomarcadores Tumorais/química , Moléculas de Adesão Celular/química , Deleção de Sequência , Animais , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Clonagem Molecular , Cristalização , Cristalografia por Raios X , Drosophila melanogaster/citologia , Drosophila melanogaster/metabolismo , Proteínas Ligadas por GPI/química , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Domínios Proteicos , 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 , Homologia de Sequência de Aminoácidos , Difração de Raios XRESUMO
Hematopoietic stem cells (HSCs) reside in bone marrow (BM) and can be induced to mobilize into the circulation for transplantation. Homing and lodgement into BM of transplanted HSCs are the first critical steps in their engraftment and involve multiple interactions between HSCs and the BM microenvironment.uPAR is a three domain receptor (DIDIIDIII) which binds urokinase, vitronectin, integrins. uPAR can be cleaved and shed from the cell surface generating full-length and cleaved soluble forms (suPAR and DIIDIII-suPAR). DIIDIII-suPAR can bind fMLF receptors through the SRSRY sequence (residues 88-92).We previously reported the involvement of soluble uPAR in HSC mobilization. We now investigate its possible role in HSC homing and engraftment.We show similar levels of circulating full-length suPAR in healthy donors and in acute myeloid leukemia (AML) patients before and after the pre-transplant conditioning regimen. By contrast, levels of circulating DIIDIII-suPAR in AML patients are higher as compared to controls and significantly decrease after the conditioning.We found that suPAR and uPAR84-95, a uPAR-derived peptide which mimics active DIIDIII-suPAR, induce a significant increase in Long Term Culture (LTC)-Initiating Cells (ICs) and in the release of clonogenic progenitors from LTCs of CD34+ HSCs. Further, suPAR increases adhesion and survival of CD34+ KG1 AML cells, whereas uPAR84-95 increases their proliferation.Thus, circulating DIIDIII-suPAR, strongly increased in HSC mobilization, is indeed down-regulated by pre-transplant conditioning, probably to favour HSC homing. BM full-length suPAR and DIIDIII-suPAR may be involved in HSC lodgement within the BM by contributing to a suitable microenvironment.
Assuntos
Células da Medula Óssea/metabolismo , Medula Óssea/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Nicho de Células-Tronco , Adulto , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Transplante de Células-Tronco Hematopoéticas/métodos , Humanos , Leucemia Mieloide Aguda/sangue , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/terapia , Pessoa de Meia-Idade , Peptídeos/farmacologia , Receptores de Ativador de Plasminogênio Tipo Uroquinase/sangue , Receptores de Ativador de Plasminogênio Tipo Uroquinase/química , Condicionamento Pré-Transplante/métodos , Adulto JovemRESUMO
BACKGROUND: Hepatitis B virus (HBV) presents an important public health problem. Liver biopsy is currently the gold standard for assessing the degree of intrahepatic inflammation and for staging liver fibrosis. However, the value of liver biopsies is limited by sampling errors, understaging and interobserver variability in interpretation. There is, therefore, a need to identify novel, non-invasive serologic biomarkers for the development of new predictive models of fibrosis. METHODS: We enrolled patients with chronic hepatitis B infection (CHB) and examined the relationships between serum soluble urokinase plasminogen activator receptor (suPAR) and interferon-induced protein-10 (IP-10), and the results of liver biopsies. Healthy volunteers with normal aminotransferase levels and negative serological results for HBV, hepatitis C virus and human immunodeficiency virus were recruited as controls. RESULTS: Mean platelet volume, serum suPAR and IP-10 were significantly elevated in patients with CHB compared with controls. Median serum suPAR and IP-10 levels were significantly higher in patients with liver fibrosis compared with patients with mild fibrosis. There was no significant difference in mean platelet volume or aspartate aminotransferase-to-platelet ratio index scores between patients with mild and significant fibrosis. CONCLUSION: suPAR and IP-10 were able to distinguish between significant and mild fibrosis with good sensitivity and specificity, and may thus represent useful biomarkers for identifying patients with significant fibrosis.
Assuntos
Quimiocina CXCL10/sangue , Hepatite B Crônica/sangue , Hepatite B Crônica/diagnóstico , Cirrose Hepática/sangue , Cirrose Hepática/diagnóstico , Receptores de Ativador de Plasminogênio Tipo Uroquinase/sangue , Adolescente , Adulto , Biomarcadores/sangue , Quimiocina CXCL10/química , Feminino , Hepatite B Crônica/complicações , Humanos , Cirrose Hepática/etiologia , Masculino , Pessoa de Meia-Idade , Receptores de Ativador de Plasminogênio Tipo Uroquinase/química , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Solubilidade , Estatística como Assunto , Adulto JovemRESUMO
Raised plasma levels of the soluble urokinase plasminogen activator receptor (suPAR) have been associated with increased incidence of cardiovascular diseases. Whether suPAR is associated with venous thromboembolism (VTE) is largely unknown. The purpose of the present study was to investigate the relationship between suPAR and incidence of VTE in a cohort study. suPAR was measured in 5,203 subjects (aged 46-68 years, 58 % women) from the general population, who participated in the Malmö Diet and Cancer (MDC) study between 1991 and 1994. Incident cases of VTE were identified from the Swedish patient register during a mean follow-up of 15.7 years. Of 5,203 subjects with measurements of suPAR, 239 had VTE during follow-up (127 venous thrombosis, 86 lung embolism, 26 both). Incidence of VTE was significantly higher in subjects with suPAR levels in the top quartile. Adjusted for age and sex, the HR (4th vs 1st quartile) was 1.74 (95 %CI: 1.2-2.6, p for trend=0.003). After adjustments for risk factors, the HR was 1.66 (95 %CI: 1.1-2.5, p for trend=0.016). High level of suPAR was a risk indicator for incidence of VTE in this population-based cohort study. The causal relationships between suPAR and VTE remain to be explored.
Assuntos
Receptores de Ativador de Plasminogênio Tipo Uroquinase/química , Tromboembolia Venosa/epidemiologia , Tromboembolia Venosa/imunologia , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/sangue , Proteína C-Reativa/química , Estudos de Coortes , Feminino , Seguimentos , Humanos , Incidência , Masculino , Pessoa de Meia-Idade , Ativadores de Plasminogênio/química , Embolia Pulmonar/imunologia , Receptores de Ativador de Plasminogênio Tipo Uroquinase/sangue , Sistema de Registros , Fatores de Risco , Suécia , Tromboembolia Venosa/sangueRESUMO
The receptor for the urokinase-type plasminogen activator (uPAR) is a widely recognized master regulator of cell migration and uPAR88-92 is the minimal sequence required to induce cell motility. We and others have previously documented that the uPAR88-92 sequence, even in the form of synthetic linear peptide (SRSRY), interacts with the formyl peptide receptor type 1 (FPR1), henceforth inducing cell migration of several cell lines, including monocytes. FPR1 is mainly expressed by mammalian phagocytic leukocytes and plays a crucial role in chemotaxis. In this study, we present evidence that the cyclization of the SRSRY sequence generates a new potent and stable inhibitor of monocyte trafficking. In rat basophilic leukaemia RBL-2H3/ETFR cells expressing high levels of constitutively activated FPR1, the cyclic SRSRY peptide ([SRSRY]) blocks FPR1 mediated cell migration by interfering with both internalization and ligand-uptake of FPR1. Similarly to RBL-2H3/ETFR cells, [SRSRY] competes with fMLF for binding to FPR1 and prevents agonist-induced FPR1 internalization in human monocyte THP-1 cells. Unlike scramble [RSSYR], [SRSRY] inhibits fMLF-directed migration of monocytes in a dose-dependent manner, with IC50 value of 0.01 nM. PMA-differentiated THP-1 cell exposure to fMLF gradient causes a marked cytoskeletal re-organization with the formation of F-actin rich pseudopodia that are prevented by the addition of [SRSRY]. Furthermore, [SRSRY] prevents migration of human primary monocytes and trans-endothelial migration of monocytes. Our findings indicate that [SRSRY] is a new FPR1 inhibitor which may suggest the development of new drugs for treating pathological conditions sustained by increased motility of monocytes, such as chronic inflammatory diseases.
Assuntos
Monócitos/efeitos dos fármacos , Monócitos/fisiologia , Peptídeos/metabolismo , Peptídeos/farmacologia , Receptores de Ativador de Plasminogênio Tipo Uroquinase/química , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Migração Transendotelial e Transepitelial/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Linhagem Celular , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão , Ciclização , Macrófagos/efeitos dos fármacos , Macrófagos/fisiologia , Peptídeos/química , Ligação Proteica , RatosRESUMO
The urokinase receptor (uPAR) is a GPI-anchored cell surface receptor that is at the center of an intricate network of protein-protein interactions. Its immediate binding partners are the serine proteinase urokinase (uPA), and vitronectin (VTN), a component of the extracellular matrix. uPA and VTN bind at distinct sites on uPAR to promote extracellular matrix degradation and integrin signaling, respectively. Here, we report the discovery of a new class of pyrrolone small-molecule inhibitors of the tight â¼1 nM uPAR·uPA protein-protein interaction. These compounds were designed to bind to the uPA pocket on uPAR. The highest affinity compound, namely 7, displaced a fluorescently labeled α-helical peptide (AE147-FAM) with an inhibition constant Ki of 0.7 µM and inhibited the tight uPAR·uPAATF interaction with an IC50 of 18 µM. Biophysical studies with surface plasmon resonance showed that VTN binding is highly dependent on uPA. This cooperative binding was confirmed as 7, which binds at the uPAR·uPA interface, also inhibited the distal VTN·uPAR interaction. In cell culture, 7 blocked the uPAR·uPA interaction in uPAR-expressing human embryonic kidney (HEK-293) cells and impaired cell adhesion to VTN, a process that is mediated by integrins. As a result, 7 inhibited integrin signaling in MDA-MB-231 cancer cells as evidenced by a decrease in focal adhesion kinase (FAK) phosphorylation and Rac1 GTPase activation. Consistent with these results, 7 blocked breast MDA-MB-231 cancer cell invasion with IC50 values similar to those observed in ELISA and surface plasmon resonance competition studies. Explicit-solvent molecular dynamics simulations show that the cooperativity between uPA and VTN is attributed to stabilization of uPAR motion by uPA. In addition, free energy calculations revealed that uPA stabilizes the VTNSMB·uPAR interaction through more favorable electrostatics and entropy. Disruption of the uPAR·VTNSMB interaction by 7 is consistent with the cooperative binding to uPAR by uPA and VTN. Interestingly, the VTNSMB·uPAR interaction was less favorable in the VTNSMB·uPAR·7 complex suggesting potential cooperativity between 7 and VTN. Compound 7 provides an excellent starting point for the development of more potent derivatives to explore uPAR biology.
Assuntos
Pirróis/farmacologia , Receptores de Ativador de Plasminogênio Tipo Uroquinase/antagonistas & inibidores , Inibidores de Serina Proteinase/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Ativador de Plasminogênio Tipo Uroquinase/antagonistas & inibidores , Vitronectina/antagonistas & inibidores , Sítios de Ligação , Ligação Competitiva , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Quinase 1 de Adesão Focal/genética , Quinase 1 de Adesão Focal/metabolismo , Regulação da Expressão Gênica , Células HEK293 , Humanos , Integrina alfaVbeta3/antagonistas & inibidores , Integrina alfaVbeta3/química , Integrina alfaVbeta3/genética , Integrina alfaVbeta3/metabolismo , Integrinas/genética , Integrinas/metabolismo , Cinética , Peptídeos/farmacologia , Ligação Proteica , Pirróis/síntese química , Receptor Cross-Talk/efeitos dos fármacos , Receptores de Ativador de Plasminogênio Tipo Uroquinase/química , Receptores de Ativador de Plasminogênio Tipo Uroquinase/genética , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Inibidores de Serina Proteinase/síntese química , Transdução de Sinais , Bibliotecas de Moléculas Pequenas/síntese química , Termodinâmica , Ativador de Plasminogênio Tipo Uroquinase/química , Ativador de Plasminogênio Tipo Uroquinase/genética , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Vitronectina/química , Vitronectina/genética , Vitronectina/metabolismo , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
Urokinase plasminogen activator receptor (uPAR) is a membrane protein which is attached to the cellular external membrane. The uPAR expression can be observed both in tumor cells and in tumor-associated stromal cells. Thus, in the present study, the human amino-terminal fragment (hATF), as a targeting element to uPAR, is used to conjugate to the surface of superparamagnetic iron nanoparticle (SPIO). Flowcytometry was used to examine the uPAR expression in different tumor cell lines. The specificity of hATF-SPIO was verified by Prussian blue stain and cell phantom test. The imaging properties of hATF-SPIO were confirmed in vivo magnetic resonance imaging (MRI) of uPAR-elevated colon tumor. Finally, the distribution of hATF-SPIO in tumor tissue was confirmed by pathological staining. Results showed that the three cells in which we screened, presented different expression characteristics, i. e., Hela cells strongly expressed uPAR, HT29 cells moderately expressed uPAR, but Lovo cells didn't express uPAR. In vitro, after incubating with Hela cells, hATF-SPIO could specifically combined to and be subsequently internalized by uPAR positive cells, which could be observed via Prussian blue staining. Meanwhile T2WI signal intensity of Hela cells, after incubation with targeted probe, significantly decreased, and otherwise no obvious changes in Lovo cells both by Prussian blue staining and MRI scans. In vivo, hATF-SPIO could be systematically delivered to HT29 xenograft and accumulated in the tumor tissue which was confirmed by Prussian Blue stain compared to Lovo xenografts. Twenty-four hours after injection of targeting probe, the signal intensity of HT29 xenografts was lower than Lovo ones which was statistically significant. This targeting nanoparticles enabled not only in vitro specifically combining to uPAR positive cells but also in vivo imaging of uPAR moderately elevated colon cancer lesions.
Assuntos
Neoplasias do Colo/diagnóstico , Nanopartículas de Magnetita/química , Imagem Molecular/métodos , Receptores de Ativador de Plasminogênio Tipo Uroquinase/química , Linhagem Celular Tumoral , Compostos Férricos , Humanos , Imageamento por Ressonância Magnética , Coloração e RotulagemRESUMO
BACKGROUND: High levels of circulating forms of the urokinase-type plasminogen activator receptor (uPAR) are significantly associated to poor prognosis in cancer patients. Our aim was to determine biological variations and reference intervals of the uPAR forms in blood, and in addition, to test the clinical relevance of using these as cut-points in colorectal cancer (CRC) prognosis. METHODS: uPAR forms were measured in citrated and EDTA plasma samples using time-resolved fluorescence immunoassays. Diurnal, intra- and inter-individual variations were assessed in plasma samples from cohorts of healthy individuals. Reference intervals were determined in plasma from healthy individuals randomly selected from a Danish multi-center cross-sectional study. A cohort of CRC patients was selected from the same cross-sectional study. RESULTS: The reference intervals showed a slight increase with age and women had ~20% higher levels. The intra- and inter-individual variations were ~10% and ~20-30%, respectively and the measured levels of the uPAR forms were within the determined 95% reference intervals. No diurnal variation was found. Applying the normal upper limit of the reference intervals as cut-point for dichotomizing CRC patients revealed significantly decreased overall survival of patients with levels above this cut-point of any uPAR form. CONCLUSIONS: The reference intervals for the different uPAR forms are valid and the upper normal limits are clinically relevant cut-points for CRC prognosis.
Assuntos
Receptores de Ativador de Plasminogênio Tipo Uroquinase/sangue , Receptores de Ativador de Plasminogênio Tipo Uroquinase/química , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Fluorimunoensaio/normas , Humanos , Masculino , Pessoa de Meia-Idade , Valores de Referência , Fatores de Tempo , Adulto JovemRESUMO
Urokinase plasminogen activator receptor (uPAR) and the epithelial integrin αvß6 are thought to individually play critical roles in cancer metastasis. These observations have been highlighted by the recent discovery (by proteomics) of an interaction between these two molecules, which are also both implicated in the epithelial-mesenchymal transition (EMT) that facilitates escape of cells from tissue barriers and is a common signature of cancer metastases. In this study, orthogonal in cellulo and in vitro functional proteomic approaches were used to better characterize the uPAR·αvß6 interaction. Proximity ligation assays (PLA) confirmed the uPAR·αvß6 interaction on OVCA429 (ovarian cancer line) and four different colon cancer cell lines including positive controls in cells with de novo ß6 subunit expression. PLA studies were then validated using peptide arrays, which also identified potential physical sites of uPAR interaction with αvß6, as well as verifying interactions with other known uPAR ligands (e.g., uPA, vitronectin) and individual integrin subunits (i.e., αv, ß1, ß3, and ß6 alone). Our data suggest that interaction with uPAR requires expression of the complete αß heterodimer (e.g., αvß6), not individual subunits (i.e., αv, ß1, ß3, or ß6). Finally, using in silico structural analyses in concert with these functional proteomics studies, we propose and demonstrate that the most likely unique sites of interaction between αvß6 and uPAR are located in uPAR domains II and III.
Assuntos
Antígenos de Neoplasias/metabolismo , Integrinas/metabolismo , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Sequência de Aminoácidos , Antígenos de Neoplasias/química , Linhagem Celular Tumoral , Transição Epitelial-Mesenquimal , Humanos , Integrinas/química , Dados de Sequência Molecular , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas , Proteômica , Receptores de Ativador de Plasminogênio Tipo Uroquinase/químicaRESUMO
The urokinase-type plasminogen activator receptor (uPAR) is implicated in cancer invasion and metastatic development in prostate cancer and provides therefore an attractive molecular target for both imaging and therapy. In this study, we provide the first in vivo data on an antimetastatic effect of uPAR radionuclide targeted therapy in such lesions and show the potential of uPAR positron emission tomography (PET) imaging for identifying small foci of metastatic cells in a mouse model of disseminating human prostate cancer. Two radiolabeled ligands were generated in high purity and specific activity: a uPAR-targeting probe ((177)Lu-DOTA-AE105) and a nonbinding control ((177)Lu-DOTA-AE105mut). Both uPAR flow cytometry and ELISA confirmed high expression levels of the target uPAR in PC-3M-LUC2.luc cells, and cell binding studies using (177)Lu-DOTA-AE105 resulted in a specific binding with an IC50 value of 100 nM in a competitive binding experiment. In vivo, uPAR targeted radionuclide therapy significantly reduced the number of metastatic lesions in the disseminated metastatic prostate cancer model, when compared to vehicle and nontargeted (177)Lu groups (p < 0.05) using bioluminescence imaging. Moreover, we found a significantly longer metastatic-free survival, with 65% of all mice without any disseminated metastatic lesions present at 65 days after first treatment dose (p = 0.047). In contrast, only 30% of all mice in the combined control groups treated with (177)Lu-DOTA-AE105mut or vehicle were without metastatic lesions. No treatment-induced toxicity was observed during the study as evaluated by observing animal weight and H&E staining of kidney tissue (dose-limiting organ). Finally, uPAR PET imaging using (64)Cu-DOTA-AE105 detected all small, disseminated metastatic foci when compared with bioluminescence imaging in a cohort of animals during the treatment study. In conclusion, uPAR targeted radiotherapy resulted in a significant reduction in the number of metastatic lesions in a human metastatic prostate cancer model. Furthermore, we have provided the first evidence of the potential for identification of small metastatic lesions using uPAR PET imaging in disseminated prostate cancer, illustrating the promising strategy of uPAR theranostics in prostate cancer.