RESUMO
Adenoviruses are used extensively as gene transfer agents, both experimentally and clinically. However, targeting of liver cells by adenoviruses compromises their potential efficacy. In cell culture, the adenovirus serotype 5 fiber protein engages the coxsackievirus and adenovirus receptor (CAR) to bind cells. Paradoxically, following intravascular delivery, CAR is not used for liver transduction, implicating alternate pathways. Recently, we demonstrated that coagulation factor (F)X directly binds adenovirus leading to liver infection. Here, we show that FX binds to the Ad5 hexon, not fiber, via an interaction between the FX Gla domain and hypervariable regions of the hexon surface. Binding occurs in multiple human adenovirus serotypes. Liver infection by the FX-Ad5 complex is mediated through a heparin-binding exosite in the FX serine protease domain. This study reveals an unanticipated function for hexon in mediating liver gene transfer in vivo.
Assuntos
Adenovírus Humanos/fisiologia , Proteínas do Capsídeo/metabolismo , Fator X/metabolismo , Fígado/virologia , Transdução Genética , Internalização do Vírus , Adenovírus Humanos/química , Adenovírus Humanos/classificação , Animais , Proteínas do Capsídeo/química , Proteínas de Transporte/metabolismo , Microscopia Crioeletrônica , Fator X/química , Hepatócitos/virologia , Humanos , Imageamento Tridimensional , Camundongos , Camundongos Transgênicos , Modelos Moleculares , Filogenia , Ligação Proteica/efeitos dos fármacos , Domínios e Motivos de Interação entre Proteínas , Ressonância de Plasmônio de Superfície , Varfarina/farmacologiaRESUMO
Long-term administration of aspirin (ASA, acetylsalicylic acid) in oncogenic patients has been related to a reduction in cancer risk incidence, but its precise mechanism of action is unclear. The activation of cancer-associated fibroblasts (CAFs) is a key element in tumor progression and can be triggered by cancer-derived extracellular vesicles (EVs). Targeting the communication between cancer cells and the surrounding tumor microenvironment (TME) may control cancer progression. Our aim was to investigate the effect of ASA on breast cancer cells, focusing on EV secretion and their effect on the biological properties of CAFs. As a result, ASA was shown to reduce the amount and alter the size distribution of EVs produced by MDA-MB-231 tumor cells. Fibroblasts stimulated with EVs derived from MDA-MB-231 treated with ASA (EV-ASA) showed a lower expression of alpha-smooth muscle actin (α-SMA), matrix metalloproteinase-2 (MMP2) but not fibroblast activation protein (FAP) in respect to the ones stimulated with EVs from untreated breast cancer cells (EV-CTR). Furthermore, invasion assays using a three-dimensional (3D) fibroblast spheroid model showed reduced MDA-MB-231 invasion towards fibroblast spheroids pretreated with EV-ASA as compared to spheroids prepared with EV-CTR-stimulated fibroblasts. This suggests that ASA partially inhibits the ability of tumor EVs to stimulate CAFs to promote cancer invasion. In conclusion, ASA can interfere with tumor communication by reducing EV secretion by breast tumor cells as well as by interfering with their capacity to stimulate fibroblasts to become CAFs.
RESUMO
Ixolaris is a potent tick salivary anticoagulant that binds coagulation factor Xa (FXa) and zymogen FX, with formation of a quaternary tissue factor (TF)/FVIIa/ FX(a)/Ixolaris inhibitory complex. Ixolaris blocks TF-induced coagulation and PAR2 signaling and prevents thrombosis, tumor growth, and immune activation. We present a high-resolution structure and dynamics of Ixolaris and describe the structural basis for recognition of FX. Ixolaris consists of 2 Kunitz domains (K1 and K2) in which K2 is strikingly dynamic and encompasses several residues involved in FX binding. This indicates that the backbone plasticity of K2 is critical for Ixolaris biological activity. Notably, a nuclear magnetic resonance-derived model reveals a mechanism for an electrostatically guided, high-affinity interaction between Ixolaris and FX heparin-binding (pro)exosite, resulting in an allosteric switch in the catalytic site. This is the first report revealing the structure-function relationship of an anticoagulant targeting a zymogen serving as a scaffold for TF inhibition.
Assuntos
Inibidores do Fator Xa/química , Inibidores do Fator Xa/farmacologia , Fator Xa/metabolismo , Proteínas e Peptídeos Salivares/química , Proteínas e Peptídeos Salivares/farmacologia , Animais , Fator Xa/química , Humanos , Simulação de Acoplamento Molecular , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , Domínios Proteicos , Carrapatos/químicaRESUMO
Psd1 is a pea plant defensin which can be actively expressed in Pichia pastoris and shows broad antifungal activity. This activity is dependent on fungal membrane glucosylceramide (GlcCer), which is also important for its internalization, nuclear localization, and endoreduplication. Certain cancer cells present a lipid metabolism imbalance resulting in the overexpression of GlcCer in their membrane. In this work, in vitroassays using B16F10 cells showed that labeled fluorescein isothiocyanate FITC-Psd1 internalized into live cultured cells and targeted the nucleus, which underwent fragmentation, exhibiting approximately 60% of cells in the sub-G0/G1 stage. This phenomenon was dependent on GlcCer, and the participation of cyclin-F was suggested. In a murine lung metastatic melanoma model, intravenous injection of Psd1 together with B16F10 cells drastically reduced the number of nodules at concentrations above 0.5 mg/kg. Additionally, the administration of 1 mg/kg Psd1 decreased the number of lung inflammatory cells to near zero without weight loss, unlike animals that received melanoma cells only. It is worth noting that 1 mg/kg Psd1 alone did not provoke inflammation in lung tissue or weight or vital signal losses over 21 days, inferring no whole animal cytotoxicity. These results suggest that Psd1 could be a promising prototype for human lung anti-metastatic melanoma therapy.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Defensinas/farmacologia , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/secundário , Pisum sativum/química , Proteínas de Plantas/farmacologia , Animais , Antineoplásicos Fitogênicos/química , Apoptose/efeitos dos fármacos , Biópsia , Linhagem Celular , Permeabilidade da Membrana Celular , Proliferação de Células/efeitos dos fármacos , Defensinas/química , Modelos Animais de Doenças , Feminino , Imunofluorescência , Glucosilceramidas/metabolismo , Imuno-Histoquímica , Neoplasias Pulmonares/tratamento farmacológico , Melanoma Experimental , Camundongos , Modelos Moleculares , Proteínas de Plantas/química , Conformação Proteica , Relação Estrutura-AtividadeRESUMO
Activated Protein C (APC) is a serine-protease that displays antithrombotic and anti-inflammatory properties. In addition, cleavage of protease-activated receptor 1 (PAR1) by APC exerts endothelial cytoprotective actions. The effects of APC on endothelial cells may be reproduced by TR47, a PAR1-based peptide that mimics the novel N-terminus of PAR1 generated upon cleavage at Arg-46 by APC. In this study we demonstrate that wild-type APC and its signaling-proficient mutant, APC-2Cys (which has dramatically reduced anticoagulant activity), display similar inhibitory effects towards the transendothelial migration of A375 human melanoma cells. Consistent with this observation, APC and APC-2Cys significantly reduced the in vivo metastatic potential of the B16F10 murine melanoma cells. TR47 recapitulated the in vitro and in vivo protective profiles of APC and APC-2Cys. Treatment of EA.hy926 endothelial cells with TR47 (20 µM) significantly decreased the A375 cell migration. In addition, treatment of C57/BL6 mice with a single TR47 dose (125 µg/animal) strongly reduced the metastatic burden of B16F10 cells. Together, our results suggest that protection of the endothelial barrier by APC/TR47-mediated signaling pathways might be a valuable therapeutic approach to prevent metastasis.
Assuntos
Movimento Celular/efeitos dos fármacos , Transformação Celular Neoplásica/efeitos dos fármacos , Melanoma/metabolismo , Melanoma/secundário , Peptídeos/administração & dosagem , Receptor PAR-1/química , Animais , Linhagem Celular Tumoral , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Humanos , Melanoma/patologia , Camundongos , Camundongos Endogâmicos C57BL , Invasividade Neoplásica/patologia , Peptídeos/químicaRESUMO
Protease-activated receptor 2 (PAR2) is a G-protein coupled receptor which is activated upon cleavage of its N-terminal region. PAR2 has been associated with many aspects regarding tumor progression, such as the production of pro-tumoral cytokines. Granulocyte colony-stimulating factor (G-CSF) is a cytokine essential to neutrophil production and maturation, and it is often overexpressed in tumors. In this study, we evaluated the ability of PAR2 to modulate G-CSF expression. PAR2 and G-CSF were significantly more expressed in metastatic (4T1 and MDA-MB-231) as compared to non-metastatic (67NR and MCF7) breast cancer cell lines. In addition, PAR2 stimulation by a synthetic agonist peptide significantly increased G-CSF gene expression in the metastatic cell lines. Knockdown of PAR2 in 4T1 cells decreased G-CSF expression and secretion. In addition, treatment of 4T1 with the commercial PAR2 antagonist, ENMD-1068, significantly decreased G-CSF expression. cBioPortal analyses of the TCGA database showed a significant co-occurrence of G-CSF and PAR2 gene overexpression in breast cancer samples. In conclusion, our data suggest that PAR2 contributes to G-CSF expression in breast cancer cells, possibly favoring tumor progression.
Assuntos
Neoplasias da Mama/metabolismo , Regulação Neoplásica da Expressão Gênica , Fator Estimulador de Colônias de Granulócitos/metabolismo , Receptor PAR-2/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animais , Linhagem Celular Tumoral , Citocinas/metabolismo , Feminino , Perfilação da Expressão Gênica , Células HEK293 , Humanos , Camundongos , Ativação Transcricional , Regulação para CimaRESUMO
Extracellular vesicles, such as microvesicles (MVs), were identified as important players in tumor progression and acquisition of an aggressive phenotype. Tissue factor (TF) is a transmembrane protein that initiates the blood coagulation cascade. In tumor cells, TF has been associated with aggressiveness and cancer progression. Previous studies demonstrate that TF is incorporated into MVs secreted by tumor cells; however, it is unknown whether TF is actively involved in the release of MVs. Here, we investigated the influence of TF expression on the release of MVs. TF silencing was achieved through CRISPR/Cas9 approaches in the human breast cancer cell line, MDA-MB-231. TF knockout in MDA-MB-231â¯cells efficiently reduced TF-dependent signaling and procoagulant activity. Remarkably, silencing of TF caused a significant decrease in the number of MVs released by MDA-MB-231â¯cells. We also observed an increase in actin-positive membrane projections in TF knockout cells and a reduction in RhoA expression when compared to TF-expressing cells. Treatment of MDA-MB-231â¯cells with the RhoA-ROCK signaling pathway inhibitor, fasudil, significantly reduced the release of MVs. Taken together, our results suggest a novel and relevant role for TF in tumor biology by playing an active role in the MVs secretion.
Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/patologia , Tromboplastina/metabolismo , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Vesículas Extracelulares/genética , Fator VIIa/análise , Fator VIIa/metabolismo , Feminino , Inativação Gênica , Humanos , Transdução de Sinais , Tromboplastina/genética , Quinases Associadas a rho/análise , Quinases Associadas a rho/metabolismo , Proteína rhoA de Ligação ao GTP/análise , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
RNA aptamers are synthetic oligonucleotide-based affinity molecules that utilize unique three-dimensional structures for their affinity and specificity to a target such as a protein. They hold the promise of numerous advantages over biologically produced antibodies; however, the binding affinity and specificity of RNA aptamers are often insufficient for successful implementation in diagnostic assays or as therapeutic agents. Strong binding affinity is important to improve the downstream applications. We report here the use of the phosphorodithioate (PS2) substitution on a single nucleotide of RNA aptamers to dramatically improve target binding affinity by â¼1000-fold (from nanomolar to picomolar). An X-ray co-crystal structure of the α-thrombin:PS2-aptamer complex reveals a localized induced-fit rearrangement of the PS2-containing nucleotide which leads to enhanced target interaction. High-level quantum mechanical calculations for model systems that mimic the PS2 moiety and phenylalanine demonstrate that an edge-on interaction between sulfur and the aromatic ring is quite favorable, and also confirm that the sulfur analogs are much more polarizable than the corresponding phosphates. This favorable interaction involving the sulfur atom is likely even more significant in the full aptamer-protein complexes than in the model systems.
Assuntos
Fosfatos/metabolismo , RNA/metabolismo , Aptâmeros de Nucleotídeos , Linhagem Celular , Humanos , Cinética , Limite de Detecção , Modelos Moleculares , Conformação de Ácido Nucleico , Ligação Proteica , Proteínas/metabolismo , Estabilidade de RNA , Padrões de Referência , Soro/metabolismo , Termodinâmica , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
BACKGROUND: Invasion of mosquito salivary glands (SGs) by Plasmodium falciparum sporozoites is an essential step in the malaria life cycle. How infection modulates gene expression, and affects hematophagy remains unclear. PRINCIPAL FINDINGS: Using Affimetrix chip microarray, we found that at least 43 genes are differentially expressed in the glands of Plasmodium falciparum-infected Anopheles gambiae mosquitoes. Among the upregulated genes, one codes for Agaphelin, a 58-amino acid protein containing a single Kazal domain with a Leu in the P1 position. Agaphelin displays high homology to orthologs present in Aedes sp and Culex sp salivary glands, indicating an evolutionarily expanded family. Kinetics and surface plasmon resonance experiments determined that chemically synthesized Agaphelin behaves as a slow and tight inhibitor of neutrophil elastase (K(D) â¼ 10 nM), but does not affect other enzymes, nor promotes vasodilation, or exhibit antimicrobial activity. TAXIscan chamber assay revealed that Agaphelin inhibits neutrophil chemotaxis toward fMLP, affecting several parameter associated with cell migration. In addition, Agaphelin reduces paw edema formation and accumulation of tissue myeloperoxidase triggered by injection of carrageenan in mice. Agaphelin also blocks elastase/cathepsin-mediated platelet aggregation, abrogates elastase-mediated cleavage of tissue factor pathway inhibitor, and attenuates neutrophil-induced coagulation. Notably, Agaphelin inhibits neutrophil extracellular traps (NETs) formation and prevents FeCl3-induced arterial thrombosis, without impairing hemostasis. CONCLUSIONS: Blockade of neutrophil elastase emerges as a novel antihemostatic mechanism in hematophagy; it also supports the notion that neutrophils and the innate immune response are targets for antithrombotic therapy. In addition, Agaphelin is the first antihemostatic whose expression is induced by Plasmodium sp infection. These results suggest that an important interplay takes place in parasite-vector-host interactions.
Assuntos
Anopheles/parasitologia , Hemostasia/fisiologia , Interações Hospedeiro-Parasita , Proteínas de Insetos/metabolismo , Neutrófilos/imunologia , Plasmodium falciparum/patogenicidade , Proteínas e Peptídeos Salivares/metabolismo , Trombose/prevenção & controle , Sequência de Aminoácidos , Animais , Anopheles/metabolismo , Dicroísmo Circular , Edema/etiologia , Edema/metabolismo , Edema/prevenção & controle , Feminino , Proteínas de Insetos/química , Proteínas de Insetos/genética , Insetos Vetores , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Glândulas Salivares/metabolismo , Glândulas Salivares/parasitologia , Proteínas e Peptídeos Salivares/química , Proteínas e Peptídeos Salivares/genética , Homologia de Sequência de Aminoácidos , Ressonância de Plasmônio de SuperfícieRESUMO
The identity of vampire bat saliva anticoagulant remained elusive for almost a century. Sequencing the salivary gland genes from the vampire bat Desmodus rotundus identified Desmolaris as a novel 21.5-kDa naturally deleted (Kunitz 1-domainless) form of tissue factor pathway inhibitor. Recombinant Desmolaris was expressed in HEK293 cells and characterized as a slow, tight, and noncompetitive inhibitor of factor (F) XIa by a mechanism modulated by heparin. Desmolaris also inhibits FXa with lower affinity, independently of protein S. In addition, Desmolaris binds kallikrein and reduces bradykinin generation in plasma activated with kaolin. Truncated and mutated forms of Desmolaris determined that Arg32 in the Kunitz-1 domain is critical for protease inhibition. Moreover, Kunitz-2 and the carboxyl-terminus domains mediate interaction of Desmolaris with heparin and are required for optimal inhibition of FXIa and FXa. Notably, Desmolaris (100 µg/kg) inhibited FeCl3-induced carotid artery thrombus without impairing hemostasis. These results imply that FXIa is the primary in vivo target for Desmolaris at antithrombotic concentrations. Desmolaris also reduces the polyphosphate-induced increase in vascular permeability and collagen- and epinephrine-mediated thromboembolism in mice. Desmolaris emerges as a novel anticoagulant targeting FXIa under conditions in which the coagulation activation, particularly the contact pathway, plays a major pathological role.
Assuntos
Anticoagulantes/química , Anticoagulantes/farmacologia , Quirópteros , Inibidores do Fator Xa , Proteínas e Peptídeos Salivares/química , Proteínas e Peptídeos Salivares/farmacologia , Trombose/tratamento farmacológico , Animais , Bradicinina/química , Bradicinina/genética , Bradicinina/metabolismo , Cloretos/efeitos adversos , Cloretos/farmacologia , Modelos Animais de Doenças , Fator Xa/química , Fator Xa/genética , Fator Xa/metabolismo , Compostos Férricos/efeitos adversos , Compostos Férricos/farmacologia , Células HEK293 , Humanos , Inflamação/tratamento farmacológico , Inflamação/genética , Inflamação/metabolismo , Calicreínas/química , Calicreínas/genética , Calicreínas/metabolismo , Camundongos , Noxas/efeitos adversos , Noxas/farmacologia , Estrutura Terciária de Proteína , Proteínas e Peptídeos Salivares/genética , Trombose/induzido quimicamente , Trombose/genética , Trombose/metabolismoRESUMO
Dengue is an infectious disease caused by dengue virus (DENV). In general, dengue is a self-limiting acute febrile illness followed by a phase of critical defervescence, in which patients may improve or progress to a severe form. Severe illness is characterized by hemodynamic disturbances, increased vascular permeability, hypovolemia, hypotension, and shock. Thrombocytopenia and platelet dysfunction are common in both cases and are related to the clinical outcome. Different mechanisms have been hypothesized to explain DENV-associated thrombocytopenia, including the suppression of bone marrow and the peripheral destruction of platelets. Studies have shown DENV-infected hematopoietic progenitors or bone marrow stromal cells. Moreover, anti-platelet antibodies would be involved in peripheral platelet destruction as platelets interact with endothelial cells, immune cells, and/or DENV. It is not yet clear whether platelets play a role in the viral spread. Here, we focus on the mechanisms of thrombocytopenia and platelet dysfunction in DENV infection. Because platelets participate in the inflammatory and immune response by promoting cytokine, chemokine, and inflammatory mediator secretion, their relevance as "immune-like effector cells" will be discussed. Finally, an implication for platelets in plasma leakage will be also regarded, as thrombocytopenia is associated with clinical outcome and higher mortality.
Assuntos
Dengue/fisiopatologia , Fibrinólise/fisiologia , Trombocitopenia/fisiopatologia , Plaquetas/fisiologia , Humanos , Mediadores da Inflamação/metabolismo , Modelos BiológicosRESUMO
Aegyptin is a mosquito salivary gland protein and potent inhibitor of platelet aggregation. Aegyptin binds to the von Willebrand factor-binding site on collagen and prevents its interaction with platelets. Because collagen also induces plasma clotting by activation of factor XII, we evaluated the effects of aegyptin on collagen-induced coagulation activation and how it interferes with thrombosis in three different in vivo models. Our results demonstrate that aegyptin abolishes collagen-induced clot formation and thrombin generation in platelet-free plasma. Aegyptin has no antithrombotic activity in the arteriovenous shunt model (collagen-independent) but it prevents laser-induced collagen-mediated thrombus formation in rats. Furthermore, aegyptin protects mice from collagen and epinephrine-induced thromboembolism. Therefore, aegyptin has a dual antithrombotic mechanism: inhibition of platelet-collagen interaction and collagen's pro-coagulant activity. This is the first description of a collagen-binding protein that also inhibits collagen-mediated coagulant activity.
Assuntos
Coagulação Sanguínea/efeitos dos fármacos , Colágeno/farmacologia , Proteínas de Insetos/farmacologia , Embolia Pulmonar/prevenção & controle , Proteínas e Peptídeos Salivares/farmacologia , Animais , Culicidae/metabolismo , Relação Dose-Resposta a Droga , Epinefrina/farmacologia , Feminino , Células HEK293 , Humanos , Proteínas de Insetos/genética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Ratos , Ratos Wistar , Proteínas Recombinantes/farmacologia , Proteínas e Peptídeos Salivares/genética , Trombina/metabolismoRESUMO
OBJECTIVE: Blood-sucking arthropods' salivary glands contain a remarkable diversity of antihemostatics. The aim of the present study was to identify the unique salivary anticoagulant of the sand fly Lutzomyia longipalpis, which remained elusive for decades. METHODS AND RESULTS: Several L. longipalpis salivary proteins were expressed in human embryonic kidney 293 cells and screened for inhibition of blood coagulation. A novel 32.4-kDa molecule, named Lufaxin, was identified as a slow, tight, noncompetitive, and reversible inhibitor of factor Xa (FXa). Notably, Lufaxin's primary sequence does not share similarity to any physiological or salivary inhibitors of coagulation reported to date. Lufaxin is specific for FXa and does not interact with FX, Dansyl-Glu-Gly-Arg-FXa, or 15 other enzymes. In addition, Lufaxin blocks prothrombinase and increases both prothrombin time and activated partial thromboplastin time. Surface plasmon resonance experiments revealed that FXa binds Lufaxin with an equilibrium constant ≈3 nM, and isothermal titration calorimetry determined a stoichiometry of 1:1. Lufaxin also prevents protease-activated receptor 2 activation by FXa in the MDA-MB-231 cell line and abrogates edema formation triggered by injection of FXa in the paw of mice. Moreover, Lufaxin prevents FeCl(3)-induced carotid artery thrombus formation and prolongs activated partial thromboplastin time ex vivo, implying that it works as an anticoagulant in vivo. Finally, salivary gland of sand flies was found to inhibit FXa and to interact with the enzyme. CONCLUSIONS: Lufaxin belongs to a novel family of slow-tight FXa inhibitors, which display antithrombotic and anti-inflammatory activities. It is a useful tool to understand FXa structural features and its role in prohemostatic and proinflammatory events.
Assuntos
Anti-Inflamatórios/farmacologia , Inibidores do Fator Xa , Fibrinolíticos/farmacologia , Inflamação/prevenção & controle , Proteínas de Insetos/farmacologia , Psychodidae/química , Receptor PAR-2/antagonistas & inibidores , Glândulas Salivares/química , Trombose/prevenção & controle , Sequência de Aminoácidos , Animais , Anti-Inflamatórios/química , Anti-Inflamatórios/isolamento & purificação , Coagulação Sanguínea/efeitos dos fármacos , Calorimetria , Linhagem Celular Tumoral , Cloretos , Clonagem Molecular , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Fator Xa/metabolismo , Feminino , Compostos Férricos , Fibrinolíticos/química , Fibrinolíticos/isolamento & purificação , Células HEK293 , Humanos , Inflamação/sangue , Inflamação/metabolismo , Proteínas de Insetos/química , Proteínas de Insetos/isolamento & purificação , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Peso Molecular , Tempo de Tromboplastina Parcial , Ligação Proteica , Tempo de Protrombina , Ratos , Receptor PAR-2/metabolismo , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/metabolismo , Ressonância de Plasmônio de Superfície , Tromboplastina/antagonistas & inibidores , Tromboplastina/metabolismo , Trombose/sangue , Trombose/induzido quimicamente , Trombose/metabolismo , Fatores de TempoRESUMO
OBJECTIVE: The coagulation-inflammation cycle has been implicated as a critical component in malaria pathogenesis. Defibrotide (DF), a mixture of DNA aptamers, displays anticoagulant, anti-inflammatory, and endothelial cell (EC)-protective activities and has been successfully used to treat comatose children with veno-occlusive disease. DF was investigated here as a drug to treat cerebral malaria. METHODS AND RESULTS: DF blocks tissue factor expression by ECs incubated with parasitized red blood cells and attenuates prothrombinase activity, platelet aggregation, and complement activation. In contrast, it does not affect nitric oxide bioavailability. We also demonstrated that Plasmodium falciparum glycosylphosphatidylinositol (Pf-GPI) induces tissue factor expression in ECs and cytokine production by dendritic cells. Notably, dendritic cells, known to modulate coagulation and inflammation systemically, were identified as a novel target for DF. Accordingly, DF inhibits Toll-like receptor ligand-dependent dendritic cells activation by a mechanism that is blocked by adenosine receptor antagonist (8-p-sulfophenyltheophylline) but not reproduced by synthetic poly-A, -C, -T, and -G. These results imply that aptameric sequences and adenosine receptor mediate dendritic cells responses to the drug. DF also prevents rosetting formation, red blood cells invasion by P. falciparum and abolishes oocysts development in Anopheles gambiae. In a murine model of cerebral malaria, DF affected parasitemia, decreased IFN-γ levels, and ameliorated clinical score (day 5) with a trend for increased survival. CONCLUSION: Therapeutic use of DF in malaria is proposed.
Assuntos
Anti-Inflamatórios/farmacologia , Anticoagulantes/farmacologia , Antimaláricos/farmacologia , Coagulação Sanguínea/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Malária Cerebral/tratamento farmacológico , Plasmodium berghei/efeitos dos fármacos , Plasmodium falciparum/efeitos dos fármacos , Polidesoxirribonucleotídeos/farmacologia , Animais , Células Cultivadas , Ativação do Complemento/efeitos dos fármacos , Citocinas/sangue , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Células Dendríticas/parasitologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Células Endoteliais/imunologia , Células Endoteliais/metabolismo , Células Endoteliais/parasitologia , Feminino , Glicosilfosfatidilinositóis/metabolismo , Hemoglobinas/metabolismo , Humanos , Mediadores da Inflamação/sangue , Malária Cerebral/sangue , Malária Cerebral/imunologia , Malária Cerebral/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Óxido Nítrico/metabolismo , Plasmodium berghei/patogenicidade , Plasmodium falciparum/crescimento & desenvolvimento , Plasmodium falciparum/metabolismo , Plasmodium falciparum/patogenicidade , Agregação Plaquetária/efeitos dos fármacos , Receptores Purinérgicos P1/efeitos dos fármacos , Receptores Purinérgicos P1/metabolismo , Índice de Gravidade de Doença , Tromboplastina/metabolismo , Fatores de TempoRESUMO
Neutrophil extracellular traps (NETs) have been implicated in several hallmarks of cancer. Among the protumor effects, NETs promote epithelial-mesenchymal transition (EMT) in different cancer models. EMT has been linked to an enhanced expression of the clotting-initiating protein, tissue factor (TF), thus favoring the metastatic potential. TF may also exert protumor effects by facilitating the activation of protease-activated receptor 2 (PAR2). Herein, we evaluated whether NETs could induce TF expression in breast cancer cells and further promote procoagulant and intracellular signaling effects via the TF/PAR2 axis. T-47D and MCF7 cell lines were treated with isolated NETs, and samples were obtained for real-time PCR, flow cytometry, Western blotting, and plasma coagulation assays. In silico analyses were performed employing RNA-seq data from breast cancer patients deposited in The Cancer Genome Atlas (TCGA) database. A positive correlation was observed between neutrophil/NETs gene signatures and TF gene expression. Neutrophils/NETs gene signatures and PAR2 gene expression also showed a significant positive correlation in the bioinformatics model. In vitro analysis showed that treatment with NETs upregulated TF gene and protein expression in breast cancer cell lines. The inhibition of ERK/JNK reduced the TF gene expression induced by NETs. Remarkably, the pharmacological or genetic inhibition of the TF/PAR2 signaling axis attenuated the NETs-induced expression of several protumor genes. Also, treatment of NETs with a neutrophil elastase inhibitor reduced the expression of metastasis-related genes. Our results suggest that the TF/PAR2 signaling axis contributes to the pro-cancer effects of NETs in human breast cancer cells.
RESUMO
The molecular mechanism of factor Xa (FXa) inhibition by Alboserpin, the major salivary gland anticoagulant from the mosquito and yellow fever vector Aedes albopictus, has been characterized. cDNA of Alboserpin predicts a 45-kDa protein that belongs to the serpin family of protease inhibitors. Recombinant Alboserpin displays stoichiometric, competitive, reversible and tight binding to FXa (picomolar range). Binding is highly specific and is not detectable for FX, catalytic site-blocked FXa, thrombin, and 12 other enzymes. Alboserpin displays high affinity binding to heparin (K(D) ~ 20 nM), but no change in FXa inhibition was observed in the presence of the cofactor, implying that bridging mechanisms did not take place. Notably, Alboserpin was also found to interact with phosphatidylcholine and phosphatidylethanolamine but not with phosphatidylserine. Further, annexin V (in the absence of Ca(2+)) or heparin outcompetes Alboserpin for binding to phospholipid vesicles, suggesting a common binding site. Consistent with its activity, Alboserpin blocks prothrombinase activity and increases both prothrombin time and activated partial thromboplastin time in vitro or ex vivo. Furthermore, Alboserpin prevents thrombus formation provoked by ferric chloride injury of the carotid artery and increases bleeding in a dose-dependent manner. Alboserpin emerges as an atypical serpin that targets FXa and displays unique phospholipid specificity. It conceivably uses heparin and phosphatidylcholine/phosphatidylethanolamine as anchors to increase protein localization and effective concentration at sites of injury, cell activation, or inflammation.
Assuntos
Aedes/química , Inibidores do Fator Xa , Fibrinolíticos/química , Heparina/química , Proteínas de Insetos/química , Insetos Vetores/química , Fosfatidilcolinas/química , Fosfatidiletanolaminas/química , Serpinas/química , Febre Amarela , Aedes/genética , Aedes/metabolismo , Aedes/virologia , Animais , Coagulação Sanguínea , Fator Xa/química , Fator Xa/genética , Fator Xa/metabolismo , Fibrinolíticos/metabolismo , Heparina/genética , Heparina/metabolismo , Humanos , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Insetos Vetores/metabolismo , Insetos Vetores/virologia , Camundongos , Fosfatidilcolinas/genética , Fosfatidilcolinas/metabolismo , Fosfatidiletanolaminas/genética , Fosfatidiletanolaminas/metabolismo , Fosfatidilserinas/química , Fosfatidilserinas/genética , Fosfatidilserinas/metabolismo , Ligação Proteica , Serpinas/genética , Serpinas/metabolismoRESUMO
Glioblastoma (GBM) is a highly aggressive cancer type characterized by intense neovascularization. Several lines of evidence indicate that blood clotting enzymes play an important role in the tumor microenvironment, mainly through the activation of protease-activated receptors (PAR). In particular, PAR1 and PAR2 isoforms may activate signal transduction pathways that promote a number of pro-tumoral responses. However, little is known concerning the role of PAR1/PAR2 in GBM progression. In this study, we investigated the expression and function of PAR1 and PAR2 in the human GBM cell lines A172 and U87-MG. We also evaluated the effect of agonist peptides for PAR1 (PAR1-AP) and PAR2 (PAR2-AP) on signaling pathways and the expression of vascular endothelial growth factor (VEGF). Immunoblotting assays showed that A172 and U87-MG constitutively express PAR1 and PAR2. Treatment of GBM cells with PAR1-AP or PAR2-AP enhanced Akt (protein kinase B) and extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation in a time-dependent manner. LY29042 and PD98059, inhibitors of the phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways, decreased PAR-mediated activation of Akt and ERK1/2, respectively. In addition, we observed that PAR2, but not PAR1, activation increased VEGF secretion in U87-MG and A172 cells. Notably, only PD98059 reduced PAR2-mediated VEGF production by GBM cells. Our results suggest that PAR2 modulates VEGF production through the MAPK/ERK1/2 pathway, and not the PI3K/Akt pathway, in human GBM cell lines. Therefore, the PAR2/MAPK signaling axis might be regarded as a relevant target for adjuvant treatment of GBM with a possible impact on tumor angiogenesis.
Assuntos
Glioblastoma/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Receptor PAR-2/metabolismo , Fator A de Crescimento do Endotélio Vascular/biossíntese , Linhagem Celular Tumoral , Humanos , Sistema de Sinalização das MAP Quinases , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor PAR-1/metabolismoRESUMO
Thrombin is a serine protease that plays fundamental roles in hemostasis. We have recently elucidated the crystal structure of thrombin in complex with suramin, evidencing the interaction through the anion binding exosite 2. Here, we show that the activity of thrombin toward natural and synthetic substrates is enhanced by suramin as well as analogs of suramin at a low micromolar range prior to an inhibitory component at higher concentrations. Suramin analogs substituted by phenyl and chlorine instead of methyl were the most efficient in promoting allosteric activation, with an enhancement of enzymatic activity of 250% and 630% respectively. We discuss the importance of exosite 2 as a regulatory site for ligands in both the procoagulant and inhibitory scenarios.
Assuntos
Modelos Químicos , Modelos Moleculares , Suramina/análogos & derivados , Trombina/química , Trombina/ultraestrutura , Sítios de Ligação , Ativação Enzimática , Humanos , Ligação Proteica , EstereoisomerismoRESUMO
Chronic Myeloid Leukemia is a neoplastic disease characterized by the abnormal expansion of hematopoietic cells with compromised functions. Leukemic cells often display a multidrug resistance phenotype, enabling them to evade a number of structurally unrelated cytotoxic compounds. One of those mechanisms relies on the high expression of efflux transporters, such as the ABC proteins, whose activity depends on the hydrolysis of ATP to reduce intracellular drug accumulation. In the present work, we employed a well-known erythroleukemia cell line, K562, and a multidrug resistant derivative cell, FEPS, to evaluate how hexokinase II, a key regulator for the rate-limiting step glycolysis, contributes to the establishment of the multidrug resistance phenotype. We found that multidrug resistant cells primarily resort to glycolysis to generate ATP. Clotrimazole reduced the expression of mitochondrial hexokinase II, which destabilized bioenergetic parameters such as reactive oxygen species production, ATP, and glutathione levels on multidrug resistant cells. This impaired the activity of ABCC1, leading to increased drug accumulation and cell death. In summary, we propose that decoupling of hexokinase II from the mitochondria emerges as a promising strategy to generate collateral sensitivity and aid in the management of chronic myeloid leukemia in chemotherapy-refractory patients.