RESUMEN
Prostate cancer is a major cause of cancer-related mortality in men in developed countries. The compound, 4-acetylantroquinonol B (4AAQB), is isolated from Antrodia cinnamomea (commonly known as Niu-Chang-Chih), which has been shown to inhibit cancer growth. However, the anticancer activity of 4AAQB has not previously been examined in prostate cancer. This study aimed to investigate the effect of 4AAQB on cancer and angiogenesis, as well as to explore its mechanism of action. Human prostate cancer cells (PC3) and human umbilical vein endothelial cells (HUVEC) were used in cell viability, cell migration, and cell cycle functional assays to evaluate the anticancer and antiangiogenic efficacy of 4AAQB in vitro. The effects of 4AAQB in vivo were determined using xenograft and angiogenesis models. The signaling events downstream of 4AAQB were also examined. The 4AAQB compound inhibited PC3 cell growth and migration, and reduced in vivo cancer growth, as shown in a subcutaneous xenograft model. Furthermore, 4AAQB inhibited HUVEC migration, tube formation, and aortic ring sprouting; it also reduced neovascularization in a Matrigel implant angiogenesis assay in vivo. The 4AAQB compound also decreased metastasis in the PC3 prostate cancer model in vivo. Serum or vascular endothelial growth factor (VEGF)-induced VEGF receptor 2 (VEGFR2), phosphoinositide 3-kinase (PI3K)/Ak strain transforming (Akt), and extracellular signal-regulated kinase ½ (ERK ½) phosphorylation were attenuated by 4AAQB in both PC3 and HUVEC. In conclusion, 4AAQB is a potential candidate for prostate cancer therapy.
Asunto(s)
4-Butirolactona/análogos & derivados , Inhibidores de la Angiogénesis/administración & dosificación , Ciclohexanonas/administración & dosificación , Neoplasias de la Próstata/tratamiento farmacológico , Transducción de Señal/efectos de los fármacos , 4-Butirolactona/administración & dosificación , 4-Butirolactona/farmacología , Inhibidores de la Angiogénesis/farmacología , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Ciclohexanonas/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana , Humanos , Masculino , Células PC-3 , Fosfatidilinositol 3-Quinasa/metabolismo , Neoplasias de la Próstata/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
OBJECTIVE: Currently prescribed antiplatelet drugs have 1 common side effect-an increased risk of hemorrhage and thrombocytopenia. On the contrary, bleeding defects associated with glycoprotein VI (GPVI) expression deficiency are usually slightly prolonged bleeding times. However, GPVI antagonists are lacking in clinic. APPROACH AND RESULTS: Using reverse-phase high-performance liquid chromatography and sequencing, we revealed the partial sequence of trowaglerix α subunit, a potent specific GPVI-targeting snaclec (snake venom C-type lectin protein). Hexapeptide (Troα6 [trowaglerix a chain hexapeptide, CKWMNV]) and decapeptide (Troα10) derived from trowaglerix specifically inhibited collagen-induced platelet aggregation through blocking platelet GPVI receptor. Computational peptide design helped to design a series of Troα6/Troα10 peptides. Protein docking studies on these decapeptides and GPVI suggest that Troα10 was bound at the lower surface of D1 domain and outer surface of D2 domain, which was at the different place of the collagen-binding site and the scFv (single-chain variable fragment) D2-binding site. The newly discovered site was confirmed by inhibitory effects of polyclonal antibodies on collagen-induced platelet aggregation. This indicates that D2 domain of GPVI is a novel and important binding epitope on GPVI-mediated platelet aggregation. Troα6/Troα10 displayed prominent inhibitory effect of thrombus formation in fluorescein sodium-induced platelet thrombus formation of mesenteric venules and ferric chloride-induced carotid artery injury thrombosis model without prolonging the in vivo bleeding time. CONCLUSIONS: We develop a novel antithrombotic peptides derived from trowaglerix that acts through GPVI antagonism with greater safety-no severe bleeding. The binding epitope of polypeptides on GPVI is novel and important. These hexa/decapeptides have therapeutic potential for developing ideal small-mass GPVI antagonists for arterial thrombogenic diseases.
Asunto(s)
Plaquetas/efectos de los fármacos , Traumatismos de las Arterias Carótidas/tratamiento farmacológico , Venenos de Crotálidos/farmacología , Fibrinolíticos/farmacología , Fragmentos de Péptidos/farmacología , Inhibidores de Agregación Plaquetaria/farmacología , Agregación Plaquetaria/efectos de los fármacos , Glicoproteínas de Membrana Plaquetaria/antagonistas & inhibidores , Trombosis/prevención & control , Animales , Sitios de Unión , Plaquetas/metabolismo , Traumatismos de las Arterias Carótidas/sangre , Traumatismos de las Arterias Carótidas/inducido químicamente , Cloruros , Diseño Asistido por Computadora , Venenos de Crotálidos/metabolismo , Venenos de Crotálidos/toxicidad , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Diseño de Fármacos , Compuestos Férricos , Fibrinolíticos/metabolismo , Fibrinolíticos/toxicidad , Fluoresceína , Hemorragia/inducido químicamente , Humanos , Lectinas Tipo C/metabolismo , Masculino , Ratones Endogámicos ICR , Simulación del Acoplamiento Molecular , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/toxicidad , Inhibidores de Agregación Plaquetaria/metabolismo , Inhibidores de Agregación Plaquetaria/toxicidad , Glicoproteínas de Membrana Plaquetaria/metabolismo , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Transducción de Señal/efectos de los fármacos , Trombosis/sangre , Trombosis/inducido químicamenteRESUMEN
Snake venoms affect blood coagulation and platelet function in a complex manner. However, two classes of venom proteins, snaclecs and disintegrins have been shown to specifically target receptors including GPIb, α2ß1, GPVI, CLEC-2 and integrins αIIbß3, αvß3, α5ß1 expressed on platelets, endothelial cells, phagocytes, tumor cells, thus affecting cell-matrices and cell-cell interactions. Here, we focus on disintegrins, a class of low molecular mass Arg-Gly-Asp(RGD)/Lys-Gly-Asp(KGD)-containing, cysteine-rich polypeptide derived from various viper snake venoms. This review describes the potential applications of disintegrins in field of integrin-related diseases, especially arterial thrombosis, angiogenesis, tumor progression and septic inflammation. In addition, a novel RGD-containing disintegrin TMV-7 is being developed as a safer antithrombotic agent with minimal side effects, such as thrombocytopenia and bleeding.
RESUMEN
Angiogenesis occurs not only during tissue growth and development but also during wound healing and tumor progression. Angiogenesis is a balanced process controlled by proangiogenic and antiangiogenic molecules. As a critical factor in the induction of angiogenesis, vascular endothelial growth factor (VEGF) has become an attractive target for antiangiogenic and cancer therapeutic agents. In an effort to develop novel inhibitors to block VEGF signaling, we selected Pj-8, a benzimidazole derivative, and investigated its inhibitory mechanisms in human umbilical vascular endothelial cells (HUVECs). Pj-8 concentration-dependently inhibited VEGF-induced proliferation, migration and tube formation of HUVECs. Pj-8 also suppressed VEGF-induced microvessel sprouting from aortic rings ex vivo and suppressed neovascularization of implanted matrigel plugs in vivo. Pj-8 inhibited VEGF-induced phosphorylation of VEGF receptor (VEGFR) 2 and the downstream protein kinases, including Akt, focal adhesion kinase, extracellular signal-regulated kinases and Src. Results from in vitro kinase assay further demonstrated that Pj-8 suppressed the kinase activity of 3-phosphoinositide-dependent kinase 1 (PDK1). Using xenograft tumor angiogenesis model, Pj-8 markedly eliminated tumor-associated angiogenesis. Taken together, our findings suggest that Pj-8 inhibits VEGF and tumor cells MDA-MB-231-induced angiogenesis, and it may be a potential drug candidate in anticancer therapy. Downregulation of VEGFR2-mediated signaling may contribute to its antiangiogenic actions.
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Bencimidazoles/farmacología , Neoplasias de la Mama/irrigación sanguínea , Neoplasias de la Mama/tratamiento farmacológico , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Receptor 2 de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Proteínas Quinasas Dependientes de 3-Fosfoinosítido , Animales , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Regulación hacia Abajo/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Neovascularización Patológica/tratamiento farmacológico , Neovascularización Patológica/metabolismo , Fosforilación/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto , Familia-src Quinasas/metabolismoRESUMEN
Cardiovascular disease (CVD) is strongly associated with the gut microbiota and its metabolites, including trimethylamine-N-oxide (TMAO), formed from metaorganismal metabolism of Ê-carnitine. Raw garlic juice, with allicin as its primary compound, exhibits considerable effects on the gut microbiota. This study validated the benefits of raw garlic juice against CVD risk via modulation of the gut microbiota and its metabolites. Allicin supplementation significantly decreased serum TMAO in Ê-carnitine-fed C57BL/6 J mice, reduced aortic lesions, and altered the fecal microbiota in carnitine-induced, atherosclerosis-prone, apolipoprotein E-deficient (ApoE-/-) mice. In human subjects exhibiting high-TMAO production, raw garlic juice intake for a week reduced TMAO formation, improved gut microbial diversity, and increased the relative abundances of beneficial bacteria. In in vitro and ex vivo studies, raw garlic juice and allicin inhibited γ-butyrobetaine (γBB) and trimethylamine production by the gut microbiota. Thus, raw garlic juice and allicin can potentially prevent cardiovascular disease by decreasing TMAO production via gut microbiota modulation.
Asunto(s)
Aterosclerosis , Ajo , Microbioma Gastrointestinal , Animales , Aterosclerosis/tratamiento farmacológico , Aterosclerosis/metabolismo , Disulfuros , Humanos , Metilaminas , Ratones , Ratones Endogámicos C57BL , Óxidos , Ácidos SulfínicosRESUMEN
Polymer polyethylene glycol (PEG), or PEGylation of polypeptides improves protein drug stability by decrease degradation and reduces renal clearance. To produce a pharmaceutical disintegrin derivative, the N-terminal PEGylation technique was used to modify the disintegrin derivative [KGDRR]trimucrin for favorable safety and pharmacokinetic profiles and antithrombotic efficacy. We compared intact [KGDRR]trimucrin (RR) and PEGylated KGDRR (PEG-RR) by in vitro and in vivo systems for their antithrombotic activities. The activity of platelet aggregation inhibition and the bleeding tendency side effect were also investigated. PEG-RR exhibited optimal potency in inhibiting platelet aggregation of human/mouse platelet-rich plasma activated by collagen or ADP with a lower IC50 than the intact derivative RR. In the illumination-induced mesenteric venous thrombosis model, RR and PEG-RR efficaciously prevented occlusive thrombosis in a dose-dependent manner. In rotational thromboelastometry assay, there was no effect of PEG-RR in human whole blood coagulation even given at a higher concentration (30 µg/mL), while RR slightly prolonged clotting time. However, RR and PEG-RR were not associated with severe thrombocytopenia or bleeding in FcγRIIa-transgenic mice at equally efficacious antithrombotic dosages. We also found the in vivo half-life of PEGylation was longer than RR (RR: 15.65 h vs. PEG-RR: 20.45 h). In conclusion, injectable PEG-RR with prolonged half-life and decreased bleeding risk is a safer anti-thrombotic agent for long-acting treatment of thrombus diseases.
Asunto(s)
Coagulación Sanguínea/efectos de los fármacos , Desintegrinas/farmacología , Fibrinolíticos/farmacología , Péptidos/farmacología , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/antagonistas & inhibidores , Polietilenglicoles/química , Trombosis/prevención & control , Animales , Modelos Animales de Enfermedad , Desintegrinas/química , Desintegrinas/toxicidad , Composición de Medicamentos , Fibrinolíticos/química , Fibrinolíticos/toxicidad , Hemorragia/inducido químicamente , Humanos , Masculino , Ratones , Ratones Endogámicos ICR , Ratones Transgénicos , Péptidos/química , Péptidos/toxicidad , Agregación Plaquetaria/efectos de los fármacos , Inhibidores de Agregación Plaquetaria/farmacología , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Receptores de IgG/genética , Receptores de IgG/metabolismo , Trombocitopenia/inducido químicamente , Trombosis/sangre , Trombosis/etiologíaRESUMEN
Subendothelial collagen supports platelet adhesion, activation, and thrombus growth at sites of vascular damage. Previous studies have shown that chalcones possess antiplatelet activity, but their mechanism of action is not fully understood. In this study, a recently synthesized chalcone, 2'-ethoxy-5'-methoxy-2-(5-methylthienyl)chalcone (EMMTC), was used to investigate chalcone effects on platelet aggregation and adhesion. We found that EMMTC potently inhibited collagen-induced platelet aggregation with an IC(50) of 1.01 micromol/l. In contrast, it did not inhibit thrombin- and ADP-induced platelet aggregation. EMMTC could inhibit arachidonic acid (AA)-induced platelet aggregation and collagen- and AA-induced thromboxane B(2) (TXB(2)) formation, suggesting that cyclooxygenase and/or thromboxane synthase were affected during this process. Moreover, EMMTC suppressed collagen-induced protein tyrosine phosphorylation, including phospholipase C-gamma2 (PLC-gamma2), phosphatidylinositol-3 kinase (PI-3K), Src, Fyn and LAT. Strikingly, EMMTC also blocked platelet adhesion to immobilized collagen and convulxin (a snake venom-derived protein that activates platelet glycoprotein VI receptor). The attenuation of phosphorylation of focal adhesion kinase (FAK) was observed during adhesion. Taken together, our results presented here demonstrate that the chalcone derivative EMMTC affects collagen-induced protein tyrosine phosphorylation and TXB(2) formation and functionally blocks collagen-induced platelet aggregation and adhesion.
Asunto(s)
Chalcona/análogos & derivados , Colágeno/metabolismo , Adhesividad Plaquetaria/efectos de los fármacos , Agregación Plaquetaria/efectos de los fármacos , Tiofenos/farmacología , Animales , Chalcona/administración & dosificación , Chalcona/farmacología , Colágeno/farmacología , Venenos de Crotálidos/metabolismo , Proteína-Tirosina Quinasas de Adhesión Focal/efectos de los fármacos , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Humanos , Concentración 50 Inhibidora , Lectinas Tipo C/metabolismo , Fosforilación/efectos de los fármacos , Prostaglandina-Endoperóxido Sintasas/metabolismo , Tiofenos/administración & dosificación , Tromboxano B2/biosíntesis , Tromboxano-A Sintasa/metabolismo , Tirosina/efectos de los fármacos , Tirosina/metabolismoRESUMEN
OBJECTIVE: Disintegrins, snake venom-derived Arg-Gly-Asp (RGD)-containing polypeptides, and GPIIb/IIIa antagonist (AP2) block fibrinogen binding to GPIIb/IIIa of activated platelets, however, the combination of these two agents caused platelet aggregation. We hypothesize that disintegrin initially binds to specific epitope of GPIIb/IIIa, causing conformational change, and the recruitment of FcgammaRII, which can be bound by AP2, and finally triggering platelet aggregation. MATERIALS AND METHODS: We prepared human platelet suspensions and measured platelet aggregation, Ca2+ mobilization, thromboxane B2 formation, and signal transduction. RESULTS: Disintegrin (e.g., accutin) and AP2 (a monoclonal antibody [mAb]-raised against GPIIb/IIIa) individually inhibited human platelet aggregation caused by collagen. However, as both accutin and AP2 were sequentially added into platelet suspension, platelet aggregation occurred. Accutin/AP2 caused shape change, cytosolic Ca2+ mobilization, P-selectin expression, and thromboxane A2 formation. Tirofiban, FcgammaRII mAb, or indomethacin completely inhibited platelet aggregation caused by accutin/AP2. Accutin/AP2 also caused tyrosine phosphorylation of signal molecules. Disintegrins enhanced AP2 binding to platelets, and AP2 also promoted disintegrin binding to platelets. FcgammaRII mAb inhibited the enhanced fluorescein isothiocyanate-disintegrin binding to platelet caused by AP2. Immunoprecipitation of the lysates of disintegrin/AP2-treated platelets using FcgammaRII Ab showed complex formation of GPIIb/IIIa and FcgammaRII. CONCLUSION: FcgammaRII mediates platelet aggregation caused by disintegrin and AP2, triggering a phospholipase C, phospholipase A2, Src-, Syk kinases, and Ca2+-dependent activation process. AP2 triggers platelet aggregation via binding to accessible FcgammaRII and the conformation-altered GPIIb/IIIa caused by disintegrin.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Plaquetas/metabolismo , Desintegrinas/farmacología , Inhibidores de Agregación Plaquetaria/farmacología , Agregación Plaquetaria/efectos de los fármacos , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Receptores de IgG/metabolismo , Animales , Plaquetas/citología , Calcio/metabolismo , Señalización del Calcio/efectos de los fármacos , Desintegrinas/química , Epítopos/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Fosfolipasas A2/metabolismo , Inhibidores de Agregación Plaquetaria/química , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/antagonistas & inhibidores , Conformación Proteica/efectos de los fármacos , Proteínas Tirosina Quinasas/metabolismo , Venenos de Serpiente/química , Quinasa Syk , Fosfolipasas de Tipo C/metabolismo , Familia-src Quinasas/metabolismoRESUMEN
Platelet-leukocyte interactions amplify inflammatory reactions, but the underlying mechanism is still unclear. CLEC5A and CLEC2 are spleen tyrosine kinase (Syk)-coupled C-type lectin receptors, abundantly expressed by leukocytes and platelets, respectively. Whereas CLEC5A is a pattern recognition receptor (PRR) to flaviviruses and bacteria, CLEC2 is the receptor for platelet-activating snake venom aggretin. Here we show that dengue virus (DV) activates platelets via CLEC2 to release extracellular vesicles (EVs), including exosomes (EXOs) and microvesicles (MVs). DV-induced EXOs (DV-EXOs) and MVs (DV-MVs) further activate CLEC5A and TLR2 on neutrophils and macrophages, thereby induce neutrophil extracellular trap (NET) formation and proinflammatory cytokine release. Compared to stat1-/- mice, simultaneous blockade of CLEC5A and TLR2 effectively attenuates DV-induced inflammatory response and increases survival rate from 30 to 90%. The identification of critical roles of CLEC2 and CLEC5A/TLR2 in platelet-leukocyte interactions will support the development of novel strategies to treat acute viral infection in the future.
Asunto(s)
Plaquetas/metabolismo , Virus del Dengue/inmunología , Dengue/inmunología , Vesículas Extracelulares/inmunología , Lectinas Tipo C/inmunología , Macrófagos/inmunología , Neutrófilos/inmunología , Receptores de Superficie Celular/inmunología , Receptor Toll-Like 2/inmunología , Animales , Micropartículas Derivadas de Células/inmunología , Micropartículas Derivadas de Células/metabolismo , Citocinas/inmunología , Dengue/virología , Exosomas/inmunología , Exosomas/metabolismo , Trampas Extracelulares/inmunología , Vesículas Extracelulares/metabolismo , Humanos , Inflamación , Lectinas Tipo C/genética , Ratones , Ratones Noqueados , Activación Plaquetaria , Receptores de Superficie Celular/genética , Factor de Transcripción STAT1/genética , Tasa de SupervivenciaRESUMEN
Snake venoms affect blood coagulation and platelet function in diverse ways. Some venom components inhibit platelet function, while other components induce platelet aggregation. Among the platelet aggregation inhibitors, disintegrins have been recognized as unique and potentially valuable tools for examining cell-matrix and cell-cell interactions and for the development of antithrombotic and antiangiogenic agents according to their anti-adhesive and anti-migration effect on tumor cells and antiangiogenesis activities. Disintegrins represent a family of low molecular weight, cysteine-rich, Arg-Gly-Asp(RGD)/Lys-Gly-Asp(KGD)-containing polypeptides, which inhibit fibrinogen binding to integrin αIIbß3 (i.e., platelet glycoprotein IIb/IIIa), as well as ligand binding to integrins αvß3, and α5ß1 expressed on cells (i.e., fibroblasts, tumor cells, and endothelial cells). This review focuses on the current efforts attained from studies using disintegrins as a tool in the field of arterial thrombosis, angiogenesis, inflammation, and tumor metastasis, and briefly describes their potential therapeutic applications and side effects in integrin-related diseases. Additionally, novel R(K)GD-containing disintegrin TMV-7 mutants are being designed as safer antithrombotics without causing thrombocytopenia and bleeding.
Asunto(s)
Desintegrinas/uso terapéutico , Fibrinolíticos/uso terapéutico , Venenos de Serpiente/química , Animales , Antiinflamatorios/uso terapéutico , Antineoplásicos/uso terapéutico , Humanos , Integrinas/antagonistas & inhibidores , Inhibidores de Agregación Plaquetaria/uso terapéuticoRESUMEN
Life-threatening thrombocytopenia and bleeding, common side effects of clinically available αIIbß3 antagonists, are associated with the induction of ligand-induced integrin conformational changes and exposure of ligand-induced binding sites (LIBSs). To address this issue, we examined intrinsic mechanisms and structure-activity relationships of purified disintegrins, from Protobothrops flavoviridis venom (i.e., Trimeresurus flavoviridis), TFV-1 and TFV-3 with distinctly different pro-hemorrhagic tendencies. TFV-1 with a different αIIbß3 binding epitope from that of TFV-3 and chimeric 7 × 103 Fab, i.e., Abciximab, decelerates αIIbß3 ligation without causing a conformational change in αIIbß3, as determined with the LIBS antibody, AP5, and the mimetic, drug-dependent antibody (DDAb), AP2, an inhibitory monoclonal antibody raised against αIIbß3. Consistent with their different binding epitopes, a combination of TFV-1 and AP2 did not induce FcγRIIa-mediated activation of the ITAM-Syk-PLCγ2 pathway and platelet aggregation, in contrast to the clinical antithrombotics, abciximab, eptifibatide, and disintegrin TFV-3. Furthermore, TFV-1 selectively inhibits Gα13-mediated platelet aggregation without affecting talin-driven clot firmness, which is responsible for physiological hemostatic processes. At equally efficacious antithrombotic dosages, TFV-1 caused neither severe thrombocytopenia nor bleeding in FcγRIIa-transgenic mice. Likewise, it did not induce hypocoagulation in human whole blood in the rotational thromboelastometry (ROTEM) assay used in perioperative situations. In contrast, TFV-3 and eptifibatide exhibited all of these hemostatic effects. Thus, the αIIbß3 antagonist, TFV-1, efficaciously prevents arterial thrombosis without adversely affecting hemostasis.
Asunto(s)
Desintegrinas/farmacología , Fibrinolíticos/efectos adversos , Fibrinolíticos/farmacología , Hemorragia/inducido químicamente , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/antagonistas & inhibidores , Venenos de Serpiente/farmacología , Abciximab/farmacología , Animales , Sitios de Unión , Tiempo de Sangría , Epítopos , Humanos , Masculino , Ratones , Ratones Endogámicos ICR , Agregación Plaquetaria , Inhibidores de Agregación Plaquetaria/farmacología , Relación Estructura-Actividad , TrimeresurusRESUMEN
BACKGROUND AND PURPOSE: Recent development in drug discovery have shown benzimidazole to be an important pharmacophore,. Benzimidazole derivatives exhibit broad-spectrum pharmacological properties including anti-microbial, anti-diabetic and anti-tumour activity. However, whether benzimidazole derivatives are effective in suppressing angiogenesis and its underlying mechanisms remain incompletely understood. In this study, we aim to characterize the anti-angiogenic mechanisms of a novel 2-aminobenzimidazole-based compound, Jzu 17, in an effort to develop novel angiogenesis inhibitor. EXPERIMENTAL APPROACH: Effects of Jzu 17 on endothelial cell proliferation, migration, invasion, and activation of signalling molecules induced by VEGF-A, were analysed by immunoblotting, MTT, BrdU, migration, and invasion assays. We performed tube formation assay, aorta ring sprouting assay, matrigel plug assay, and a mouse model of metastasis to evaluate ex vivo and in vivo anti-angiogenic effects of Jzu 17. KEY RESULTS: Jzu 17 inhibited VEGF-A-induced cell proliferation, migration, invasion, and endothelial tube formation of HUVECs. Jzu 17 suppressed VEGF-A-induced microvessel sprouting ex vivo and attenuated VEGF-A- or tumour cell-induced neovascularization in vivo. Jzu 17 also reduced B16F10 melanoma lung metastasis. In addition, Jzu 17 inhibited the phosphorylation of VEGFR-2 and its downstream signalling molecules in VEGF-A-stimulated HUVECs. Results from computer modelling further showed that Jzu 17 binds to VEGFR-2 with high affinity. CONCLUSIONS AND IMPLICATIONS: Jzu 17 may inhibit endothelial remodelling and suppress angiogenesis through targeting VEGF-A-VEGFR-2 signalling. These results also suggest Jzu 17 as a potential lead compound and warrant the clinical development of similar agents in the treatment of cancer and angiogenesis-related diseases.
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Antineoplásicos/farmacología , Neoplasias Pulmonares/tratamiento farmacológico , Melanoma/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Transducción de Señal/efectos de los fármacos , Receptor 2 de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Inhibidores de la Angiogénesis/química , Animales , Antineoplásicos/química , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundario , Masculino , Melanoma/metabolismo , Melanoma/patología , Ratones , Ratones Endogámicos C57BL , Simulación del Acoplamiento Molecular , Estructura Molecular , Neovascularización Patológica/tratamiento farmacológico , Neovascularización Patológica/metabolismo , Neovascularización Patológica/patología , Inhibidores de Proteínas Quinasas/química , Ratas , Ratas Sprague-Dawley , Relación Estructura-Actividad , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Inflammatory mediators such as TNF-alpha and interleukin (IL)-1beta, and IL-8, which can enhance binding of low-density lipoprotein (LDL) to endothelium and upregulate expression of leukocyte adhesion molecules on endothelium during atherogenesis. Lycopene, a natural carotenoid from tomato and other sources, has been shown to prevent cardiovascular diseases in epidemiological studies. However, its anti-inflammatory action mechanism remains unclear. In the present study, we studied the effect of lycopene on TNF-alpha-induced signaling in human umbilical endothelial cells (HUVECs). We found that TNF-alpha-induced intercellular adhesion molecule-1 (ICAM-1) expression in HUVECs was inhibited by lycopene, whereas cyclooxygenase-2 (COX-2) and platelet-endothelial cell adhesion molecule (PECAM-1) expression were not affected. A further analysis indicated that lycopene attenuated TNF-alpha-induced IkappaB phosphorylation, NF-kappaB expression, and NF-kappaB p65 translocation from cytosol to nucleus. In line with this, TNF-alpha-induced NF-kappaB-DNA but not AP1-DNA complexes formation was inhibited by lycopene, as determined by the electrophoretic mobility shift assay (EMSA). On the other hand, lycopene did not affect TNF-alpha-induced p38 and extracellular matrix-regulated kinase1/2 (ERK1/2) phosphorylation and interferon-gamma (IFN-gamma)-induced signaling, suggesting that lycopene primarily affects TNF-alpha-induced NF-kappaB signaling pathway. In a functional study, lycopene dose-dependently attenuated monocyte adhesion to endothelial monolayer but not that adhesion to extracellular matrix. Taken together, we provided here the first evidence showing that lycopene is able to inhibit TNF-alpha-induced NF-kappaB activation, ICAM-1 expression, and monocyte-endothelial interaction, suggesting an anti-inflammatory role of lycopene and possibly explaining in part why lycopene can prevent cardiovascular diseases.
Asunto(s)
Antioxidantes/farmacología , Carotenoides/farmacología , Adhesión Celular/efectos de los fármacos , Molécula 1 de Adhesión Intercelular/biosíntesis , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Western Blotting , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Ensayo de Cambio de Movilidad Electroforética , Células Endoteliales/efectos de los fármacos , Humanos , Quinasa I-kappa B/fisiología , Interferón gamma/farmacología , Licopeno , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Monocitos/efectos de los fármacos , ARN Mensajero/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacos , Factor de Necrosis Tumoral alfa/farmacología , Molécula 1 de Adhesión Celular Vascular/biosíntesis , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
In continuing search for novel antiplatelet agents, the highly potent agent 2-chloro-3-methoxycarbonylethylcarboxamido-1,4-naphthoquinone 2 was selected as lead compound. Structure-activity relationships in this series were examined. Some of these compounds showed significant antiplatelet activities. Further studies on the action mechanism showed that 2-acetamido-3-chloro-1,4-naphthoquinone 4 has a direct inhibitory action on cytosolic phospholipase A(2) (cPLA(2)) activity in platelets.
Asunto(s)
Diseño de Fármacos , Naftoquinonas/síntesis química , Inhibidores de Agregación Plaquetaria/síntesis química , Animales , Plaquetas/citología , Plaquetas/efectos de los fármacos , Plaquetas/enzimología , Células Cultivadas , Citosol/efectos de los fármacos , Citosol/enzimología , Estructura Molecular , Naftoquinonas/química , Naftoquinonas/farmacología , Fosfolipasas A2 Citosólicas/antagonistas & inhibidores , Agregación Plaquetaria/efectos de los fármacos , Inhibidores de Agregación Plaquetaria/química , Inhibidores de Agregación Plaquetaria/farmacología , Relación Estructura-ActividadRESUMEN
Thromboxane A2 (TXA2) activation of TP receptor has been shown contributing to the progression and acute complications of atherosclerosis including endothelial dysfunction, platelet hyperactivity and inflammation. Growing evidence suggests that TP receptor may represent as a therapeutic target in atherosclerosis and related cardiovascular diseases. We investigated whether nstpbp5185, an orally active TP receptor antagonist, exhibits protective effects against atherosclerotic progression. Nstpbp5185 and aspirin were orally administered daily for 12 weeks in high-cholesterol-fed ApoE-deficient mice to examine their anti-atherosclerosis effects. Total cholesterol, low-density lipoprotein cholesterol and triglycerides were slightly decreased in nstpbp5185-treated mice. However, nstpbp5185 significantly reduced neointima formation and aortic atherosclerotic lesion area. Nstpbp5185 increased serum paraoxonase 1 activity. In contrast, plasma levels of interleukin-6 and tumour necrosis factor-α were reduced in nstpbp5185-treated mice. Plasma level of TXA2 metabolite, TXB2, was lower in both aspirin- and nstpbp5185-treated mice, while the urinary 2,3-dinor-6-keto PGF1α (a PGI2 metabolite) and plasma iPF2α-III were not altered. Moreover, nstpbp5185 neither caused gastric ulceration nor affected the haemostatic response. Nstpbp5185 also inhibited U46619-induced endothelial NF-kB activation, ICAM-1 and VCAM-1 expression, as well as monocyte adhesion to endothelial cells. In conclusion, nstpbp5185 may represent as an ideal, safe and efficacious agent for preventing atherosclerotic progression through its antiplatelet, anti-inflammatory and antioxidative activities.
Asunto(s)
Aterosclerosis/tratamiento farmacológico , Bencimidazoles/farmacología , Receptores de Tromboxano A2 y Prostaglandina H2/antagonistas & inhibidores , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacología , Animales , Aorta/metabolismo , Aterosclerosis/genética , Adhesión Celular , Supervivencia Celular , Células Endoteliales de la Vena Umbilical Humana , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados para ApoE , Especies Reactivas de Oxígeno/metabolismoRESUMEN
We previously reported that the fibrinogen-binding segment (residues 221-550) of Staphylococcus aureus clumping factor A (ClfA), which binds to fibrinogen gamma chain C-terminus, exerted inhibitory effects on platelet aggregation and fibrin clot formation in vitro. Here, we further demonstrated the effectiveness of using ClfA221-550 to inhibit platelet-rich thrombus formation in vivo. Platelet-rich thrombi were formed in the mesenteric venules of fluorescein-loaded mice by filtered light illumination. It grew rapidly and ultimately resulted in the cessation of blood flow due to vessel occlusion. Given by intravenous bolus injection, ClfA221-550 delayed occlusive thrombi formation in a dose-dependent manner: 2-, 3- and 4.5-fold prolongations of vessel occlusion time were attained with 0.69, 6.9 and 34.5 mg/kg of ClfA221-550, respectively. Reduced fibrin clot formation at the late phase with plasmas, which were prepared from ClfA221-550-treated mice, was also dose-dependent. The suppression of fibrin formation ex vivo coincided with the delay of occlusive thrombus formation in vivo, suggesting that the antithrombotic effect of ClfA221-550 may result from the blockade of fibrinogen gamma chain C-terminal functions, in mediating platelet aggregation and fibrin clot formation. Administration of ClfA221-550 also lengthened the tail bleeding of mice; however, significant effect was achieved only with a higher dosage, namely 34.5 mg/kg. These results together showed that blockade of fibrinogen gamma chain C-terminus with ClfA221-550 preferentially affected platelet-rich thrombus formation rather than normal haemostasis, thus providing a rationale for selecting fibrinogen gamma chain C-terminus as a new target for thrombotic intervention.
Asunto(s)
Plaquetas/efectos de los fármacos , Coagulantes/farmacología , Coagulasa/farmacología , Fibrinógeno/metabolismo , Agregación Plaquetaria/efectos de los fármacos , Staphylococcus aureus/fisiología , Animales , Plaquetas/fisiología , Relación Dosis-Respuesta a Droga , Fibrinógeno/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos ICR , Agregación Plaquetaria/fisiología , Unión Proteica , Trombosis/metabolismoRESUMEN
Trimucrin, a novel small-mass Arg-Gly-Asp (RGD)-containing disintegrin, has been demonstrated to possess anti-platelet and anti-inflammatory effect through blockade of platelet αIIbß3 and phagocyte αvß3 integrin. In this study, we found that the platelet-rich plasma prepared from trimucrin-treated rats platelet aggregation was diminished in response to adenosine diphosphate (ADP). We tried to determine whether trimucrin is cardioprotective in rats subjected to myocardial ischemia-reperfusion (I-R) injury. The left anterior descending coronary artery of anesthetized rats was subjected to 1h occlusion and 3h reperfusion. The animals received intravenous trimucrin or saline, and the severities of I-R-induced arrhythmia and infarction were compared. Trimucrin significantly reduced I-R-induced arrhythmias and reduced mortality, as well as infarct volume, troponin-I levels, creatine kinase, and lactate dehydrogenase activity in carotid blood compared with vehicle-treated animals during the same period. Trimucrin also improved cardiac function and survival rates after I-R injury. In addition, trimucrin concentration-dependently inhibited platelet adhesion on collagen- and fibrinogen-coated surfaces without affecting platelet counts. Trimucrin also significantly reduced neutrophil infiltration into heart tissues after I-R compared with controls. Furthermore, trimucrin treatment caused significant downregulation of Bax, Caspase-3 apoptotic proteins and upregulation of anti-apoptotic Bcl-2 protein. These results demonstrate that trimucrin exerts cardioprotective property against myocardial I-R injury mediated through antiplatele, anti-inflammatory, anti-apoptotic mechanism, as well as improvements in cardiac function.
Asunto(s)
Plaquetas/efectos de los fármacos , Plaquetas/fisiología , Desintegrinas/química , Daño por Reperfusión Miocárdica/tratamiento farmacológico , Daño por Reperfusión Miocárdica/fisiopatología , Oligopéptidos , Péptidos/farmacología , Animales , Apoptosis/efectos de los fármacos , Cardiotónicos/química , Cardiotónicos/farmacología , Cardiotónicos/uso terapéutico , Hemodinámica/efectos de los fármacos , Masculino , Ratones , Daño por Reperfusión Miocárdica/patología , Miocardio/patología , Péptidos/química , Péptidos/uso terapéutico , Adhesividad Plaquetaria/efectos de los fármacos , Ratas , Ratas Sprague-DawleyRESUMEN
VEGF and VEGFR antibodies have been used as a therapeutic strategy to inhibit angiogenesis in many diseases; however, frequent and repeated administration of these antibodies to patients induces immunogenicity. In previous studies, we demonstrated that aggretin, a heterodimeric snake venom C-type lectin, exhibits pro-angiogenic activities via integrin α2ß1 ligation. We hypothesised that small-mass aggretin fragments may bind integrin α2ß1 and act as antagonists of angiogenesis. In this study, the anti-angiogenic efficacy of a synthesised aggretin α-chain C-terminus (AACT, residue 106-136) was evaluated in both in vitro and in vivo angiogenesis models. The AACT demonstrated inhibitory effects on collagen-induced platelet aggregation and HUVEC adhesion to immobilised collagen. These results indicated that AACT may block integrin α2ß1-collagen interaction. AACT also inhibited HUVEC migration and tube formation. Aortic ring sprouting and Matrigel implant models demonstrated that AACT markedly inhibited VEGF-induced neovascularisation. In addition, induction of FAK/PI3K/ERK1/2 tyrosine phosphorylation and talin 1/2 associated with integrin ß1 which are induced by VEGF were blocked by AACT. Similarly, tyrosine phosphorylation of VEFGR2 and ERK1/2 induced by VEGF was diminished in integrin α2-silenced endothelial cells. Our results demonstrate that AACT is a potential therapeutic candidate for angiogenesis related-diseases via integrin α2ß1 blockade.
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Integrina alfa2beta1/antagonistas & inhibidores , Péptidos/farmacología , Venenos de Serpiente/farmacología , Inhibidores de la Angiogénesis/química , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Colágeno/metabolismo , Combinación de Medicamentos , Quinasa 1 de Adhesión Focal/metabolismo , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Laminina , Lectinas Tipo C , Neovascularización Fisiológica/efectos de los fármacos , Péptidos/química , Fosfatidilinositol 3-Quinasas/metabolismo , Agregación Plaquetaria/efectos de los fármacos , Unión Proteica , Proteoglicanos , Transducción de Señal/efectos de los fármacos , Venenos de Serpiente/química , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
As angiogenesis is required for tumor growth and metastasis, suppressing angiogenesis is a promising strategy in limiting tumor progression. Vascular endothelial growth factor (VEGF)-A, a critical pro-angiogenic factor, has thus become an attractive target for therapeutic interventions in cancer. In this study, we explored the underlying mechanisms of a novel anthraquinone derivative DDA in suppressing angiogenesis. DDA inhibited VEGF-A-induced proliferation, migration and tube formation of human umbilical vein endothelial cells (HUVECs). DDA also reduced VEGF-A-induced microvessel sprouting from aortic rings ex vivo and suppressed neovascularization in vivo. VEGF-A-induced VEGFR1, VEGFR2, FAK, Akt, ERK1/2 or STAT3 phosphorylation was reduced in the presence of DDA. In addition, NRP-1 siRNA reduced VEGF-A's enhancing effects in VEGFR2, FAK and Akt phosphorylation and cell proliferation in HUVECs. DDA disrupted VEGF-A-induced complex formation between NRP-1 and VEGFR2. Furthermore, systemic administration of DDA was shown to suppress tumor angiogenesis and growth in in vivo mouse xenograft models. Taken together, we demonstrated in this study that DDA exhibits anti-angiogenic properties through suppressing VEGF-A signaling. These observations also suggest that DDA might be a potential drug candidate for developing anti-angiogenic agent in the field of cancer and angiogenesis-related diseases.