RESUMO
Melanoma is a highly metastatic and rapidly progressing cancer, a leading cause of mortality among skin cancers. The melanoma microenvironment, formed from the activity of malignant cells on the extracellular matrix and the recruitment of immune cells, plays an active role in the development of drug resistance and tumor recurrence, which are clinical challenges in cancer treatment. These tumoral metabolic processes are affected by proteins, including Galectin-3 (Gal-3), which is extensively involved in cancer development. Previously, we characterized a partially methylated mannogalactan (MG-Pe) with antimelanoma activities. In vivo models of melanoma were used to observe MG-Pe effects in survival, spontaneous, and experimental metastases and in tissue oxidative stress. Analytical assays for the molecular interaction of MG-Pe and Gal-3 were performed using a quartz crystal microbalance, atomic force microscopy, and contact angle tensiometer. MG-Pe exhibits an additive effect when administered together with the chemotherapeutic agent dacarbazine, leading to increased survival of treated mice, metastases reduction, and the modulation of oxidative stress. MG-Pe binds to galectin-3. Furthermore, MG-Pe antitumor effects were substantially reduced in Gal-3/KO mice. Our results showed that the novel Gal-3 ligand, MG-Pe, has both antitumor and antimetastatic effects, alone or in combination with chemotherapy.
Assuntos
Antineoplásicos , Galectina 3 , Melanoma , Neoplasias Cutâneas , Animais , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Dacarbazina/metabolismo , Dacarbazina/farmacologia , Dacarbazina/uso terapêutico , Galectina 3/metabolismo , Galectina 3/farmacologia , Galectina 3/uso terapêutico , Ligantes , Melanoma/tratamento farmacológico , Melanoma/metabolismo , Camundongos , Recidiva Local de Neoplasia , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/metabolismo , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/fisiologiaRESUMO
Advanced melanoma patients that are not included in common genetic classificatory groups lack effective and safe therapeutic options. Chemotherapy and immunotherapy show unsatisfactory results and devastating adverse effects for these called triple wild-type patients. New approaches exploring the intrinsic antitumor properties of gold nanoparticles might reverse this scenario as a safer and more effective alternative. Therefore, we investigated the efficacy and safety of a composite made of gum arabic-functionalized gold nanorods (GA-AuNRs) against triple wild-type melanoma. The natural polymer gum arabic successfully stabilized the nanorods in the biological environment and was essential to improve their biocompatibility. In vivo results obtained from treating triple wild-type melanoma-bearing mice showed that GA-AuNRs remarkably reduced primary tumor growth by 45%. Furthermore, GA-AuNRs induced tumor histological features associated with better prognosis while also reducing superficial lung metastasis depth and the incidence of intrapulmonary metastasis. GA-AuNRs' efficacy comes from their capacity to reduce melanoma cells ability to invade the extracellular matrix and grow into colonies, in addition to a likely immunomodulatory effect induced by gum arabic. Additionally, a broad safety investigation found no evidence of adverse effects after GA-AuNRs treatment. Therefore, this study unprecedentedly reports GA-AuNRs as a potential nanomedicine for advanced triple wild-type melanomas.
Assuntos
Ouro/administração & dosagem , Goma Arábica/química , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/secundário , Melanoma/tratamento farmacológico , Animais , Células 3T3 BALB , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Ouro/química , Ouro/farmacologia , Humanos , Neoplasias Pulmonares/metabolismo , Melanoma/metabolismo , Nanopartículas Metálicas , Camundongos , Resultado do Tratamento , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The increase in the number of deaths from infections caused by multidrug-resistant bacteria and cancer diseases highlights the need for new molecules with biological activity. Actinobacteria represent a potential source of new compounds, as these microorganisms have already produced a great diversity of clinically employed antibiotics. Endophytes from unexplored biomes, such as the Pantanal (the largest wetland in the world), can be a source of new molecules. Hymenachne amplexicaulis is among the unexplored native plants of the Pantanal in terms of its endophytic community. This plant is considered a weed in other countries due to its ability to adapt and compete with native plants, and there is evidence to suggest that the endophytic community of H. amplexicaulis plays an important role in this competitiveness. To explore its therapeutic potential, the present study isolated, identified (using partial sequence of the 16S rDNA) and bioprospected H. amplexicaulis endophytic actinobacteria. Ten isolates belonging to the genera Streptomyces, Microbispora, Leifsonia, and Verrucosispora were obtained from root fragments. The susceptibility profile of the isolates to the different classes of antibiotics was evaluated, with 80 % of the isolates showing resistance to the antibiotics Nalidixic Acid, Ampicillin, Chloramphenicol, Oxacillin, and Rifampicin. To assess antibacterial and antitumor activities, methanolic extracts were obtained by fermentation in SG culture medium at 36 °C at 180 rpm for 10 days. The extract produced from the S. albidoflavus CMRP4854 isolate was the only one to show activity against the Gram-negative bacterium Acinetobacter baumanii. Due to the great clinical importance of this pathogen and the difficulty in obtaining active compounds against it, the CMRP4854 isolate should be further investigated for the identification of active compounds and mode of action. We also emphasize the results obtained by the extract of the isolates Streptomyces albidoflavus CMRP4852 and Verrucosispora sp. CMRP4860 that presented antibacterial effect against Methicilin-resistant Staphylococcus aureus (MRSA) (MIC: 1.5 µg/mL and 13 µg/mL, respectively) and Vancomycin-resistant Enterococcus (VRE) (MIC: 40 µg/mL for both extracts). Extracts (200 µg/mL) of these two endophytes also showed selective cytotoxicity action against murine B16-F10 melanoma cells. However, the CMRP4852 extract also affected the density of normal cells. Due to these results, the crude extract of isolate CMRP4860 Verrucosispora sp., which was the only one that presented cytotoxicity and reduced cell density only in tumor cells, was selected for subsequent analysis involving scale-up fermentation of the CMRP4860 resulting in 9 fractions that were tested against both bacteria and tumor cells, with particular fractions showing promise and meriting further investigation. Taken together, the results of this study not only show for the first time that the endophytic community of H. amplexicaulis actinobacteria can produce secondary metabolites that potentially possess important antibacterial and cytotoxic properties, but also reinforce the pressing need to conserve biomes such as the Brazilian Pantanal.
Assuntos
Actinobacteria/química , Antibacterianos/farmacologia , Antineoplásicos/farmacologia , Endófitos/química , Poaceae/microbiologia , Actinobacteria/classificação , Actinobacteria/isolamento & purificação , Actinobacteria/metabolismo , Animais , Antibacterianos/química , Antibacterianos/metabolismo , Antineoplásicos/química , Antineoplásicos/metabolismo , Brasil , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Endófitos/classificação , Endófitos/isolamento & purificação , Endófitos/metabolismo , Enterococcus/efeitos dos fármacos , Enterococcus/crescimento & desenvolvimento , Humanos , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/crescimento & desenvolvimento , Camundongos , Áreas AlagadasRESUMO
Natural killer (NK) cells are innate lymphocytes responsible for the elimination of infected or transformed cells. The activation or inhibition of NK cells is determined by the balance of target cell ligand recognition by stimulatory and inhibitory receptors on their surface. Previous reports have suggested that the glycosaminoglycan heparin is a ligand for the natural cytotoxicity receptors NKp30, NKp44 (human), and NKp46 (both human and mouse). However, the effects of heparin on NK cell homeostasis and function remain unclear. Here, we show that heparin does not enhance NK cell proliferation or killing through NK cell activation. Alternatively, in mice models, heparin promoted NK cell survival in vitro and controlled B16-F10 melanoma metastasis development in vivo. In human NK cells, heparin promisingly increased interferon (IFN)-γ production in synergy with IL-12, although the mechanism remains elusive. Our data showed that heparin is not able to increase NK cell cytotoxicity.
RESUMO
The tumor microenvironment (TME) is composed of multiple infiltrating host cells (e.g., endothelial cells, fibroblasts, lymphocytes, and myeloid cells), extracellular matrix, and various secreted or cell membrane-presented molecules. Group 1 innate lymphoid cells (ILCs), which includes natural killer (NK) cells and ILC1, contribute to protecting the host against cancer and infection. Both subsets are able to quickly produce cytokines such as interferon gamma (IFN-γ), chemokines, and other growth factors in response to activating signals. However, the TME provides many molecules that can prevent the potential effector function of these cells, thereby protecting the tumor. For example, TME-derived tumor growth factor (TGF)-ß and associated members of the superfamily downregulate NK cell cytotoxicity, cytokine secretion, metabolism, proliferation, and induce effector NK cells to upregulate ILC1-like characteristics. In concert, a family of carbohydrate-binding proteins called galectins, which can be produced by different cells composing the TME, can downregulate NK cell function. Matrix metalloproteinase (MMP) and a disintegrin and metalloproteinase (ADAM) are also enzymes that can remodel the extracellular matrix and shred receptors from the tumor cell surface, impairing the activation of NK cells and leading to less effective effector functions. Gaining a better understanding of the characteristics of the TME and its associated factors, such as infiltrating cells and extracellular matrix, could lead to tailoring of new personalized immunotherapy approaches. This review provides an overview of our current knowledge on the impact of the TME and extracellular matrix-associated components on differentiation, impairment, and function of NK cells.
Assuntos
Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Microambiente Tumoral/imunologia , Diferenciação Celular , Quimiocinas/metabolismo , Citocinas/metabolismo , Células Endoteliais/metabolismo , Matriz Extracelular/metabolismo , Galectinas , Glicosaminoglicanos , Humanos , Imunoterapia , Interferon gama/metabolismo , ProteoglicanasRESUMO
Metastasis is responsible for the majority of deaths among patients with malignant melanoma. Despite recent advances, the majority of current and modern therapies are ineffective and/or financially unfeasible. Thus, in this study, we investigated two lowcost highlydiluted natural complexes (HDNCs) that have been shown to be effective against malignant melanoma in a murine model in vivo. The aim of this study was to determine the mechanisms through which these HDNCs directly affect melanoma cells, either alone or in an artificial tumor microenvironment, suppressing the metastatic phenotype, thus explaining previous in vivo effects. For this purpose, HDNC in vitro treatments of B16F10 melanoma cells, alone or in coculture with Balb/3T3 fibroblasts, were carried out. Molecular biology techniques and standard functional assays were used to assess the changes in molecule expression and in cell behaviors related to the metastatic phenotype. Melanoma progression features were found to be regulated by HDNCs. Molecules related to cell adhesion (Ncadherin, ß1integrin and CD44), and migration, extracellular matrix remodeling and angiogenesis were modulated. The cell migratory, invasive and clonogenic capacities were reduced by the HDNCs. No loss of cell proliferation or viability were observed. On the whole, the findings of this study indicate that HDNCs directly reprogram, molecularly and functionally, melanoma cells in vitro, modulating their metastatic phenotype. Such findings are likely to be responsible for the attenuation of tumor growth and lung colonization previously observed in vivo.
Assuntos
Antígenos CD/metabolismo , Caderinas/metabolismo , Receptores de Hialuronatos/metabolismo , Integrina beta1/metabolismo , Melanoma/metabolismo , Extratos Vegetais/farmacologia , Neoplasias Cutâneas/metabolismo , Animais , Células 3T3 BALB , Adesão Celular , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Ensaios de Seleção de Medicamentos Antitumorais , Regulação Neoplásica da Expressão Gênica , Humanos , Melanoma/tratamento farmacológico , Camundongos , Metástase Neoplásica , Extratos Vegetais/química , Plantas/química , Neoplasias Cutâneas/tratamento farmacológico , Melanoma Maligno CutâneoRESUMO
World fisheries and aquaculture production totaled 167â¯millionâ¯tons in 2014. This high fish production generates a lot of waste that could be used as raw material for extraction of substances of pharmacological interest. In this work, we extract and characterize glycosaminoglycans (GAGs) present in the viscera of Nile tilapia (Oreochromis niloticus) and Pacu (Piaractus mesopotamicus), which are among the most vastly produced fishes in inland aquaculture in Brazil. Moreover, the anticoagulant activity of the GAGs fractions was evaluated. GAGs were obtained from total defatted viscera, after proteolysis, precipitation with ethanol, anion exchange chromatography and treatment with chondroitinase. Chondroitin sulfate (CS), dermatan sulfate (DS) and heparan sulfate (HS) were identified by agarose gel electrophoresis and NMR analyses. CS, DS and HS were identified in equivalent fractions obtained from both fishes, and all GAGs fractions showed anticoagulant activity.
Assuntos
Anticoagulantes/isolamento & purificação , Anticoagulantes/farmacologia , Caraciformes/anatomia & histologia , Ciclídeos/anatomia & histologia , Glicosaminoglicanos/isolamento & purificação , Glicosaminoglicanos/farmacologia , Vísceras/química , AnimaisRESUMO
The fungal genus Fonsecaea comprises etiological agents of human chromoblastomycosis, a chronic implantation skin disease. The current hypothesis is that patients acquire the infection through an injury from plant material. The present study aimed to evaluate a model of infection in plant and animal hosts to understand the parameters of trans-kingdom pathogenicity. Clinical strains of causative agents of chromoblastomycosis (Fonsecaea pedrosoi and Fonsecaea monophora) were compared with a strain of Fonsecaea erecta isolated from a living plant. The clinical strains of F. monophora and F. pedrosoi remained concentrated near the epidermis, whereas F. erecta colonized deeper plant tissues, resembling an endophytic behavior. In an invertebrate infection model with larvae of a beetle, Tenebrio molitor, F. erecta exhibited the lowest survival rates. However, F. pedrosoi produced dark, spherical to ovoidal cells that resembled muriform cells, the invasive form of human chromoblastomycosis confirming the role of muriform cells as a pathogenic adaptation in animal tissues. An immunologic assay in BALB/c mice demonstrated the high virulence of saprobic species in animal models was subsequently controlled via host higher immune response.
RESUMO
An aqueous extract containing polysaccharides was obtained from the giant mushroom Macrocybe titans, and it was purified by amylase treatment, freeze-thawing process and dialysis. The purified fraction (ESP) was analyzed by HPSEC and GC-MS which showed a homogenous polysaccharide with Mw 14.2â¯×â¯103â¯g/mol composed by galactose and fucose. NMR and methylation analysis of ESP confirmed the presence of a fucogalactan with a (1â¯ââ¯6)-linked α-d-Galp main chain partially substituted at O-2 by non reducing end units of α-l-Fucp residues in the side chain. Its biological activity was evaluated against murine melanoma cells B16-F10. The fucogalactan did not alter the viability, proliferative capacity and morphology of cells. However, this polysaccharide was able to reduce the cell migration in vitro at 40% (100⯵g/mL) and 33% (250⯵g/mL). The results obtained showed that Macrocybe titans fucogalactan is a promising agent capable of altering melanoma cell migration without decrease the cell viability.
Assuntos
Agaricales/química , Movimento Celular/efeitos dos fármacos , Galactanos/farmacologia , Melanoma Experimental/patologia , Melanoma/patologia , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Galactanos/químicaRESUMO
Hemoplasmas were detected in two apparently healthy captive South American coatis (Nasua nasua) from southern Brazil during an investigation for vector-borne pathogens. Blood was subjected to packed cell volume (PCV) determination, a commercial real-time PCR panel for the detection of Anaplasma spp., Babesia spp., Bartonella spp., Hepatozoon spp., Leishmania spp., Mycoplasma haemofelis, 'Candidatus Mycoplasma turicensis', 'Candidatus Mycoplasma haemominutum', Neorickettsia risticii, Rickettsia rickettsii and Leptospira spp., and a pan-hemoplasma conventional PCR assay. PCV was normal, but both coatis tested positive for hemoplasmas and negative for all the remaining pathogens tested. Using different techniques for microscopy (light, confocal or SEM), structures compatible with hemoplasmas were identified. Sequencing of the 16S rRNA gene identified an organism resembling Mycoplasma haemofelis and another hemotropic Mycoplasma sp., with a sequence identity of 96.8% to a Mycoplasma sp. previously detected in capybaras.
Assuntos
Infecções por Mycoplasma/veterinária , Mycoplasma/isolamento & purificação , Procyonidae/microbiologia , Animais , Animais Domésticos , Animais Selvagens/microbiologia , Brasil/epidemiologia , DNA Bacteriano/genética , Feminino , Masculino , Microscopia , Microscopia Confocal , Microscopia Eletrônica de Varredura/métodos , Mycoplasma/genética , Mycoplasma/ultraestrutura , Infecções por Mycoplasma/epidemiologia , Infecções por Mycoplasma/microbiologia , Infecções por Mycoplasma/transmissão , RNA Ribossômico 16S/genética , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de DNARESUMO
The non-steroidal anti-inflammatory drugs are emerging contaminants in aquatic ecosystems. This study aimed to evaluate toxic effects of some representative drugs of this pharmaceutical group on primary culture of monocytic lineage of Hoplias malabaricus anterior kidney. The effects of diclofenac, acetaminophen and ibuprofen in cell viability, lipopolysaccharide (LPS)-induced NO production and genotoxicity were evaluated. Cytometry analysis CD11b(+) cells showed 71.5% of stem cells, 19.5% of macrophages and 9% of monocytes. Cell viability was lower in the ficoll compared to percoll separation. LPS-induced NO production by these cells was blocked after treatment with dexamethasone and NG-Methyl-L-Arginine (L-NMMA). Exposure of the cells to diclofenac (0.2-200 ng/mL), acetaminophen (0.025-250 ng/mL) ibuprofen (10-1000 ng/mL) reduced basal NO production and inhibited LPS-induced NO production at all concentrations after 24 h of exposure. Genotoxicity occurred at the highest concentration of diclofenac and at the intermediary concentration of acetaminophen. Genotoxicity was also observed by ibuprofen. In summary, the pharmaceuticals influenced NO production and caused DNA damage in monocytic cells suggesting that these drugs can induce immunosuppression and genotoxicity in fish.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Characidae/metabolismo , Animais , Técnicas de Cultura de Células , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Diclofenaco/farmacologia , Ibuprofeno/farmacologia , Lipopolissacarídeos/farmacologia , Testes de Mutagenicidade , Óxido Nítrico/metabolismoRESUMO
Galactofuranoside derivatives were synthesised by the classic Fischer glycosydation method, and their immune modulation properties were studied in vitro and in vivo. NMR spectroscopic and ESI-MS analyses confirmed the purity and authenticity of all derivatives. Their phagocyte capacities were tested in resident macrophages. Methyl ß-galactofuranoside (GFB-Me) and n-octyl ß-galactofuranoside (GFB-O) had an immune stimulant effect at 25µmolml(-1) with an enhancement of 35.12%±0.06 SD and 17.49%±0.11 SD, respectively, but Methyl α-galactofuranoside (GFA-Me) and n-octyl α-galactofuranoside (GFA-O) gave a low immune response. Methyl α-galactofuranoside 5,6-O-isopropylidene (GFA-IP) and Methyl ß-galactofuranoside 5,6-O-isopropylidene (GFB-IP) had negative values relative to the control group of minus 4.96%±0.10 SD and -40.72%±0.07 SD, respectively. Furthermore, GFB-Me and GFB-Me-IP were evaluated in vivo on the lethality induced by cecal ligation and puncture. Cytokine levels and iNOS expression were determined and correlated to mortality data. The results showed that the free HO-5 and HO-6 and the ß-configuration are essential for the induction of phagocytic activity by the galactofuranosyl units. The methyl ß-galactofuranosides also enhanced lethality during sepsis, increasing the levels of pro-inflammatory cytokines and iNOS expression.
Assuntos
Glicosídeos/farmacologia , Fatores Imunológicos/farmacologia , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/imunologia , Sepse/imunologia , Animais , Citocinas/biossíntese , Glicosídeos/química , Glicosídeos/toxicidade , Peróxido de Hidrogênio/metabolismo , Íleo/efeitos dos fármacos , Íleo/imunologia , Íleo/metabolismo , Fatores Imunológicos/química , Fatores Imunológicos/toxicidade , Espectroscopia de Ressonância Magnética , Masculino , Camundongos , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase Tipo II/metabolismo , Fagocitose/efeitos dos fármacos , Sepse/etiologia , Sepse/metabolismo , Superóxidos/metabolismoRESUMO
In recent years, the antitumoral activity of antimicrobial peptides (AMPs) has been the goal of many research studies. Among AMPs, gomesin (Gm) displays antitumor activity by unknown mechanisms. Herein, we studied the cytotoxicity of Gm in the Chinese hamster ovary (CHO) cell line. Furthermore, we investigated the temporal ordering of organelle changes and the dynamics of Ca(2+) signaling during Gm-induced cell death. The results indicated that Gm binds to the plasma membrane and rapidly translocates into the cytoplasm. Moreover, 20 µM Gm increases the cytosolic Ca(2+) and induces membrane permeabilization after 30 min of treatment. Direct Ca(2+) measurements in CHO cells transfected with the genetically encoded D1-cameleon to the endoplasmic reticulum (ER) revealed that Gm induces ER Ca(2+) depletion, which in turn resulted in oscillatory mitochondrial Ca(2+) signal, as measured in cells expressing the genetically encoded probe to the mitochondrial matrix (mit)Pericam. This leads to mitochondria disruption, loss of mitochondrial membrane potential and increased reactive oxygen species prior to membrane permeabilization. Gm-induced membrane permeabilization by a Ca(2+)-dependent pathway involving Gm translocation into the cell, ER Ca(2+) depletion and disruption, mitochondrial Ca(2+) overload and oxidative stress.
Assuntos
Peptídeos Catiônicos Antimicrobianos/farmacologia , Cálcio/metabolismo , Morte Celular/efeitos dos fármacos , Permeabilidade da Membrana Celular/efeitos dos fármacos , Animais , Células CHO , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Cricetinae , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Heparin and its derivatives are known to regulate a variety of pathophysiological events related to vascular biology. In the present manuscript we examine a variety of heparinomimetics biochemically (electrophoretic behavior and enzymatic degradation) and pharmacologically (in vitro anticoagulant activity and in vivo hemorrhagic and antithrombotic tests) as well as their interactions with cells from the vessel wall using a time resolved fluorometric method and confocal microscopy. Data were determined for unfractionated heparin (UFH), enoxaparin, synthetic heparin pentasaccharide, C3 heparin derived oligosaccharides and phosphosulfomannan (PI-88). While being structurally distinct from UFH, all compounds exhibited anticoagulant, antithrombotic and hemorrhagic activities. In addition, besides the pentasaccharide, they all stimulated the synthesis of an antithrombotic heparan sulfate present at the cell surface and secreted by endothelial cells. Also, like UFH, they interacted with both endothelial and smooth muscle cells and dislodged UFH from its binding sites in a dose dependent manner but, with distinct saturable curves showing that the binding of polymeric structures to extracellular matrix (ECM) proteins does not depend on a glycosaminoglycan backbone. The data also suggest a common pathway, which does not depend on the presence of the conventionally accepted antithrombin binding pentasaccharide, for ECM dependent activity of the heparinomimetic stimulated synthesis of antithrombotic heparan sulfate. Notably, although of similar molecular weight as well as polymeric backbone, the synthetic heparin pentasaccharide showed significant hemorrhagic action and negligible antithrombotic activity in a venous thrombosis model, contrasting with C3, that displayed negligible hemorrhagic effect and potent antithrombotic action. These results provide evidence that structurally unrelated polymers can elicit similar hemostatic activities and show that polymeric sequence is not always crucial for certain activities. The results also suggest that non-GAG structures may provide an alternative route for the pharmaceutical control of hemostasis.
Assuntos
Matriz Extracelular/efeitos dos fármacos , Hemostasia , Heparina/análogos & derivados , Heparina/farmacologia , Animais , Anticoagulantes/farmacologia , Sítios de Ligação , Relação Dose-Resposta a Droga , Células Endoteliais/química , Células Endoteliais/efeitos dos fármacos , Matriz Extracelular/química , Fibrinolíticos/farmacologia , Heparina Liase/química , Peso Molecular , Miócitos de Músculo Liso/química , Miócitos de Músculo Liso/efeitos dos fármacos , Oligossacarídeos/farmacologia , Ligação Proteica , Proteólise , Coelhos , Ratos , Especificidade por SubstratoRESUMO
Brown spider (Loxosceles sp.) venom affects the endothelium of vessels and triggers disruptive activity in the subendothelial matrix. The vascular disorders observed after venom exposure include leukocyte and platelet activation, disseminated intravascular coagulation, an increase in vessel permeability and hemorrhage into the dermis. In this study, we report additional evidence regarding the mechanism of endothelial cell cytotoxicity induced by Loxosceles intermedia venom. Exposure to venom led to endothelial cell detachment in a time-dependent manner. Loss of cell anchorage and cell-cell adhesion following venom exposure was accompanied by changes in the distribution of the α5ß1 integrin and VE-cadherin. An ultrastructural analysis of cells treated with venom revealed morphological alterations characteristic of apoptosis. Moreover, after venom exposure, the ratio between Bax and Bcl-2 proteins was disturbed in favor of Bax. In addition, late apoptosis was only observed in cells detached by the action of venom. Accordingly, there was no increase in apoptosis when cells were exposed to L. intermedia venom in suspension, suggesting that the loss of cell anchorage provides the signal to initiate apoptosis. Thus, L. intermedia venom likely triggers endothelial cell death indirectly through an apoptotic mechanism known as anoikis.
Assuntos
Anoikis/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Venenos de Aranha/farmacologia , Aranhas/metabolismo , Animais , Antígenos CD/metabolismo , Aorta Torácica/citologia , Aorta Torácica/efeitos dos fármacos , Aorta Torácica/metabolismo , Aorta Torácica/ultraestrutura , Proteínas Reguladoras de Apoptose/metabolismo , Brasil , Caderinas/metabolismo , Adesão Celular/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Células Cultivadas , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Endotélio Vascular/ultraestrutura , Integrina alfa5beta1/metabolismo , Cinética , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , CoelhosRESUMO
Endothelial cells (ECs) are a source of physiologically important molecules that are synthesized and released to the blood and/or to the subendothelial extracellular matrix such as a heparan sulfate proteoglycan (HSPG) with antithrombotic properties. Previously, we have shown that heparin stimulates the synthesis and modifies the sulfation pattern of this HSPG. Here the molecular mechanisms involved in the up-regulation of HSPG synthesis by heparin in endothelial cells were decoded. The cells were stimulated with heparin and the expression of HSPG and intracellular pathways were evaluated by a combination of methods involving confocal microscopy, flow cytometry, Western blotting analyses, and [(35) S]-sulfate metabolically labeling of the cells. We observed that the up-regulation of HSPG synthesis evoked by heparin is dependent on the interaction of heparin with integrin since RGD peptide abolishes the effect. The activation of integrin leads to tyrosine-phosphorylation of focal adhesion-associated proteins such as FAK, Src, and paxillin. In addition, heparin induces ERK1/2 phosphorylation and inhibitors of Ras and MEK decreased heparin-dependent HSPG synthesis. Moreover, heparin also induced intracellular Ca(2+) release, PLCγ1 (phospholipase Cγ1) and CaMKII (calcium calmodulin kinase II) activation, as well as an increase in nitric oxide (NO) production. Finally, an intracellular Ca(2+) chelator, Ca(2+) signaling inhibitors, and an endothelial NO synthase inhibitor were all able to abolish the effect in heparan sulfate synthesis. In conclusion, the heparin-induced up-regulation of HSPG expression is associated with the phosphorylation of focal adhesion proteins and Ras/Raf/MEK/ERK MAP and Ca(2+) /NO pathways.
Assuntos
Células Endoteliais/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Heparina/metabolismo , Heparitina Sulfato/metabolismo , Integrinas/metabolismo , Animais , Anticoagulantes/metabolismo , Anticoagulantes/farmacologia , Western Blotting , Cálcio/metabolismo , Sinalização do Cálcio , Adesão Celular , Fibronectinas/química , Fibronectinas/metabolismo , Citometria de Fluxo , Heparina/farmacologia , Heparitina Sulfato/genética , Microscopia Confocal , Óxido Nítrico , Fosforilação , Ligação Proteica , Coelhos , Regulação para CimaRESUMO
Previous reports have shown that heparin may promote human hypotension and vascular relaxation by elevation of NO levels through unclear mechanisms. We hypothesized that endothelial muscarinic M(3) receptor activation mediates the heparin-induced vasodilation of rat aortic rings. The experiments were carried out using unfractionated heparin extracted from bovine intestinal mucosa, which elicited an endothelium and NO-dependent relaxation of aortic segments with maximal potency and efficacy (EC(50): 100±10 µmol/L; E(max): 41±3%). Atropine and 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide inhibitors reduced the heparin-dependent relaxation, indicating that M(3) muscarinic receptor is involved in this phenomenon. However, no direct binding of heparin to muscarinic receptors was observed. More importantly, studies performed using the arginine-glycine-aspartic acid peptide and 1-(1,1-dimethylethyl)-3-(1-naphthalenyl)-1H-pyrazolo[3,4-day]pyrimidin-4-amine, an Src family inhibitor, reduced by 51% and 73% the heparin-dependent relaxation, respectively, suggesting the coupling of heparin and M(3) receptor through extracellular matrix molecules and integrin. Furthermore, unfractionated heparin induced activation of focal adhesion protein kinase, Src, and paxillin. Finally, fluorescence resonance energy transfer approach confirmed the interaction of the M(3) receptor to integrin. Taken together, these data demonstrate the participation of M(3) receptor and integrin in heparin-dependent relaxation of vascular smooth muscle. These results provide new insights into the molecular mechanism and potential pharmacological action of heparin in vascular physiology.
Assuntos
Aorta Torácica/efeitos dos fármacos , Heparina/farmacologia , Integrinas/metabolismo , Receptor Muscarínico M3/metabolismo , Vasodilatação/efeitos dos fármacos , Acetilcolina/farmacologia , Animais , Anticoagulantes/farmacologia , Aorta Torácica/metabolismo , Aorta Torácica/fisiologia , Atropina/farmacologia , Western Blotting , Bovinos , Linhagem Celular , Relação Dose-Resposta a Droga , Endotélio Vascular/metabolismo , Endotélio Vascular/fisiologia , Transferência Ressonante de Energia de Fluorescência , Técnicas In Vitro , Masculino , Óxido Nítrico/biossíntese , Oligopeptídeos/farmacologia , Paxilina/metabolismo , Ligação Proteica , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , Ratos , Ratos Wistar , Vasodilatadores/farmacologiaRESUMO
Bites from the Loxosceles genus (brown spiders) cause severe clinical symptoms, including dermonecrotic injury, hemorrhage, hemolysis, platelet aggregation and renal failure. Histological findings of dermonecrotic lesions in animals exposed to Loxosceles intermedia venom show numerous vascular alterations. Study of the hemorrhagic consequences of the venom in endothelial cells has demonstrated that the degeneration of blood vessels results not only from degradation of the extracellular matrix molecule or massive leukocyte infiltration, but also from a direct and primary activity of the venom on endothelial cells. Exposure of an endothelial cell line in vitro to L. intermedia venom induce morphological alterations, such as cell retraction and disadhesion to the extracellular matrix. The aim of the present study was to investigate the interaction between the venom toxins and the endothelial cell surface and their possible internalization, in order to illuminate the information about the deleterious effect triggered by venom. After treating endothelial cells with venom toxins, we observed that the venom interacts with cell surface. Venom treatment also can cause a reduction of cell surface glycoconjugates. When cells were permeabilized, it was possible to verify that some venom toxins were internalized by the endothelial cells. The venom internalization involves endocytic vesicles and the venom was detected in the lysosomes. However, no damage to lysosomal integrity was observed, suggesting that the cytotoxic effect evoked by L. intermedia venom on endothelial cells is not mediated by venom internalization.
Assuntos
Membrana Celular/efeitos dos fármacos , Endocitose , Venenos de Aranha/farmacologia , Aranhas/química , Animais , Linhagem Celular , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Lisossomos/química , Lisossomos/efeitos dos fármacos , Coelhos , Venenos de Aranha/análise , Venenos de Aranha/químicaRESUMO
BACKGROUND: Melanoma is the most aggressive form of skin cancer and the most rapidly expanding cancer in terms of worldwide incidence. Chemotherapeutic approaches to treat melanoma have had only marginal success. Previous studies in mice demonstrated that a high diluted complex derived from Calcarea carbonica (M8) stimulated the tumoricidal response of activated lymphocytes against B16F10 melanoma cells in vitro. METHODS: Here we describe the in vitro inhibition of invasion and the in vivo anti-metastatic potential after M8 treatment by inhalation in the B16F10 lung metastasis model. RESULTS: We found that M8 has at least two functions, acting as both an inhibitor of cancer cell adhesion and invasion and as a perlecan expression antagonist, which are strongly correlated with several metastatic, angiogenic and invasive factors in melanoma tumors. CONCLUSION: The findings suggest that this medication is a promising non-toxic therapy candidate by improving the immune response against tumor cells or even induce direct dormancy in malignancies.
Assuntos
Materia Medica/farmacologia , Melanoma Experimental/tratamento farmacológico , Animais , Células da Medula Óssea/patologia , Neoplasias Colorretais/tratamento farmacológico , Células HT29 , Humanos , Imunofenotipagem , Neoplasias Pulmonares/sangue , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/secundário , Masculino , Melanoma Experimental/sangue , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Gangliosides are known to be important in many biological processes. However, details concerning the exact function of these glycosphingolipids in cell physiology are poorly understood. In this study, the role of gangliosides present on the surface of rodent mast cells in maintaining cell structure was examined using RBL-2H3 mast cells and two mutant cell lines (E5 and D1) deficient in the gangliosides, GM(1) and the alpha-galactosyl derivatives of the ganglioside GD(1b). The two deficient cell lines were morphologically different from each other as well as from the parental RBL-2H3 cells. Actin filaments in RBL-2H3 and E5 cells were under the plasma membrane following the spindle shape of the cells, whereas in D1 cells, they were concentrated in large membrane ruffles. Microtubules in RBL-2H3 and E5 cells radiated from the centrosome and were organized into long, straight bundles. The bundles in D1 cells were thicker and organized circumferentially under the plasma membrane. The endoplasmic reticulum, the Golgi complex, and the secretory granule matrix were also altered in the mutant cell lines. These results suggest that the mast cell-specific alpha-galactosyl derivatives of ganglioside GD(1b) and GM(1) are important in maintaining normal cell morphology.