RESUMO
This study demonstrates the potential of gelatin nanoparticles as a nanodelivery system for antagonists of nicotinic acetylcholine receptors (nAChRs) to improve chemotherapy efficacy and reduce off-target effects. Too often, chemotherapy for lung cancer does not lead to satisfactory results. Therefore, new approaches directed at multiple pharmacological targets in cancer therapy are being developed. Following the activation of nAChRs (e.g. by nicotine), cancer cells begin to proliferate and become more resistant to chemotherapy-induced apoptosis. This work shows that the 3-alkylpyridinium salt, APS7, a synthetic analog of a toxin from the marine sponge Haliclona (Rhizoneira) sarai, acts as an nAChR antagonist that inhibits the pro-proliferative and anti-apoptotic effects of nicotine on A549 human lung adenocarcinoma cells. In this study, gelatin-based nanoparticles filled with APS7 (APS7-GNPs) were prepared and their effects on A549 cells were compared with that of free APS7. Both APS7 and APS7-GNPs inhibited Ca2+ influx and increased the efficacy of cisplatin chemotherapy in nicotine-stimulated A549 cells. However, significant benefits from APS7-GNPs were observed - a stronger reduction in the proliferation of A549 lung cancer cells and a much higher selectivity in cytotoxicity towards cancer cells compared with non-tumorigenic lung epithelial BEAS-2B cells.
Assuntos
Proliferação de Células , Gelatina , Neoplasias Pulmonares , Nanopartículas , Humanos , Gelatina/química , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/metabolismo , Células A549 , Nanopartículas/química , Proliferação de Células/efeitos dos fármacos , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Compostos de Piridínio/farmacologia , Compostos de Piridínio/química , Cisplatino/farmacologia , Nicotina/farmacologia , Antagonistas Nicotínicos/farmacologia , Antagonistas Nicotínicos/química , Linhagem Celular TumoralRESUMO
Nicotine activates nicotinic acetylcholine receptors (nAChRs), which are overexpressed in numerous cancer types, leading to signaling pathways that increase lung cancer invasiveness and resistance to chemotherapeutic agents. In this study, the effects of APS12-2, a synthetic analog of marine sponge toxin that acts as an antagonist of nAChRs, was investigated in vitro on A549 human lung adenocarcinoma cells and non-tumorigenic human lung epithelial BEAS-2B cells. In addition, gelatin nanoparticles (GNPs) loaded with APS12-2 (APS12-2-GNPs) were prepared and their effects were compared with those of free APS12-2. Nicotine reduced cytotoxicity, the formation of reactive oxygen species, and the formation of lipid droplets caused by cisplatin on A549 cells. The effects of nicotine on the decreased efficacy of cisplatin were reduced by APS12-2 and APS12-2-GNPs. APS12-2-GNPs showed a substantial advantage compared with free APS12-2; the cytotoxicity of APS12-2 on BEAS-2B cells was greatly reduced when APS12-2 was loaded in GNPs, whereas the cytotoxicity on A549 cells was only slightly reduced. Our results suggest that both APS12-2 and APS12-2-GNPs hold promise as supportive agents in the cisplatin-based chemotherapy of lung cancer.
RESUMO
Nicotine binds to nicotinic acetylcholine receptors (nAChRs) that are overexpressed in different cancer cells, promoting tumor growth and resistance to chemotherapy. In this study, we aimed to investigate the potential of APS7-2 and APS8-2, synthetic analogs of a marine sponge toxin, to inhibit nicotine-mediated effects on A549 human lung cancer cells. Our electrophysiological measurements confirmed that APS7-2 and APS8-2 act as α7 nAChR antagonists. APS8-2 showed no cytotoxicity in A549 cells, while APS7-2 showed concentration-dependent cytotoxicity in A549 cells. The different cytotoxic responses of APS7-2 and APS8-2 emphasize the importance of the chemical structure in determining their cytotoxicity on cancer cells. Nicotine-mediated effects include increased cell viability and proliferation, elevated intracellular calcium levels, and reduced cisplatin-induced cytotoxicity and reactive oxygen species production (ROS) in A549 cells. These effects of nicotine were effectively attenuated by APS8-2, whereas APS7-2 was less effective. Our results suggest that APS8-2 is a promising new therapeutic agent in the chemotherapy of lung cancer.
Assuntos
Antineoplásicos , Sobrevivência Celular , Neoplasias Pulmonares , Nicotina , Espécies Reativas de Oxigênio , Receptor Nicotínico de Acetilcolina alfa7 , Humanos , Receptor Nicotínico de Acetilcolina alfa7/metabolismo , Células A549 , Nicotina/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Espécies Reativas de Oxigênio/metabolismo , Antineoplásicos/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Animais , Antagonistas Nicotínicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Cisplatino/farmacologia , Cálcio/metabolismo , Poríferos/químicaRESUMO
Covering: Published between 1974 up to 2018Inhibition of cholinesterases is a common approach for the management of several disease states. Most notably, cholinesterase inhibitors are used to alleviate the symptoms of neurological disorders like dementia and Alzheimer's disease and treat myasthenia gravis and glaucoma. Historically, most drugs of natural origin have been isolated from terrestrial sources and inhibitors of cholinesterases are no exception. However, the last 50 years have seen a rise in the quantity of marine natural products with close to 25 000 reported in the scientific literature. A number of marine natural products with potent cholinesterase inhibitory properties have also been reported; isolated from a variety of marine sources from algae to ascidians. Representing a diverse range of structural classes, these compounds provide inspirational leads that could aid the development of therapeutics. The current paper aims to, for the first time, comprehensively summarize the literature pertaining to cholinesterase inhibitors derived from marine sources, including the first papers published in 1974 up to 2018. The review does not report bioactive extracts, only isolated compounds, and a specific focus lies on compounds with reported dose-response data. In vivo and mechanistic data is included for compounds where this is reported. In total 185 marine cholinesterase inhibitors and selected analogs have been identified and reported and some of the compounds display inhibitory activities comparable or superior to cholinesterase inhibitors in clinical use.
Assuntos
Organismos Aquáticos/química , Inibidores da Colinesterase/farmacologia , Animais , Produtos Biológicos , Inibidores da Colinesterase/isolamento & purificação , Inibidores da Colinesterase/toxicidade , Relação Dose-Resposta a Droga , Humanos , Estrutura MolecularRESUMO
The alkylpyridinium polymer APS8, a potent antagonist of α7 nicotinic acetylcholine receptors (nAChRs), selectively induces apoptosis in non-small cell lung cancer cells but not in normal lung fibroblasts. To explore the potential therapeutic value of APS8 for at least certain types of lung cancer, we determined its systemic and organ-specific toxicity in mice, evaluated its antitumor activity against adenocarcinoma xenograft models, and examined the in-vitro mechanisms of APS8 in terms of apoptosis, cytotoxicity, and viability. We also measured Ca2+ influx into cells, and evaluated the effects of APS8 on Ca2+ uptake while siRNA silencing of the gene for α7 nAChRs, CHRNA7. APS8 was not toxic to mice up to 5 mg/kg i.v., and no significant histological changes were observed in mice that survived APS8 treatment. Repetitive intratumoral injections of APS8 (4 mg/kg) significantly delayed growth of A549 cell tumors, and generally prevented regrowth of tumors, but were less effective in reducing growth of HT29 cell tumors. APS8 impaired the viability of A549 cells in a dose-dependent manner and induced apoptosis at micro molar concentrations. Nano molar APS8 caused minor cytotoxic effects, while cell lysis occurred at APS8 >3 µM. Furthermore, Ca2+ uptake was significantly reduced in APS8-treated A549 cells. Observed differences in response to APS8 can be attributed to the number of α7 nAChRs expressed in these cells, with those with more AChRs (i.e., A549 cells) being more sensitive to nAChR antagonists like APS8. We conclude that α7 nAChR antagonists like APS8 have potential to be used as therapeutics for tumors expressing large numbers of α7 nAChRs.
Assuntos
Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Neoplasias Pulmonares/tratamento farmacológico , Polímeros/farmacologia , Compostos de Piridínio/farmacologia , Receptor Nicotínico de Acetilcolina alfa7/metabolismo , Células A549 , Adenocarcinoma de Pulmão/tratamento farmacológico , Adenocarcinoma de Pulmão/metabolismo , Animais , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias Pulmonares/metabolismo , Camundongos , RNA Interferente Pequeno/metabolismoRESUMO
The heteronemertine Parborlasia corrugatus contains a cytolytic protein, parborlysin, which after extensive purification was found by Edman sequencing to be a mixture of several homologues. To investigate this microheterogeneity and enable the analysis of single toxins, we have obtained seven parborlysin isoform genes from P. corrugatus collected in Antarctica. Total RNA was isolated from the homogenized head region and parborlysin genes were identified from a cDNA library using degenerate primers. The translated sequences reveal that the isoforms are â¼ 10 kDa basic (pI â¼ 10) proteins of which all but one harbour six cysteine residues. We generated a model of the three dimensional structure of parborlysins, which suggests that they are composed of five alpha-helical segments that include large, exposed hydrophobic surfaces. Finally, we constructed plasmids and inserted them into Escherichia coli to obtain overexpressed amino- or carboxy-terminal polyhistidine-tagged parborlysin isoforms fused to the third domain of the E. coli periplasmic-protein TolA to facilitate toxin isolation. One of the isoforms adversely affected growth in the E. coli expressing it. Although we succeeded in isolating one of the recombinant parborlysin constructs, it lacked haemolytic activity.
Assuntos
Invertebrados/genética , Toxinas Marinhas/química , Sequência de Aminoácidos , Animais , Clonagem Molecular , Escherichia coli/genética , Invertebrados/metabolismo , Toxinas Marinhas/genética , Toxinas Marinhas/toxicidade , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Análise de Sequência de ProteínaRESUMO
Patients suffering from tauopathies including frontotemporal dementia (FTD) and Alzheimer's disease (AD) present with intra-neuronal aggregation of microtubule-associated protein Tau. During the disease process, Tau undergoes excessive phosphorylation, dissociates from microtubules and aggregates into insoluble neurofibrillary tangles (NFTs), accumulating in the soma. While many aspects of the disease pathology have been replicated in transgenic mouse models, a region-specific non-transgenic expression model is missing. Complementing existing models, we here report a novel region-specific approach to modelling Tau pathology. Local co-administration of the pore-former polymeric 1,3-alkylpyridinium salts (Poly-APS) extracted from marine sponges, and synthetic full-length 4R recombinant human Tau (hTau) was performed in vitro and in vivo. At low doses, Poly-APS was non-toxic and cultured cells exposed to Poly-APS (0.5 µg/ml) and hTau (1 µg/ml; ~22 µM) had normal input resistance, resting-state membrane potentials and Ca(2+) transients induced either by glutamate or KCl, as did cells exposed to a low concentration of the phosphatase inhibitor Okadaic acid (OA; 1 nM, 24 h). Combined hTau loading and phosphatase inhibition resulted in a collapse of the membrane potential, suppressed excitation and diminished glutamate and KCl-stimulated Ca(2+) transients. Stereotaxic infusions of Poly-APS (0.005 µg/ml) and hTau (1 µg/ml) bilaterally into the dorsal hippocampus at multiple sites resulted in hTau loading of neurons in rats. A separate cohort received an additional 7-day minipump infusion of OA (1.2 nM) intrahippocampally. When tested 2 weeks after surgery, rats treated with Poly-APS+hTau+OA presented with subtle learning deficits, but were also impaired in cognitive flexibility and recall. Hippocampal plasticity recorded from slices ex vivo was diminished in Poly-APS+hTau+OA subjects, but not in other treatment groups. Histological sections confirmed the intracellular accumulation of hTau in CA1 pyramidal cells and along their processes; phosphorylated Tau was present only within somata. This study demonstrates that cognitive, physiological and pathological symptoms reminiscent of tauopathies can be induced following non-mutant hTau delivery into CA1 in rats, but functional consequences hinge on increased Tau phosphorylation. Collectively, these data validate a novel model of locally infused recombinant hTau protein as an inducer of Tau pathology in the hippocampus of normal rats; future studies will provide insights into the pathological spread and maturation of Tau pathology.
Assuntos
Hipocampo/citologia , Plasticidade Neuronal/fisiologia , Neurônios/efeitos dos fármacos , Polímeros/administração & dosagem , Compostos de Piridínio/administração & dosagem , Proteínas tau/farmacologia , Animais , Comportamento Animal/efeitos dos fármacos , Sinalização do Cálcio/efeitos dos fármacos , Células Cultivadas , Modelos Animais de Doenças , Eletrofisiologia/métodos , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Humanos , Deficiências da Aprendizagem/tratamento farmacológico , Masculino , Neurônios/metabolismo , Neurônios/fisiologia , Fosforilação , Polímeros/farmacologia , Compostos de Piridínio/farmacologia , Ratos Sprague-Dawley , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Tauopatias/metabolismo , Proteínas tau/administração & dosagem , Proteínas tau/metabolismoRESUMO
The cell membrane is crucial for protection of the cell from its environment. MACPF/CDC proteins are a large superfamily known to be essential for bacterial pathogenesis and proper functioning of the immune system. The three most studied groups of MACPF/CDC proteins are cholesterol-dependent cytolysins from bacteria, the membrane attack complex of complement and human perforin. Their primary function is to form transmembrane pores in target cell membranes. The common mechanism of action comprises water-soluble monomeric proteins binding to the host cell membrane, oligomerization, and formation of a functional pore. This causes a disturbance in gradients of ions and other molecules across the membrane and can lead to cell death. Cells react to this form of attack in a complex manner. Responses can be general, like removing the perforated part of the membrane, or more specific, in many cases depending on binding of proteins to specific receptors to trigger various signalling cascades.
Assuntos
Membrana Celular/metabolismo , Complexo de Ataque à Membrana do Sistema Complemento/metabolismo , Citotoxinas/metabolismo , Perforina/metabolismo , Animais , Membrana Celular/química , Colesterol/metabolismo , Complexo de Ataque à Membrana do Sistema Complemento/química , Citotoxinas/química , Humanos , Lipídeos de Membrana/química , Lipídeos de Membrana/metabolismo , Perforina/química , Proteínas Citotóxicas Formadoras de Poros/química , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Ligação ProteicaRESUMO
Polymeric alkylpyridinium salts (poly-APS) isolated from the Mediterranean marine sponge, Haliclona (Rhizoniera) sarai, effectively inhibit barnacle larva settlement and natural marine biofilm formation through a non-toxic and reversible mechanism. Potential use of poly-APS-like compounds as antifouling agents led to the chemical synthesis of monomeric and oligomeric 3-alkylpyridinium analogues. However, these are less efficient in settlement assays and have greater toxicity than the natural polymers. Recently, a new chemical synthesis method enabled the production of poly-APS analogues with antibacterial, antifungal and anti-acetylcholinesterase activities. The present study examines the antifouling properties and toxicity of six of these synthetic poly-APS using the barnacle (Amphibalanus amphitrite) as a model (cyprids and II stage nauplii larvae) in settlement, acute and sub-acute toxicity assays. Two compounds, APS8 and APS12-3, show antifouling effects very similar to natural poly-APS, with an anti-settlement effective concentration that inhibits 50% of the cyprid population settlement (EC50) after 24 h of 0.32 mg/L and 0.89 mg/L, respectively. The toxicity of APS8 is negligible, while APS12-3 is three-fold more toxic (24-h LC50: nauplii, 11.60 mg/L; cyprids, 61.13 mg/L) than natural poly-APS. This toxicity of APS12-3 towards nauplii is, however, 60-fold and 1200-fold lower than that of the common co-biocides, Zn- and Cu-pyrithione, respectively. Additionally, exposure to APS12-3 for 24 and 48 h inhibits the naupliar swimming ability with respective IC50 of 4.83 and 1.86 mg/L.
Assuntos
Incrustação Biológica/prevenção & controle , Haliclona/metabolismo , Polímeros/farmacologia , Compostos de Piridínio/farmacologia , Thoracica/efeitos dos fármacos , Animais , Concentração Inibidora 50 , Larva , Mar Mediterrâneo , Polímeros/síntese química , Polímeros/isolamento & purificação , Compostos de Piridínio/síntese química , Compostos de Piridínio/isolamento & purificação , Testes de Toxicidade Aguda , Testes de Toxicidade SubagudaRESUMO
Naturally occurring 3-alkylpyridinium polymers (poly-APS) from the marine sponge Reniera sarai, consisting of monomers containing polar pyridinium and nonpolar alkyl chain moieties, have been demonstrated to exert a wide range of biological activities, including a selective cytotoxicity against non-small cell lung cancer (NSCLC) cells. APS8, an analog of poly-APS with defined alkyl chain length and molecular size, non-competitively inhibits α7 nicotinic acetylcholine receptors (nAChRs) at nanomolar concentrations that are too low to be acetylcholinesterase (AChE) inhibitory or generally cytotoxic. In the present study we show that APS8 inhibits NSCLC tumor cell growth and activates apoptotic pathways. APS8 was not toxic for normal lung fibroblasts. Furthermore, in NSCLC cells, APS8 reduced the adverse anti-apoptotic, proliferative effects of nicotine. Our results suggest that APS8 or similar compounds might be considered as lead compounds to develop antitumor therapeutic agents for at least certain types of lung cancer.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Fatores Biológicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Polímeros/farmacologia , Compostos de Piridínio/farmacologia , Receptor Nicotínico de Acetilcolina alfa7/antagonistas & inibidores , Acetilcolinesterase/metabolismo , Animais , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Caspase 9/metabolismo , Linhagem Celular Tumoral , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Neoplasias Pulmonares/metabolismo , Poríferos/química , Receptor Nicotínico de Acetilcolina alfa7/metabolismoRESUMO
We report on the screening of ethanolic extracts from 33 deep-sea Antarctic marine sponges for different biological activities. We monitored hemolysis, inhibition of acetylcholinesterase, cytotoxicity towards normal and transformed cells and growth inhibition of laboratory, commensal and clinically and ecologically relevant bacteria. The most prominent activities were associated with the extracts from sponges belonging to the genus Latrunculia, which show all of these activities. While most of these activities are associated to already known secondary metabolites, the extremely strong acetylcholinesterase inhibitory potential appears to be related to a compound unknown to date. Extracts from Tetilla leptoderma, Bathydorus cf. spinosus, Xestospongia sp., Rossella sp., Rossella cf. racovitzae and Halichondria osculum were hemolytic, with the last two also showing moderate cytotoxic potential. The antibacterial tests showed significantly greater activities of the extracts of these Antarctic sponges towards ecologically relevant bacteria from sea water and from Arctic ice. This indicates their ecological relevance for inhibition of bacterial microfouling.
Assuntos
Antibacterianos/farmacologia , Inibidores da Colinesterase/farmacologia , Poríferos/química , Extratos de Tecidos/farmacologia , Acetilcolinesterase/efeitos dos fármacos , Acetilcolinesterase/metabolismo , Animais , Regiões Antárticas , Antibacterianos/isolamento & purificação , Antibacterianos/toxicidade , Bactérias/efeitos dos fármacos , Células CACO-2 , Bovinos , Linhagem Celular , Inibidores da Colinesterase/isolamento & purificação , Inibidores da Colinesterase/toxicidade , Etanol/química , Hemólise/efeitos dos fármacos , Humanos , Extratos de Tecidos/isolamento & purificaçãoRESUMO
Giant unilamellar vesicles (GUVs) are an attractive experimental model for studying various membrane-related phenomena. The procedure for GUV electroformation from erythrocyte ghosts under physiological conditions was introduced recently; however, it allows preparation of a limited number of GUVs. Here we describe an efficient, reliable, and simple method for electroformation of GUVs from native erythrocyte membranes at low salt concentration, which enables the formation of higher amounts of large, spherical GUVs. GUVs prepared according to the new procedure may not retain original lipid asymmetry; however, they preserved native proteins, lipids, and oligosaccharide heterogeneity and could be a suitable system for functional studies for which larger amounts of GUVs of complex composition are needed.
Assuntos
Técnicas Eletroquímicas , Membrana Eritrocítica/química , Sais/química , Lipossomas Unilamelares/química , Eletrodos , Membrana Eritrocítica/metabolismo , Glicoforinas/química , Glicoforinas/metabolismo , Humanos , Lipídeos/química , Microscopia de Fluorescência , Oligossacarídeos/química , Oligossacarídeos/metabolismo , Lipossomas Unilamelares/metabolismoRESUMO
The in vivo and in vitro toxic effects of the synthetic polymeric 3-alkylpyridinium salt (APS3), from the Mediterranean marine sponge Reniera sarai, were evaluated on mammals, with emphasis to determine its mode of action. The median lethal doses of APS3 were 7.25 and higher that 20mg/kg in mouse and rat, respectively. Intravenous administration of 7.25 and 20mg/kg APS3 to rat caused a significant fall followed by an increase in mean arterial blood pressure accompanied by tachycardia. In addition, cumulative doses of APS3 (up to 60 mg/kg) inhibited rat nerve-evoked skeletal muscle contraction in vivo, with a median inhibitory dose (ID(50)) of 37.25mg/kg. When administrated locally by intramuscular injection to mouse, APS3 decreased the compound muscle action potential recorded in response to in vivo nerve stimulation, with an ID(50) of 0.5mg/kg. In vitro experiments confirmed the inhibitory effect of APS3 on mouse hemidiaphragm nerve-evoked muscle contraction with a median inhibitory concentration (IC(50)) of 20.3 µM, without affecting directly elicited muscle contraction. The compound inhibited also miniature endplate potentials and nerve-evoked endplate potentials with an IC(50) of 7.28 µM in mouse hemidiaphragm. Finally, APS3 efficiently blocked acetylcholine-activated membrane inward currents flowing through Torpedo nicotinic acetylcholine receptors (nAChRs) incorporated to Xenopus oocytes, with an IC(50) of 0.19 µM. In conclusion, our results strongly suggest that APS3 blocks muscle-type nAChRs, and show for the first time that in vivo toxicity of APS3 is likely to occur through an antagonist action of the compound on these receptors.
Assuntos
Antagonistas Nicotínicos/toxicidade , Polímeros/toxicidade , Poríferos/química , Compostos de Piridínio/toxicidade , Receptores Nicotínicos/efeitos dos fármacos , Animais , Pressão Sanguínea/efeitos dos fármacos , Bovinos , Relação Dose-Resposta a Droga , Feminino , Concentração Inibidora 50 , Injeções Intramusculares , Injeções Intravenosas , Dose Letal Mediana , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Contração Muscular/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Antagonistas Nicotínicos/administração & dosagem , Antagonistas Nicotínicos/isolamento & purificação , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Polímeros/administração & dosagem , Polímeros/isolamento & purificação , Compostos de Piridínio/administração & dosagem , Compostos de Piridínio/isolamento & purificação , Ratos , Ratos Wistar , Receptores Nicotínicos/metabolismo , Xenopus laevisRESUMO
APS12-2, a non-competitive acetylcholinesterase inhibitor, is one of the synthetic analogs of polymeric alkylpyridinium salts (poly-APS) isolated from the marine sponge Reniera sarai. In the present work the effects of APS12-2 were studied on isolated mouse phrenic nerve-hemidiaphragm muscle preparations, using twitch tension measurements and electrophysiological recordings. APS12-2 in a concentration-dependent manner blocked nerve-evoked isometric muscle contraction (IC(50)=0.74 µM), without affecting directly-elicited twitch tension up to 2.72 µM. The compound (0.007-3.40 µM) decreased the amplitude of miniature endplate potentials until a complete block by concentrations higher than 0.68 µM, without affecting their frequency. Full size endplate potentials, recorded after blocking voltage-gated muscle sodium channels, were inhibited by APS12-2 in a concentration-dependent manner (IC(50)=0.36 µM) without significant change in the resting membrane potential of the muscle fibers up to 3.40 µM. The compound also blocked acetylcholine-evoked inward currents in Xenopus oocytes in which Torpedo (α1(2)ß1γδ) muscle-type nicotinic acetylcholine receptors (nAChRs) have been incorporated (IC(50)=0.0005 µM), indicating a higher affinity of the compound for Torpedo (α1(2)ß1γδ) than for the mouse (α1(2)ß1γε) nAChR. Our data show for the first time that APS12-2 blocks neuromuscular transmission by a non-depolarizing mechanism through an action on postsynaptic nAChRs of the skeletal neuromuscular junction.
Assuntos
Inibidores da Colinesterase/farmacologia , Contração Muscular/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Compostos de Piridínio/farmacologia , Receptores Nicotínicos/metabolismo , Animais , Relação Dose-Resposta a Droga , Feminino , Técnicas In Vitro , Concentração Inibidora 50 , Masculino , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Potenciais Pós-Sinápticos em Miniatura/efeitos dos fármacos , Músculo Esquelético/metabolismo , Junção Neuromuscular/efeitos dos fármacos , Antagonistas Nicotínicos , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Análise de Regressão , XenopusRESUMO
Naturally occurring 3-alkylpyridinium polymers from the marine sponge Reniera sarai are membrane-active compounds exerting a selective cytotoxicity towards non small cell lung cancer cells, and stable transfection of nucleated mammalian cells. In view of their possible use as chemotherapeutics and/or transfection tools, three poly-APS based synthetic compounds were tested on their activity using natural and artificial lipid membranes. The tested compounds were found to be very stable over a wide range of temperature, ionic strength, and pH, and to prefer the solid-ordered membrane state. Their membrane-damaging activity increases with the length of their alkyl chains and the degree of polymerization.
Assuntos
Bicamadas Lipídicas/química , Polímeros/química , Compostos de Piridínio/química , Animais , Bicamadas Lipídicas/metabolismo , Polímeros/metabolismo , Poríferos , Compostos de Piridínio/metabolismo , TransfecçãoRESUMO
Six new steroidal saponins, pandarosides K-M (1-3) and their methyl esters (4-6), were isolated as minor components, after a careful chemical reinvestigation of the Caribbean sponge Pandaros acanthifolium. Their structures were established on the basis of spectroscopic analyses and comparison with the data obtained from previous metabolites of this family. All new compounds showed moderate to weak activity against four parasitic protozoa. Additionally, these compounds and previously reported pandarosides and acanthifoliosides were tested on three human tumour cell lines, and their haemolytic and liposome permeabilizing activity were assessed. Two pandarosides exhibited moderate to strong cytotoxic effect, while three acanthifoliosides showed strong haemolytic activity.
Assuntos
Anticarcinógenos/farmacologia , Antiprotozoários/farmacologia , Hemolíticos/farmacologia , Poríferos/química , Saponinas/farmacologia , Esteroides/farmacologia , Animais , Anticarcinógenos/química , Antiprotozoários/química , Antiprotozoários/isolamento & purificação , Região do Caribe , Linhagem Celular Tumoral , Hemolíticos/química , Hemolíticos/isolamento & purificação , Humanos , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Ratos , Saponinas/química , Saponinas/isolamento & purificação , Esteroides/química , Esteroides/isolamento & purificaçãoRESUMO
APS12-2 is one in a series of synthetic analogs of the polymeric alkylpyridinium salts isolated from the marine sponge Reniera sarai. As it is a potential candidate for treating non small cell lung cancer (NSCLC), we have studied its possible toxic and lethal effects in vivo. The median lethal dose (LD(50)) of APS12-2 in mice was determined to be 11.5mg/kg. Electrocardiograms, arterial blood pressure and respiratory activity were recorded under general anesthesia in untreated, pharmacologically vagotomized and artificially ventilated rats injected with APS12-2. In one group, the in vivo effects of APS12-2 were studied on nerve-evoked muscle contraction. Administration of APS12-2 at a dose of 8mg/kg caused a progressive reduction of arterial blood pressure to a mid-circulatory value, accompanied by bradycardia, myocardial ischemia, ventricular extrasystoles, and second degree atrio-ventricular block. Similar electrocardiogram and arterial blood pressure changes caused by APS12-2 (8mg/kg) were observed in animals pretreated with atropine and in artificially ventilated animals, indicating that hypoxia and cholinergic effects do not play a crucial role in the toxicity of APS12-2. Application of APS12-2 at sublethal doses (4 and 5.5mg/kg) caused a decrease of arterial blood pressure, followed by an increase slightly above control values. We found that APS12-2 causes lysis of rat erythrocytes in vitro, therefore it is reasonable to expect the same effect in vivo. Indeed, hyperkalemia was observed in the blood of experimental animals. Hyperkalemia probably plays an important role in APS12-2 cardiotoxicity since no evident changes in histopathology of the heart were found. However, acute lesions were observed in the pulmonary vessels of rats after application of 8mg/kg APS12-2. Predominant effects were dilation of interalveolar blood vessels and lysis of aggregated erythrocytes within their lumina.
Assuntos
Compostos de Piridínio/toxicidade , Acetilcolinesterase/sangue , Animais , Pressão Sanguínea/efeitos dos fármacos , Eletrocardiografia/efeitos dos fármacos , Hemólise/efeitos dos fármacos , Dose Letal Mediana , Pulmão/efeitos dos fármacos , Pulmão/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Ratos , Ratos Wistar , Respiração/efeitos dos fármacosRESUMO
Eggs of the common barbel (Barbus barbus) cause intoxication in humans after ingestion. In this work, the chemical composition of the haemolytically active fraction from methanolic barbel roe extract was analyzed. Compounds showing haemolytic activity and cytotoxicity towards normal and transformed cell lines were isolated and identified as polyunsaturated fatty acids, using online liquid chromatography-electrospray ionization mass spectrometry through tandem fragmentation experiments (HPLC-MS/MS). Arachidonic acid (C20:4), docosahexaenoic acid (C22:6) and eicosapentaenoic acid (C20:5) proved to be the three most abundant members of a complex series of free fatty acids ranging from C14:0 to C24:5.
Assuntos
Cyprinidae/metabolismo , Ácidos Graxos Insaturados/análise , Hemólise , Óvulo/metabolismo , Animais , Ácido Araquidônico/análise , Ácido Araquidônico/química , Ácido Araquidônico/metabolismo , Linhagem Celular Transformada , Cromatografia Líquida de Alta Pressão , Ácidos Docosa-Hexaenoicos/análise , Ácidos Docosa-Hexaenoicos/química , Ácidos Docosa-Hexaenoicos/metabolismo , Ácido Eicosapentaenoico/análise , Ácido Eicosapentaenoico/química , Ácido Eicosapentaenoico/metabolismo , Estudos de Avaliação como Assunto , Ácidos Graxos Insaturados/química , Ácidos Graxos Insaturados/metabolismo , Feminino , Humanos , Metanol/química , Óvulo/química , Espectrometria de Massas por Ionização por Electrospray , Testes de ToxicidadeRESUMO
Cnidarians, mostly soft-bodied water organisms, produce several classes of toxins deployed in biological warfare or signalling. Cytolytic toxins, that form pores in cell membranes, form a significant part of their "weaponry". Here, we describe the physiological relevance of membrane permeabilization, and present basic data on those proteinaceous cnidarian cytolysins proven or presumed to damage cell membranes by pore formation. We describe cytolysins that have been at least partially characterized, both functionally and structurally.
RESUMO
We report on screening tests of 66 extracts obtained from 35 marine sponge species from the Caribbean Sea (Curaçao) and from eight species from the Great Barrier Reef (Lizard Island). Extracts were prepared in aqueous and organic solvents and were tested for hemolytic, hemagglutinating, antibacterial and anti-acetylcholinesterase (AChE) activities, as well as their ability to inhibit or activate cell protein phosphatase 1 (PP1). The most interesting activities were obtained from extracts of Ircinia felix, Pandaros acanthifolium, Topsentia ophiraphidites, Verongula rigida and Neofibularia nolitangere. Aqueous and organic extracts of I. felix and V. rigida showed strong antibacterial activity. Topsentia aqueous and some organic extracts were strongly hemolytic, as were all organic extracts from I. felix. The strongest hemolytic activity was observed in aqueous extracts from P. acanthifolium. Organic extracts of N. nolitangere and I. felix inhibited PP1. The aqueous extract from Myrmekioderma styx possessed the strongest hemagglutinating activity, whilst AChE inhibiting activity was found only in a few sponges and was generally weak, except in the methanolic extract of T. ophiraphidites.