RESUMO
Glioblastoma multiforme (GBM) is the most aggressive type of brain cancer, with a poor prognosis. GBM cells, which develop in the environment of neural tissue, often exploit neurotransmitters and their receptors to promote their own growth and invasion. Nicotinic acetylcholine receptors (nAChRs), which play a crucial role in central nervous system signal transmission, are widely represented in the brain, and GBM cells express several subtypes of nAChRs that are suggested to transmit signals from neurons, promoting tumor invasion and growth. Analysis of published GBM transcriptomes revealed spatial heterogeneity in nAChR subtype expression, and functional nAChRs of α1*, α7, and α9 subtypes are demonstrated in our work on several patient-derived GBM microsphere cultures and on the U87MG GBM cell line using subtype-selective neurotoxins and fluorescent calcium mobilization assay. The U87MG cell line shows reactions to nicotinic agonists similar to those of GBM patient-derived culture. Selective α1*, α7, and α9 nAChR neurotoxins stimulated cell growth in the presence of nicotinic agonists. Several cultivating conditions with varying growth factor content have been proposed and tested. The use of selective neurotoxins confirmed that cell cultures obtained from patients are representative GBM models, but the use of media containing fetal bovine serum can lead to alterations in nAChR expression and functioning.
Assuntos
Glioblastoma , Receptores Nicotínicos , Humanos , Receptores Nicotínicos/genética , Receptores Nicotínicos/metabolismo , Receptor Nicotínico de Acetilcolina alfa7/genética , Receptor Nicotínico de Acetilcolina alfa7/metabolismo , Agonistas Nicotínicos/farmacologia , Proteínas/metabolismo , Peptídeos/farmacologia , Linhagem Celular , Proliferação de Células , Antagonistas Nicotínicos/farmacologiaRESUMO
Evidence to date indicates that activation of nicotinic acetylcholine receptors (nAChRs) can reduce cardiac injury from ischemia and subsequent reperfusion. The use of nAChR agonists in various animal models leads to a reduction in reperfusion injury. Earlier this effect was shown for the agonists of α7 nAChR subtype. In this work, we demonstrated the expression of mRNA encoding α4, α6 and ß2 nAChR subunits in the left ventricle of rat heart. In a rat model of myocardial ischemia, we studied the effect of α4ß2 nAChR agonists cytisine and varenicline, medicines used for the treatment of nicotine addiction, and found them to significantly reduce myocardium ischemia-reperfusion injury, varenicline manifesting a higher protection. Dihydro-ß-erythroidine, antagonist of α4ß2 nAChR, as well as methyllycaconitine, antagonist of α7 and α6ß2-containing nAChR, prevented protective effect of varenicline. This together with the presence of α4, α6 and ß2 subunit mRNA in the left ventricule of rat heart raises the possibility that the varenicline effect is mediated by α4ß2 as well as by α7 and/or α6ß2-containing receptors. Our results point to a new way for the use of cytisine and varenicline as cardioprotective agents.
Assuntos
Alcaloides , Isquemia Miocárdica , Receptores Nicotínicos , Traumatismo por Reperfusão , Ratos , Animais , Vareniclina/farmacologia , Antagonistas Nicotínicos/uso terapêutico , Agonistas Nicotínicos/farmacologia , Agonistas Nicotínicos/uso terapêutico , Alcaloides/farmacologia , Alcaloides/uso terapêutico , Receptores Nicotínicos/genética , Reperfusão , Isquemia Miocárdica/tratamento farmacológico , Traumatismo por Reperfusão/tratamento farmacológico , RNA Mensageiro/genéticaRESUMO
Cardiotoxins (CaTx) of the three-finger toxin family are one of the main components of cobra venoms. Depending on the structure of the N-terminal or the central polypeptide loop, they are classified into either group I and II or P- and S-types, respectively, and toxins of different groups or types interact with lipid membranes variably. While their main target in the organism is the cardiovascular system, there is no data on the effects of CaTxs from different groups or types on cardiomyocytes. To evaluate these effects, a fluorescence measurement of intracellular Ca2+ concentration and an assessment of the rat cardiomyocytes' shape were used. The obtained results showed that CaTxs of group I containing two adjacent proline residues in the N-terminal loop were less toxic to cardiomyocytes than group II toxins and that CaTxs of S-type were less active than P-type ones. The highest activity was observed for Naja oxiana cobra cardiotoxin 2, which is of P-type and belongs to group II. For the first time, the effects of CaTxs of different groups and types on the cardiomyocytes were studied, and the data obtained showed that the CaTx toxicity to cardiomyocytes depends on the structures both of the N-terminal and central polypeptide loops.
Assuntos
Proteínas Cardiotóxicas de Elapídeos , Contratura , Toxinas Biológicas , Ratos , Animais , Proteínas Cardiotóxicas de Elapídeos/farmacologia , Proteínas Cardiotóxicas de Elapídeos/toxicidade , Cálcio , Miócitos Cardíacos , Venenos Elapídicos/química , Peptídeos , Cálcio da DietaRESUMO
Snake venoms as tools for hunting are primarily aimed at the most vital systems of the prey, especially the nervous and circulatory systems. In general, snakes of the Elapidae family produce neurotoxic venoms comprising of toxins targeting the nervous system, while snakes of the Viperidae family and most rear-fanged snakes produce hemotoxic venoms directed mainly on blood coagulation. However, it is not all so clear. Some bites by viperids results in neurotoxic signs and it is now known that hemotoxic venoms do contain neurotoxic components. For example, viperid phospholipases A2 may manifest pre- or/and postsynaptic activity and be involved in pain and analgesia. There are other neurotoxins belonging to diverse families ranging from large multi-subunit proteins (e.g., C-type lectin-like proteins) to short peptide neurotoxins (e.g., waglerins and azemiopsin), which are found in hemotoxic venoms. Other neurotoxins from hemotoxic venoms include baptides, crotamine, cysteine-rich secretory proteins, Kunitz-type protease inhibitors, sarafotoxins and three-finger toxins. Some of these toxins exhibit postsynaptic activity, while others affect the functioning of voltage-dependent ion channels. This review represents the first attempt to systematize data on the neurotoxins from "non-neurotoxic" snake venom. The structural and functional characteristic of these neurotoxins affecting diverse targets in the nervous system are considered.
Assuntos
Neurotoxinas , Toxinas Biológicas , Animais , Humanos , Neurotoxinas/toxicidade , Venenos de Serpentes/toxicidade , Venenos de Serpentes/metabolismo , Elapidae/metabolismo , Fosfolipases A2 , Venenos Elapídicos/químicaRESUMO
The method based on the combination of Raman spectroscopy and principal component analysis (PCA) was applied to the set of peptide and protein toxins from animal venoms and to synthetic analogues of peptides. The study demonstrated the possibility of toxin classification according to their primary and secondary structures based on Raman spectroscopy. The method described here allows discrimination of snake venom three-finger toxins from predatory marine mollusks α-conotoxins. Moreover, PCA of the Raman spectra of toxins revealed differences within the group of three-finger toxins and also within the group of conotoxins, related to their spatial structure. In particular, on the basis of the developed technique it is possible to distinguish the disulfide isomers of the same peptide toxin. The results obtained have been confirmed by bioinformatic methods. So, we have proposed a method for the rapid analysis of newly discovered venom-derived protein or peptide toxins by establishing their similarity with other already studied toxins by referring to a particular class. Taking into account a low specimen consumption by Raman spectroscopy, the proposed method could represent a first step in the study of toxins from rare and/or endangered venomous animals. The ability to distinguish configuration of disulfide bonds allows to synthesize the correct isomer of the toxin.
Assuntos
Dissulfetos , Análise Espectral Raman , Animais , Dissulfetos/química , Peptídeos/química , Análise de Componente PrincipalRESUMO
The purpose of our review is to briefly show what different compounds of marine origin, from low molecular weight ones to peptides and proteins, offer for understanding the structure and mechanism of action of nicotinic acetylcholine receptors (nAChRs) and for finding novel drugs to combat the diseases where nAChRs may be involved. The importance of the mentioned classes of ligands has changed with time; a protein from the marine snake venom was the first excellent tool to characterize the muscle-type nAChRs from the electric ray, while at present, muscle and α7 receptors are labeled with the radioactive or fluorescent derivatives prepared from α-bungarotoxin isolated from the many-banded krait. The most sophisticated instruments to distinguish muscle from neuronal nAChRs, and especially distinct subtypes within the latter, are α-conotoxins. Such information is crucial for fundamental studies on the nAChR revealing the properties of their orthosteric and allosteric binding sites and mechanisms of the channel opening and closure. Similar data are provided by low-molecular weight compounds of marine origin, but here the main purpose is drug design. In our review we tried to show what has been obtained in the last decade when the listed classes of compounds were used in the nAChR research, applying computer modeling, synthetic analogues and receptor mutants, X-ray and electron-microscopy analyses of complexes with the nAChRs, and their models which are acetylcholine-binding proteins and heterologously-expressed ligand-binding domains.
Assuntos
Conotoxinas , Receptores Nicotínicos , Conotoxinas/química , Conotoxinas/farmacologia , Ligantes , Antagonistas Nicotínicos/química , Antagonistas Nicotínicos/farmacologia , Peptídeos/química , Receptores Nicotínicos/metabolismoRESUMO
Cobra cytotoxins (CTs) belong to the three-fingered protein family and possess membrane activity. Here, we studied cytotoxin 13 from Naja naja cobra venom (CT13Nn). For the first time, a spatial model of CT13Nn with both "water" and "membrane" conformations of the central loop (loop-2) were determined by X-ray crystallography. The "water" conformation of the loop was frequently observed. It was similar to the structure of loop-2 of numerous CTs, determined by either NMR spectroscopy in aqueous solution, or the X-ray method. The "membrane" conformation is rare one and, to date has only been observed by NMR for a single cytotoxin 1 from N. oxiana (CT1No) in detergent micelle. Both CT13Nn and CT1No are S-type CTs. Membrane-binding of these CTs probably involves an additional step-the conformational transformation of the loop-2. To confirm this suggestion, we conducted molecular dynamics simulations of both CT1No and CT13Nn in the Highly Mimetic Membrane Model of palmitoiloleoylphosphatidylglycerol, starting with their "water" NMR models. We found that the both toxins transform their "water" conformation of loop-2 into the "membrane" one during the insertion process. This supports the hypothesis that the S-type CTs, unlike their P-type counterparts, require conformational adaptation of loop-2 during interaction with lipid membranes.
Assuntos
Proteínas Cardiotóxicas de Elapídeos/química , Cristalografia por Raios X/métodos , Citotoxinas/química , Modelos Moleculares , Estrutura Molecular , Conformação ProteicaRESUMO
In aqueous solutions, cobra cytotoxins (CTX), three-finger folded proteins, exhibit conformational equilibrium between conformers with either cis or trans peptide bonds in the N-terminal loop (loop-I). The equilibrium is shifted to the cis form in toxins with a pair of adjacent Pro residues in this loop. It is known that CTX with a single Pro residue in loop-I and a cis peptide bond do not interact with lipid membranes. Thus, if a cis peptide bond is present in loop-I, as in a Pro-Pro containing CTX, this should weaken its lipid interactions and likely cytotoxic activities. To test this, we have isolated seven CTX from Naja naja and N. haje cobra venoms. Antibacterial and cytotoxic activities of these CTX, as well as their capability to induce calcein leakage from phospholipid liposomes, were evaluated. We have found that CTX with a Pro-Pro peptide bond indeed exhibit attenuated membrane-perturbing activity in model membranes and lower cytotoxic/antibacterial activity compared to their counterparts with a single Pro residue in loop-I.
Assuntos
Proteínas Cardiotóxicas de Elapídeos , Elapidae , Animais , Elapidae/metabolismo , Proteínas Cardiotóxicas de Elapídeos/toxicidade , Proteínas Cardiotóxicas de Elapídeos/química , Citotoxinas/toxicidade , Citotoxinas/química , Conformação Proteica , Venenos Elapídicos/toxicidade , Venenos Elapídicos/química , Fosfolipídeos/metabolismo , Peptídeos/toxicidadeRESUMO
Animal venoms comprise numerous toxin families, consisting mainly of peptides and proteins [...].
Assuntos
Peptídeos , Peçonhas , Animais , Humanos , Peptídeos/farmacologia , Peptídeos/uso terapêutico , Peptídeos/toxicidade , Peçonhas/farmacologia , Peçonhas/uso terapêutico , Peçonhas/toxicidadeRESUMO
Cobra cytotoxins (CTs), the three-fingered proteins, feature high amino acid sequence homology in the beta-strands and variations in the loop regions. We selected a pair of cytotoxins from Naja kaouthia crude venom to clarify the sequence-structure relationships. Using chromatography and mass spectroscopy, we separated and identified the mixture of cytotoxins 2 and 3, differentiated by the only Val 41/Ala 41 substitution. Here, using natural abundance 13C, 15N NMR-spectroscopy we performed chemical shift assignments of the signals of the both toxins in aqueous solution in the major and minor forms. Combining NOE and chemical shift data, the toxins' spatial structure was determined. Finally, we proved that the tip of the "finger"-2, or the loop-2 of cytotoxins adopts the shape of an omega-loop with a tightly-bound water molecule in its cavity. Comparison with other NMR and X-ray structures of cytotoxins possessing different amino acid sequences reveals spatial similarity in this family of proteins, including the loop-2 region, previously considered to be flexible.
Assuntos
Proteínas Cardiotóxicas de Elapídeos/química , Proteínas Cardiotóxicas de Elapídeos/genética , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Proteínas Cardiotóxicas de Elapídeos/classificação , Venenos Elapídicos/química , Venenos Elapídicos/genética , Elapidae/genética , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Conformação ProteicaRESUMO
Among the brain tumors, glioma is the most common. In general, different biochemical mechanisms, involving nicotinic acetylcholine receptors (nAChRs) and the arachidonic acid cascade are involved in oncogenesis. Although the engagement of the latter in survival and proliferation of rat C6 glioma has been shown, there are practically no data about the presence and the role of nAChRs in C6 cells. In this work we studied the effects of nAChR antagonists, marine snail α-conotoxins and snake α-cobratoxin, on the survival and proliferation of C6 glioma cells. The effects of the lipoxygenase and cyclooxygenase inhibitors either alone or together with α-conotoxins and α-cobratoxin were studied in parallel. It was found that α-conotoxins and α-cobratoxin promoted the proliferation of C6 glioma cells, while nicotine had practically no effect at concentrations below 1 µL/mL. Nordihydroguaiaretic acid, a nonspecific lipoxygenase inhibitor, and baicalein, a 12-lipoxygenase inhibitor, exerted antiproliferative and cytotoxic effects on C6 cells. nAChR inhibitors weaken this effect after 24 h cultivation but produced no effects at longer times. Quantitative real-time polymerase chain reaction showed that mRNA for α4, α7, ß2 and ß4 subunits of nAChR were expressed in C6 glioma cells. This is the first indication for involvement of nAChRs in mechanisms of glioma cell proliferation.
Assuntos
Proteínas Neurotóxicas de Elapídeos/farmacologia , Conotoxinas/farmacologia , Glioma/tratamento farmacológico , Antagonistas Nicotínicos/farmacologia , Animais , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Inibidores de Ciclo-Oxigenase/farmacologia , Glioma/patologia , Inibidores de Lipoxigenase/farmacologia , Nicotina/farmacologia , Ratos , Receptores Nicotínicos/efeitos dos fármacos , Receptores Nicotínicos/metabolismo , Fatores de TempoRESUMO
Cobra venoms contain three-finger toxins (TFT) including α-neurotoxins efficiently binding nicotinic acetylcholine receptors (nAChRs). As shown recently, several TFTs block GABAA receptors (GABAARs) with different efficacy, an important role of the TFTs central loop in binding to these receptors being demonstrated. We supposed that the positive charge (Arg36) in this loop of α-cobratoxin may explain its high affinity to GABAAR and here studied α-neurotoxins from African cobra N. melanoleuca venom for their ability to interact with GABAARs and nAChRs. Three α-neurotoxins, close homologues of the known N. melanoleuca long neurotoxins 1 and 2, were isolated and sequenced. Their analysis on Torpedocalifornica and α7 nAChRs, as well as on acetylcholine binding proteins and on several subtypes of GABAARs, showed that all toxins interacted with the GABAAR much weaker than with the nAChR: one neurotoxin was almost as active as α-cobratoxin, while others manifested lower activity. The earlier hypothesis about the essential role of Arg36 as the determinant of high affinity to GABAAR was not confirmed, but the results obtained suggest that the toxin loop III may contribute to the efficient interaction of some long-chain neurotoxins with GABAAR. One of isolated toxins manifested different affinity to two binding sites on Torpedo nAChR.
Assuntos
Colinérgicos/farmacologia , Proteínas Neurotóxicas de Elapídeos/farmacologia , Venenos Elapídicos/metabolismo , Antagonistas de Receptores de GABA-A/farmacologia , Naja , Receptores de GABA/efeitos dos fármacos , Receptor Nicotínico de Acetilcolina alfa7/efeitos dos fármacos , Animais , Sítios de Ligação , Ligação Competitiva , Linhagem Celular Tumoral , Colinérgicos/metabolismo , Proteínas Neurotóxicas de Elapídeos/metabolismo , Antagonistas de Receptores de GABA-A/metabolismo , Potenciais da Membrana , Camundongos , Ligação Proteica , Conformação Proteica , Receptores de GABA/genética , Receptores de GABA/metabolismo , Relação Estrutura-Atividade , Torpedo , Xenopus laevis , Receptor Nicotínico de Acetilcolina alfa7/metabolismoRESUMO
Lynx1, membrane-bound protein co-localized with the nicotinic acetylcholine receptors (nAChRs) and regulates their function, is a three-finger protein (TFP) made of three ß-structural loops, similarly to snake venom α-neurotoxin TFPs. Since the central loop II of α-neurotoxins is involved in binding to nAChRs, we have recently synthesized the fragments of Lynx1 central loop, including those with the disulfide between Cys residues introduced at N- and C-termini, some of them inhibiting muscle-type nAChR similarly to the whole-size water-soluble Lynx1 (ws-Lynx1). Literature shows that the main fragment interacting with TFPs is the C-loop of both nAChRs and acetylcholine binding proteins (AChBPs) while some ligand-binding capacity is preserved by analogs of this loop, for example, by high-affinity peptide HAP. Here we analyzed the structural organization of these peptide models of ligands and receptors and its role in binding. Thus, fragments of Lynx1 loop II, loop C from the Lymnaea stagnalis AChBP and HAP were synthesized in linear and Cys-cyclized forms and structurally (CD and NMR) and functionally (radioligand assay on Torpedo nAChR) characterized. Connecting the C- and N-termini by disulfide in the ws-Lynx1 fragment stabilized its conformation which became similar to the loop II within the 1H-NMR structure of ws-Lynx1, the activity being higher than for starting linear fragment but lower than for peptide with free cysteines. Introduced disulfides did not considerably change the structure of HAP and of loop C fragments, the former preserving high affinity for α-bungarotoxin, while, surprisingly, no binding was detected with loop C and its analogs.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Bungarotoxinas/química , Proteínas de Transporte/ultraestrutura , Receptores Nicotínicos/química , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/ultraestrutura , Sítios de Ligação , Proteínas de Transporte/química , Humanos , Ligantes , Lymnaea/química , Lymnaea/genética , Modelos Moleculares , Neurotoxinas/química , Peptídeos/química , Ligação Proteica/genética , Conformação Proteica em Folha beta , Receptores Nicotínicos/ultraestruturaRESUMO
Nerve growth factor (NGF) is a minor and neglected component of snake venom. Present study describes the purification and characterization of a NGF isoform (RVV-NGFa) from Indian Russell's viper venom (RVV). RVV-NGFa showed a protonated molecular ion [MH+] at m/z 17388.725 Da. The RVV-NGFa induced neuritogenesis in PC-12 cells but did not show cytotoxicity in mammalian cells, hemolytic activity, platelet modulation, and interference in blood coagulation system which are the characteristic pharmacological properties of RVV. By ELISA and immunofluorescence microplate reader assay, the RVV-NGFa showed appreciable binding to TrkA receptor expressed in breast cancer MDA-MB-231 and MCF-7 cells; nevertheless, pre-incubation of cells with anti-TrkA (and not TrkB or TrkC) or anti-p75NTR antibody significantly decreased (p < 0.05) this binding. The RVV-NGFa demonstrated insignificant binding with non-cancerous cells (HEK-293, L6) lacking TrkA receptor. The binding of RVV-NGFa to TrkA receptor of breast cancer cells resulted in internalization of ligand (RVV-NGFa)-receptor (TrkA) complex to cell cytoplasm in a time-dependent manner. The spectrofluorometric study demonstrated an interaction between RVV-NGFa and cytosolic domain of the purified TrkA receptor. The fluorescence (FITC)-tagged RVV-NGFa depicted a strong fluorescence signal that was observed under a fluorescence microscope and determined by fluorescence microplate reader assay post binding to breast cancer cells; but no fluorescence signal was detected after incubating FITC-RVV-NGFa with non-cancerous L6 and HEK-293 cells. The clinical application of FITC/fluorescence nanoparticle tagged RVV-NGFa for the ex vivo and in vivo diagnosis of breast cancer is highly promising.
Assuntos
Neoplasias da Mama , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Nanopartículas , Proteínas de Neoplasias/biossíntese , Fator de Crescimento Neural , Imagem Óptica , Receptor trkA/biossíntese , Venenos de Víboras , Animais , Neoplasias da Mama/diagnóstico por imagem , Neoplasias da Mama/enzimologia , Feminino , Fluoresceína-5-Isotiocianato/química , Fluoresceína-5-Isotiocianato/farmacologia , Células HEK293 , Humanos , Células MCF-7 , Fator de Crescimento Neural/química , Fator de Crescimento Neural/farmacologia , Células PC12 , Ratos , Coloração e Rotulagem , Venenos de Víboras/química , Venenos de Víboras/farmacologiaRESUMO
Snake venoms possess lethal activities against different organisms, ranging from bacteria to higher vertebrates. Several venoms were shown to be active against protozoa, however, data about the anti-protozoan activity of cobra and viper venoms are very scarce. We tested the effects of venoms from several snake species on the ciliate Tetrahymena pyriformis. The venoms tested induced T. pyriformis immobilization, followed by death, the most pronounced effect being observed for cobra Naja sumatrana venom. The active polypeptides were isolated from this venom by a combination of gel-filtration, ion exchange and reversed-phase HPLC and analyzed by mass spectrometry. It was found that these were cytotoxins of the three-finger toxin family. The cytotoxins from several cobra species were tested and manifested toxicity for infusorians. Light microscopy revealed that, because of the cytotoxin action, the infusorians' morphology was changed greatly, from teardrop-like to an almost spherical shape, this alteration being accompanied by a leakage of cell contents. Fluorescence microscopy showed that the fluorescently labelled cytotoxin 2 from cobra N. oxiana was localized mainly at the membrane of killed infusorians, indicating that cytotoxins may kill T. pyriformis by causing membrane rupture. This work is the first evidence of the antiprotozoal activity of cobra venom cytotoxins, as demonstrated by the example of the ciliate T. pyriformis.
Assuntos
Antiprotozoários/farmacologia , Citotoxinas/farmacologia , Venenos Elapídicos/química , Peptídeos/farmacologia , Tetrahymena pyriformis/efeitos dos fármacos , Antiprotozoários/isolamento & purificação , Citotoxinas/isolamento & purificação , Peptídeos/isolamento & purificaçãoRESUMO
Immune response during sepsis is characterized by hyper-inflammation followed by immunosuppression. The crucial role of macrophages is well-known for both septic stages, since they are involved in immune homeostasis and inflammation, their dysfunction being implicated in immunosuppression. The cholinergic anti-inflammatory pathway mediated by macrophage α7 nicotinic acetylcholine receptor (nAChR) represents possible drug target. Although α7 nAChR activation on macrophages reduces the production of proinflammatory cytokines, the role of these receptors in immunological changes at the cellular level is not fully understood. Using α7 nAChR selective agonist PNU 282,987, we investigated the influence of α7 nAChR activation on the expression of cytokines and, for the first time, of the macrophage membrane markers: cluster of differentiation 14 (CD14), human leukocyte antigen-DR (HLA-DR), CD11b, and CD54. Application of PNU 282,987 to THP-1MÏ (THP-1 derived macrophages) cells led to inward ion currents and Ca2+ increase in cytoplasm showing the presence of functionally active α7 nAChR. Production of cytokines tumor necrosis factor-α (TNF-α), interleukin (IL)-6, and IL-10 was estimated in classically activated macrophages (M1) and treatment with PNU 282,987 diminished IL-10 expression. α7 nAChR activation on THP-1MÏ, THP-1M1, and monocyte-derived macrophages (MDMs) increased the expression of HLA-DR, CD54, and CD11b molecules, but decreased CD14 receptor expression, these effects being blocked by alpha (α)-bungarotoxin. Thus, PNU 282,987 enhances the macrophage-mediated immunity via α7 nAChR by regulating expression of their membrane receptors and of cytokines, both playing an important role in preventing immunosuppressive states.
Assuntos
Imunidade Adaptativa/fisiologia , Antígenos HLA-DR/imunologia , Tolerância Imunológica/efeitos dos fármacos , Macrófagos/metabolismo , Receptor Nicotínico de Acetilcolina alfa7/metabolismo , Imunidade Adaptativa/efeitos dos fármacos , Benzamidas/farmacologia , Compostos Bicíclicos com Pontes/farmacologia , Citocinas/metabolismo , Humanos , Tolerância Imunológica/imunologia , Interleucina-10/metabolismo , Receptores de Lipopolissacarídeos/metabolismo , Lipopolissacarídeos/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Agonistas Nicotínicos/farmacologia , Técnicas de Patch-Clamp , Células THP-1 , Receptor Nicotínico de Acetilcolina alfa7/genética , Receptor Nicotínico de Acetilcolina alfa7/imunologiaRESUMO
Several biochemical mechanisms, including the arachidonic acid cascade and activation of nicotinic acetylcholine receptors (nAChRs), are involved in increased tumor survival. Combined application of inhibitors acting on these two pathways may result in a more pronounced antitumor effect. Here, we show that baicalein (selective 12-lipoxygenase inhibitor), nordihydroguaiaretic acid (non-selective lipoxygenase inhibitor), and indomethacin (non-selective cyclooxygenase inhibitor) are cytotoxic to Ehrlich carcinoma cells in vitro. Marine snail α-conotoxins PnIA, RgIA and ArIB11L16D, blockers of α3ß2/α6ß2, α9α10 and α7 nAChR subtypes, respectively, as well as α-cobratoxin, a blocker of α7 and muscle subtype nAChRs, exhibit low cytotoxicity, but enhance the antitumor effect of baicalein 1.4-fold after 24 h and that of nordihydroguaiaretic acid 1.8-3.9-fold after 48 h of cell cultivation. α-Conotoxin MII, a blocker of α6-containing and α3ß2 nAChR subtypes, increases the cytotoxic effect of indomethacin 1.9-fold after 48 h of cultivation. In vivo, baicalein, α-conotoxins MII and PnIA inhibit Ehrlich carcinoma growth and increase mouse survival; these effects are greatly enhanced by the combined application of α-conotoxin MII with indomethacin or conotoxin PnIA with baicalein. Thus, we show, for the first time, antitumor synergism of α-conotoxins and arachidonic acid cascade inhibitors.
Assuntos
Carcinoma de Ehrlich/tratamento farmacológico , Sobrevivência Celular/efeitos dos fármacos , Conotoxinas/farmacologia , Inibidores de Ciclo-Oxigenase/farmacologia , Inibidores de Lipoxigenase/farmacologia , Antagonistas Nicotínicos/farmacologia , Animais , Ácido Araquidônico/antagonistas & inibidores , Carcinoma/tratamento farmacológico , Proteínas Neurotóxicas de Elapídeos/farmacologia , Sinergismo Farmacológico , Flavanonas/farmacologia , Indometacina/farmacologia , Masoprocol/farmacologia , Camundongos , Receptores NicotínicosRESUMO
Excitatory α7 neuronal nicotinic receptors (nAChR) are widely expressed in the central and peripheral nervous and immune systems and are important for learning, memory, and immune response regulation. Specific α7 nAChR ligands, including positive allosteric modulators are promising to treat cognitive disorders, inflammatory processes, and pain. One of them, PNU-120596, highly increased the neuron response to α7 agonists and retarded desensitization, showing selectivity for α7 as compared to heteromeric nAChRs, but was not examined at the inhibitory ligand-gated channels. We studied PNU-120596 action on anion-conducting channels using voltage-clamp techniques: it slightly potentiated the response of human glycine receptors expressed in PC12 cells, of rat GABAA receptors in cerebellar Purkinje cells and mouse GABAA Rs heterologously expressed in Xenopus oocytes. On the contrary, PNU-120596 exerted an inhibitory effect on the receptors mediating anion currents in Lymnaea stagnalis neurons: two nAChR subtypes, GABA and glutamate receptors. Acceleration of the current decay, contrary to slowing down desensitization in mammalian α7 nAChR, was observed in L. stagnalis neurons predominantly expressing one of the two nAChR subtypes. Thus, PNU-120596 effect on these anion-selective nAChRs was just opposite to the action on the mammalian cation-selective α7 nAChRs. A comparison of PNU-120596 molecule docked to the models of transmembrane domains of the human α7 AChR and two subunits of L. stagnalis nAChR demonstrated some differences in contacts with the amino acid residues important for PNU-120596 action on the α7 nAChR. Thus, our results show that PNU-120596 action depends on a particular subtype of these Cys-loop receptors.
Assuntos
Canais de Cloreto/metabolismo , Isoxazóis/farmacologia , Canais Iônicos de Abertura Ativada por Ligante/metabolismo , Compostos de Fenilureia/farmacologia , Receptor Nicotínico de Acetilcolina alfa7/agonistas , Receptor Nicotínico de Acetilcolina alfa7/metabolismo , Regulação Alostérica/efeitos dos fármacos , Regulação Alostérica/fisiologia , Sequência de Aminoácidos , Animais , Células Cultivadas , Canais de Cloreto/antagonistas & inibidores , Canais de Cloreto/genética , Feminino , Humanos , Canais Iônicos de Abertura Ativada por Ligante/antagonistas & inibidores , Canais Iônicos de Abertura Ativada por Ligante/genética , Lymnaea , Células PC12 , Ratos , Ratos Wistar , Xenopus laevis , Receptor Nicotínico de Acetilcolina alfa7/genéticaRESUMO
Antibacterial activity of the three-finger toxins from cobra venom, including cytotoxin 3 from N. kaouthia, cardiotoxin-like basic polypeptide A5 from N. naja (CLBP), and alpha-neurotoxin from N. oxiana venom, was investigated. All toxins failed to influence Gram-negative bacteria. The most pronounced activity against Bacillus subtilis was demonstrated by CLBP. The latter is ascribed to the presence of additional Lys-residues within the membrane-binding motif of this toxin.
Assuntos
Antibacterianos/química , Venenos Elapídicos/metabolismo , Peptídeos/química , Sequência de Aminoácidos , Animais , Antibacterianos/isolamento & purificação , Antibacterianos/farmacologia , Bacillus subtilis/efeitos dos fármacos , Cardiotoxinas/química , Elapidae/metabolismo , Escherichia coli/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Peptídeos/isolamento & purificação , Peptídeos/farmacologia , Estrutura Terciária de Proteína , Staphylococcus aureus/efeitos dos fármacosRESUMO
BACKGROUND: Snake venoms are the complex mixtures of different compounds manifesting a wide array of biological activities. The venoms of kraits (genus Bungarus, family Elapidae) induce mainly neurological symptoms; however, these venoms show a cytotoxicity against cancer cells as well. This study was conducted to identify in Bungarus fasciatus venom an active compound(s) exerting cytotoxic effects toward MCF7 human breast cancer cells and A549 human lung cancer cells. METHODS: The crude venom of B. fasciatus was separated by gel-filtration on Superdex HR 75 column and reversed phase HPLC on C18 column. The fractions obtained were screened for cytotoxic effect against MCF7, A549, and HK2 cell lines using colorimetric assay with the tetrazolium dye MTT- 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. The primary structure of active protein was established by ultra high resolution LC-MS/MS. The molecular mechanism of the isolated protein action on MCF7 cells was elucidated by flow cytometry. RESULTS: MTT cell viability assays of cancer cells incubated with fractions isolated from B. fasciatus venom revealed a protein with molecular mass of about 13 kDa possessing significant cytotoxicity. This protein manifested the dose and time dependent cytotoxicity for MCF7 and A549 cell lines while showed no toxic effect on human normal kidney HK2 cells. In MCF7, flow cytometry analysis revealed a decrease in the proportion of Ki-67 positive cells. As Ki-67 protein is a cellular marker for proliferation, its decline indicates the reduction in the proliferation of MCF7 cells treated with the protein. Flow cytometry analysis of MCF7 cells stained with propidium iodide and Annexin V conjugated with allophycocyanin showed that a probable mechanism of cell death is apoptosis. Mass spectrometric studies showed that the cytotoxic protein was phospholipase A2. The amino acid sequence of this enzyme earlier was deduced from cloned cDNA, and in this work it was isolated from the venom as a protein for the first time. It is also the first krait phospholipase A2 manifesting the cytotoxicity for cancer cells.