RESUMO
The dysfunction of adipose tissue with aging and the accumulation of senescent cells has been implicated in the pathophysiology of chronic diseases. Recently interventions capable of reducing the burden of senescent cells and in particular the identification of a new class of drugs termed senolytics have been object of extensive investigation. We used an in vitro model of induced senescence by treating both pre-adipocytes as well as mature adipocytes with hydrogen peroxide (H2O2) at a sub-lethal concentration for 3 h for three consecutive days, and hereafter with 20 uM quercetin at a dose that in preliminary experiments resulted to be senolytic without cytotoxicity. H2O2 treated pre-adipocytes and adipocytes showed typical senescence-associated features including increased beta-galactosidase activity (SA-ß-gal) and p21, activation of ROS and increased expression of pro-inflammatory cytokines. The treatment with quercetin in senescent pre-adipocytes and adipocytes was associated to a significant decrease in the number of the SA-ß-gal positive cells along with the suppression of ROS and of inflammatory cytokines. Besides, quercetin treatment decreased miR-155-5p expression in both models, with down-regulation of p65 and a trend toward an up-regulation of SIRT-1 in complete cell extracts. The senolytic compound quercetin could affect AT ageing by reducing senescence, induced in our in vitro model by oxidative stress. The downregulation of miRNA-155-5p, possibly through the modulation of NF-κB and SIRT-1, could have a key role in the effects of quercetin on both pre-adipocytes and adipocytes.
Assuntos
Adipócitos/efeitos dos fármacos , Quercetina/farmacologia , Senoterapia/farmacologia , Células 3T3-L1 , Envelhecimento/efeitos dos fármacos , Animais , Senescência Celular/efeitos dos fármacos , Peróxido de Hidrogênio , Camundongos , MicroRNAs/metabolismo , Estresse Oxidativo/efeitos dos fármacosRESUMO
Aging is a physiological process characterized by an age-progressive decline in intrinsic physiological functions, with an increased risk of developing chronic metabolic conditions, such as insulin resistance and diabetes. Furthermore, from a physiopathological point of view, several authors describe an association between oxidative stress, hypoxia and these metabolic conditions. It had been suggested that adipose tissue (AT) dysfunction, senescent cell accumulation and proinflammatory pathways may be involved in this processes. The purpose of this study was to develop an in vitro model to study the progressive morphological and functional changes of adipocytes with aging, in standard culture conditions and after severe hypoxia and hydrogen peroxide treatment. We evaluated the degree of apoptosis and intracellular reactive oxygen species (ROS) accumulation as well as the gene expression profile of aging adipocytes. Our results show that aged adipocytes become senescent, undergo apoptosis, accumulate ROS, and present an inflammatory profile with an increase in mRNA expression level of key proteins related to the remodeling of the extracellular matrix (ECM). Aged adipocytes present increased levels of p53, p21 and p16, key regulators of senescence, and a decrease in SIRT-1 protein compared to younger cells. Moreover, adipocytes aged in hypoxia or in oxidative stress conditions represent a model of accelerated aging with a decrease in their area, a greater proportion of apoptotic and of intracellular ROS accumulation compared to controls. This study characterizes the progressive morphological and functional changes in aging adipocytes during prolonged cell cultures and explores the addictive effects of hypoxia and oxidation, given at different stages of cellular maturation and senescence.
Assuntos
Adipócitos/fisiologia , Hipóxia Celular/fisiologia , Senescência Celular/fisiologia , Estresse Oxidativo/fisiologia , Apoptose/fisiologia , Células Cultivadas , Humanos , Técnicas In Vitro , Oxirredução , Espécies Reativas de Oxigênio/metabolismoRESUMO
α-Bisabolol (BSB) is a plant-derived sesquiterpene alcohol able to trigger regulated cell death in transformed cells, while deprived of the general toxicity in several mouse models. Here, we investigated the involvement of lysosomal and mitochondrial compartments in the cytotoxic effects of BSB, with a specific focus on the BH3-only activator protein BID. We found that BSB particularly accumulated in cancer cell lines, displaying a higher amount of lipid rafts as compared to normal blood cells. By means of western blotting and microscopy techniques, we documented rapid BSB-induced BID translocation to lysosomes and mitochondria, both of them becoming dysfunctional. Lysosomal membranes were permeabilized, thus blocking the cytoprotective autophagic flux and provoking cathepsin B leakage into the cytosol. Multiple flow cytometry-based experiments demonstrated the loss of mitochondrial membrane potential due to pore formation across the lipid bilayer. These parallel events converged on neoplastic cell death, an outcome significantly prevented by BID knockdown. Therefore, BSB promoted BID redistribution to the cell death executioner organelles, which in turn activated anti-autophagic and proapoptotic mechanisms. This is an example of how xenohormesis can be exploited to modulate basic cellular programs in cancer.
Assuntos
Apoptose/efeitos dos fármacos , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Lisossomos/metabolismo , Mitocôndrias/metabolismo , Sesquiterpenos Monocíclicos/farmacologia , Autofagia/efeitos dos fármacos , Benzimidazóis/farmacologia , Carbocianinas/farmacologia , Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Gangliosídeo G(M1)/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Lisossomos/efeitos dos fármacos , Microdomínios da Membrana/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Proteínas de Membrana/metabolismo , Mitocôndrias/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacosRESUMO
In a precedent report we showed that alpha-bisabolol, a sesquiterpene present widely in the plant kingdom, exerts a rapid and efficient apoptosis-inducing action selectively towards human and murine malignant glioblastoma cell lines through mitochondrial damage. The present study extends these data demonstrating the apoptosis-inducing action of alpha-bisabolol towards highly malignant human pancreatic carcinoma cell lines without affecting human fibroblast viability. The present study further shows the preferential incorporation of alpha-bisabolol to transformed cells through lipid rafts on plasma membranes and, thereafter, direct interaction between alpha-bisabolol and Bid protein, one of pro-apoptotic Bcl-2 family proteins, analyzed either by Surface Plasmon Resonance method or by intrinsic fluorescence measurement. Notions that lipid rafts are rich in plasma membranes of transformed cells and that Bid, richly present in lipid rafts, is deeply involved in lipid transport make highly credible the hypothesis that the molecular mechanism of alpha-bisabolol action may include its capacity to interact with Bid protein.
Assuntos
Apoptose/efeitos dos fármacos , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Microdomínios da Membrana/metabolismo , Sesquiterpenos/farmacologia , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/química , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/genética , Caspase 3/análise , Caspase 3/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Humanos , Potenciais da Membrana/efeitos dos fármacos , Mitocôndrias/metabolismo , Modelos Moleculares , Estrutura Molecular , Sesquiterpenos Monocíclicos , Neoplasias Pancreáticas/patologia , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Sesquiterpenos/química , Ressonância de Plasmônio de Superfície , Fatores de TempoRESUMO
AIMS: Beclomethasone/formoterol (BDP/FOR) has been reported to be more effective than its separate components in airway disease control and in airway inflammation improvement. However, BDP/FOR effects on cytokine-induced inflammation in structural cells have not been described and whether these effects occur in a cell- and mediator-dependent manner has not been fully elucidated. We sought to evaluate BDP and/or FOR effects on endothelial ICAM-1, E-selectin, IL-8 and on bronchial epithelial ICAM-1 and IL-8. Specific intracellular signaling pathways were also investigated. MATERIALS AND METHODS: Surface adhesion molecule expression and IL-8 release induced by TNF-alpha were measured by ELISA. Intracellular signaling pathways were investigated by a) EMSA and Western blot analysis to evaluate NF-κB DNA-binding and MAPK-p38 phosphorylation; b) PDTC/SB203580 as NF-κB/p38 inhibitors; c) forskolin/H-89 as PKA activator/inhibitor. KEY FINDINGS: BDP/FOR additively reduced endothelial E-selectin and IL-8 as well as bronchial epithelial ICAM-1 and IL-8. BDP/FOR and SB203580 showed the highest inhibitory effect on epithelial IL-8, whereas endothelial ICAM-1 was never affected by BDP/FOR and PDTC. TNF-alpha-induced NF-κB DNA-binding and MAPK-p38 phosphorylation were not influenced by BDP/FOR. Forskolin mimicked FOR effects; H-89 partially reversed the BDP/FOR inhibition in a mediator-dependent manner. SIGNIFICANCE: The BDP/FOR inhibition degree was related to the inflammatory mediator- and cell-type considered. FOR additively enhanced BDP effects by partially involving both dependent- and independent-PKA mechanisms. Our results might contribute to highlight the strong relationship between specific molecular pathways and different sensitivity to the corticosteroid/ß2-agonist effects and to clarify the molecular mechanisms underlying the BDP/FOR anti-inflammatory activity in vivo.
Assuntos
Beclometasona/farmacologia , Brônquios/efeitos dos fármacos , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Sinergismo Farmacológico , Fumarato de Formoterol/farmacologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , NF-kappa B/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Anti-Inflamatórios/farmacologia , Brônquios/metabolismo , Broncodilatadores/farmacologia , Adesão Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Feminino , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Transdução de Sinais/efeitos dos fármacosRESUMO
Despite enormous scientific and economic effort tumour still is one of the most terrible pathologies among human population all over the world. Products derived from the plant kingdom have often offered an opportunity to counteract or alleviate this illness. Here, we summarize the short story of the study of an extraordinary effect of one plant compound towards transformed cells derived from highly malignant tumours. Alpha-bisabolol, a sesquiterpene widely present in plants, selectively kills transformed cells by apoptosis without affecting the viability of normal cells. One of its intracellular targets seems to be situated on mitochondria and is possibly identified as the permeability transition pore, as judged from rapid mitochondrial membrane potential dissipation induced by alpha-bisabolol and the failure to kill cells in the presence of cyclosporine A. Preferential adsorption of alpha-bisabolol into lipid rafts, rich in tumour cells, may explain the selective action of this compounds towards tumour cells. Furthermore, Surface Plasmon Resonance analysis indicates that alpha-bisabolol directly interacts with Bid protein, a member of the Bcl2 family deeply involved in apoptosis, suggesting a possibility that Bid, or similar protein(s), may be involved in a putative intracellular transport system of alpha-bisabolol from plasma membrane to mitochondria. Experiments with animals indicate that alpha-bisabolol is not toxic and is accumulated, through blood flow, in every tissues examined. Further animal studies to test its effect are currently under way.
Assuntos
Apoptose/efeitos dos fármacos , Neoplasias/prevenção & controle , Extratos Vegetais/farmacologia , Sesquiterpenos/farmacologia , Animais , Apoptose/fisiologia , Linhagem Celular Tumoral , Humanos , Modelos Biológicos , Sesquiterpenos Monocíclicos , Neoplasias/patologia , Neoplasias/fisiopatologia , Extratos Vegetais/química , Sesquiterpenos/química , Transdução de SinaisRESUMO
OBJECTIVE: The role of peritumoral adipose tissue (AT) has not been extensively studied in colorectal cancer (CRC). METHODS: This study was conducted in 20 male subjects undergoing elective surgery for CRC. The differences between the peritumoral visceral adipose tissue (P-VAT), visceral adipose tissue (VAT), and subcutaneous adipose tissue (SAT) of the patients were described via immunohistochemistry and molecular biology analyses. The interactions between adipocytes and a colon cancer cell line were also investigated by using an in vitro coculture system. RESULTS: The analyses revealed that adipocytes near the tumor were significantly smaller than the adipocytes from other sites. The P-VAT was preferentially infiltrated by a CD68+/CD163+/IDO- macrophage subset with a prevalent reparative inflammatory response, while the macrophages identified in VAT and SAT mainly presented inflammatory features. Furthermore, the P-VAT presented a higher expression of adiponectin compared with other sites. Morphological analysis in vitro showed that after a few days of coculture, 3T3-L1 adipocytes were reduced in number and size with an increase in lipolysis rate and dedifferentiation phenomena. CONCLUSIONS: This study reveals important morphological and functional changes in the AT surrounding the tumor as an increase in lipolysis and in adiponectin-producing adipocytes, preferentially infiltrated by a macrophage subset, with prevalent reparative inflammatory response.
Assuntos
Tecido Adiposo/metabolismo , Neoplasias Colorretais/patologia , Idoso , Idoso de 80 Anos ou mais , Humanos , Gordura Intra-Abdominal/metabolismo , Masculino , Pessoa de Meia-Idade , Gordura Subcutânea/metabolismoRESUMO
A significant epidemiological association between obesity and pancreatic ductal adenocarcinoma (PDAC) has previously been described, as well as a correlation between the degree of pancreatic steatosis, PDAC risk and prognosis. The underlying mechanisms are still not completely known.After co-culture of 3T3-L1 adipocytes and MiaPaCa2 with an in vitro transwell system we observed the appearance of fibroblast-like cells, along with a decrease in number and size of remaining adipocytes. RT-PCR analyses of 3T3-L1 adipocytes in co-culture showed a decrease in gene expression of typical markers of mature adipocytes, in parallel with an increased expression of fibroblast-specific and reprogramming genes. We found an increased WNT5a gene and protein expression early in MiaPaCa2 cells in co-culture. Additionally, EMSA of c-Jun and AP1 in 3T3-L1 demonstrated an increased activation in adipocytes after co-culture. Treatment with WNT5a neutralizing antibody completely reverted the activation of c-Jun and AP1 observed in co-cultured adipocytes.Increasing doses of recombinant SFRP-5, a competitive inhibitor for WNT5a receptor, added to the co-culture medium, were able to block the dedifferentiation of adipocytes in co-culture.These data support a WNT5a-mediated dedifferentiation process with adipocytes reprogramming toward fibroblast-like cells that might profoundly influence cancer microenvironment.
Assuntos
Adipócitos/patologia , Desdiferenciação Celular , Fibroblastos/patologia , Neoplasias Pancreáticas/patologia , Proteína Wnt-5a/metabolismo , Células 3T3-L1 , Adipócitos/metabolismo , Animais , Apoptose , Proliferação de Células , Células Cultivadas , Técnicas de Cocultura , Fibroblastos/metabolismo , Camundongos , Neoplasias Pancreáticas/metabolismo , Microambiente TumoralRESUMO
Cyclooxygenase (COX) is widely considered as the molecular target of non-steroid anti-inflammatory drugs (NSAIDs). However, due to the harmful side effect frequently observed following chronic use, the development of new anti-inflammatory agents is the matter of many studies. Signal transducers and activators of transcription (STAT) are a family of nuclear proteins mediating the action of a number of cytokines. Among them, STAT1 plays a critical role in the signal transduction pathway of interferon-gamma (IFN-gamma) and growth hormone. STAT1 cascade is one major signalling pathway converting the IFN-gamma signal into gene expression, such as inducible nitric oxide synthase (iNOS), COX, vascular cell adhesion molecules (VCAM) and intercellular cell adhesion molecules (ICAM), critically involved in different pathologies correlated to the inflammatory process. This review focuses the attention on an alternative approach to the development of novel drugs based on inhibition of STAT1 pathway. In this context, a growing interest has been focused on natural compounds. We have recently reported a several data indicating that green tea extract (GTE), St. John's Wort extract and epigallocatechin-3-gallate (EGCG) exhibit a specific and strong anti-STAT1 activity which is independent of their acclaimed strong anti-oxidant activity. More recently, GTE has been shown to protect heart damage from ischaemia/reperfusion in rats, suggesting that the protective effect of green tea might be correlated to its anti-STAT1 activity. The present review is aimed at providing data that STAT1 may potentially be claimed as a new molecular target of an anti-inflammatory treatment and that among natural compounds there are a number of anti-STAT1 substances.
Assuntos
Anti-Inflamatórios/farmacologia , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Fator de Transcrição STAT1/antagonistas & inibidores , Fator de Transcrição STAT1/metabolismo , Animais , Anti-Inflamatórios/química , Anti-Inflamatórios/uso terapêutico , Doença Crônica , HumanosRESUMO
Our previous studies showed that (-)-epigallocatechin-3-gallate (EGCG) inhibits signal transducer activator of transcription 1 (STAT1) activation. Since EGCG may be a promising lead compound for new anti-STAT1 drug design, 15 synthetic catechins, characterized by the (-)-gallocatechin-3-gallate stereochemistry, were studied in the human mammary MDA-MB-231 cell line to identify the minimal structural features that preserve the anti-STAT1 activity. We demonstrate that the presence of three hydroxyl groups of B ring and one hydroxyl group in D ring is essential to preserve their inhibitory action. Moreover, a possible molecular target of these compounds in the STAT1 pathway was investigated. Our results demonstrate a direct interaction between STAT1 protein and catechins displaying anti-STAT1 activity. In particular, surface plasmon resonance (SPR) analysis and molecular modeling indicate the presence of two putative binding sites (a and b) with different affinity. Based on docking data, site-directed mutagenesis was performed, and interaction of the most active catechins with STAT1 was studied with SPR to test whether Gln518 on site a and His568 on site b could be important for the catechin-STAT1 interaction. Data indicate that site b has higher affinity for catechins than site a as the highest affinity constant disappears in the H568A-STAT1 mutant. Furthermore, Janus kinase 2 (JAK2) kinase assay data suggest that the contemporary presence in vitro of STAT1 and catechins inhibits JAK2-elicited STAT1 phosphorylation. The very tight catechin-STAT1 interaction prevents STAT1 phosphorylation and represents a novel, specific and efficient molecular mechanism for the inhibition of STAT1 activation.
Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Catequina/análogos & derivados , Desenho de Fármacos , Modelos Moleculares , Proteínas de Neoplasias/antagonistas & inibidores , Fator de Transcrição STAT1/antagonistas & inibidores , Substituição de Aminoácidos , Antineoplásicos/química , Antineoplásicos/metabolismo , Sítios de Ligação , Neoplasias da Mama/metabolismo , Catequina/química , Catequina/metabolismo , Catequina/farmacologia , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Conformação Molecular , Proteínas Mutantes/antagonistas & inibidores , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Fragmentos de Peptídeos/antagonistas & inibidores , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Fosforilação/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Fator de Transcrição STAT1/química , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/metabolismo , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
STAT3 is a latent transcription factor that promotes cell survival and proliferation and is often constitutively active in cancers. Although many reports provide evidence that STAT3 is a direct target of oxidative stress, its redox regulation is poorly understood. Under oxidative conditions STAT3 activity can be modulated by S-glutathionylation, a reversible redox modification of cysteine residues. This suggests the possible cross-talk between phosphorylation and glutathionylation and points out that STAT3 is susceptible to redox regulation. Recently, we reported that decreasing the GSH content in different cell lines induces inhibition of STAT3 activity through the reversible oxidation of thiol groups. In the present work, we demonstrate that GSH/diamide treatment induces S-glutathionylation of STAT3 in the recombinant purified form. This effect was completely reversed by treatment with the reducing agent dithiothreitol, indicating that S-glutathionylation of STAT3 was related to formation of protein-mixed disulfides. Moreover, addition of the bulky negatively charged GSH moiety impairs JAK2-mediated STAT3 phosphorylation, very likely interfering with tyrosine accessibility and thus affecting protein structure and function. Mass mapping analysis identifies two glutathionylated cysteine residues, Cys328 and Cys542, within the DNA-binding domain and the linker domain, respectively. Site direct mutagenesis and in vitro kinase assay confirm the importance of both cysteine residues in the complex redox regulatory mechanism of STAT3. Cells expressing mutant were resistant in this regard. The data presented herein confirmed the occurrence of a redox-dependent regulation of STAT3, identified the more redox-sensitive cysteines within STAT3 structure, and may have important implications for development of new drugs.
Assuntos
Cisteína/metabolismo , Glutationa/metabolismo , Fator de Transcrição STAT3/metabolismo , Células HeLa , Humanos , Oxirredução , Fosforilação , Fator de Transcrição STAT3/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
STAT3 is a transcription factor constitutively activated in a variety of cancers that has a critical role in the inhibition of apoptosis and induction of chemoresistance. Inhibition of the STAT3 signaling pathway suppresses cell survival signals and leads to apoptosis in cancer cells, suggesting that direct inhibition of STAT3 function is a viable therapeutic approach. Herein, we identify the naturally occurring sesquiterpene lactone cynaropicrin as a potent inhibitor of both IL-6-inducible and constitutive STAT3 activation (IC50=12 µM). Cynaropicrin, which contains an α-ß-unsaturated carbonyl moiety and acts as potent Michael reaction acceptor, induces a rapid drop in intracellular glutathione (GSH) concentration, thereby triggering S-glutathionylation of STAT3. Furthermore, glutathione ethylene ester, the cell permeable form of GSH, reverts the inhibitory action of cynaropicrin on STAT3 tyrosine phosphorylation. These findings suggest that this sesquiterpene lactone is able to induce redox-dependent post-translational modification of cysteine residues of STAT3 protein to regulate its function. STAT3 inhibition led to the suppression of two anti-apoptotic genes, Bcl-2 and survivin, in DU145 cells that constitutively express active STAT3. This event may be responsible for the decline in cell viability after cynaropicrin treatment. As revealed by PI/annexin-V staining, PARP cleavage, and DNA ladder formation, cynaropicrin cytotoxicity is mediated by apoptosis. Finally, cynaropicrin displayed a slight to strong synergism with two well-established chemotherapeutic drugs, cisplatin and docetaxel. Taken together our studies suggest that cynaropicrin suppresses the STAT3 pathway, leading to the down-regulation of STAT3-dependent gene expression and chemosensitization of tumour cells to chemotherapy.
Assuntos
Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Citotoxinas/farmacologia , Lactonas/farmacologia , Fator de Transcrição STAT3/antagonistas & inibidores , Sesquiterpenos/farmacologia , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cisplatino/farmacologia , Docetaxel , Resistencia a Medicamentos Antineoplásicos , Sinergismo Farmacológico , Glutationa/análogos & derivados , Glutationa/análise , Glutationa/metabolismo , Humanos , Proteínas Inibidoras de Apoptose/antagonistas & inibidores , Estresse Oxidativo , Fosforilação/efeitos dos fármacos , Poli(ADP-Ribose) Polimerases/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Espécies Reativas de Oxigênio/metabolismo , Fator de Transcrição STAT3/química , Transdução de Sinais , Survivina , Taxoides/farmacologiaRESUMO
From the bark of Guazuma ulmifolia, a plant used as anti-inflammatory remedy, the flavanocoumarin epiphyllocoumarin (1) along with epiphyllocoumarin-[4ßâ8]-(-)-epicatechin (2) and epiphyllocoumarin-[4ßâ8]-(-)-epicatechin-[4ßâ8]-(-)-epicatechin (3), never reported before, have been isolated. The structures of the proantocyanidins 2-3 have been elucidated by extensive NMR and ESI-MS analysis. On the basis of the flavane nature of the isolated compounds and considering the strong anti-STAT1 activity reported for epigallocatechin 3-O-gallate (EGCG), the affinity of compounds 1-3 for STAT1 has been evaluated by Surface Plasmon Resonance. Compounds 1-2 showed affinity for STAT1 comparable to that exerted by EGCG. In order to confirm this result, molecular docking studies to evaluate the interactions of compounds 1-3, phyllocoumarin (4), the reference compound EGCG (5), and its related compound (+)-gallocatechin 3-O-gallate (6) with STAT1 have been carried out. Furthermore the ability of compounds 1-3 to inhibit STAT1 activation has been evaluated using a nuclear extract obtained from the human monocytic leukemia cell line TPH-1. Flavanocoumarins 1-2 inhibited STAT1-DNA binding.
Assuntos
Cumarínicos/química , Cumarínicos/farmacologia , DNA/metabolismo , Malvaceae/química , Casca de Planta/química , Fator de Transcrição STAT1/metabolismo , Linhagem Celular , Humanos , Ligação Proteica/efeitos dos fármacos , Ressonância de Plasmônio de SuperfícieRESUMO
The main purpose of the present study is to envisage the molecular mechanism of inhibitory action of dehydrocostuslactone (DCE) and costunolide (CS), two naturally occurring sesquiterpene lactones, towards the activation of signal transducer and activator of transcription 3 (STAT3). We report that, in human THP-1 cell line, they inhibit IL-6-elicited tyrosine phosphorylation of STAT3 and its DNA binding activity with EC(50) of 10 µM with concomitant down-regulation of the phosphorylation of the tyrosine Janus kinases JAK1, JAK2 and Tyk2. Furthermore, these compounds that contain an α-ß-unsaturated carbonyl moiety and function as potent Michael reaction acceptor, induce a rapid drop in intracellular glutathione (GSH) concentration by direct interaction with it, thereby triggering S-glutathionylation of STAT3. Dehydrocostunolide (HCS), the reduced form of CS lacking only the α-ß-unsaturated carbonyl group, fails to exert any inhibitory action. Finally, the glutathione ethylene ester (GEE), the cell permeable GSH form, reverts the inhibitory action of DCE and CS on STAT3 tyrosine phosphorylation. We conclude that these two sesquiterpene lactones are able to induce redox-dependent post-translational modification of cysteine residues of STAT3 protein in order to regulate its function.
Assuntos
Glutationa/metabolismo , Lactonas/farmacologia , Fator de Transcrição STAT3/metabolismo , Sesquiterpenos/farmacologia , Sequência de Bases , Western Blotting , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão , Primers do DNA , Ensaio de Desvio de Mobilidade Eletroforética , Humanos , Fosforilação , Reação em Cadeia da Polimerase , Espécies Reativas de Oxigênio/metabolismoRESUMO
Nitric oxide (NO) homeostasis maintained by neuronal/endothelial nitric oxide (NO) synthase (n/eNOS) contributes to regulate cardiac function under physiological conditions. At the early stages of ischaemia, NO homeostasis is disturbed due to Ca2+-dependent e/nNOS activation. In endothelial cells, successive drop in NO concentration may occur due to both uncoupling of eNOS and/or successive inhibition of nNOS catalytic activity mediated by arachidonic acid-induced tyrosine phosphorylation of this enzyme. The reduced NO bioavailability triggers nuclear factor (NF)-kB activation followed by the induction of inducible NOS (iNOS) expression. In cardiomyocytes ischaemia also triggers the induction of iNOS expression during reperfusion. The massive amounts of NO which are subsequently produced following iNOS induction may exert on cardiomyocytes and the other cell types of cells of the heart, such as endothelial and smooth muscle cells, macrophages and neutrophils, opposing effects, either beneficial or toxic. The balance between these two double-faced actions may contribute to the final clinical outcomes, determining the degree of functional adaptation of the heart to ischaemia/reperfusion injury. In the light of this new vision on the critical role played by the cross-talk between n/eNOS and iNOS as well as the non enzymatic NO production by nitrite, we have reason to believe that new pharmacological measurements or experimental interventions, such as ischaemic preconditioning, aimed at counteracting the drop in NO levels beyond the normal range of NO homeostasis during early reperfusion can represent an efficient strategy to reduce the extent of functional impairment and cardiac damage in the heart exposed to ischaemia/reperfusion injury.
Assuntos
Inflamação/metabolismo , Traumatismo por Reperfusão Miocárdica/metabolismo , Miocárdio/metabolismo , Óxido Nítrico/metabolismo , Animais , Fármacos Cardiovasculares/uso terapêutico , Homeostase , Humanos , Inflamação/imunologia , Mediadores da Inflamação/metabolismo , Precondicionamento Isquêmico Miocárdico , Traumatismo por Reperfusão Miocárdica/imunologia , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Miocárdio/enzimologia , Óxido Nítrico Sintase Tipo I/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Transdução de SinaisRESUMO
The present review describes the role of the putative cross-talk between two neurotransmitters, nitric oxide (NO) and D-serine, in the brain. Under physiological conditions NO homeostasis guarantees the correct function of NO in a number of events in the brain such as neurotransmission and vascular tone regulation. D-serine, produced in astrocytes, acts synergistically with glutamate at NMDA receptors on postsynaptic neurons. Neuronal and endothelial NO synthase (nNOS and eNOS) in astrocytes cross-talk with serine racemase (SR) and D-amino acid oxydase (DAAO), catalyzing the synthesis and degradation of D-serine, respectively. SR is inhibited by NO which activates DAAO. D-serine inhibits nNOS but not eNOS and activates SR. Astrocytes and neurons also cross-talk through NO/D-serine system. D-serine released from astrocytes induces a rapid increase in NO contents in postsynaptic neurons. Overall, D-serine production in astrocytes is negatively regulated by NO. Under inflammatory conditions, pro-inflammatory cytokines or Abeta induce, first, a drop in NO contents and an increase in the amounts of D-serine in astrocytes. Together with enhanced glutamate release from presynaptic neurons, D-serine induces an increase in Ca(2+) up-take into presynaptic neurons. In astrocytes an initial drop in NO contents triggers NF-kappaB activation followed by inducible NOS (iNOS) expression. iNOS-derived massive amounts of NO may potentially be toxic. Under schizophrenic conditions, D-serine production is down-regulated. Together with reduced glutamate release, this situation leads to the decreased NO production in postsynaptic neurons. In astrocytes induction of iNOS expression becomes predominant. Initial drop in nNOS-derived NO is potentially toxic in this scenario.
Assuntos
Astrócitos/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Óxido Nítrico/metabolismo , Receptor Cross-Talk/efeitos dos fármacos , Serina/farmacologia , Envelhecimento , Animais , Animais Recém-Nascidos , Astrócitos/metabolismo , Encéfalo/citologia , Neoplasias Encefálicas/etiologia , Células Cultivadas , Citocinas/farmacologia , Ácido Glutâmico/farmacologia , Plasticidade Neuronal/efeitos dos fármacos , Neurônios/metabolismo , Óxido Nítrico/farmacologia , Óxido Nítrico Sintase Tipo III , Ratos , Ratos Endogâmicos Lew , Ratos Sprague-Dawley , Ratos Wistar , Serina/química , Estresse FisiológicoRESUMO
Alpha-bisabolol is a natural monocyclic sesquiterpene alcohol. It has been used in cosmetics for hundreds of years because of its perceived skin-healing properties. Alpha-bisabolol is known to have anti-irritant, anti-inflammatory and antimicrobial properties. In precedent studies, we described how alpha-bisabolol exerts a selective pro-apoptotic action towards transformed cells [Cavalieri E et al. (2004) Biochem Biophys Res Commun 315, 589-594] and its uptake is mediated by lipid rafts on the plasma membrane [Darra E et al. (2008) Arch Biochem Biophys 476, 113-123]. In this study, we hypothesize that the intracellular target of alpha-bisabolol may be the mitochondrial permeability transition pore (mPTP). To evaluate this hypothesis, we used one transformed cell line (human glioma T67) in comparison with a nontransformed one (human fibroblasts). We assessed the effect of a specific mPTP inhibitor (cyclosporine A) on the toxic action of alpha-bisabolol. Results show that the alpha-bisabolol-induced decrease in oxygen consumption is abolished by the addition of cyclosporine A in T67 cells, indicating that alpha-bisabolol may target mPTP. The central role of mitochondria was also demonstrated by using galactose to force cells to a more aerobic metabolism. In this condition, we observed higher alpha-bisabolol toxicity. Furthermore, we studied the effect of alpha-bisabolol on isolated rat liver mitochondria. This study expands the notion of the specific action of alpha-bisabolol on transformed cells and suggests that it may act by disturbing the structure and function of the mPTP. Alpha-bisabolol toxicity is clearly related to its cellular uptake, which is higher in transformed cell lines.
Assuntos
Apoptose/efeitos dos fármacos , Proteínas de Transporte da Membrana Mitocondrial/fisiologia , Sesquiterpenos/farmacologia , Neoplasias da Mama , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/fisiologia , Glioma , Humanos , Masculino , Proteínas de Transporte da Membrana Mitocondrial/efeitos dos fármacos , Poro de Transição de Permeabilidade Mitocondrial , Sesquiterpenos Monocíclicos , Neutrófilos/efeitos dos fármacos , Neutrófilos/fisiologia , Neoplasias da Próstata , Linfócitos T/efeitos dos fármacos , Linfócitos T/fisiologiaRESUMO
Flavonoids exhibit a variety of beneficial effects in cardiovascular diseases. Although their therapeutic properties have been attributed mainly to their antioxidant action, they have additional protective mechanisms such as inhibition of signal transducer and activator of transcription 1 (STAT1) activation. Here, we have investigated the cardioprotective mechanisms of strong antioxidant flavonoids such as quercetin, myricetin and delphinidin. Although all of them protect the heart from ischemia/reperfusion-injury, myricetin and delphinidin exert a more pronounced protective action than quercetin by their capacity to inhibit STAT1 activation. Biochemical and computer modeling analysis indicated the direct interaction between STAT1 and flavonoids with anti-STAT1 activity.
Assuntos
Antocianinas/uso terapêutico , Flavonoides/uso terapêutico , Traumatismo por Reperfusão Miocárdica/tratamento farmacológico , Traumatismo por Reperfusão Miocárdica/metabolismo , Fator de Transcrição STAT1/metabolismo , Animais , Antioxidantes/uso terapêutico , Linhagem Celular Tumoral , Humanos , Masculino , Modelos Moleculares , Estrutura Molecular , Traumatismo por Reperfusão Miocárdica/patologia , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Ratos , Ratos Sprague-Dawley , Relação Estrutura-AtividadeRESUMO
In the present study, we show that an aqueous extract of Arbutus unedo L. (AuE), a Mediterranean endemic plant widely employed as an astringent, diuretic and urinary anti-septic, in vitro down-regulates the expression of some inflammatory genes, such as those encoding inducible nitric oxide synthase (iNOS) and intracellular adhesion molecule-(ICAM)-1, exerting a inhibitory action on both IFN-gamma-elicited STAT1 activation and IL-6-elicited STAT3 activation. To evaluate further the effect of AuE in animal models of acute inflammation, we examined whether AuE administration attenuates inflammatory response of murine induced by intrapleural injection of carrageenan. For this purpose we studied: (1) STAT1/3 activation, (2) TNF-alpha, IL-1beta and IL-6 production in pleural exudate, (3) lung iNOS, COX-2 and ICAM-1 expression, (4) neutrophil infiltration, (5) the nitration of cellular proteins by peroxynitrite, (6) lipid peroxidation, (7) prostaglandin E2 and nitrite/nitrate levels and (8) lung injury. We show that AuE strongly down-regulates STAT3 activation induced in the lung by carrageenan with concomitant attenuation of all parameters examined associated with inflammation, suggesting that STAT3 should be a new molecular target for anti-inflammatory treatment. This study demonstrates that acute lung inflammation is significantly attenuated by the treatment with AuE.
Assuntos
Anti-Inflamatórios/uso terapêutico , Ericaceae/química , Pneumonia/tratamento farmacológico , Animais , Carragenina , Linhagem Celular , Dinoprostona/metabolismo , Humanos , Molécula 1 de Adesão Intercelular/biossíntese , Interferon gama/biossíntese , Interleucina-6/biossíntese , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Camundongos , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase Tipo II/metabolismo , Extratos Vegetais/uso terapêutico , Folhas de Planta/química , Pleurisia/induzido quimicamente , Pleurisia/tratamento farmacológico , Pleurisia/patologia , Pneumonia/induzido quimicamente , Pneumonia/metabolismo , Fator de Transcrição STAT1/metabolismo , Fator de Transcrição STAT3/metabolismo , Fator de Necrose Tumoral alfa/biossíntese , Tirosina/análogos & derivados , Tirosina/biossínteseRESUMO
The beneficial effect of naturally occurring flavonoids in health is believed to be due to their strong antioxidant activity. However, recent laboratory evidence indicates the involvement of a more specific action. Here, we present evidence that, among a number of catechins present in green tea extract, only epigallocatechin-3-gallate (EGCG) exerts a strong inhibitory action on interferon-gamma-elicited activation of signal transducer and activator of transcription 1 (STAT1). Protective action of EGCG in ischemia/reperfusion injury in the heart and the molecular mechanism of action, which has nothing to do with its anti-oxidant capacity are described.