RESUMO
Decrease in caspase activity is a common phenomenon in drug resistance. For effective therapeutic intervention, detection of such agents, which affects other pathway independent of caspases to promote cell death, might be important. Oleandrin, a polyphenolic glycoside induced cell death through activation of caspases in a variety of human tumour cells. In this report we provide evidence that besides caspases activation, oleandrin interacts with plasma membrane, changes fluidity of the membrane, disrupts Na(+)/K(+)-ATPase pump, enhances intracellular free Ca(2+) and thereby activates calcineurin. Calcineurin, in turns, activates nuclear transcription factor NF-AT and its dependent genes such as FasL, which induces cell death as a late response of oleandrin. Cell death at early stages is mediated by caspases where inhibitors partially protected oleandrin-mediated cell death in vector-transfected cells, but almost completely in Bcl-xL-overexpressed cells. Overall, our data suggest that oleandrin might be important therapeutic molecule in case of tumors where cell death pathway occurs due to deregulation of caspase-mediated pathway.
Assuntos
Apoptose/efeitos dos fármacos , Calcineurina/metabolismo , Cardenolídeos/farmacologia , Caspases/metabolismo , Proteína Ligante Fas/metabolismo , Fatores de Transcrição NFATC/metabolismo , Anticorpos/farmacologia , Inibidores de Calcineurina , Cálcio/metabolismo , Inibidores de Caspase , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Ciclosporina/farmacologia , DNA/metabolismo , Dactinomicina/farmacologia , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Proteína Ligante Fas/genética , Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Lipídeos/farmacologia , Fluidez de Membrana/efeitos dos fármacos , Necrose , Oligopeptídeos , Transporte Proteico/efeitos dos fármacos , ATPase Trocadora de Sódio-Potássio/metabolismo , Proteína bcl-X/genética , Proteína bcl-X/metabolismoRESUMO
Common practice to evaluate the efficacy of any compound as drug is done in cell-based in vitro system followed by in vivo murine model prior to clinical trial in human. Cardiac glycosides are very effective to kill human cells, but not murine cells. In this report, we describe the comparative molecular mechanism of oleandrin, a cardiac glycoside action in human and murine cells. Treatment with oleandrin facilitated nuclear translocation of FKHR in human, but not murine cells by dephosphorylating Akt. It activated MAPK and JNK in human, but not in murine cells and also induced expression of FasL leads to apoptosis in human cells as detected by assaying caspases activation, PARP cleavage, nuclear fragmentation, and annexin staining. Oleandrin interacted with human plasma membrane as evaluated by HPLC, altered its fluidity as detected by DPH binding, inhibited Na+/K+-ATPase activity, and increased intracellular free Ca2+ level followed by calcineurin activity only in human, but not in murine cells. Results suggest that human plasma membrane might be different than murine, which interact with oleandrin that disturb Na+/K+-ATPase pump resulting in the calcification followed by induction of Ca2+-dependent cellular responses such as apoptosis.
Assuntos
Apoptose/efeitos dos fármacos , Cardenolídeos/farmacologia , Proteína Ligante Fas/genética , Fluidez de Membrana/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Calcineurina/metabolismo , Cálcio/metabolismo , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão , Proteína Ligante Fas/metabolismo , Proteína Forkhead Box O1 , Fatores de Transcrição Forkhead/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Camundongos , Células NIH 3T3 , Fosforilação/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismoRESUMO
Chemotherapeutic agent is characterized by its concentration in tumor cells with minimum side effects. Oleandrin, a polyphenolic cardiac glycoside is known to induce apoptosis in tumor cells. However, no report is available on its efficacy in primary cells. In this report we are providing the evidence that oleandrin induces apoptosis, not necrosis in tumor cells but not in primary cells like peripheral blood mononuclear cells (PBMC) and neutrophils. Oleandrin inhibited NF-kappaB activation in tumor cells but not in primary cells. It induced cell death in NF-kappaB-overexpressed tumor cells. Oleandrin induced Fas expression thereby inducing apoptosis in tumor cells but not in primary cells. Dominant negative FADD inhibited oleandrin-induced cell death in tumor cells. Overall, these results suggest that oleandrin mediates apoptosis in tumor cells by inducing Fas but not in primary cells indicating its potential anti-cancer property with no or slight side effect.
Assuntos
Apoptose/efeitos dos fármacos , Cardenolídeos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Receptor fas/genética , Antineoplásicos/farmacologia , Células Sanguíneas/efeitos dos fármacos , Linhagem Celular Tumoral , Células Cultivadas , Humanos , NF-kappa B/antagonistas & inibidores , Neoplasias/patologiaRESUMO
Considering the role of interleukin-8 (IL-8) in a large number of acute and chronic inflammatory diseases, the regulation of IL-8-mediated biological responses is important. Alpha-melanocyte-stimulating hormone (alpha-MSH), a tridecapeptide, inhibits most forms of inflammation by an unknown mechanism. In the present study, we have found that alpha-MSH interacts predominantly with melanocortin-1 receptors and inhibits several IL-8-induced biological responses in macrophages and neutrophils. It down-regulated receptors for IL-8 but not for TNF, IL-4, IL-13 or TNF-related apoptosis-inducing ligand (TRAIL) in neutrophils. It down-regulated CXCR type 1 and 2 but not mRNA levels. alpha-MSH did not inhibit IL-8 binding in purified cell membrane or affinity-purified CXCR. IL-8 or anti-CXCR Ab protected against alpha-MSH-mediated inhibition of IL-8 binding. The level of neutrophil elastase, a specific serine protease, but not cathepsin G or proteinase 3 increased in alpha-MSH-treated cells, and restoration of CXCR by specific neutrophil elastase or serine protease inhibitors indicates the involvement of elastase in alpha-MSH-induced down-regulation of CXCR. These studies suggest that alpha-MSH inhibits IL-8-mediated biological responses by down-regulating CXCR through induction of serine protease and that alpha-MSH acts as a potent immunomodulator in neutrophil-driven inflammatory distress.
Assuntos
Regulação para Baixo/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Elastase de Leucócito/imunologia , Receptor Tipo 1 de Melanocortina/imunologia , Receptores de Quimiocinas/imunologia , alfa-MSH/farmacologia , Doença Aguda , Catepsina G , Catepsinas/imunologia , Catepsinas/metabolismo , Doença Crônica , Citocinas/imunologia , Regulação para Baixo/imunologia , Regulação Enzimológica da Expressão Gênica/imunologia , Células HL-60 , Humanos , Inflamação/imunologia , Elastase de Leucócito/metabolismo , Macrófagos/enzimologia , Macrófagos/imunologia , Mieloblastina , Neutrófilos/enzimologia , Neutrófilos/imunologia , Receptor Tipo 1 de Melanocortina/metabolismo , Receptores de Quimiocinas/biossíntese , Serina Endopeptidases/imunologia , Serina Endopeptidases/metabolismo , alfa-MSH/imunologiaRESUMO
Considering the potential role of interleukin-8 (IL-8) in inflammation, angiogenesis, tumorogenesis, and metastasis, and the involvement of different cell types especially neutrophils and macrophages in those processes, the regulation of IL-8-mediated biological responses is important. In this report we provide evidences that oleandrin, a cardiac glycoside potentially inhibited IL-8-, formyl peptide (FMLP)-, EGF-, or nerve growth factor (NGF)-, but not IL-1- or TNF-induced NF-kappaB activation in macrophages. Oleandrin inhibited IL-8-, but not TNF-induced NF-kappaB-dependent genes expression. Oleandrin inhibited the binding of IL-8, EGF, or NGF, but not IL-1 or TNF. It decreased almost 79% IL-8 binding without altering affinity towards IL-8 receptors and this inhibition of IL-8 binding was observed in isolated membrane. The IL-8, anti-IL-8Rs antibodies, or protease inhibitors were unable to protect oleandrin-mediated inhibition of IL-8 binding. Phospholipids significantly protected oleandrin-mediated inhibition of IL-8 binding thereby restoring IL-8-induced NF-kappaB activation. Oleandrin altered the membrane fluidity as detected by microviscosity parameter and a decrease in diphenylhexatriene, a lipid binding fluorophore binding in a dose-dependent manner. Overall, our results suggest that oleandrin inhibits IL-8-mediated biological responses in diverse cell types by modulating IL-8Rs through altering membrane fluidity and microviscosity. The study might help to regulate IL-8-mediated biological responses involved in inflammation, metastasis, and neovascularization.
Assuntos
Cardenolídeos/farmacologia , Interleucina-8/farmacologia , Fluidez de Membrana/efeitos dos fármacos , Receptores de Interleucina/metabolismo , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Anticorpos/imunologia , Anticorpos/farmacologia , Glicosídeos Cardíacos/farmacologia , Linhagem Celular , Linhagem Celular Tumoral , Membrana Celular/química , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Colesterol/farmacologia , Ciclo-Oxigenase 2/metabolismo , Difenilexatrieno/metabolismo , Relação Dose-Resposta a Droga , Regulação para Baixo/efeitos dos fármacos , Fator de Crescimento Epidérmico/metabolismo , Fator de Crescimento Epidérmico/farmacologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HL-60 , Humanos , Quinase I-kappa B/metabolismo , Proteínas I-kappa B/metabolismo , Molécula 1 de Adesão Intercelular/metabolismo , Interleucina-1/metabolismo , Interleucina-1/farmacologia , Interleucina-8/imunologia , Interleucina-8/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Proteínas de Membrana/metabolismo , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Inibidor de NF-kappaB alfa , NF-kappa B/genética , NF-kappa B/metabolismo , Subunidade p50 de NF-kappa B/metabolismo , Fator de Crescimento Neural/metabolismo , Fator de Crescimento Neural/farmacologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Fosfolipídeos/farmacologia , Fosforilação/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Inibidores de Proteases/farmacologia , Receptores de Superfície Celular/metabolismo , Receptores de Interleucina/imunologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Esfingosina/farmacologia , Fator de Transcrição RelA/metabolismoRESUMO
Mangiferin, a natural polyphenol is known to exhibit anti-inflammatory, antioxidant, and antiviral effects. However the molecular mechanism underlying these effects has not been well characterized. Because NF-kappaB plays an important role in these processes, it is possible that mangiferin modulates NF-kappaB activation. Our results show that mangiferin blocks tumor necrosis factor (TNF)-induced NF-kappaB activation and NF-kappaB-dependent genes like ICAM1 and COX2. The effect was mediated through inhibition of IKK activation and subsequent blocking of phosphorylation and degradation of IkappaBalpha. In addition, mangiferin inhibits TNF-induced p65 phosphorylation as well as translocation to the nucleus and also inhibits NF-kappaB activation induced by other inflammatory agents like PMA, ceramide, and SA-LPS. Mangiferin, similar to the other known antioxidants, NAC and PDTC, inhibits TNF-induced reactive oxygen intermediate (ROI) generation. Since intracellular glutathione (GSH) levels are known to modulate NF-kappaB levels, we measured the levels of GSH. Mangiferin enhances glutathione level by almost 2-fold more than other anti-oxidants, and at the same time it decreases the levels of GSSG and increases the activity of catalase. Depletion of GSH by buthionine sulfoximine led to a significant reversal of mangiferin effect. Hence mangiferin with its ability to inhibit NF-kappaB and increase the intracellular GSH levels may prove to be a potent drug for anti-inflammatory and antioxidant therapy. Mangiferin-mediated down-regulation of NF-kappaB also potentiates chemotherapeutic agent-mediated cell death, suggesting a role in combination therapy for cancer.
Assuntos
Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , NF-kappa B/fisiologia , Fator de Necrose Tumoral alfa/farmacologia , Xantonas/farmacologia , Animais , Anti-Infecciosos , Antineoplásicos/farmacologia , Neoplasias da Mama , Butionina Sulfoximina/farmacologia , Proteínas de Ligação ao Cálcio/metabolismo , Catalase/metabolismo , Linhagem Celular , Ciclo-Oxigenase 2 , Sinergismo Farmacológico , Ativação Enzimática/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Glutamato-Cisteína Ligase/genética , Glutationa/análise , Células HeLa , Humanos , Quinase I-kappa B , Molécula 1 de Adesão Intercelular/genética , Isoenzimas/genética , Células Jurkat , Linfoma Difuso de Grandes Células B , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana , Camundongos , NF-kappa B/antagonistas & inibidores , Proteínas do Tecido Nervoso/metabolismo , Fosforilação , Prostaglandina-Endoperóxido Sintases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Sinaptotagmina I , Sinaptotagminas , Células Tumorais CultivadasRESUMO
Ceramide (N-acetyl-D-sphingosine), a second messenger for cell signaling induces transcription factors, like nuclear factor-kappa B (NF-kappa B), and activator protein-1 (AP-1) and is involved in inflammation and apoptosis. Agents that can suppress these transcription factors may be able to block tumorigenesis and inflammation. Oleandrin (trans-3,4',5-trihydroxystilbene), a polyphenolic cardiac glycoside derived from the leaves of Nerium oleander, has been used in the treatment of cardiac abnormalities in Russia and China for years. We investigated the effect of oleandrin on NF-kappa B and AP-1 activation and apoptosis induced by ceramide. Oleandrin blocked ceramide-induced NF-kappa B activation. Oleandrin-mediated suppression of NF-kappa B was not restricted to human epithelial cells; it was also observed in human lymphoid, insect, and murine macrophage cells. The suppression of NF-kappa B coincided with suppression of AP-1. Ceramide-induced reactive intermediates generation, lipid peroxidation, cytotoxicity, caspase activation, and DNA fragmentation were potentiated by oleandrin. Oleandrin did not show its activity in primary cells. Oleandrin's anticarcinogenic, anti-inflammatory, and growth-modulatory effects may thus be partially ascribed to the inhibition of activation of NF-kappa B and AP-1 and potentiation of apoptosis.
Assuntos
Apoptose/efeitos dos fármacos , Cardenolídeos/farmacologia , Ceramidas/farmacologia , NF-kappa B/antagonistas & inibidores , NF-kappa B/metabolismo , Fator de Transcrição AP-1/antagonistas & inibidores , Animais , Apoptose/fisiologia , Linhagem Celular , Ceramidas/antagonistas & inibidores , Chlorocebus aethiops , Sinergismo Farmacológico , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Células HeLa , Humanos , Células Jurkat , Camundongos , Transdução de Sinais/efeitos dos fármacos , Fator de Transcrição AP-1/metabolismoRESUMO
Monocytes/macrophages are the first cells involved in inflammation. alpha-Melanocyte-stimulating hormone (alpha-MSH) is known to possess an anti-inflammatory role induced by a variety of stimuli; however, the molecular mechanisms underlying these effects are not clearly defined. In this report we provide evidence that alpha-MSH inhibited serum-activated lipopolysaccharide (SA-LPS)-induced proteolytic enzyme release, oxidative burst response, reactive oxygen intermediate generation, nitric oxide production, and adhesion molecule expression in monocyte-derived macrophages. alpha-MSH also inhibited SA-LPS-induced nuclear transcription factor kappaB activation not only in macrophages, but also in a T-cell line and human neutrophils isolated from fresh blood. alpha-MSH downregulated CD14, but not interleukin-1 receptor, tumor necrosis factor receptor 1 or 2 from the surface of macrophages. Anti-CD14 antibody was unable to protect alpha-MSH-mediated downregulation of CD14. Overall, our results suggest that alpha-MSH exerts its anti-inflammatory effect by a novel mechanism in macrophages through downregulating of the endotoxin receptor CD14.
Assuntos
Receptores de Lipopolissacarídeos/metabolismo , Lipopolissacarídeos/antagonistas & inibidores , Macrófagos/metabolismo , alfa-MSH/farmacologia , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Animais , Regulação para Baixo , Interações Medicamentosas , Células HeLa , Humanos , Inflamação/metabolismo , Células Jurkat , Receptores de Lipopolissacarídeos/imunologia , Lipopolissacarídeos/farmacologia , Macrófagos/citologia , Macrófagos/imunologia , Camundongos , NF-kappa B/genética , NF-kappa B/metabolismo , Células NIH 3T3 , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Óxido Nítrico/biossíntese , Espécies Reativas de Oxigênio/metabolismo , Receptores de Interleucina-1/metabolismo , Receptores do Fator de Necrose Tumoral/metabolismo , Explosão Respiratória/efeitos dos fármacos , Explosão Respiratória/fisiologia , Células Tumorais Cultivadas , Células U937RESUMO
Mast cells play a major role in the initiation of inflammation and allergic reactions. As cell numbers are tightly controlled by the interplay of factors affecting cell proliferation, development, and death the regulation of mast cell number may be important. Melanocyte-stimulating hormone inhibits most forms of inflammation by an unknown mechanism. In the present study, we have found that the alpha-melanocyte-stimulating hormone (alpha-MSH) inhibited endotoxin-mediated nuclear transcription factor kappaB (NF-kappaB) activation in different cells correlated with the expression of alpha-MSH receptors. We have also found for the first time that it induces cell death alone or in endotoxin-stimulated mast cells. alpha-MSH-mediated apoptosis was not observed in NF-kappaB overexpressed cells. The inhibitory effect of alpha-MSH was mediated through generation of cAMP, as inhibitors of adenylate cyclase and of protein kinase A reversed its inhibitory effect. Overall, our results suggest that NF-kappaB is the key molecule involved in alpha-MSH-mediated cell death and this may help to regulate mast cell-mediated inflammation.
Assuntos
Mastócitos/citologia , NF-kappa B/antagonistas & inibidores , alfa-MSH/farmacologia , Animais , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , AMP Cíclico/metabolismo , Endotoxinas/farmacologia , Inibidores Enzimáticos/farmacologia , Humanos , Inflamação/patologia , Mastócitos/efeitos dos fármacos , Mastócitos/metabolismo , Camundongos , NF-kappa B/fisiologia , Receptores do Hormônio Hipofisário/análiseRESUMO
Nuclear transcription factor kappa B (NF-kappaB) has been shown both to block apoptosis and to promote cell proliferation, and hence has been considered an important target for anticancer drug development. The pyrimidine analogue cytosine arabinoside (araC) is among the most effective agents used in the treatment of acute leukemia, and we demonstrate in this study that its chemotherapeutic activity may be mediated by its inhibition of NF-kappaB. We found that in Jurkat cells, although tumor necrosis factor (TNF), araC, or ceramide induced NF-kappaB, the induction was only transient in the case of araC. In both HuT-78 and serum-activated LPS-stimulated Jurkat (SA-LPS/Jkt) cells that expressed NF-kappaB, TNF or ceramide treatments did not affect the NF-kappaB expression whereas araC downregulated it. AraC, but not TNF or ceramide was able to induce apoptosis in these cells as detected by assays for lipid peroxidation, reactive oxygen intermediates generation, caspase activation, cytotoxicity, Bcl-2 degradation, and DNA fragmentation. AraC also potentiated apoptosis mediated by cis-platin, etoposide, or taxol in these cells. AraC was able to induce protein phosphatases (PP) 2A and 2B-A, and phosphorylation of p65 subunit of NF-kappaB in the HuT-78 and SA-LPS/Jkt cells was downregulated by araC treatment. Furthermore, calyculin A, a specific phospho-serine/phospho-threonine phosphatase inhibitor, protected HuT-78 and SA-LPS/Jkt cells from araC-mediated NF-kappaB downregulation and apoptosis. These observations collectively suggest that araC induces apoptosis in NF-kappaB-expressing cells by dephosphorylating the p65 subunit of NF-kappaB.