RESUMO
Human cervical cancer is caused by high-risk types of human papillomavirus (HPV) such as HPV16 and HPV18, which possess the E6 and E7 oncogenes, whose concurrent expression is a prerequisite for cancer development and maintaining malignant phenotypes. Silencing these oncogenes is considered to be applicable in molecular therapies of human cervical cancer. However, it remains to be determined whether E6, E7, or both should be silenced to obtain most efficient antitumor activity by an HPV small-interfering RNA (siRNA). Herein, we report two types of siRNAs targeting HPV18 E6, that exerted a negative growth effect on HPV18-positive cervical cancer cells (HeLa and SW756), in part, inducing cell death. One siRNA (Ex-18E6), designed to target both E6-E7 mRNA and its splicing variant, E6*I-E7 mRNA, efficiently knocked down both E6 and E7 expression. The other (Sp-18E6), designed to specifically target E6-E7 mRNA but not E6*I-E7 mRNA, suppressed E6 to a similar level as Ex-18E6; however, it less efficiently inhibited E7 as compared to Ex-18E6. Although both siRNAs induced cell death, Sp-18E6 siRNA induced more prominent cell death than Ex-18E6. Our results suggest that E6-specific suppression may induce more potent anticancer activity than simultaneous E6 and E7 suppression, and that E6-specific targeting is a promising strategy for siRNA-based therapy for HPV-positive cervical cancer.
Assuntos
Apoptose , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Inativação Gênica , Proteínas Oncogênicas Virais/genética , Proteínas Oncogênicas Virais/fisiologia , RNA Interferente Pequeno/farmacologia , Neoplasias do Colo do Útero/patologia , Neoplasias do Colo do Útero/virologia , Animais , Células COS , Chlorocebus aethiops , Feminino , Regulação Viral da Expressão Gênica , Células HeLa , Humanos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , TransfecçãoRESUMO
Nitrogen monoxide (NO) has diverse physiological roles and also contributes to the immune defense against viruses, bacteria, and other parasites. However, excess production of NO is associated with various diseases such arthritis, diabetes, stroke, septic shock, autoimmune, chronic inflammatory diseases, and atheriosclerosis. Cells respond to activating or depressing stimuli by enhancing or inhibiting the expression of the enzymatic machinery that produce NO. Thus, maintenance of a tight regulation of NO production is important for human health. Phytochemicals have been traditionally utilized in ways to treat a family of pathologies that have in common the disregulation of NO production. Here we report the scavenging activity of Pycnogenol (the polyphenols containing extract of the bark from Pinus maritima) against reactive oxygen and nitrogen species, and its effects on NO metabolism in the murine macrophages cell line RAW 264.7. Macrophages were activated by the bacterial wall components lipopolysaccharide (LPS) and interferon (IFN-gamma), which induces the expression of large amounts of the enzyme nitric oxide synthase (iNOS). Preincubation of cells with physiological concentrations of Pycnogenol significantly decreased NO generation. It was found that this effect was due to the combination of several different biological activities, i.e., its ROS and NO scavenging activity, inhibition of iNOS activity, and inhibition of iNOS-mRNA expression. These data begin to provide the basis for the conceptual understanding of the biological activity of Pycnogenol and possibly other polyphenolic compounds as therapeutic agents in various human disorders.
Assuntos
Flavonoides/farmacologia , Sequestradores de Radicais Livres/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Óxido Nítrico/metabolismo , Animais , Antioxidantes/isolamento & purificação , Antioxidantes/farmacologia , Linhagem Celular , Proteínas de Ligação a DNA/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Inibidores Enzimáticos/farmacologia , Flavonoides/isolamento & purificação , Sequestradores de Radicais Livres/isolamento & purificação , Humanos , Radical Hidroxila/metabolismo , Fator Regulador 1 de Interferon , Ativação de Macrófagos , Camundongos , NF-kappa B/metabolismo , Nitratos/metabolismo , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase Tipo II , Nitritos/metabolismo , Fosfoproteínas/metabolismo , Extratos Vegetais , RNA Mensageiro/genética , Superóxidos/metabolismo , Árvores/químicaRESUMO
Regulation of adhesion molecule expression and function by reactive oxygen species via specific redox sensitive mechanisms have been reported. The effects of clinically safe antioxidants in the regulation of adhesion molecule expression in human endothelial cells (ECV), and adherence of human Jurkat T cells to ECV cells were investigated. The thiol antioxidant, alpha-lipoate, at clinically relevant doses down-regulated phorbol 12-myristate 13-acetate (PMA)-induced adhesion molecule expression and cell-cell adhesion. Inhibition of PMA-induced ICAM-1 and VCAM-1 expression as well as PMA-induced adhesion of Jurkat T-cells to ECV cells by alpha-lipoate was dose dependent (50-250 microM). The effect was significant for ICAM-1 (p < .01) and VCAM-1 (p < .01) expression in cells pretreated with 100 microM alpha-lipoate compared to PMA-activated untreated cells. Inhibition of PMA-induced adhesion molecule expression and cell-cell adhesion was more pronounced when a combination of antioxidants, alpha-lipoate and alpha-tocopherol, were used compared to the use of either of these antioxidant alone. The regulation of adhesion molecule expression and function by low concentration of antioxidants investigated does not appear to be NF-kappaB regulated or transcription dependent because no change in the mRNA response was observed. Protein kinase C (PKC) has been suggested to regulate PMA-induced adhesion molecule expression by post-transcriptional stabilization of adhesion molecule mRNA. Alpha-lipoate pretreatment did not influence the response of PKC activity to PMA. Oxidants are known to be involved in the regulation of cell adhesion processes. Treatment of ECV cells with PMA induced generation of intracellular oxidants. Alpha-lipoate (100 or 250 microM) treatment decreased PMA-induced generation of intracellular oxidants. The inhibitory effect of low concentration of alpha-lipaote alone or in combination with alpha-tocopherol on agonist-induced adhesion processes observed in this study may be of potential therapeutic value.
Assuntos
Antioxidantes/farmacologia , Endotélio Vascular/efeitos dos fármacos , Linfócitos T/efeitos dos fármacos , Acetato de Tetradecanoilforbol/farmacologia , Adesão Celular/efeitos dos fármacos , Linhagem Celular Transformada , Regulação para Baixo/efeitos dos fármacos , Sinergismo Farmacológico , Endotélio Vascular/citologia , Humanos , Molécula 1 de Adesão Intercelular/biossíntese , Molécula 1 de Adesão Intercelular/efeitos dos fármacos , Molécula 1 de Adesão Intercelular/genética , Líquido Intracelular/química , Líquido Intracelular/efeitos dos fármacos , Células Jurkat , NF-kappa B/efeitos dos fármacos , NF-kappa B/metabolismo , Peróxidos/análise , Proteína Quinase C/efeitos dos fármacos , Proteína Quinase C/metabolismo , RNA Mensageiro/análise , RNA Mensageiro/biossíntese , RNA Mensageiro/efeitos dos fármacos , Linfócitos T/citologia , Acetato de Tetradecanoilforbol/antagonistas & inibidores , Ácido Tióctico/farmacologia , Células Tumorais Cultivadas , Molécula 1 de Adesão de Célula Vascular/biossíntese , Molécula 1 de Adesão de Célula Vascular/efeitos dos fármacos , Molécula 1 de Adesão de Célula Vascular/genética , Vitamina E/farmacologiaRESUMO
To investigate the role of superoxide dismutase (SOD) in the ovulatory process, SOD isozymes and their mRNAs were determined in the ovary of 22-day-old rats. After treatment with pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG), ovarian activity of Mn-SOD decreased markedly while Cu/Zn-SOD remained unchanged. However, the ovarian level of mRNA for Mn-SOD markedly increased after hCG-treatment while that for Cu/Zn-SOD decreased only slightly. Ovulation was inhibited by intravenous injection of a long-acting SOD. These results suggested that superoxide radicals in the ovary might play a critical role in the mechanism for hCG-induced ovulation.
Assuntos
Isoenzimas/metabolismo , Ovário/enzimologia , Ovulação/fisiologia , Superóxido Dismutase/metabolismo , Animais , Northern Blotting , Feminino , Isoenzimas/genética , Ovário/fisiologia , RNA Mensageiro/metabolismo , Ratos , Ratos Endogâmicos , Superóxido Dismutase/genéticaRESUMO
The cDNA encoding lipocortin-like 39 kDa protein in guinea pig neutrophils was cloned into a yeast expression vector and the constructed plasmid was introduced into a yeast. The gene was expressed in an eukaryotic cell, yeast Saccharomyces cerevisiae and the recombinant protein was purified and characterized. The purified protein was identical with the native one with respect to the antigenicity and several biochemical properties, such as inhibitory action against phospholipase A2, Ca2+-dependent binding to acidic-phospholipids and F-actin and availability as a substrate for tyrosine kinase (EGF receptor/kinase) and protein kinase C.
Assuntos
Proteínas de Ligação ao Cálcio/genética , Clonagem Molecular , DNA/genética , Neutrófilos/metabolismo , Fosfolipases/antagonistas & inibidores , Saccharomyces cerevisiae/genética , Animais , Anexinas , Proteínas de Ligação ao Cálcio/sangue , Proteínas de Ligação ao Cálcio/isolamento & purificação , Vetores Genéticos , Cobaias , Peso Molecular , Plasmídeos , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
The present study was conducted to evaluate the effect of Ginkgo biloba extract (EGb 761) on the synthesis of nitric oxide (NO) induced by lipopolysaccharide (LPS) plus interferon-gamma (IFN-gamma) in the mouse macrophage cell line RAW 264.7. EGb 761 inhibited nitrite and nitrate production, taken as an index for NO, in a concentration-dependent fashion. The IC50 for inhibition of nitrite production by activated macrophages was about 100 micrograms/mL EGb 761. The inducible NO synthase (iNOS) enzyme activity of cytosolic preparations from activated RAW 264.7 cells was inhibited by treatment with EGb 761. In addition, reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed that the expression of iNOS mRNA in activated macrophages was suppressed by high concentrations of EGb 761. However, NF-kappa B DNA binding activity induced by activation with LPS/IFN-gamma was not inhibited by EGb 761. These findings indicate that not only does EGb 761 directly act as an NO scavenger but also that it inhibits NO production in LPS/IFN-gamma-activated macrophages by concomitant inhibition of induction of iNOS mRNA and the enzyme activity of iNOS. Thus, EGb 761 may act as a potent inhibitor of NO production under tissue-damaging inflammatory conditions.
Assuntos
Sequestradores de Radicais Livres/farmacologia , Macrófagos/metabolismo , Óxido Nítrico/biossíntese , Extratos Vegetais/farmacologia , Animais , Linhagem Celular , DNA/metabolismo , Ginkgo biloba , Camundongos , NF-kappa B/metabolismo , Nitratos/metabolismo , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase/genética , Nitritos/metabolismo , RNA Mensageiro/análiseRESUMO
The biochemical properties and specificity of n-3 and n-6 polyunsaturated fatty acids (PUFAs) are not well known. Because PUFAs induce apoptosis of different cells, we studied the effect of various PUFAs, such as arachidonic acid (AA), eicosapentaenoic acid (EPA), and docosapentaenoic acid (DPA), on the fate of cultured human promyelocytic leukemia cells (HL-60) to elucidate the mechanism of apoptosis and the difference in action between n-3 and n-6 PUFAs. Fairly low concentrations of PUFAs inhibited the growth of HL-60 cells and induced their apoptosis by a mechanism that is sensitive to DMSO, an antioxidant, and z-Val-Ala-Asp(OMe)-fluoromethylketone (z-VAD-fmk), a pan-caspase inhibitor. PUFAs stimulated the generation of reactive oxygen species (ROS) and activated various types of caspase-like proteases, such as caspase-3, -6, -8, and -9, but not caspase-1. In addition, PUFAs triggered the reaction leading to the cleavage of Bid, a death agonist member of the Bcl-2 family, and also released cytochrome c from mitochondria into the cytosol. PUFAs also decreased the mitochondrial membrane potential of intact HL-60 cells. All of these actions of n-3 PUFAs were stronger than those of AA, an n-6 PUFA, although the mechanism is not known. PUFAs stimulate swelling and membrane depolarization of isolated mitochondria in a cyclosporin A-sensitive manner. The results indicated that PUFA-induced apoptosis of HL-60 cells may be caused, in part, by direct action on the cells and by activation of the caspase cascade through cytochrome c release coupled with mitochondrial membrane depolarization.
Assuntos
Apoptose , Ácidos Graxos Insaturados/farmacologia , Células HL-60/efeitos dos fármacos , Triglicerídeos/farmacologia , Clorometilcetonas de Aminoácidos/farmacologia , Caspases/metabolismo , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Inibidores de Cisteína Proteinase/farmacologia , Grupo dos Citocromos c/metabolismo , DNA/efeitos dos fármacos , DNA/metabolismo , Fragmentação do DNA/efeitos dos fármacos , Ácidos Graxos Ômega-3 , Ácidos Graxos Ômega-6 , Células HL-60/patologia , Humanos , Potenciais da Membrana/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/enzimologiaRESUMO
Upon stimulation by various ligands, freshly isolated human peripheral neutrophils (PMN) respond in a variety of ways, such as superoxide (O2-.) generation, phagocytosis enzyme release, migration etc. Chemotactic peptide formylmethionyl-leucyl-phenylalanine (FMLP) and opsonized zymosan activate neutrophils by a receptor-mediated mechanism, while phorbol myristate acetate and dioctanoylglycerol activate the cells by a mechanism involving Ca(2+)-and phospholipid-dependent protein kinase (PKC). Receptor-mediated but not PKC-mediated O2-. generation in PMN was enhanced by the priming of recombinant human granulocyte colony stimulating factor (G-CSF). FMLP-dependent luminol chemiluminescence was also enhanced by G-CSF. However, no appreciable enhancement was observed in FMLP-induced intracellular calcium ion concentration ([Ca2+]i). Enhancement of FMLP-induced generation of O2-. by G-CSF was inhibited by genistein or alpha-cyano-3-ethoxy-4-hydroxy-5-phenylthiomethylcinnamamide (ST 638), inhibitors of tyrosine kinase (TK), and was stimulated by staurosporine and 1-(5-isoquinolinesulfonyl)-3-methyl-piperazine (H-7), inhibitors of PKC. The ED50 values of genistein and ST 638 for the inhibition of the FMLP-induced O2-. generation from G-CSF were 0.5 and 5 microM, respectively. In contrast, O2-. generation by PKC activation without G-CSF priming was inhibited by stauroporine and H-7, but was stimulated by genistein and ST 638. These results suggested that the enhancing effect of G-CSF on receptor-mediated generation of the O2-. might be regulated by protein kinases, such as TK and PKC, and that the TK inhibitor selectively inhibited the G-CSF-primed receptor-mediated O2-. generation of neutrophils.
Assuntos
Fator Estimulador de Colônias de Granulócitos/farmacologia , Neutrófilos/efeitos dos fármacos , Inibidores de Proteínas Quinases , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina , Alcaloides/farmacologia , Cálcio/análise , Cinamatos/farmacologia , Genisteína , Humanos , Isoflavonas/farmacologia , Isoquinolinas/farmacologia , Luminol , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Neutrófilos/metabolismo , Piperazinas/farmacologia , Estaurosporina , Sulfetos/farmacologia , Superóxidos/metabolismoRESUMO
Dibucaine, a local anesthetic, inhibited the growth of promyelocytic leukemia cells (HL-60) without inducing arrest of the cell cycle and differentiation to granulocytes. Typical DNA fragmentation and DNA ladder formation were induced in a concentration- and time-dependent manner. The half-maximal concentration of dibucaine required to induce apoptosis was 100 microM. These effects were prevented completely by the pan-caspase inhibitor z-Val-Ala-Asp-(OMe)-fluoromethylketone (z-VAD-fmk), thereby implicating the cysteine aspartase (caspase) cascade in the process. Dibucaine activated various caspases, such as caspase-3, -6, -8, and -9 (-like) activities, but not caspase-1 (-like) activity, and induced mitochondrial membrane depolarization and the release of cytochrome c (Cyt.c) from mitochondria into the cytosol. Processing of pro-caspase-3, -8, and -9 by dibucaine was confirmed by western blot analysis. Bid, a death agonist member of the Bcl-2 family, was processed by caspases following exposure of cells to dibucaine. However, 100 microM dibucaine scarcely inhibited oxidative phosphorylation, but it induced membrane permeability transition in isolated rat liver mitochondria. Taken together, these data suggest that dibucaine induced apoptosis of HL-60 cells through activation of the caspase cascade in conjunction with Cyt.c release induced by a processed product of Bid and depolarization of the mitochondrial membrane potential.
Assuntos
Apoptose , Dibucaína/farmacologia , Anestésicos Locais/farmacologia , Caspases/metabolismo , Ciclo Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Tamanho Celular/efeitos dos fármacos , Grupo dos Citocromos c/metabolismo , Fragmentação do DNA/efeitos dos fármacos , Células HL-60 , Humanos , Membranas Intracelulares/efeitos dos fármacos , Membranas Intracelulares/metabolismo , Leucemia Promielocítica Aguda , Potenciais da Membrana/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/fisiologia , Dilatação Mitocondrial/efeitos dos fármacos , Oxirredução/efeitos dos fármacos , Fosforilação/efeitos dos fármacosRESUMO
Effect of biscoclaurine alkaloids, such as cepharanthine, on active oxygen production of neutrophils was investigated. Cepharanthine inhibited both superoxide generation and luminol-dependent chemiluminescence (CL) induced by either formylmethionyl-leucyl-phenylalanine, opsonized zymosan, arachidonic acid or by phorbol myristate acetate. Ca2(+)- and phospholipid-dependent protein kinase (PKC) activity and the phosphorylation of cytoplasmic protein including 47 kDa proteins of neutrophils were also inhibited by cepharanthine; dose dependent inhibition of CL was quite similar to that of PKC. Among various biscoclaurines tested, the inhibitory effect of cepharanthine, tetrandrine and isotetrandrine was strong, but that of berbamine and cycreanine was weak; the inhibitory action of the former on lipid peroxidation and platelet aggregation were also stronger than those of the latter. These and other observations indicated that these alkaloids inhibited the active oxygen generation by way of stabilizing plasma membrane and inhibiting PKC and NADPH oxidase activation.
Assuntos
Alcaloides/farmacologia , Neutrófilos/efeitos dos fármacos , Oxigênio/metabolismo , Animais , Ácido Araquidônico , Ácidos Araquidônicos/farmacologia , Benzilisoquinolinas , Radicais Livres , Cobaias , Medições Luminescentes , Luminol , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Neutrófilos/metabolismo , Proteínas Quinases/metabolismo , Superóxidos/metabolismo , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
Blepharismin is an endogenous photosensitizing pigment found in the protozoan Blepharisma. This pigment inhibited the generation of superoxide anion (O2-.) in neutrophils not only via a diacylglycerol-induced protein kinase C (PKC)-dependent reaction but also by an arachidonate-induced PKC-independent reaction. The inhibition was light and concentration dependent for both reactions. Light-activated inhibition was strong at wavelengths between 520 and 570 nm but not above 610 nm. PKC activity in neutrophils and from rat brain was inhibited by blepharismin in a light- and concentration-dependent manner. Moreover, arachidonate-activated NADPH oxidase activity in a cell-free system was also inhibited by the pigment in a light- and concentration-dependent manner. These results suggest that blepharismin inhibits NADPH oxidase activation through the non-specific inhibition of various membrane-bound enzymes and that this inhibition may also be correlated with that of PKC.
Assuntos
Eucariotos/química , Neutrófilos/efeitos dos fármacos , Perileno/análogos & derivados , Pigmentos Biológicos/farmacologia , Proteína Quinase C/antagonistas & inibidores , Superóxidos/metabolismo , Animais , Sistema Livre de Células , Luz , NADH NADPH Oxirredutases/antagonistas & inibidores , NADPH Oxidases , Neutrófilos/metabolismo , Perileno/isolamento & purificação , Perileno/farmacologia , Fosforilação/efeitos dos fármacos , Fotoquímica , Pigmentos Biológicos/isolamento & purificaçãoRESUMO
Selective induction of apoptosis in tumor cells is important for treating patients with cancer. Because oxidative stress plays an important role in the process of apoptosis, we studied the effect of alpha-tocopheryl succinate (VES) on the fate of cultured human promyelocytic leukemia cells (HL-60). The presence of fairly low concentrations of VES inhibited the growth and DNA synthesis of HL-60 cells, and also induced their apoptosis via a mechanism that was inhibited by z-VAD-fluoromethylketone (z-VAD-fmk), an inhibitor of pan-caspases. VES activated various types of caspases, including caspase-3, 6, 8, and 9, but not caspase-1. VES triggered the reaction leading to the cleavage of Bid, a member of the death agonist Bcl-2 family, and released cytochrome c (Cyt.c) from the mitochondria into the cytosol by a z-VAD-fmk-inhibitable mechanism. VES transiently increased the intracellular calcium level [Ca2+]i and stimulated the release of Cyt.c in the presence of inorganic phosphate (Pi). However, high concentrations of VES (approximately 100 microM) hardly induced swelling of isolated mitochondria but depolarized the mitochondrial membrane potential by a cyclosporin A (CsA)-insensitive mechanism. These results indicate that VES-induced apoptosis of HL-60 cells might be caused by activation of the caspase cascade coupled with modulation of mitochondrial membrane function.
Assuntos
Apoptose/efeitos dos fármacos , Leucemia Promielocítica Aguda/patologia , Vitamina E/análogos & derivados , Vitamina E/farmacologia , Clorometilcetonas de Aminoácidos/farmacologia , Animais , Inibidores de Caspase , Caspases/metabolismo , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Inibidores de Cisteína Proteinase/farmacologia , DNA/biossíntese , Fragmentação do DNA/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Humanos , Leucemia Promielocítica Aguda/metabolismo , Masculino , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/fisiologia , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Tocoferóis , Células Tumorais Cultivadas , Vitamina E/administração & dosagemRESUMO
Alpha-tocopherol but not 2-carboxy-2,5,7,8-tetramethyl-6-chromanol (trolox or CTMC) and 2,2,5,7,8 pentamethyl-6-hydroxy chromane (PMC), derivatives of alpha-tocopherol, inhibited the superoxide (O2-.) generation of rat peritoneal neutrophils (RPMN) induced by phorbol 12-myrisate 13-acetate (PMA). ID50 for neutrophils obtained from the peritoneal cavity of rat and guinea pig was about 1microM. This concentration, however, was much lower than that for the inhibition of PMA-activated phospholipid-dependent protein kinase (PKC) (ID50 = 30 microM). The alpha-tocopherol sensitive O2-. generation was also observed in neutrophils induced by dioctanoylglycerol (diC8) and calcium ionophore A23187 but not by formylmethionyl-leucyl-phenylalanine (FMLP), opsonized zymosan (OZ) and sodium dodecyl sulfate (SDS). The pattern of inhibition by alpha-tocopherol was quite similar to that of staurosporine, a specific inhibitor of PKC. The alpha-tocopherol content of RPMN was 12 ng/10(6) cells and a linear increase to 200 ng/10(6) cells by addition of alpha-tocopherol to the cell suspension corresponded with an increased inhibition of O2-. generation. These results indicate that both the chemical structure and the content of alpha-tocopherol might be important factors in O2-. generation by neutrophils.
Assuntos
Neutrófilos/efeitos dos fármacos , Superóxidos/metabolismo , Vitamina E/farmacologia , Animais , Antioxidantes/farmacologia , Cromanos/farmacologia , Cobaias , Peso Molecular , Neutrófilos/metabolismo , Fosforilação , Ratos , Estimulação Química , Acetato de Tetradecanoilforbol/antagonistas & inibidores , Vitamina E/análogos & derivadosRESUMO
The present study has been undertaken to examine the effect of cell hybridization of Bloom syndrome (BS) B-lymphoblastoid cell lines (LCLs) and various cell lines from lymphoid malignancies in order to clarify the relationship between sister-chromatoid exchange (SCE) and malignant conditions. Cell hybridization studies have shown that though BS high-SCE frequencies were completed by fusion with normal cells, fusion with various malignant cell lines did not result in complete normalization of BS SCEs, with 15-30 SCEs remaining per hybrid cell, demonstrating possibly common defects in DNA of BS and malignant cells. These findings strongly support the idea that the characteristic high SCE frequency in BS cells has some connection with the malignant condition, and that at least one step in carcinogenesis is either accompanied by the production of SCEs, or that SCEs themselves cause such a step to occur.
Assuntos
Síndrome de Bloom/genética , Leucemia/genética , Linfoma/genética , Troca de Cromátide Irmã , Linfócitos B/fisiologia , Fusão Celular , Teste de Complementação Genética , Humanos , Células Híbridas , Técnicas In VitroRESUMO
Lipoic acid (thiotic acid) is being used as a dietary supplement, and as a therapeutic agent, and is reported to have beneficial effects in disorders associated with oxidative stress, but its mechanism of action remains unclear. We present evidence that lipoic acid induces a substantial increase in cellular reduced glutathione in cultured human Jurkat T cells human erythrocytes, C6 glial cells, NB41A3 neuroblastoma cells, and peripheral blood lymphocytes. The effect depends on metabolic reduction of lipoic acid to dihydrolipoic acid. Dihydrolipoic acid is released into the culture medium where it reduces cystine. Cysteine thus formed is readily taken up by the neutral amino acid transport system and utilized for glutathione synthesis. By this mechanism lipoic acid enables cystine to bypass the xc- transport system, which is weakly expressed in lymphocytes and inhibited by glutamate. Thereby lipoic acid enables the key enzyme of glutathione synthesis, gamma-glutamylcysteine synthetase, which is regulated by uptake-limited cysteine supply, to work at optimum conditions. Flow cytometric analysis of freshly prepared human peripheral blood lymphocytes, using monobromobimane labeling of cellular thiols, reveals that lipoic acid acts mainly to normalize a subpopulation of cells severely compromised in thiol status rather than to increase thiol content beyond physiological levels. Hence lipoic acid may have clinical relevance in restoration of severely glutathione deficient cells.
Assuntos
Cistina/metabolismo , Glutationa/biossíntese , Ácido Tióctico/farmacologia , Animais , Eritrócitos/metabolismo , Citometria de Fluxo , Glutamato-Cisteína Ligase/metabolismo , Ácido Glutâmico/farmacologia , Humanos , Cinética , Leucemia-Linfoma de Células T do Adulto/metabolismo , Camundongos , Neuroblastoma/metabolismo , Neuroglia/metabolismo , Oxirredução , Ratos , Ácido Tióctico/análogos & derivados , Ácido Tióctico/metabolismo , Células Tumorais CultivadasRESUMO
It is now generally accepted that human tumor necrosis factor-alpha (hTNF-alpha) affects not only tumor cells but also normal cells, providing critical tissue damage. hTNF-alpha also enhanced the response of polymorphonuclear neutrophils (PMN) by its priming action and resulted in the increased generation of active oxygen which in turn may be responsible for the tissue injury. Seeking a conventional drug to attenuate the cytolytic activity of tumor necrosis factor (TNF-alpha) and thereby prevent excessive tissue injury, we focused on the cytolytic action of hTNF-alpha against L929 cells, which are sensitive to TNF-alpha, and found that flecainide acetate [N-(2-piperidylmethyl) 1,5-bis-(2,2,2-trifluoroethoxy) benzamide acetate] inhibited specifically the cytolytic action of hTNF-alpha against L929 cells. Flecainide acetate also specifically inhibited the priming action of hTNF-alpha which enhance the formylmethionyl-leucyl-phenylalanine (FMLP)-induced receptor-mediated superoxide (O.2-) generation of human peripheral polymorphonuclear neutrophils (hPMN). The ID50 values for hTNF-alpha induced cytotoxicity in L929 cells and hTNF-alpha primed FMLP-induced O.2- generation of hPMN were 30 and 50-60 microM, respectively. However, the drug does not inhibit the FMLP- or phorbol myristate acetate (PMA)-induced O.2- generation of nonprimed hPMN and has a weak cytotoxic effect on L929 cells. From these results, it is concluded that flecainide acetate suppressed specifically the action of hTNF-alpha.
Assuntos
Antiarrítmicos/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Flecainida/farmacologia , Neutrófilos/fisiologia , Superóxidos/metabolismo , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Animais , Relação Dose-Resposta a Droga , Humanos , Técnicas In Vitro , Células L , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Camundongos , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Neutrófilos/efeitos dos fármacos , Fosfoproteínas/sangue , Fosfoproteínas/isolamento & purificação , Fosfotirosina/análise , Acetato de Tetradecanoilforbol/farmacologia , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Receptor-mediated superoxide (O2-.)-generation in human peripheral neutrophils (HPPMN) is enhanced by various priming agents, such as granulocyte colony stimulating factor (G-CSF) and tumor necrosis factor (TNF-alpha). We previously reported that this enhancement occurred in parallel with the priming agent-induced increase in protein tyrosyl phosphorylation which is sensitive to tyrosine kinase (TK) inhibitors [Akimaru K. et al. Arch. Biochem. Biophys. 298:703, 1992]. We describe here that nonsteroidal anti-inflammatory drugs (NSAIDs) such as indomethacin and aspirin, modulate the priming and tyrosine phosphorylation of HPPMN. The enhancement by TNF-alpha of formylmethionyl-leucyl-phenylalanine (FMLP)-induced O2-. generation was not inhibited by 5 microM azide, whereas that of FMLP-induced luminol chemiluminescence (LCL) was sensitive. Enhancement of FMLP-induced O2-. generation and LCL by priming agents was inhibited by both aspirin and indomethacin in a concentration dependent manner. This inhibition by the NSAIDs was much stronger than that for FMLP-induced O2-. generation without priming. The half-inhibition dose of indomethacin and aspirin were 50 microM and 1.5 mM, respectively. The priming-induced enhancement of tyrosyl phosphorylation of some neutrophil proteins, such as that of 108 and 115 kDa, was also inhibited by aspirin and indomethacin in a dose-dependent manner. However, dose dependent inhibition of the enhanced O2-. generation by the NSAIDs was not completely similar to that of enhanced tyrosyl phosphorylation. However, PKC-mediated O2-. generation, which has little sensitivity to TNF-alpha or G-CSF, was rather stimulated by the NSAIDs. These findings suggest that aspirin and indomethacin inhibit also the early steps of neutrophil activation as reflected by their ability to inhibit priming and the related tyrosyl phosphorylation of neutrophil proteins.
Assuntos
Aspirina/farmacologia , Indometacina/farmacologia , Neutrófilos/metabolismo , Fosfoproteínas/sangue , Fator de Necrose Tumoral alfa/farmacologia , Tirosina/análogos & derivados , Animais , Azidas/farmacologia , Eletroforese em Gel de Poliacrilamida , Fator Estimulador de Colônias de Granulócitos/farmacologia , Cobaias , Humanos , Técnicas In Vitro , Cinética , Medições Luminescentes , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Neutrófilos/efeitos dos fármacos , Fosfoproteínas/isolamento & purificação , Fosforilação , Fosfotirosina , Azida Sódica , Superóxidos/sangue , Acetato de Tetradecanoilforbol/farmacologia , Tirosina/sangueRESUMO
The induction of leukemic cell differentiation is a hopeful therapeutic modality. We studied the effects of monochloramine (NH2Cl) on erythroleukemic K562 cell differentiation, and compared the effects observed with those of U0126 and staurosporine, which are known inducers of erythroid and megakaryocytic differentiation, respectively. CD235 (glycophorin) expression, a marker of erythroid differentiation, was significantly increased by NH2Cl and U0126, along with an increase in cd235 mRNA levels. Other erythroid markers such as γ-globin and CD71 (transferrin receptor) were also increased by NH2Cl and U0126. In contrast, CD61 (integrin ß3) and CD42b (GP1bα) expression, markers of megakaryocytic differentiation, was increased by staurosporine, but did not change significantly by NH2Cl and U0126. NH2Cl retarded cell proliferation without a marked loss of viability. When ERK phosphorylation (T202/Y204) and CD235 expression were compared using various chemicals, a strong negative correlation was observed (r = -0.76). Paradoxically, NH2Cl and staurosporine, but not U0126, induced large cells with multiple or lobulated nuclei, which was characteristic to megakaryocytes. NH2Cl increased the mRNA levels of gata1 and scl, decreased that of gata2, and did not change those of pu.1 and klf1. The changes observed in mRNA expression were different from those of U0126 or staurosporine. These results suggest that NH2Cl induces the bidirectional differentiation of K562. Oxidative stress may be effective in inducing leukemic cell differentiation.