RESUMO
Calmodulin (CaM) antagonists have been shown to inhibit tumor cell invasion and metastasis and to induce apoptosis in various tumor models, but the molecular mechanism of CaM antagonist-mediated apoptosis is poorly understood. Here, we demonstrate that interferon (IFN)-gamma induces susceptibility to CaM antagonist-mediated apoptosis in human cholangiocarcinoma cells weakly expressing Fas (Fas-low cells). During CaM antagonist-mediated apoptosis in IFN-gamma-pretreated Fas-low cells, cleavage of caspases-8, -9, and -3 and Bid, release of cytochrome c from the mitochondria and an increase in the free cytosolic calcium concentration were observed. CaM antagonists also caused depolarization of the mitochondrial membrane independent of caspase activation. Although a broad-range caspase inhibitor partially blocked CaM antagonist-mediated apoptosis, the neutralizing Fas antibody had no effect, suggesting that CaM antagonist-mediated apoptosis does not require interaction between CaM antagonists and surface Fas. CaM antagonists induce apoptosis via mechanisms other than inhibition of CaM-dependent protein kinase II and calcineurin, as their inhibitors, KN93 and cyclosporine A, had no effect on apoptosis. Taken together, these results indicate that CaM antagonists induce apoptosis in both caspase-dependent and -independent manners, and that susceptibility to CaM antagonists is modulated by IFN-gamma. The combination of IFN-gamma and CaM antagonists, including tamoxifen, may be a potential therapeutic modality for cholangiocarcinoma and possibly other malignancies.
Assuntos
Apoptose/efeitos dos fármacos , Calmodulina/antagonistas & inibidores , Caspases/metabolismo , Interferon gama/farmacologia , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3 , Western Blotting , Proteínas de Transporte/efeitos dos fármacos , Proteínas de Transporte/metabolismo , Inibidores de Caspase , Linhagem Celular Tumoral , Colangiocarcinoma/metabolismo , Colangiocarcinoma/patologia , Grupo dos Citocromos c/efeitos dos fármacos , Grupo dos Citocromos c/metabolismo , Inibidores Enzimáticos/farmacologia , Humanos , Potenciais da Membrana/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Receptor fas/genéticaRESUMO
Glucocorticoids are widely used as anti-inflammatory and chemotherapeutic agents. However, prolonged use of glucocorticoids leads to osteoporosis. This study was designed to examine the mechanism of dexamethasone (DEX)-induced apoptosis in murine osteoblastic MC3T3-E1 cells. Total RNA was extracted from MC3T3-E1 cells treated with 10(-7) M DEX for 6 h. DEX exerted a variety of effects on apoptotic gene expression in osteoblasts. Ribonuclease protection assays (RPA) revealed that DEX upregulated mRNA levels of caspases-1, -3, -6, -8, -11, -12, and bcl-XL. Western blot analysis showed enhanced processing of these caspases, with the appearance of their activated enzymes 8 h after DEX treatment. In addition, DEX also induced the activation of caspase-9. DEX elevated the levels of cleaved poly(ADP-ribose) polymerase and lamin A, a caspase-3 and a caspase-6 substrate, respectively. Expression of bcl-XL protein level was upregulated by DEX. Cytochrome c release was detected in the cytosol of DEX-treated cells. Furthermore, caspase-3 enzyme activity was elevated by 2-fold after DEX treatment for 7 h. Finally, early apoptotic cells were detected in cells treated with DEX for 3 h. Our results demonstrate that DEX-induced apoptosis involves gene activation of a number of caspases.
Assuntos
Apoptose/efeitos dos fármacos , Caspases/genética , Dexametasona/farmacologia , Indução Enzimática/efeitos dos fármacos , Osteoblastos/efeitos dos fármacos , Animais , Apoptose/fisiologia , Grupo dos Citocromos c/efeitos dos fármacos , Grupo dos Citocromos c/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/genética , Lamina Tipo A/efeitos dos fármacos , Lamina Tipo A/metabolismo , Camundongos , Osteoblastos/enzimologia , Osteoporose/induzido quimicamente , Osteoporose/enzimologia , Osteoporose/genética , Poli(ADP-Ribose) Polimerase-1 , Poli(ADP-Ribose) Polimerases , Proteínas/efeitos dos fármacos , Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Tempo de Reação/efeitos dos fármacos , Tempo de Reação/fisiologia , Ativação Transcricional , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genética , Proteína bcl-XRESUMO
BACKGROUND AND PURPOSE: Ischemic injury and reperfusion increases superoxide (O2-) production and reduces the ability of neurons to scavenge free radicals, leading to the release of cytochrome c and apoptosis. Here we test whether overexpression with the use of gene therapy of the antioxidant glutathione peroxidase (Gpx), delivered before or after experimental stroke, is protective against ischemic injury. METHODS: Sixty-two rats underwent middle cerebral artery occlusion for 1 hour. Defective herpes simplex viral vectors expressing Gpx/lacZ or lacZ alone (control) were delivered into each striatum 12 hours before or 2 or 5 hours after ischemia onset. RESULTS: Striatal neuron survival at 2 days was improved by 36% when Gpx was delivered 12 hours before ischemia onset, 26% with a 2-hour delay, and 25% when delayed 5 hours. After ischemia, Gpx overexpression significantly reduced cytosolic translocation of cytochrome c and increased the proportion of Bcl-2-positive cells compared with cells transfected with control vector. Bax and activated caspase-3, while present in control-transfected neurons after ischemia, were rarely noted in Gpx-transfected cells. CONCLUSIONS: Expression from these herpes simplex viral vectors begins 4 to 6 hours after injection, which suggests a 9- to 11-hour temporal therapeutic window for Gpx. This is the first study to show that overexpression of Gpx with the use of gene therapy protects against experimental stroke, even with postischemic transfection, and the neuroprotective mechanism involves attenuation of apoptosis-related events.
Assuntos
Apoptose/fisiologia , Grupo dos Citocromos c/metabolismo , Glutationa Peroxidase/biossíntese , Neurônios/metabolismo , Acidente Vascular Cerebral/metabolismo , Animais , Apoptose/efeitos dos fármacos , Caspase 3 , Caspases/efeitos dos fármacos , Caspases/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/metabolismo , Corpo Estriado/patologia , Grupo dos Citocromos c/efeitos dos fármacos , Citoproteção/genética , Modelos Animais de Doenças , Esquema de Medicação , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Glutationa Peroxidase/administração & dosagem , Glutationa Peroxidase/genética , Masculino , Neurônios/efeitos dos fármacos , Neurônios/patologia , Fármacos Neuroprotetores/administração & dosagem , Proteínas Proto-Oncogênicas/efeitos dos fármacos , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos , Ratos Sprague-Dawley , Simplexvirus/genética , Acidente Vascular Cerebral/patologia , Acidente Vascular Cerebral/terapia , Fatores de Tempo , Proteína X Associada a bcl-2RESUMO
N-(4-hydroxyphenyl)retinamide (4-HPR, fenretinide) is a potent chemopreventive agent whose effect has been suggested to involve apoptosis induction. 4-HPR induces a loss of the mitochondrial transmembrane potential and the mitochondrial release of cytochrome c before caspase activation. Inhibition of mitochondrial membrane permeabilization (MMP) by transfection with Bcl-2 or the Cytomegalovirus UL37 gene product vMIA prevented caspase activation and cell death. In contrast to other retinoid derivatives, 4-HPR has no direct MMP-inducing effects when added to isolated mitochondria or when added to proteoliposomes containing the MMP-regulatory permeability transition pore complex (PTPC). Moreover, although reactive oxygen species (ROS) overproduction appears to be instrumental for 4-HPR-induced MMP and apoptosis, inhibition of the NF-kappaB or p53-mediated signal transduction pathways failed to modulate 4-HPR-induced apoptosis. 4-HPR was found to cause an antioxidant-inhibitable conformational change of both Bax and Bak, leading to the exposure of their N-termini and to the mitochondrial relocalization of Bax. Cells with a Bax(-/-) Bak(-/-) genotype were resistant against the 4-HPR-induced MMP, overproduction of ROS and cell death. Altogether, these data indicate that 4-HPR induces MMP through an ROS-mediated pathway that involves the obligatory contribution of the proapopotic Bcl-2 family members Bax and/or Bak.
Assuntos
Anticarcinógenos/farmacologia , Apoptose/efeitos dos fármacos , Fenretinida/farmacologia , Mitocôndrias/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Inibidores de Caspase , Caspases/metabolismo , Grupo dos Citocromos c/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Células HeLa , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mitocôndrias/efeitos dos fármacos , Permeabilidade/efeitos dos fármacos , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Transdução de Sinais , Células Tumorais Cultivadas , Proteína Killer-Antagonista Homóloga a bcl-2 , Proteína X Associada a bcl-2RESUMO
Effects of the PI-3 kinase inhibitor LY294002 (LY) have been examined in relation to responses of human leukemia cells to histone deacetylase inhibitors (HDIs). Coexposure of U937 cells for 24 h to marginally toxic concentrations of LY294002 (e.g., 30 microM) and sodium butyrate (SB; 1 mM) resulted in a marked increase in mitochondrial damage (e.g., cytochrome c and Smac/DIABLO release, loss of DeltaPsi(m)), caspase activation, and apoptosis. Similar results were observed in Jurkat, HL-60, and K562 leukemic cells and with other HDIs (e.g., SAHA, MS-275). Exposure of cells to SB/LY was associated with Bcl-2 and Bid cleavage, XIAP and Mcl-1 downregulation, and diminished CD11b expression. While LY blocked SB-mediated Akt activation, enforced expression of a constitutively active (myristolated) Akt failed to attenuate SB/LY-mediated lethality. Unexpectedly, treatment of cells with SB+/-LY resulted in a marked reduction in phosphorylation (activation) of p44/42 mitogen-activated protein (MAP) kinase. Moreover, enforced expression of a constitutively active MEK1 construct partially but significantly attenuated SB/LY-induced apoptosis. Lastly, cotreatment with LY blocked SB-mediated induction of p21(CIP1/WAF1); moreover, enforced expression of p21(CIP1/WAF1) significantly reduced SB/LY-mediated apoptosis. Together, these findings indicate that LY promotes SB-mediated apoptosis through an AKT-independent process that involves MEK/MAP kinase inactivation and interference with p21(CIP1/WAF1) induction.
Assuntos
Apoptose/efeitos dos fármacos , Ciclinas/metabolismo , Inibidores de Histona Desacetilases , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Benzamidas/farmacologia , Butiratos/farmacologia , Cromonas/farmacologia , Complexo de Ataque à Membrana do Sistema Complemento , Proteínas do Sistema Complemento , Inibidor de Quinase Dependente de Ciclina p21 , Grupo dos Citocromos c/efeitos dos fármacos , Sinergismo Farmacológico , Inibidores Enzimáticos/farmacologia , Glicoproteínas/efeitos dos fármacos , Células HL-60 , Humanos , Células Jurkat , Células K562 , Leucemia Mielogênica Crônica BCR-ABL Positiva/enzimologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Mitocôndrias/efeitos dos fármacos , Morfolinas/farmacologia , Proteínas de Plantas , Piridinas/farmacologia , Células U937RESUMO
We examined the temporal and causal relationship between Smac/DIABLO release, cytochrome c (cyt-c) release, and caspase activation at the single cell level during apoptosis. Cells treated with the broad-spectrum caspase inhibitor z-VAD-fmk, caspase-3 (Casp-3)-deficient MCF-7 cells, as well as Bax-deficient DU-145 cells released Smac/DIABLO and cyt-c in response to proapoptotic agents. Real-time confocal imaging of MCF-7 cells stably expressing Smac/DIABLO-yellow fluorescent protein (YFP) revealed that the average duration of Smac/DIABLO-YFP release was greater than that of cyt-c-green fluorescent protein (GFP). However, there was no significant difference in the time to the onset of release, and both cyt-c-GFP and Smac/DIABLO-YFP release coincided with mitochondrial membrane potential depolarization. We also observed no significant differences in the Smac/DIABLO-YFP release kinetics when z-VAD-fmk-sensitive caspases were inhibited or Casp-3 was reintroduced. Simultaneous measurement of DEVDase activation and Smac/DIABLO-YFP release demonstrated that DEVDase activation occurred within 10 min of release, even in the absence of Casp-3.
Assuntos
Apoptose/fisiologia , Proteínas de Transporte/metabolismo , Células Eucarióticas/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2 , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose , Proteínas de Bactérias , Proteínas de Transporte/efeitos dos fármacos , Caspase 3 , Inibidores de Caspase , Caspases/deficiência , Caspases/genética , Caspases/metabolismo , Linhagem Celular , Grupo dos Citocromos c/efeitos dos fármacos , Grupo dos Citocromos c/metabolismo , Inibidores Enzimáticos/farmacologia , Células Eucarióticas/citologia , Células Eucarióticas/efeitos dos fármacos , Proteínas de Fluorescência Verde , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas Luminescentes , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteínas Mitocondriais/efeitos dos fármacos , Peptídeo Hidrolases/efeitos dos fármacos , Peptídeo Hidrolases/metabolismo , Proteínas Proto-Oncogênicas/deficiência , Proteínas Proto-Oncogênicas/genética , Fatores de Tempo , Proteína X Associada a bcl-2RESUMO
15-deoxy-Delta(12,14)-prostaglandin J(2) (15-d-PGJ(2)) and troglitazone have been shown to induce apoptosis in several carcinoma cell lines. However, apoptotic signaling pathways of these agents are poorly understood. We tested the hypothesis that peroxisome proliferator-activated receptor-gamma ligands such as these two agents will induce caspase-mediated apoptosis in human oral squamous cell carcinomas (SCC). Treatment of these cell lines with 15-d-PGJ(2) or troglitazone decreased cell viability in a time- and dose-dependent manner. 15-d-PGJ(2), but not troglitazone, induced apoptosis, and this effect was time-dependent. Exposure of cells to 20 micro M of 15-d-PGJ(2) initiated early cytochrome c release, followed by late caspase activation. Furthermore, co-treatment with caspase inhibitors such as Z-VAD-FMK or Z-DEVD-FMK of oral SCC cells that had been treated with 20 micro M of 15-d-PGJ(2) blocked apoptosis. Our study demonstrates that treatment with 15-d-PGJ(2), but not troglitazone, induces apoptosis in human SCC cell lines, and 15-d-PGJ(2) appears to work through cytochrome c release and caspase activation.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Carcinoma de Células Escamosas/patologia , Cromanos/farmacologia , Fatores Imunológicos/farmacologia , Neoplasias Bucais/patologia , Prostaglandina D2/farmacologia , Tiazóis/farmacologia , Tiazolidinedionas , Clorometilcetonas de Aminoácidos/farmacologia , Análise de Variância , Antineoplásicos/administração & dosagem , Inibidores de Caspase , Caspases/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cromanos/administração & dosagem , Inibidores de Cisteína Proteinase/farmacologia , Grupo dos Citocromos c/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Humanos , Fatores Imunológicos/administração & dosagem , Oligopeptídeos/farmacologia , Prostaglandina D2/administração & dosagem , Prostaglandina D2/análogos & derivados , Transdução de Sinais/efeitos dos fármacos , Tiazóis/administração & dosagem , Fatores de Tempo , Troglitazona , Células Tumorais CultivadasRESUMO
BACKGROUND: The incidence of nonmelanoma skin cancer, basal cell carcinomas (BCCs), and squamous cell carcinomas (SCCs) is increasing, representing a major medical and economic problem. Imiquimod, a topical small-molecule immune response modifier, has shown efficacy toward BCC and actinic keratoses in clinical trials. Imiquimod acts both indirectly, via cytokine-mediated stimulation of cellular immune responses, and directly, through unknown mechanisms against tumor cells. We examined the mechanism by which imiquimod induces apoptosis in cancer cells. METHODS: Apoptosis was assessed by enzyme-linked immunosorbent assay, western blot analysis, and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assays in five SCC cell lines, HaCaT cells (a spontaneously immortalized human keratinocyte cell line), and normal keratinocytes treated with imiquimod, with its analog resiquimod, or with neither. Expression of death receptors, caspases, and cytochrome c in the apoptotic signaling cascade was analyzed using western blot and flow cytometric analyses. The functional relevance of imiquimod-induced cytochrome c release was assessed by transfection of HaCaT cells with Bcl-2. Apoptosis in BCCs in vivo was assessed by TUNEL assays of imiquimod-treated and untreated tumors from three patients. Differences between treated and untreated cells and tumors were determined using a two-tailed Student's t test. RESULTS: Imiquimod, but not resiquimod, induced apoptosis in all SCC cell lines and HaCaT cells. This induction involved activation of several caspases and Bcl-2-dependent cytosolic translocation of cytochrome c but was independent of the membrane-bound death receptors Fas, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-R1-R4 receptors, and tumor necrosis factor-R1 and -R2 receptors. Topical application of imiquimod to BCC tumors in vivo induced apoptosis. CONCLUSION: Imiquimod has the potential to induce apoptosis in skin cancer cells, possibly by circumventing mechanisms developed by malignant tumors to resist apoptotic signals.
Assuntos
Aminoquinolinas/farmacologia , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/imunologia , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/imunologia , Imidazóis/farmacologia , Fatores Imunológicos/farmacologia , Western Blotting , Carcinoma Basocelular/tratamento farmacológico , Carcinoma Basocelular/imunologia , Carcinoma de Células Escamosas/metabolismo , Caspase 3 , Caspases/efeitos dos fármacos , Grupo dos Citocromos c/efeitos dos fármacos , Fragmentação do DNA/efeitos dos fármacos , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Imiquimode , Marcação In Situ das Extremidades Cortadas , Queratinócitos/efeitos dos fármacos , Queratinócitos/imunologia , Proteínas Proto-Oncogênicas c-bcl-2/efeitos dos fármacos , Receptores do Fator de Necrose Tumoral/efeitos dos fármacos , Células Tumorais Cultivadas , Regulação para Cima/efeitos dos fármacosRESUMO
Various stimuli including anticancer drugs are capable of initiating the apoptotic death program in human tumor cells via activation of caspases. Mitochondria play an essential role for cell apoptotic commitment. Previous studies have shown a potential role of calpain activation in apoptosis, however, the involved molecular mechanisms remain to be defined. In the current study, we have examined the expression and activation of mitochondrial calpain in Jurkat T leukemia cells, MCF-7 breast carcinoma and LNCaP prostate cancer cells during apoptosis induced by an anticancer drug (VP-16, tamoxifen) or the specific p38 kinase inhibitor PD-169316. Our results suggest that increased expression and autolysis of the mitochondrial calpain small subunit are tightly associated with calpain activation in an early stage of apoptosis. In contrast, there were no correlations observed between the early calpain activation and changes in levels of mitochondrial calpain large subunit and the endogenous calpain inhibitor calpastatin. Furthermore, pretreatment with the specific pharmacological calpain inhibitor calpeptin blocked the drug-induced calpain small subunit autolysis and calpain activation in mitochondria and inhibited apoptosis-associated caspase-3 activation, demonstrating that mitochondrial calpain activation through small subunit cleavage is an essential step for inducing tumor cell apoptosis by various anticancer drugs.
Assuntos
Apoptose/fisiologia , Calpaína/metabolismo , Mitocôndrias/metabolismo , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Autólise , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Grupo dos Citocromos c/efeitos dos fármacos , Grupo dos Citocromos c/metabolismo , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Etoposídeo/farmacologia , Feminino , Humanos , Imidazóis/farmacologia , Células Jurkat/efeitos dos fármacos , Masculino , Mitocôndrias/enzimologia , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Subunidades Proteicas , Tamoxifeno/farmacologia , Células Tumorais Cultivadas , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
We have recently reported that the cytokine interferon-alpha (IFNalpha), commonly used in the treatment of cancer, induced a caspase-dependent apoptosis in tumor cell lines. The signaling mechanisms involved have not been defined. Here, we show that both proapoptotic Bcl-2 family members Bak and Bax were activated by IFNalpha, strictly in correlation with the induction of apoptosis. Using double stainings, we demonstrated that Bak was activated prior to cytochrome c (cyt c) release and caspase-3 activation, whereas activated Bax was only found in cells with released cyt c, mitochondrial depolarization, as well as activated caspase-3. Furthermore, IFNalpha-induced activation of Bak, and to a large extent also of Bax, was dependent on caspase activity. With the use of a panel of specific caspase inhibitors we found, however, that none of caspases-1 to -10 were responsible for this activation. Neither was the Ca(2+)-dependent protease calpain nor the stress-activated p38 SAPK pathway significantly involved. Overexpression of Bcl-2 blocked apoptosis induced by IFNalpha totally abolished Bak activation, as well as decreased the amount of activated Bax. We conclude that IFNalpha induces Bak and Bax activation via distinct mechanisms involving an unknown protease, and that their activation is regulated by Bcl-2.
Assuntos
Apoptose/efeitos dos fármacos , Linfoma de Burkitt/metabolismo , Interferon-alfa/farmacologia , Proteínas de Membrana/metabolismo , Mieloma Múltiplo/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Apoptose/fisiologia , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3 , Linfoma de Burkitt/tratamento farmacológico , Linfoma de Burkitt/patologia , Calpaína/antagonistas & inibidores , Calpaína/metabolismo , Proteínas de Transporte/efeitos dos fármacos , Proteínas de Transporte/metabolismo , Inibidores de Caspase , Caspases/metabolismo , Inibidores de Cisteína Proteinase/farmacologia , Grupo dos Citocromos c/efeitos dos fármacos , Grupo dos Citocromos c/metabolismo , Inibidores Enzimáticos/farmacologia , Humanos , Potenciais da Membrana/efeitos dos fármacos , Proteínas de Membrana/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/patologia , Proteínas Proto-Oncogênicas/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/genética , Células Tumorais Cultivadas , Proteína Killer-Antagonista Homóloga a bcl-2 , Proteína X Associada a bcl-2 , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
In Parkinson's disease, neuroprotective therapy to rescue dopamine neurons has been proposed. Ginsenoside Rg1, one of the biologically active ingredients of ginseng, may be a candidate neuroprotective drug. In the present study, the mechanism underlying the neuroprotection provided by ginsenosde Rg1 was studied against apoptosis induced by exogenous dopamine in PC12 cells. Pretreatment with ginsenoside Rg1 markedly reduced the generation of dopamine-induced reactive oxygen species and the release of mitochondrial cytochrome c into the cytosol, and subsequently inhibited the activation of caspase-3. In addition, Rg1 pretreatment also reduced inducible nitric oxide (NO) synthase protein level and NO production. These results suggested that ginsenoside Rg1 may attenuate dopamine-induced apoptotic cell death through suppression of intracellular oxidative stress, and that it may rescue or protect dopamine neurons in Parkinson's disease.
Assuntos
Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Dopamina/metabolismo , Ginsenosídeos/farmacologia , Fármacos Neuroprotetores/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Animais , Western Blotting , Caspase 3 , Inibidores de Caspase , Caspases/metabolismo , Grupo dos Citocromos c/efeitos dos fármacos , Grupo dos Citocromos c/metabolismo , Fragmentação do DNA , Dopamina/farmacologia , Citometria de Fluxo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo II , Células PC12 , Panax/química , RatosRESUMO
Many pro-apoptotic signals activate caspase-9, an initiator protease that activates caspase-3 and downstream caspases to initiate cellular destruction. However, survival signals can impinge on this pathway and suppress apoptosis. Activation of the Ras-Raf-MEK-ERK mitogen-activated protein kinase (MAPK) pathway is associated with protection of cells from apoptosis and inhibition of caspase-3 activation, although the targets are unknown. Here, we show that the ERK MAPK pathway inhibits caspase-9 activity by direct phosphorylation. In mammalian cell extracts, cytochrome c-induced activation of caspases-9 and -3 requires okadaic-acid-sensitive protein phosphatase activity. The opposing protein kinase activity is overcome by treatment with the broad-specificity kinase inhibitor staurosporine or with inhibitors of MEK1/2. Caspase-9 is phosphorylated at Thr 125, a conserved MAPK consensus site targeted by ERK2 in vitro, in a MEK-dependent manner in cells stimulated with epidermal growth factor (EGF) or 12-O-tetradecanoylphorbol-13-acetate (TPA). Phosphorylation at Thr 125 is sufficient to block caspase-9 processing and subsequent caspase-3 activation. We suggest that phosphorylation and inhibition of caspase-9 by ERK promotes cell survival during development and tissue homeostasis. This mechanism may also contribute to tumorigenesis when the ERK MAPK pathway is constitutively activated.
Assuntos
Apoptose/fisiologia , Caspases/metabolismo , Sobrevivência Celular/fisiologia , Transformação Celular Neoplásica/metabolismo , Células Eucarióticas/enzimologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Células 3T3 , Animais , Apoptose/efeitos dos fármacos , Sequência de Bases/genética , Caspase 3 , Caspase 9 , Sobrevivência Celular/efeitos dos fármacos , Transformação Celular Neoplásica/genética , Grupo dos Citocromos c/efeitos dos fármacos , Grupo dos Citocromos c/metabolismo , Inibidores Enzimáticos/farmacologia , Fator de Crescimento Epidérmico/farmacologia , Células Eucarióticas/efeitos dos fármacos , Células HeLa , Humanos , MAP Quinase Quinase 1 , Camundongos , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Dados de Sequência Molecular , Fosforilação/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/metabolismo , Piridinas/farmacologia , Proteínas Recombinantes de Fusão , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Treonina/metabolismoRESUMO
Trehalose, a naturally occurring osmolyte, is known to be an exceptional stabilizer of proteins and helps retain the activity of enzymes in solution as well as in the freeze-dried state. To understand the mechanism of action of trehalose in detail, we have conducted a thorough investigation of its effect on the thermal stability in aqueous solutions of five well characterized proteins differing in their various physico-chemical properties. Among them, RNase A has been used as a model enzyme to investigate the effect of trehalose on the retention of enzymatic activity upon incubation at high temperatures. 2 m trehalose was observed to raise the transition temperature, Tm of RNase A by as much as 18 degrees C and Gibbs free energy by 4.8 kcal mol-1 at pH 2.5. There is a decrease in the heat capacity of protein denaturation (DeltaCp) in trehalose solutions for all the studied proteins. An increase in the DeltaG and a decrease in the DeltaCp values for all the proteins points toward a general mechanism of stabilization due to the elevation and broadening of the stability curve (DeltaG versus T). A direct correlation of the surface tension of trehalose solutions and the thermal stability of various proteins has been observed. Wyman linkage analysis indicates that at 1.5 m concentration 4-7 molecules of trehalose are excluded from the vicinity of protein molecules upon denaturation. We further show that an increase in the stability of proteins in the presence of trehalose depends upon the length of the polypeptide chain. The pH dependence data suggest that even though the charge status of a protein contributes significantly, trehalose can be expected to work as a universal stabilizer of protein conformation due to its exceptional effect on the structure and properties of solvent water compared with other sugars and polyols.
Assuntos
Excipientes/farmacologia , Proteínas/química , Proteínas/efeitos dos fármacos , Trealose/farmacologia , Animais , Bovinos , Fenômenos Químicos , Físico-Química , Quimotripsinogênio/química , Quimotripsinogênio/efeitos dos fármacos , Quimotripsinogênio/metabolismo , Grupo dos Citocromos c/química , Grupo dos Citocromos c/efeitos dos fármacos , Grupo dos Citocromos c/metabolismo , Estabilidade de Medicamentos , Estabilidade Enzimática/efeitos dos fármacos , Técnicas In Vitro , Cinética , Muramidase/química , Muramidase/efeitos dos fármacos , Muramidase/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/efeitos dos fármacos , Proteínas de Plantas/metabolismo , Desnaturação Proteica/efeitos dos fármacos , Proteínas/metabolismo , Ribonuclease Pancreático/química , Ribonuclease Pancreático/efeitos dos fármacos , Ribonuclease Pancreático/metabolismo , Soluções , Tensão Superficial , Temperatura , Termodinâmica , Inibidores da Tripsina , alfa-Amilases/antagonistas & inibidoresRESUMO
Reactive oxygen species (ROS) appear to play an important role in regulating growth and survival of prostate cancer. However, the sources for ROS production in prostate cancer cells have not been determined. We report that ROS are generated by intact American Type Culture Collection DU 145 cells and by their membranes through a mechanism blocked by NAD(P)H oxidase inhibitors. ROS are critical for growth in these cells, because NAD(P)H oxidase inhibitors and antioxidants blocked proliferation. Components of the human phagocyte NAD(P)H oxidase, p22phox and gp91phox, as well as the Ca2+ concentration-responsive gp91phox homolog NOX5 were demonstrated in DU 145 cells by RT-PCR and sequencing. Although the protein product for p22phox was not detectable, both gp91phox and NOX5 were identified throughout the cell by immunostaining and confocal microscopy and NOX5 immunostaining was enhanced in a perinuclear location, corresponding to enhanced ROS production adjacent to the nuclear membrane imaged by 2',7'-dichlorofluorescin diacetate oxidation. The calcium ionophore ionomycin dramatically stimulated ferricytochrome c reduction in cell media, further supporting the importance of NOX5 for ROS production. Antisense oligonucleotides for NOX5 inhibited ROS production and cell proliferation in DU 145 cells. In contrast, antisense oligonucleotides to p22phox or gp91phox did not impair cell growth. Inhibition of ROS generation with antioxidants or NAD(P)H oxidase inhibitors increased apoptosis in cells. These results indicate that ROS generated by the newly described NOX5 oxidase are essential for prostate cancer growth, possibly by providing trophic intracellular oxidant tone that retards programmed cell death.
Assuntos
Apoptose/genética , Carcinoma/enzimologia , Divisão Celular/genética , Proteínas de Membrana/metabolismo , NADPH Oxidases/metabolismo , Neoplasias da Próstata/enzimologia , Espécies Reativas de Oxigênio/metabolismo , Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/metabolismo , Grupo dos Citocromos c/efeitos dos fármacos , Grupo dos Citocromos c/metabolismo , Inibidores Enzimáticos/farmacologia , Humanos , Ionóforos/farmacologia , Masculino , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , NADPH Oxidase 2 , NADPH Oxidase 5 , NADPH Oxidases/antagonistas & inibidores , NADPH Oxidases/genética , Membrana Nuclear/metabolismo , Oligorribonucleotídeos Antissenso/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Células Tumorais CultivadasRESUMO
Low-risk myelodysplastic syndromes (MDS), including refractory anemia and sideroblastic anemia, are characterized by increased apoptotic death of erythroid progenitors. The signaling pathways that elicit this pathologic cell death in MDS have, however, remained unclear. Treatment with erythropoietin in combination with granulocyte colony-stimulating factor (G-CSF) may synergistically improve the anemia in patients with MDS, with a concomitant decrease in the number of apoptotic bone marrow precursors. Moreover, we have previously reported that G-CSF inhibits Fas-induced caspase activation in sideroblastic anemia (RARS). The present data demonstrate that almost 50% of erythroid progenitor cells derived from patients with MDS exhibit spontaneous release of cytochrome c from mitochondria with ensuing activation of caspase-9, whereas normal erythroid progenitors display neither of these features. G-CSF significantly inhibited cytochrome c release and suppressed apoptosis, most noticeably in cells from patients with sideroblastic anemia. Furthermore, inhibition of caspase-9 suppressed both spontaneous and Fas-mediated apoptosis of erythroid progenitors in all low-risk MDS cases studied. We propose that the increased sensitivity of MDS progenitor cells to death receptor stimulation is due to a constitutive activation of the mitochondrial axis of the apoptotic signaling pathway in these cells. These studies yield a mechanistic explanation for the beneficial clinical effects of growth factor administration in patients with MDS, and provide a model for the study of growth factor-mediated suppression of apoptosis in other bone marrow disorders.
Assuntos
Apoptose/efeitos dos fármacos , Grupo dos Citocromos c/metabolismo , Fator Estimulador de Colônias de Granulócitos/farmacologia , Mitocôndrias/fisiologia , Síndromes Mielodisplásicas/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Inibidores de Caspase , Grupo dos Citocromos c/efeitos dos fármacos , Células Precursoras Eritroides/efeitos dos fármacos , Células Precursoras Eritroides/patologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/patologia , Humanos , Pessoa de Meia-Idade , Mitocôndrias/metabolismo , Transdução de Sinais/efeitos dos fármacos , Receptor fas/farmacologiaRESUMO
The current study examines the association between glucose induction of reactive oxygen species (ROS), mitochondrial (Mt) depolarization, and programmed cell death in primary neurons. In primary dorsal root ganglion (DRG) neurons, 45 mM glucose rapidly induces a peak rise in ROS corresponding to a 50% increase in mean Mt size at 6 h (P<0.001). This is coupled with loss of regulation of the Mt membrane potential (Mt membrane hyperpolarization, followed by depolarization, MMD), partial depletion of ATP, and activation of caspase-3 and -9. Glucose-induced activation of ROS, MMD, and caspase-3 and -9 activation is inhibited by myxothiazole and thenoyltrifluoroacetone (P<0.001), which inhibit specific components of the Mt electron transfer chain. Similarly, MMD and caspase-3 activation are inhibited by 100 microM bongkrekic acid (an inhibitor of the adenosine nucleotide translocase ANT). These results indicate that mild increases in glucose induce ROS and Mt swelling that precedes neuronal apoptosis. Glucotoxicity is blocked by inhibiting ROS induction, MMD, or caspase cleavage by specific inhibitors of electron transfer, or by stabilizing the ANT.
Assuntos
Glucose/farmacologia , Mitocôndrias/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Apoptose/efeitos dos fármacos , Caspase 3 , Caspase 9 , Inibidores de Caspase , Caspases/metabolismo , Células Cultivadas , Grupo dos Citocromos c/efeitos dos fármacos , Grupo dos Citocromos c/metabolismo , Relação Dose-Resposta a Droga , Gânglios Espinais/citologia , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/metabolismo , Marcação In Situ das Extremidades Cortadas , Membranas Intracelulares/efeitos dos fármacos , Membranas Intracelulares/fisiologia , Potenciais da Membrana/efeitos dos fármacos , Microscopia Confocal , Mitocôndrias/fisiologia , Neurônios/metabolismo , Estresse Oxidativo/efeitos dos fármacos , RatosRESUMO
The retinoblastoma tumour suppressor protein RB is cleaved by caspases during apoptosis. Here we have mutated the caspase cleavage site in the carboxy terminus of the murine Rb protein in the mouse germ line to create the Rb-MI allele. After endotoxic shock, expression of Rb-MI inhibits apoptosis in the intestines, but not in the spleen, and promotes the survival of male mice. Fibroblasts expressing Rb-MI protein are protected from apoptosis induced by the tumour-necrosis factor-alpha type I receptor (TNFRI) but remain sensitive to cell death induced by DNA damage. Correspondingly, the release of cytochrome c and the activation of caspase-3 induced by TNFRI, but not by DNA damage, are defective in cells expressing Rb-MI. Our results highlight the importance of Rb cleavage in TNFRI-induced apoptosis.
Assuntos
Antígenos CD/metabolismo , Apoptose/genética , Caspases/metabolismo , Células Eucarióticas/metabolismo , Receptores do Fator de Necrose Tumoral/metabolismo , Proteína do Retinoblastoma/deficiência , Proteína do Retinoblastoma/genética , Células 3T3 , Alelos , Animais , Animais Recém-Nascidos , Apoptose/efeitos dos fármacos , Caspase 3 , Grupo dos Citocromos c/efeitos dos fármacos , Grupo dos Citocromos c/metabolismo , Células Eucarióticas/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Lipopolissacarídeos , Camundongos , Camundongos Knockout , Mutação/genética , Receptores do Fator de Necrose Tumoral/antagonistas & inibidores , Receptores Tipo I de Fatores de Necrose Tumoral , Receptores Tipo II do Fator de Necrose Tumoral , Retina/crescimento & desenvolvimento , Retina/metabolismo , Retina/efeitos da radiação , Choque Séptico/induzido quimicamente , Choque Séptico/genética , Choque Séptico/metabolismo , Transdução de Sinais/genética , Toxinas Biológicas , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder characterized by motor neuron involvement. Mutations in the human Cu/Zn superoxide dismutase (SOD1) gene are found in some cases of familial ALS. Many studies have reported SOD1 mutation-related neurodegeneration. However, whether or not a mutant SOD1 affects neural development has not been demonstrated. We developed motor neuron-neuroblastoma hybrid cells that expressed a mutant (G93A) or the wild type (WT) SOD1. Cells were differentiated by dibutyryl cAMP and aphidicolin. The mutant showed a defect in neurite outgrowth and had decreased viability. Cytochrome c released and nuclear fragmentation were observed. Western blot analysis showed that the amount of neurofilament and microtubule associated proteins-2 (MAP-2) decreased during differentiation. These results suggest that the defect in neurite outgrowth of mutant SOD1 cells is a cytoskeletal defect and is associated with neuronal death.
Assuntos
Esclerose Lateral Amiotrófica/enzimologia , Diferenciação Celular/genética , Sobrevivência Celular/genética , Neurônios Motores/enzimologia , Mutação/genética , Neuritos/enzimologia , Superóxido Dismutase/deficiência , Esclerose Lateral Amiotrófica/genética , Animais , Afidicolina , Diferenciação Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , AMP Cíclico , Grupo dos Citocromos c/efeitos dos fármacos , Grupo dos Citocromos c/metabolismo , Fragmentação do DNA/efeitos dos fármacos , Fragmentação do DNA/genética , Inibidores Enzimáticos , Humanos , Proteínas Associadas aos Microtúbulos/efeitos dos fármacos , Proteínas Associadas aos Microtúbulos/metabolismo , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/patologia , Neuritos/efeitos dos fármacos , Neuritos/patologia , Proteínas de Neurofilamentos/efeitos dos fármacos , Proteínas de Neurofilamentos/metabolismo , Superóxido Dismutase/genética , Células Tumorais CultivadasRESUMO
Indoleamine 2,3-dioxygenase (IDO) is a tryptophan-catabolizing enzyme that, expressed by different cell types, has regulatory effects on T cells resulting from tryptophan depletion in specific local tissue microenvironments. Different mechanisms, however, might contribute to IDO-dependent immune regulation. We show here that tryptophan metabolites in the kynurenine pathway, such as 3-hydroxyanthranilic and quinolinic acids, will induce the selective apoptosis in vitro of murine thymocytes and of Th1 but not Th2 cells. T cell apoptosis was observed at relatively low concentrations of kynurenines, did not require Fas/Fas ligand interactions, and was associated with the activation of caspase-8 and the release of cytochrome c from mitochondria. When administered in vivo, the two kynurenines caused depletion of specific thymocyte subsets in a fashion qualitatively similar to dexamethasone. These data suggest that the selective deletion of T lymphocytes may be a major mechanism whereby tryptophan metabolism affects immunity under physiopathologic conditions.
Assuntos
Apoptose/fisiologia , Doenças Autoimunes/imunologia , Cinurenina/metabolismo , Tolerância a Antígenos Próprios/imunologia , Linfócitos T/metabolismo , Triptofano/metabolismo , Ácido 3-Hidroxiantranílico/metabolismo , Ácido 3-Hidroxiantranílico/farmacologia , Animais , Apoptose/efeitos dos fármacos , Caspase 8 , Caspase 9 , Inibidores de Caspase , Caspases/metabolismo , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Células Cultivadas , Grupo dos Citocromos c/efeitos dos fármacos , Grupo dos Citocromos c/metabolismo , Citosol/efeitos dos fármacos , Citosol/metabolismo , Relação Dose-Resposta a Droga , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Microscopia Eletrônica , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Ácido Quinolínico/metabolismo , Ácido Quinolínico/farmacologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/ultraestrutura , Células Th1/efeitos dos fármacos , Células Th1/metabolismo , Células Th1/ultraestrutura , Timo/efeitos dos fármacos , Timo/metabolismo , Timo/ultraestrutura , Receptor fas/genética , Receptor fas/metabolismoRESUMO
Exposure to methoxyacetic acid (MAA), a major byproduct of the paint industry, causes testicular atrophy in multiple species. This study demonstrates DNA breakdown in rat germ cells after exposure to MAA in vivo within 12 h, leading to 40% germ cell death by 24 h. Within 4 h of treatment, cytochrome c is released from the mitochondria into the cytosol without the involvement of mitochondrial potential loss, reactive oxygen species generation or lipid peroxidation events. Peak activation of caspase-9 and caspase-3 is detectable post treatment at 4 and 8 h respectively. There is a decrease in germ cell glutathione levels within 2 h of MAA treatment. Replenishment of glutathione by pretreatment of the animals with the antioxidant N-acetylcysteine prior to MAA treatment could prevent the release of cytochrome c, DNA fragmentation and cell death.