RESUMO
The role of the 18-kDa Translocator Protein (TSPO) in cell death induced by NH4Cl (1-50 mM) for 24-72 hours to human glioblastoma U118MG cells was investigated. Cell death was already observed after 48 hours of treatment with NH4Cl at 5 mM. Dose and time-responses curves indicated that 15 mM of NH4Cl applied for 72 hours was the optimal condition for our viability assays. For example, 72 hours of 15 mM of NH4Cl caused a 50.3% increase in propidium iodide uptake, and lactate dehydrogenase release was 41.2% of the positive control, indicating significant increases in cell death. Furthermore, compared to vehicle control, these experimental conditions resulted in a significant decrease of 44.9% of the mitochondrial activity, a 62.3% increase in incidence of collapse of mitochondrial membrane potential, and an increase of 49.0% of cardiolipin peroxidation. In addition, a significant 4.3 fold increase in the maximal binding capacity (Bmax) of TSPO was found in NH4Cl-exposed cells. Surprisingly, western blot analysis and real-time PCR did not demonstrate changes in TSPO expression. We also found that neither NH4Cl nor glutamine (a metabolic product of enhanced NH4Cl levels) inhibited binding of the TSPO ligand [(3)H]PK 11195. Interestingly, we observed a bimodal effect of the TSPO ligands PK 11195, Ro5-4864, and FGIN-1-27 on the toxicity of NH4Cl; such that 1-100 nM concentrations of TSPO ligands were protective, while concentrations above 1 µM enhanced NH4Cl-induced cell death processes. In conclusion, TSPO takes part in a bimodal way in the lethal effects induced by NH4Cl in glial type cells.
Assuntos
Cloreto de Amônio/toxicidade , Morte Celular/efeitos dos fármacos , Morte Celular/fisiologia , Hiperamonemia/fisiopatologia , Receptores de GABA/metabolismo , Benzodiazepinonas/farmacologia , Cardiolipinas/metabolismo , Linhagem Celular Tumoral , Forma Celular , Relação Dose-Resposta a Droga , GABAérgicos/farmacologia , Glutamina/metabolismo , Humanos , Hiperamonemia/induzido quimicamente , Hiperamonemia/tratamento farmacológico , Ácidos Indolacéticos/farmacologia , Isoquinolinas/metabolismo , Isoquinolinas/farmacologia , L-Lactato Desidrogenase/metabolismo , Ligantes , Peroxidação de Lipídeos/efeitos dos fármacos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Potencial da Membrana Mitocondrial/fisiologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/fisiologia , Fármacos Neuroprotetores/farmacologia , Propídio/químicaRESUMO
Various studies have shown that several lethal agents induce cell death via the mitochondrial 18 kDa Translocator Protein (TSPO). In this study we tested the possibility that nitric oxide (NO) is the signaling component inducing the TSPO to initiate cell death process. Cell viability assays included Trypan blue uptake, propidium iodide uptake, lactate dehydrogenase release, and DNA fragmentation. These assays showed that application of the specific TSPO ligand PK 11195 reduced these parameters for the lethal effects of the NO donor sodium nitroprusside (SNP) by 41, 27, 40, and 42 %, respectively. TSPO silencing by siRNA also reduced the measured lethal effects of SNP by 50 % for all of these four assays. With 2,3-bis[2-methoxy-4-nitro-5-sulphophenyl]-2H-tetrazolium-5-carboxyanilide (XTT) changes in metabolic activity were detected. PK 11195 and TSPO knockdown fully prevented the reductions in XTT signal otherwise induced by SNP. Collapse of the mitochondrial membrane potential was studied with the aid of JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-benzimidazolylcarbocyanine chloride). PK 11195 and TSPO knockdown reduced, respectively by 36 and 100 %, the incidence of collapse of the mitochondrial membrane potential otherwise induced by SNP. 10-N-Nonyl-Acridine Orange (NAO) was used to detect mitochondrial reactive oxygen species generation due to SNP. PK 11195 and TSPO knockdown reduced this effect of SNP by 65 and 100 %, respectively. SNP did not affect TSPO protein expression and binding characteristics, and also did not cause TSPO S-nitrosylation. However, ß-actin and various other proteins (not further defined) were S-nitrosylated. In conclusion, TSPO is required for the lethal and metabolic effects of the NO donor SNP, but TSPO itself is not S-nitrosylated.
Assuntos
Proteínas de Transporte/metabolismo , Doadores de Óxido Nítrico/farmacologia , Nitroprussiato/farmacologia , Receptores de GABA-A/metabolismo , Receptores de GABA/metabolismo , Animais , Western Blotting , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Técnicas de Silenciamento de Genes , Humanos , Isoquinolinas/farmacologia , Ligantes , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Peso Molecular , Nitrosação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Ratos , Canais de Ânion Dependentes de Voltagem/metabolismoRESUMO
The role of the TSPO in metabolism of human osteoblasts is unknown. We hypothesized that human osteoblast metabolism may be modulated by the TSPO. Therefore we evaluated the presence of TSPO in human osteoblast-like cells and the effect of its synthetic ligand PK 11195 on these cells. The presence of TSPO was determined by [(3)H]PK 11195 binding using Scatchard analysis: Bmax 7682 fmol/mg, Kd 9.24 nM. PK 11195 did not affect significantly cell proliferation, cell death, cellular viability, maturation, [(18)F]-FDG incorporation and hexokinase 2 gene expression or protein levels. PK 11195 exerted a suppressive effect on VDAC1 and caused an increase in TSPO gene expression or protein levels. In parallel there was an increase in mitochondrial mass, mitochondrial ATP content and a reduction in ΔΨm collapse. Thus, it appears that PK11195 (10(-5) M) stimulates mitochondrial activity in human osteoblast-like cells without affecting glycolytic activity and cell death.
Assuntos
Antineoplásicos/farmacologia , Isoquinolinas/farmacologia , Mitocôndrias/metabolismo , Proteínas Mitocondriais/biossíntese , Receptores de GABA/biossíntese , Trifosfato de Adenosina/metabolismo , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Glicólise/efeitos dos fármacos , Hexoquinase/biossíntese , Humanos , Osteoblastos , Canal de Ânion 1 Dependente de Voltagem/biossínteseRESUMO
7,12-Dimethylbenz[a]anthracene (DMBA) presents a pollutant implicated in various toxicological effects. The aim of this experiment was to study the effects of DMBA administration on oxidative stress, histopathological signs, and 18 kDa translocator protein (TSPO) binding characteristics in rat liver. We also studied the effects of dose stoichiometry, dose frequency, and duration of protocol of DMBA administration. In this study, rats surviving eighteen weeks after DMBA exposure showed mild to moderate histopathological changes in the liver, mainly characterized by glossy appearance of hepatocytes, heterochromatic nuclei, and glycogen overload in the midzonal region of the hepatic lobe. These changes were accompanied by significant rises in oxidant levels, along with declines in nonenzymic antioxidants, indicating that DMBA induced oxidative stress in the liver. This finding correlated well with decreases in TSPO binding capacity in the liver of the rats in our study. Other studies have shown that TSPO can be affected by oxidative stress, as well as contribute to oxidative stress at mitochondrial levels. Further studies are needed to assay whether the decreases in TSPO density in the liver are part of the damaging effects caused by DMBA or a compensatory response to the oxidative stress induced by DMBA.
Assuntos
9,10-Dimetil-1,2-benzantraceno/toxicidade , Carcinógenos/toxicidade , Proteínas de Transporte/efeitos dos fármacos , Fígado/efeitos dos fármacos , Mitocôndrias Hepáticas/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Receptores de GABA-A/efeitos dos fármacos , Animais , Proteínas de Transporte/metabolismo , Fígado/metabolismo , Fígado/patologia , Masculino , Mitocôndrias Hepáticas/metabolismo , Proteínas Mitocondriais/efeitos dos fármacos , Proteínas Mitocondriais/metabolismo , Ligação Proteica/efeitos dos fármacos , Ratos , Ratos Wistar , Receptores de GABA-A/metabolismoRESUMO
In this study, rats surviving 18 weeks after 7,12-dimethylbenz[a]anthracene (DMBA) exposure showed robust pathological changes in the aorta. This correlated well with decreases in 18-kDa translocator protein (TSPO) binding capacity in this tissue. As expected, markers for oxidative stress, including thiobarbituric-acid-reactive substances, and advanced oxidation protein products, showed that the applied DMBA exposure increased oxidative stress in the aorta. Our study suggests that TSPO may be involved in toxic DMBA effects in the aorta, including inflammatory responses and reactive oxygen species generation.
Assuntos
9,10-Dimetil-1,2-benzantraceno/toxicidade , Aorta/efeitos dos fármacos , Proteínas de Transporte/metabolismo , Mitocôndrias/efeitos dos fármacos , Proteínas Mitocondriais/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Receptores de GABA-A/metabolismo , Animais , Aorta/metabolismo , Aorta/patologia , Ligantes , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Mitocôndrias/metabolismo , Ligação Proteica , Ratos , Ratos Wistar , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismo , Fatores de TempoRESUMO
BACKGROUND: Cigarette smoke (CS) is the major risk factor for aerodigestive tract cancers such as lung and oral cancers. METHODS: In in vitro models of lung and oral cancers, we found D-penicillamine (PenA) to be a most potent protector against CS, both in the absence and presence of saliva (a highly pro-oxidative condition). RESULTS: The survival rate of lung cancer cells and oral cancer cells was reduced by CS in the absence of saliva by 39-45% (p < 0.01) and by 55-60% (p < 0.01) in the presence of saliva. The addition of 5 mM PenA to cell medium prior to CS exposure limited cell loss to 22-25% only (p < 0.01). Similarly, the iron chelator desferal protected the cells only in the presence of saliva. PenA also protected against a CS-induced increase in carbonyls (oxidized proteins) and decrease in p53 levels (in the presence of saliva) and mitochondrial membrane potential (a hallmark of CS-induced apoptotic cell death). Malfunctioning p53 often characterizes carcinogenesis of CS-induced cancers. CONCLUSIONS: Redox-active iron and copper in pleural fluid and saliva, upon encounter with CS, may be responsible for this carcinogenesis, mediated via alteration of p53 function. Chelation of redox-active metals may be an efficient tool for prevention of CS-induced lung and oral cancers. The superiority of PenA results from its copper-chelating action as well as its antialdehyde and anti-inflammatory capabilities.
Assuntos
Anticarcinógenos/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Bucais/tratamento farmacológico , Penicilamina/farmacologia , Fumaça/efeitos adversos , Fumar/efeitos adversos , Anticarcinógenos/uso terapêutico , Linhagem Celular Tumoral , Humanos , Penicilamina/uso terapêutico , SalivaRESUMO
Oral cancer features high rates of mortality and morbidity, and is in dire need for new approaches. In the present study we analyzed 18 kDa translocator protein (TSPO) expression in oral (tongue) cancer tumors by immunohistochemistry. We also assayed TSPO binding in human tongue cancer cell lines and in the cellular fraction of saliva from tongue cancer patients, heavy cigarette smokers, and non-smoking healthy people as controls. Concurrently, TSPO protein levels, cell viability, mitochondrial membrane potential (Deltapsi(m)), and general protein levels were analyzed. TSPO expression could be significantly enhanced in oral cancer tumors, compared to unaffected adjacent tissue. We also found that five-year survival probability dropped from 65% in patients with TSPO negative tumors to 7% in patients with highly expressed TSPO (p<0.001). TSPO binding capacity was also pronounced in the human oral cancer cell lines SCC-25 and SCC-15 (3133+/-643 fmol/mg protein and 6956+/-549 fmol/mg protein, respectively). Binding decreased by 56% and 72%, in the SCC-25 and SCC-15 cell lines, respectively (p<0.05) following CS exposure in cell culture. In the cellular fraction of saliva of heavy smokers TSPO binding was lower than in non-smokers (by 53%, p<0.05). Also the cellular fraction of saliva exposed to CS in vitro showed decreased TSPO binding compared to unexposed saliva (by 30%, p<0.001). Interestingly, oral cancer patients also displayed significantly lower TSPO binding in the cellular fraction of saliva compared to healthy controls (by 40%, p<0.01). Our results suggest that low TSPO binding found in the cellular fraction of saliva may depend on genetic background as well as result from exposure to CS. We suggest that this may be related to a predisposition for occurrence of oral cancer.
Assuntos
Neoplasias Bucais/metabolismo , Receptores de GABA/metabolismo , Saliva/metabolismo , Fumar , Idoso , Estudos de Casos e Controles , Sobrevivência Celular/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Feminino , Humanos , Técnicas Imunoenzimáticas , Técnicas In Vitro , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Neoplasias Bucais/patologia , Proteínas Quinases Associadas a Fase S/metabolismo , Células Tumorais CultivadasRESUMO
It is well known that high fat and high cholesterol levels present a contributing factor to pathologies including fatty liver and atherosclerosis. Oxidative stress is also considered to play a role in these pathologies. The 18 kDa Translocator Protein (TSPO), formerly known as the peripheral-type benzodiazepine receptor, is known to be involved in cholesterol metabolism, oxidative stress, and cardiovascular pathology. We applied a high fat high cholesterol atherogenic (HFHC) diet to rats to study correlations between cardiovascular and liver pathology, oxidative stress, and TSPO expression in the liver and the cardiovascular system. This study corroborates the presence of increased oxidative stress markers and decreased anti-oxidants in liver and aorta. In addition, it appeared that induction of oxidative stress in the liver and aorta by atherogenic HFHC diet was accompanied by a reduction in TSPO binding density in both these tissues. Our data suggest that involvement of TSPO in oxidative stress and ROS generation, as reported in other studies, may also take part in atherogenesis as induced by HFHC diet. Presently, it is not clear whether this TSPO response is compensatory for the stress induced by HFHC diet or is a participant in the induction of oxidative stress.
Assuntos
Aorta Torácica/metabolismo , Proteínas de Transporte/metabolismo , Colesterol na Dieta/efeitos adversos , Gorduras na Dieta/efeitos adversos , Fígado/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Receptores de GABA-A/metabolismo , Animais , Antioxidantes/metabolismo , Aorta Torácica/efeitos dos fármacos , Aorta Torácica/patologia , Biomarcadores , Doenças Cardiovasculares/induzido quimicamente , Doenças Cardiovasculares/metabolismo , Doenças Cardiovasculares/patologia , Dieta , Dieta Aterogênica , Endotélio Vascular/patologia , Produtos Finais de Glicação Avançada/metabolismo , Fígado/efeitos dos fármacos , Fígado/patologia , Masculino , Oxirredução , Carbonilação Proteica/efeitos dos fármacos , Ratos , Ratos WistarRESUMO
We examined the effect of ovariectomy, with and without estradiol treatment, on 18 kDa translocator protein (TSPO) gene expression and its binding density in the uterus and kidney of rats. Ovariectomy causes a significant decrease in uterine, but not renal TSPO binding density, while estradiol treatment of ovariectomized rats restored TSPO binding density in the uterus. These TSPO density levels did not correlate with steady state or new RNA transcription. Our in vivo study suggests that estradiol is responsible for the maintenance of uterine TSPO density via transcriptional mechanisms. Our in vivo study also suggests that in the kidney estradiol appears to operate via post-transcriptional mechanisms to maintain TSPO density.
Assuntos
Proteínas de Transporte/genética , Estradiol/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Rim/efeitos dos fármacos , Rim/metabolismo , Receptores de GABA-A/genética , Útero/efeitos dos fármacos , Útero/metabolismo , Animais , Feminino , Peso Molecular , Ovariectomia , Ligação Proteica/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Transcrição Gênica/efeitos dos fármacosRESUMO
The 18 kDa translocator protein (TSPO), formerly known as the peripheral-type benzodiazepine receptor, has been reported to be closely associated with the mitochondrial permeability transition pore (MPTP). TSPO is believed to exert pro-apoptotic functions via modulation of MPTP opening. Cobalt chloride (CoCl(2)), which is sometimes used as a hypoxia mimicking agent, is also known to be able to induce apoptosis. One of our questions was whether CoCl(2) may induce apoptosis via the TSPO. To address this question, we used the U118MG human glioblastoma cell line. We applied the specific TSPO ligand, PK 11195, as well as TSPO knockdown with siRNA and studied their influence on the effects of CoCl(2) on cell death, including activation of the mitochondrial apoptosis pathway. To assay TSPO expression, we applied binding assays and Western blotting to whole cell homogenates and mitochondrial fractions. To assay activation of the mitochondrial apoptosis pathway, including some of the cellular mechanisms involved, we determined the incidence of collapse of the mitochondrial membrane potential (Deltapsi(m)) and cardiolipin oxidation and measured the level of DNA fragmentation to assay apoptotic rates. We found that the TSPO ligand, PK 11195, significantly counteracted induction of cell death by 0.4 mM CoCl(2), including apoptosis, collapse of the Deltapsi(m), and cardiolipin oxidation. Moreover, we found that TSPO knockdown with siRNA fully protected against mentioned cell death mechanisms. Thus, we found that the TSPO is required for cell death induction by CoCl(2), including apoptosis. In conclusion, our studies show that activation of TSPO by CoCl(2) application is required for ROS generation, leading to cardiolipin oxidation, and collapse of the Deltapsi(m), as induced by CoCl(2).
Assuntos
Apoptose/efeitos dos fármacos , Cobalto/farmacologia , Glioblastoma/patologia , Receptores de GABA/metabolismo , Cardiolipinas/metabolismo , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Técnicas de Silenciamento de Genes , Humanos , Isoquinolinas/farmacologia , Ligantes , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Poro de Transição de Permeabilidade Mitocondrial , Oxirredução , RNA Interferente Pequeno/genética , Espécies Reativas de Oxigênio/metabolismo , Receptores de GABA/genética , Transdução de Sinais/efeitos dos fármacosRESUMO
OBJECTIVE: The involvement of the 18-kDa translocator protein (TSPO), formerly known as the peripheral-type benzodiazepine receptor, in apoptosis regulation of HT29 colorectal cancer cells was studied in-vitro. In-vivo TSPO involvement in tumor growth of HT29 cells xenografted into SCID mice was studied. METHODS: Knockdown of TSPO expression in the human HT29 cell line was established by stable transfection with vectors containing the TSPO gene in the antisense direction. Successful TSPO knockdown was characterized by reduction of 20% in TSPO RNA levels, 50% in protein expression of the TSPO, and 50% in binding with the TSPO ligand, [3H]PK 11195. Subsequently, in-vitro cell viability and proliferation assays were applied. In addition, transient transfecton with short interfering RNA (siRNA) directed against human TSPO was studied in this way. Furthermore, we also grafted HT29 cells subcutaneously into the right thighs of SCID mice to examine the effects of the putative TSPO agonist, FGIN-1-27, on tumor growth in-vivo. RESULTS: In-vitro TSPO knockdown established by stable transfection of TSPO antisense gene resulted in HT29 clones displaying significantly lower levels of cell death as determined with trypan blue (50% less), lower apoptotic rates (28% less), and higher proliferation rates (48% more one week after seeding and 27% more two weeks after seeding). Transient transfection with anti-human TSPO siRNA resulted in similar viability and antiapoptotic effects. In-vivo, the proapoptotic TSPO ligand, FGIN-1-27 significantly reduced the growth rate of grafted tumors (40% less), in comparison with vehicle-treated mice. CONCLUSION: TSPO knockdown by genetic manipulation transforms the human HT29 cancer line to a more malignant type in-vitro. In-vivo pharmacological treatment with the putative TSPO agonist FGIN-1-27 reduces tumor growth of the HT29 cell line. These data suggest that TSPO involvement in apoptosis provides a target for anticancer treatment.
Assuntos
Antineoplásicos/metabolismo , Apoptose , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Receptores de GABA/metabolismo , Animais , Apoptose/efeitos dos fármacos , Bioensaio , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cloranfenicol O-Acetiltransferase/metabolismo , Neoplasias Colorretais/genética , Elementos Facilitadores Genéticos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HT29 , Humanos , Ácidos Indolacéticos/farmacologia , Ligantes , Camundongos , Camundongos SCID , Peso Molecular , Regiões Promotoras Genéticas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Receptores de GABA/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: We have previously shown that the anti-neoplastic agent erucylphosphohomocholine (ErPC3) requires the mitochondrial 18 kDa Translocator protein (TSPO), formerly known as the peripheral-type benzodiazepine receptor (PBR), to induce cell death via the mitochondrial apoptosis pathway. METHODS: With the aid of the dye JC-1 and cyclosporin A, applied to glioblastoma cells, we now investigated the significance of opening of the mitochondrial permeability transition pore (MPTP) for ErPC3-induced apoptosis in interaction with the TSPO ligands, PK 11195 and Ro5 4864. Furthermore, we measured cytochrome c release, and caspase-9 and -3 activation in this paradigm. RESULTS: The human glioblastoma cell lines, U87MG, A172 and U118MG express the MPTP-associated TSPO, voltage-dependent anion channel and adenine nucleotide transporter. Indeed, ErPC3-induced apoptosis was inhibited by the MPTP blocker cyclosporin A and by PK 11195 and Ro5 4864 in a concentration-dependent manner. Furthermore, PK 11195 and Ro5 4864 inhibited collapse of the mitochondrial membrane potential, cytochrome c release, and caspase-9 and -3 activation caused by ErPC3 treatment. CONCLUSIONS: This study shows that PK 11195 and Ro5 4864 inhibit the pro-apoptotic function of ErPC3 by blocking its capacity to cause a collapse of the mitochondrial membrane potential. Thus, the TSPO may serve to open the MPTP in response to anti-cancer drugs such as ErPC3.
Assuntos
Antineoplásicos/farmacologia , Apoptose/fisiologia , Ácidos Erúcicos/farmacologia , Glioblastoma/metabolismo , Fosforilcolina/análogos & derivados , Receptores de GABA/metabolismo , Apoptose/efeitos dos fármacos , Benzodiazepinonas/farmacologia , Western Blotting , Caspases/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Citocromos c/efeitos dos fármacos , Humanos , Isoquinolinas/farmacologia , Ligantes , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Proteínas de Transporte da Membrana Mitocondrial/efeitos dos fármacos , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Poro de Transição de Permeabilidade Mitocondrial , Fosforilcolina/farmacologiaRESUMO
It has been shown that the atypical antipsychotic drug clozapine increases the levels of the neurosteroid allopregnanolone in the rat brain. The 18 kDa translocator protein (TSPO), formerly known as the peripheral-type benzodiazepine receptor, has been demonstrated to be involved in the process of steroid biosynthesis, in peripheral steroidogenic tissues as well as in glia cells in the brain. In the current study, we investigated the influence of chronic treatment with clozapine and other antipsychotics (thioridazine,sulpiride and risperidone) on TSPO binding in cell cultures and rat tissues. Clozapine significantly increased TSPO binding density in C6 rat glioma cells and in MA-10 mouse Leydig tumor cells, while the antipsychotic sulpiride had no effect on TSPO binding density in both cell lines. In addition, clozapine, but not sulpiride, significantly increased progesterone synthesis by MA-10 Leydig tumor cells. In an animal experiment, male Sprague-Dawley rats were treated with clozapine (20 mg/kg), risperidone (0.5 mg/kg), thioridazine (20 mg/kg), or sulpiride (20 mg/kg) for 21 days, followed by 7 days of withdrawal. Clozapine induced significant increases in TSPO binding in brain and peripheral steroidogenic tissues, whereas the other antipsychotics did not show such pronounced effects on TSPO binding. Our results suggest that TSPO may be involved in the modulation of steroidogenesis by clozapine.
Assuntos
Antipsicóticos/farmacologia , Clozapina/farmacologia , Receptores de GABA/metabolismo , Esteroides/biossíntese , Glândulas Suprarrenais/efeitos dos fármacos , Glândulas Suprarrenais/metabolismo , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Linhagem Celular , Corticosterona/sangue , Humanos , Isoquinolinas/metabolismo , Masculino , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Radioimunoensaio , Ratos , Ratos Sprague-Dawley , Risperidona/farmacologia , Sulpirida/farmacologia , Testículo/efeitos dos fármacos , Testículo/metabolismo , Testosterona/sangue , Tioridazina/farmacologiaRESUMO
A previous study by us indicated that peripheral-type benzodiazepine receptor (PBR) density may be increased in the ovaries and uterus of pregnant rats (Weizman R, Dagan E, Snyder SH, Gavish M. Impact of pregnancy and lactation on GABAA receptor and central-type and peripheral-type benzodiazepine receptors. Brain Res 1997;752:7-14). In the present study, the effects of prostaglandin F2alpha (PGF2alpha) on PBR density in the ovary and uterus of pseudopregnant rats were assayed. Pseudopregnancy was induced on day 29 post-partum (PP) by s.c. injection of 50IU pregnant mare serum gonadotropin (PMSG) and 3 days later by s.c. injection of 20IU human chorionic gonadotropin (hCG). PBR ligand binding density was assayed with the specific PBR ligand [3H]PK 11195. A two-fold increase in ovarian PBR density was observed 2 days after hCG administration compared with vehicle control rats and this effect was maintained for 3 weeks. In the uterus, a three-fold increase in PBR density was observed and this increase was maintained for 1 week after hCG administration. Pseudopregnancy did not appear to affect renal PBR density or affinity. Treatment with PGF2alpha, which causes luteolysis, resulted in an approximately 50% reduction of PBR density in the ovaries of pseudopregnant rats at day 53 PP compared to pseudopregnant control rats. Treatment with indomethacin, which prevents the formation of PGF2alpha, caused the PBR density in the uterus of pseudopregnant rats at day 53 PP to be twice as high as in pseudopregnant control rats. All the above treatments did not affect the affinity of [3H]PK 11195 to ovarian and uterine PBR. These data suggest that PBR density in corpora lutea and uterus during pseudopregnancy is regulated by PGF2alpha.
Assuntos
Proteínas de Transporte/metabolismo , Dinoprosta/farmacologia , Ovário/efeitos dos fármacos , Pseudogravidez/fisiopatologia , Receptores de GABA-A/metabolismo , Útero/efeitos dos fármacos , Animais , Corpo Lúteo/efeitos dos fármacos , Corpo Lúteo/fisiologia , Feminino , Gonadotropinas Equinas/farmacologia , Indometacina/farmacologia , Isoquinolinas/farmacologia , Ovário/metabolismo , Gravidez , Progesterona/sangue , Ratos , Ratos Sprague-Dawley , Útero/metabolismoRESUMO
Peripheral-type benzodiazepine receptors (PBR) are constituted by three protein components, the isoquinoline binding protein (IBP), the voltage-dependent anion channel (VDAC), and the adenine nucleotide transporter (ANT). Recently, we found that high levels of PBR ligand binding in glioma cell lines correlate with in vitro tumorigenicity. To study whether enhanced PBR expression is causative or in response to cancer, we genetically modified C6 glioma cells. Antisense knockdown of the IBP resulted in more than 50% reductions in PBR ligand binding both in the mitochondrial and whole cell fractions, accompanied by similar reductions in IBP levels in these respective fractions. The IBP knockdown was accompanied by a 25% increase in cell number in confluent cultures. This correlated with an 8-fold increase in in vitro tumorigenicity, as assessed by anchorage independent growth. Cell cycle analysis indicated that knockdown of the IBP resulted in a 60% reduction in the number of cells in the pre-G1 apoptosis phase. This paralleled the reduction seen in apoptosis and cell death shown by DNA fragmentation and Trypan blue assays, respectively. Furthermore, knockdown of the IBP appeared to prevent induction of apoptosis by the antineoplastic agent, erucylphosphocholine. In addition, IBP knockdown prevented processing of the caspase 3 component of the apoptosis cascade by the erucylphosphocholine congener, erucylphospho-N,N,N-trimethylammonium. In conclusion, our results suggest that enhanced IBP expression, including enhanced PBR ligand binding, such as occurring in untreated C6 glioma cells, may provide a mechanism to increase apoptotic rates of cancer cells.
Assuntos
Proteínas de Transporte/química , Proteínas de Transporte/genética , Receptores de GABA-A/química , Receptores de GABA-A/genética , Animais , Apoptose/genética , Western Blotting , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/biossíntese , Caspase 3 , Caspases/metabolismo , Contagem de Células , Linhagem Celular Tumoral , Células Clonais , DNA Antissenso/química , Antagonistas de Receptores de GABA-A , Fosforilcolina/análogos & derivados , Fosforilcolina/química , Processamento de Proteína Pós-Traducional , Ratos , Receptores de GABA-A/biossíntese , Transfecção , Azul TripanoRESUMO
Peripheral benzodiazepine receptors (PBR), first described more than 20 years ago, have been attributed with many putative functions including ones in cellular proliferation and cellular respiration. Hence, it is quite conceivable that deregulation of this receptor could lead to pathology. We and others have reported the existence of PBR overexpression in different human and nonhuman malignancies, but it has never been made clear whether this aberrant malignant PBR expression is a cause or consequence of the cancer. In the current study we induced PBR underexpression by downregulating one critical subunit of the PBR complex, the isoquinoline-binding protein (IBP), using the stable antisense knockout approach, in the MA-10 Leydig cell line. Resultant clones, showing PBR deregulation, also demonstrated increased tumorigenicity, using both in vitro (loss of contact inhibition and growth in soft agar) and in vivo (increased mortality on grafting back into isogenic mice) assays. We suggest that this type of deregulation could be a later event in natural tumor progression. Consequently, PBR deregulation should be more closely studied in human malignancy.
Assuntos
Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Células Intersticiais do Testículo/metabolismo , Células Intersticiais do Testículo/patologia , Neoplasias/patologia , Receptores de GABA-A/genética , Receptores de GABA-A/metabolismo , Animais , Sequência de Bases , Contagem de Células , Divisão Celular , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Masculino , Camundongos , Dados de Sequência Molecular , Transplante de Neoplasias , Neoplasias/genética , Ratos , Receptores de GABA-A/biossíntese , Alinhamento de Sequência , Taxa de Sobrevida , TransfecçãoRESUMO
The peripheral-type benzodiazepine receptor is found primarily on the outer mitochondrial membrane and consists of three subunits: the 18kDa isoquinoline binding protein, the 32kDa voltage-dependent anion channel, and the 30kDa adenine nucleotide transporter. The current study evaluates the potential importance of peripheral-type benzodiazepine receptor expression in glioma cell tumorigenicity. While previous studies have suggested that peripheral-type benzodiazepine receptor-binding may be relatively increased in tumor tissue and cells, so far, little is known about the relationships between peripheral-type benzodiazepine receptor density and factors underlying tumorigenicity. In the present study, we found in glioma cell lines (C6, U87MG, and T98G), that peripheral-type benzodiazepine receptor ligand-binding density is relatively high for C6 and low for T98G, while U87MG displays intermediate levels. Cell growth of these cell lines in soft agar indicated that high levels of peripheral-type benzodiazepine receptor-binding were associated with increased colony size, indicative of their ability to establish anchorage independent cell proliferation. Potential causes for differences in tumorigenicity between these cell lines were suggested by various cell death and proliferation assays. Cell death, including apoptosis, appeared to be low in C6, and high in T98G, while U87MG displayed intermediate levels in this respect. Cell proliferation appeared to be high in C6, low in T98G, and intermediate in U87MG. In conclusion, our study suggests that relatively high peripheral-type benzodiazepine receptor-binding density is associated with enhanced tumorigenicity and cell proliferation rate. In particular, apoptosis appears to be an important tumorigenic determinant in these glioma cell lines. Moreover, application of PBR-specific ligands indicated that PBR indeed are functionally involved in apoptosis in glioma cells.
Assuntos
Apoptose/fisiologia , Receptores de GABA-A/metabolismo , Animais , Testes de Carcinogenicidade , Ciclo Celular/fisiologia , Divisão Celular/fisiologia , Glioma , Humanos , Ratos , Receptores de GABA-A/fisiologia , Células Tumorais CultivadasRESUMO
Peripheral-type benzodiazepine receptors (PBR) have been shown to be sensitive to stressful conditions. This study aimed to explore a possible association of platelets PBR binding with aggressive behavior and homicidal history in schizophrenia patients. The authors compared [(3)H] PK 11195 binding to platelet membrane among 11 currently aggressive schizophrenia patients, 15 schizophrenia patients with homicidal history, 14 nonaggressive schizophrenia patients, and 15 healthy volunteers. Subjects were assessed for aggressive behavior, psychopathology, anxiety, anger, and emotional distress using standardized instruments. We found that currently aggressive patients had significantly lower (-30%) platelet PBR density (B(max)), and scored significantly higher on hostility, anxiety, state anger, and emotional distress compared to homicidal and nonaggressive schizophrenia patients and healthy controls. Predominance of positive or negative symptoms, homicidal or suicidal attempt history, emotional distress levels, and conventional or atypical antipsychotic therapy is not associated with the expression of platelet PBR binding sites. Significant negative correlations emerged between PBR density and scores for aggressive behavior, hostility and anxiety. Thus, decreased platelet PBR density in aggressive schizophrenia patients is associated with higher scores for overt aggression, hostility and anxiety, but independent of illness subtype, homicidal and suicidal attempt history, distress level and type of antipsychotic treatment.
Assuntos
Receptores de GABA-A/metabolismo , Esquizofrenia/sangue , Violência/psicologia , Adulto , Antipsicóticos/uso terapêutico , Plaquetas/citologia , Plaquetas/metabolismo , Contagem de Células , Doença Crônica , Manual Diagnóstico e Estatístico de Transtornos Mentais , Feminino , Humanos , Incidência , Masculino , Pessoa de Meia-Idade , Periodicidade , Psicometria , Esquizofrenia/diagnóstico , Esquizofrenia/tratamento farmacológico , Índice de Gravidade de DoençaRESUMO
UNLABELLED: In an effort to clarify the mechanism of action of isoflurane, we studied the effect of flumazenil on mice chronically treated with isoflurane or diazepam. Mice were pretreated with diazepam, isoflurane, or saline, with and without flumazenil. After 2 wk, responses to isoflurane and diazepam were assessed, and central benzodiazepine receptor (CBR) binding characteristics were assayed. Mice pretreated with isoflurane failed the horizontal wire test at a larger isoflurane concentration (0.5%) compared with saline-pretreated mice (0.4%) (P < 0.05). These differences did not occur when flumazenil was added to the pretreatment. After the administration of diazepam, 20% of diazepam- and 11% of isoflurane-pretreated mice failed the horizontal wire test, versus 50% and 44% when flumazenil was added to either drug (P < 0.002) and 80% and 100% in the saline and saline plus flumazenil-treated mice. The increased CBR density due to flumazenil was attenuated by the coadministration of isoflurane or diazepam. Flumazenil attenuated the development of tolerance to diazepam after chronic treatment with diazepam or isoflurane and attenuated the development of tolerance to isoflurane. Isoflurane, like diazepam, attenuated the effect of flumazenil on CBR ligand binding. These findings suggest that isoflurane shares a mechanism of action with diazepam, probably via the gamma-aminobutyric acid system, most probably the CBR. IMPLICATIONS: Flumazenil attenuates the development of tolerance to isoflurane and diazepam after chronic isoflurane pretreatment. Isoflurane, like diazepam, attenuates the increase in central benzodiazepine receptor (CBR) density caused by flumazenil. These findings suggest that isoflurane and diazepam share a mechanism of action, most probably via the gamma-aminobutyric acid system and the CBR.
Assuntos
Anestésicos Inalatórios/farmacologia , Ansiolíticos/farmacologia , Diazepam/farmacologia , Flumazenil/farmacologia , Moduladores GABAérgicos/farmacologia , Isoflurano/farmacologia , Animais , Comportamento Animal/efeitos dos fármacos , Sítios de Ligação/efeitos dos fármacos , Tolerância a Medicamentos , Camundongos , Equilíbrio Postural/efeitos dos fármacos , Receptores de GABA-A/efeitos dos fármacosRESUMO
Behavioral animal paradigms and experimental neuroendocrinological and neurochemical studies have shown that early environmental manipulations have profound effects on the late response to stress. The aim of the present study was to investigate the interactive effects of environmental manipulation (early handling) and experimentally induced behavioral differences on the peripheral benzodiazepine receptor (PBR) system, which is known to be involved in the response to stressors. Adult early-handled (EH) and nonhandled (NH; control) Wistar rats were placed in a two-way active avoidance/latent inhibition (LI) paradigm, and PBR densities in the adrenal glands, kidneys, and gonads were assessed. In line with previous studies, overall avoidance learning improved in the EH group, and LI was disrupted in the NH group (primarily in males). PBR densities were up-regulated in EH subjects, and more so in females than males. However, PBR densities did not correlate with any of the behavioral measures. These findings strengthen the hypothesis that differences in PBR densities between EH and NH rats are a reflection of trait rather than state, and they suggest that the PBR system is characterized by a highly stressor-specific response.