RESUMO
Two free operant conditioning experiments with rats examined the impact of conducting a large amount of extinction training on situations that enhance the ABC renewal effect (ABC super renewal). In Experiment 1, ABC renewal was strengthened by conducting acquisition in multiple contexts. All rats were trained to press a lever for food. One group was trained in one context, while the other two groups were trained in three contexts. Then, all rats received extinction in context B. For two groups this phase lasted 4 sessions, whereas it lasted 36 sessions for the other group. In Experiment 2, ABC renewal was strengthened by using a large number of acquisition sessions. Rats were trained to perform an operant response to obtain food in context A. One group received a moderate amount of training, while the rest of the rats received a larger number of acquisition sessions. Responses underwent extinction in context B. Two groups received 4 sessions, while 36 extinction sessions were used for the remaining group. In both experiments, rats were tested in context B (extinction context) and C (renewal context). Greater ABC renewal occurred both when acquisition training was conducted in multiple contexts (Experiment 1) and by increasing the amount of acquisition training (Experiment 2). Nevertheless, we found that conducting a large number of extinction sessions reduced ABC super renewal in Experiment 1 only.
Assuntos
Condicionamento Operante , Extinção Psicológica , Ratos , Animais , Ratos Wistar , Extinção Psicológica/fisiologia , Condicionamento Operante/fisiologia , Alimentos , Proteínas Repressoras/farmacologiaRESUMO
Berberine, a constituent of some traditional Chinese medicinal plants, has been reported to have cytotoxicity effects on different human cancer cell lines. There is no available information about the effects and mechanism of action of berberine on human colon cancer cell line HCT-8. In this paper, the cytotoxicity of berberine on HCT-8 cancer cells was investigated by MTT assay, fluorescence microscopy and flow cytometry analysis. Our data revealed that berberine could significantly inhibit the growth of HCT-8 cells in a dose- and time-dependent manner. Morphology of apoptotic cells was studied with acridine orange/ethidium bromide staining. The concentrations of lactate dehydrogenase and both acid and alkaline phosphatases were significantly increased in cell supernatants after berberine treatment, suggesting cell death. Furthermore, flow cytometry analysis showed that berberine could arrest HCT-8 cells at S phase in a time-dependent manner. To further investigate the apoptotic molecular mechanism, reverse transcription-polymerase chain reaction (RT-PCR) and western blotting methods were used. The up-regulated mRNA and/or protein expressions of Fas, FasL, TNF-a, caspase-3 and down-regulation of pro-caspase-3 suggest that the death receptor pathway may be involved in the apoptotic pathway induced by berberine. Decrease of Bcl-2 and increase of Bax in mRNA and/or protein expressions showed that the Bcl-2 family proteins were involved in berberine-induced apoptosis. We also found that berberine-induced apoptosis was associated with an up-regulated expressions of p53 and prohibitin (PHB), and decreased vimentin expression. These results suggest that berberine can suppress cell growth and reduce cell survival by arresting the cell-cycle and by inducing apoptosis of HCT-8 cells.(AU)
Assuntos
Humanos , Berberina/farmacologia , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/metabolismo , Apoptose , Berberina/metabolismo , Ciclo Celular , Linhagem Celular Tumoral , Citometria de Fluxo , L-Lactato Desidrogenase/metabolismo , Medicina Tradicional Chinesa , Microscopia de Fluorescência , RNA Mensageiro/metabolismo , Proteínas Repressoras/farmacologia , Fase S , Sais de Tetrazólio/farmacologia , Tiazóis/farmacologia , Fatores de Tempo , Proteína Supressora de Tumor p53/metabolismo , Vimentina/metabolismo , Proteína X Associada a bcl-2/metabolismoRESUMO
Berberine, a constituent of some traditional Chinese medicinal plants, has been reported to have cytotoxicity effects on different human cancer cell lines. There is no available information about the effects and mechanism of action of berberine on human colon cancer cell line HCT-8. In this paper, the cytotoxicity of berberine on HCT-8 cancer cells was investigated by MTT assay, fluorescence microscopy and flow cytometry analysis. Our data revealed that berberine could significantly inhibit the growth of HCT-8 cells in a dose- and time-dependent manner. Morphology of apoptotic cells was studied with acridine orange/ethidium bromide staining. The concentrations of lactate dehydrogenase and both acid and alkaline phosphatases were significantly increased in cell supernatants after berberine treatment, suggesting cell death. Furthermore, flow cytometry analysis showed that berberine could arrest HCT-8 cells at S phase in a time-dependent manner. To further investigate the apoptotic molecular mechanism, reverse transcription-polymerase chain reaction (RT-PCR) and western blotting methods were used. The up-regulated mRNA and/or protein expressions of Fas, FasL, TNF-a, caspase-3 and down-regulation of pro-caspase-3 suggest that the death receptor pathway may be involved in the apoptotic pathway induced by berberine. Decrease of Bcl-2 and increase of Bax in mRNA and/or protein expressions showed that the Bcl-2 family proteins were involved in berberine-induced apoptosis. We also found that berberine-induced apoptosis was associated with an up-regulated expressions of p53 and prohibitin (PHB), and decreased vimentin expression. These results suggest that berberine can suppress cell growth and reduce cell survival by arresting the cell-cycle and by inducing apoptosis of HCT-8 cells.
Assuntos
Humanos , Berberina/farmacologia , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/metabolismo , Apoptose , Berberina/metabolismo , Ciclo Celular , Linhagem Celular Tumoral , Citometria de Fluxo , L-Lactato Desidrogenase/metabolismo , Medicina Tradicional Chinesa , Microscopia de Fluorescência , RNA Mensageiro/metabolismo , Proteínas Repressoras/farmacologia , Fase S , Fatores de Tempo , Sais de Tetrazólio/farmacologia , Tiazóis/farmacologia , /metabolismo , Vimentina/metabolismo , /metabolismoRESUMO
Follicle-stimulating hormone receptor (FSHR) and luteinizing hormone receptor (LHR) belong to the super-family of G protein-coupled receptors (GPCR); GPCRs are negatively regulated by RGS ("regulators of G protein signaling") proteins. In this study we evaluated the effects of RGS3 and RGS10 on FSHR and LHR ligand binding and effector coupling. FSHR and LHR ligand binding were unchanged in the presence of RGS3 or RGS10. However, signaling by FSHR and LHR was diminished by RGS3 but not by RGS10. This constitutes the first demonstration of an interaction between RGS proteins and LH and FSH signaling pathways and identifies a mechanism for negative regulation of RGS3 on FSHR and LHR signaling.
Assuntos
Proteínas RGS/farmacologia , Receptores do FSH/metabolismo , Receptores do LH/metabolismo , Proteínas Repressoras/farmacologia , Transdução de Sinais/fisiologia , Animais , Linhagem Celular , Feminino , Humanos , Ligantes , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacosRESUMO
We have reported that interferon-alpha inhibits HPV-18 mRNA in HeLa cells. Here we examine mechanisms by which IFN could modulate HPV expression. In northern blot experiments, we observed that interferon-alpha 2b treatment reduced HPV-18 mRNA levels in a time- and dose-dependent manner, with a maximal effect achieved at 48 h. Simultaneously, induction of 2-5A synthetase mRNA was verified as indicative of IFN action. The IFN regulatory effect on HPV-18 mRNA at 48 h required de novo protein synthesis. We performed run-on experiments to determine whether the IFN regulatory effect was at the transcriptional level. HPV-18 endogenous transcription was repressed using 200 and 1000 IU/ml. Interferon treatment did not affect HPV-18 mRNA stability, at least under our experimental conditions. To verify whether HPV-18 enhancer sequences were involved in the interferon effect, we transfected a construct containing the chloramphenicol acetyltransferase driven by the HPV-18 upstream regulatory region. The enzyme activity was unmodified on human keratinocytes and HeLa cells by interferon exposition. Our data demonstrate that interferon-alpha downregulates HPV-18 mRNA levels on HeLa cells by repressing nascent viral transcripts, possibly through regulatory cellular flanking regions.