RESUMO
AIM: To determine the role of c-Jun N-terminal kinase (JNK) activity in ethanol-induced apoptosis and the modulation of this signaling cascade by S-Adenosyl-methionine (AdoMet). METHODS: Primary hepatocyte cultures were pretreated with 100 micromol/L SP600125, a selective JNK inhibitor, 1 mL/L DMSO or 4 mmol/L AdoMet and then exposed to 100 mmo/L ethanol. Hepatocyte apoptosis was determined by the TUNEL and DNA ladder assays. JNK activity and its inhibition by SP600125 and AdoMet were determined by Western blot analysis of c-jun phosphorylation and Bid fragmentation. SP600125 and AdoMet effects on the apoptotic signaling pathway were determined by Western blot analysis of cytochrome c release and pro-caspase 3 fragmentation. The AdoMet effect on glutathione levels was measured by Ellman's method and reactive oxygen species (ROS) generation by cell cytometry. RESULTS: The exposure of hepatocytes to ethanol induced JNK activation, c-jun phosphorylation, Bid fragmentation, cytochrome c release and pro-caspase 3 cleavage; these effects were diminished by SP600125, and caused a significant decrease in ethanol-induced apoptosis (P< 0.05). AdoMet exerted an antioxidant effect maintaining glutathione levels and decreasing ROS generation, without a significant effect on JNK activity, and prevented cytochrome c release and pro-caspase 3 cleavage. CONCLUSION: The JNK signaling cascade is a key component of the proapoptotic signaling pathway induced by ethanol. JNK activation may be independent from ROS generation, since AdoMet which exerted antioxidant properties did not have a significant effect on JNK activity. JNK pathway modulator agents and AdoMet may be components of promising therapies for alcoholic liver disease (ALD) treatment.
Assuntos
Apoptose/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Proteínas Quinases JNK Ativadas por Mitógeno/fisiologia , S-Adenosilmetionina/farmacologia , Transdução de Sinais/efeitos dos fármacos , Animais , Antracenos/farmacologia , Apoptose/fisiologia , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Caspase 3 , Caspases/metabolismo , Células Cultivadas , Citocromos c/metabolismo , Etanol/farmacologia , Glutationa/metabolismo , Hepatócitos/fisiologia , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Masculino , Mitocôndrias , Ratos , Ratos Endogâmicos F344 , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/fisiologiaRESUMO
BACKGROUND/AIMS: Several studies suggest that cyclooxygenase-2 (COX-2) inhibitors are chemopreventive agents against colon, breast and skin cancer. In this study, we evaluated the chemopreventive effect of celecoxib, a specific COX-2 inhibitor, on the development of liver preneoplastic lesions in rats. METHODS: Male Sprague-Dawley rats were fed during 5 weeks either a control or an experimental diet containing 1500 ppm celecoxib on a medium-term hepatocarcinogenesis protocol. Livers were collected and evaluated by histological and biochemical assays. RESULTS: A reduction by 80 and 90% both in the number and size of altered hepatic foci was observed in the group treated with celecoxib during hepatocarcinogenesis treatment, respectively. No evidence of apoptosis was observed in our present study, however, the expression of the proliferation markers such as PCNA and Ki-67 was drastically reduced. Interestingly, neither COX-2 expression nor prostaglandin-E2 (PGE2) production were altered by the hepatocarcinogenic treatment or celecoxib treatment. Finally, celecoxib inhibited the translocation of Rel A/p65 to the nucleus with significant effect on stability of the repressor IkappaB-alpha. CONCLUSIONS: This is the first demonstration that a specific COX-2 inhibitor, celecoxib, possesses striking chemopreventive activity, inhibiting preneoplastic lesions during hepatocarcinogenesis in vivo, suggesting that celecoxib effects are mediated by PGE2-independent mechanisms.
Assuntos
Inibidores de Ciclo-Oxigenase/uso terapêutico , Neoplasias Hepáticas/prevenção & controle , Lesões Pré-Cancerosas/prevenção & controle , Pirazóis/uso terapêutico , Sulfonamidas/uso terapêutico , Animais , Celecoxib , Neoplasias Hepáticas Experimentais/prevenção & controle , Masculino , Modelos Animais , Ratos , Ratos Sprague-DawleyRESUMO
In this work, we investigated the effects of Casiopeina II-gly (Cas IIgly)--a new copper compound exhibiting antineoplastic activity--on glioma C6 cells under both in vitro and in vivo conditions, as an approach to identify potential therapeutic agents against malignant glioma. The exposure of C6 cells to Cas IIgly significantly inhibited cell proliferation, increased reactive oxygen species (ROS) formation, and induced apoptosis in a dose-dependent manner. In cultured C6 cells, Cas IIgly caused mitochondrio-nuclear translocation of apoptosis induction factor (AIF) and endonuclease G at all concentrations tested; in contrast, fragmentation of nucleosomal DNA, cytochrome c release, and caspase-3 activation were observed at high concentrations. Administration of N-acetyl-L-cystein, an antioxidant, resulted in significant inhibition of AIF translocation, nucleosomal DNA fragmentation, and caspase-3 activation induced by Cas IIgly. These results suggest that caspase-dependent and caspase-independent pathways both participate in apoptotic events elicited by Cas IIgly. ROS formation induced by Cas IIgly might also be involved in the mitochondrio-nuclear translocation of AIF and apoptosis. In addition, treatment of glioma C6-positive rats with Cas IIgly reduced tumor volume and mitotic and cell proliferation indexes, and increased apoptotic index. Our findings support the use of Cas IIgly for the treatment of malignant gliomas.