RESUMO
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising drug for the treatment of tumors; however, a number of cancer cells are resistant to this cytokine. Among the mechanisms of resistance of small cell lung carcinomas (SCLCs) to TRAIL is the lack of caspase-8 expression. Although methylation of the caspase-8 promoter has been suggested as the main mechanism of caspase-8 silencing, we showed that reduction of the enzymes involved in DNA methylation, DNA methyltransferases (DNMT) 1, 3a and 3b, was not sufficient to significantly restore caspase-8 expression in SCLC cells, signifying that other mechanisms are involved in caspase-8 silencing. We found that combination of the DNMT inhibitor decitabine with an inhibitor of histone deacetylase (HDAC) significantly increased caspase-8 expression in SCLC cells at the RNA and protein levels. Among all studied HDAC inhibitors, valproic acid (VPA) and CI-994 showed prolonged effects on histone acetylation, while combination with decitabine produced the most prominent effects on caspase-8 re-expression. Moreover, a significant reduction of survivin and cIAP-1 proteins level was observed after treatment with VPA. The combination of two drugs sensitized SCLC cells to TRAIL-induced apoptosis, involving mitochondrial apoptotic pathway and was accompanied by Bid cleavage, activation of Bax, and release of cytochrome c. Both initiator caspase-8 and -9 were required for the sensitization of SCLC cells to TRAIL. Thus, efficient restoration of caspase-8 expression in SCLC cells is achieved when a combination of DNMT and HDAC inhibitors is used, suggesting a combination of decitabine and VPA or CI-994 as a potential treatment for sensitization of SCLC cells lacking caspase-8 to TRAIL.
Assuntos
Caspase 8/metabolismo , DNA (Citosina-5-)-Metiltransferases/antagonistas & inibidores , Histona Desacetilases/química , Neoplasias Pulmonares/prevenção & controle , Carcinoma de Pequenas Células do Pulmão/prevenção & controle , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Western Blotting , Caspase 8/genética , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , DNA Metiltransferase 3A , Decitabina , Inibidores Enzimáticos/farmacologia , Imunofluorescência , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Humanos , Proteínas Inibidoras de Apoptose/genética , Proteínas Inibidoras de Apoptose/metabolismo , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/patologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Carcinoma de Pequenas Células do Pulmão/enzimologia , Carcinoma de Pequenas Células do Pulmão/patologia , Survivina , Ligante Indutor de Apoptose Relacionado a TNF/genética , Células Tumorais Cultivadas , Ácido Valproico/farmacologia , DNA Metiltransferase 3BRESUMO
BACKGROUND: Recent data suggest that low concentrations of proteasome inhibitors (PIs) are cytoprotective in models of ischemia-reperfusion injury, but the underlying mechanisms of this effect still remain unclear. AIM: To investigate the effect of 100 nM of clasto-lactacystin beta-lactone on cell death and gene expression in neonatal rat cardiomyocytes exposed to anoxia-reoxygenation. METHODS: Fluorescent microscopy and real-time polymerase chain reaction were used to detect different types of cell death and gene expression, respectively, in neonatal rat cardiomyocyte cultures exposed to anoxia-reoxygenation. RESULTS: It was shown that a low dose of clasto-lactacystin beta-lactone protected the cells against anoxia-reoxygenation injury by a reduction in the number of necrotic and apoptotic cells. The number of autophagic cells was greatly increased by proteasomal inhibition. The PI increased the heat shock protein 70 messenger RNA expression twofold and slightly reduced the expression of heat shock protein 90 gene. The expression of the FK506 binding protein 12-rapamycin associated protein gene was increased 1.57-fold on PI application. The B-cell lymphoma 2 gene expression was unaffected by the use of clasto-lactacystin beta-lactone in low dose. CONCLUSION: Although PIs are injurious, they may be cardioprotective in low doses; ie, they do not result in cell death. Moreover, PIs initiate the protective mechanisms that prevent cell damage by changing the expression of several genes.
RESUMO
BACKGROUND: The critical role of microRNAs (miRNAs) in the global control of gene expression in the heart has recently been postulated; however, the mechanisms of miRNA regulation in cardiac pathology are not clear. OBJECTIVE: To evaluate the levels of miR-1, miR-208a and miR-29a expressed in neonatal rat cardiomyocytes during anoxia-reoxygenation (AR). METHODS: Reverse transcription coupled with real-time polymerase chain reaction was used to evaluate the level of mature and immature miRNAs in cardiomyocyte culture during AR. RESULTS: THE INITIAL LEVELS OF THE MATURE AND IMMATURE MIRNAS WERE DIFFERENT: mature - miR-1 7.46±4.440, miR-208a 0.02±0.015 and miR-29a 5.60±2.060; immature - miR-1 0.02±0.007, miR-208a 0.05±0.029 and miR-29a 0.01±0.008. The most prominent changes were observed for immature miRNAs during AR, with immature miR-1 and miR-29a expressed at significantly higher levels during remote reoxygenation (AR [0.5 h/24 h]) compared with control, while the level of expressed immature miR-208a was significantly decreased during acute reoxygenation (AR [0.5 h /1 h]) and returned to control levels during remote reoxygenation (AR [0.5h /24 h]). Also, the ratios of mature to immature miRNAs were significantly increased during acute reoxygenation for miR-1 and miR-208a, returning to control levels during remote reoxygenation, while for miR-29a, this ratio had the progressive tendency to decrease under AR. CONCLUSION: The discordance between the estimated levels of mature and immature miRNA during AR supports the hypothesis that transcriptional and post-transcriptional regulatory mechanisms at the miRNA level play a role in the response of cardiomyocytes to AR, and could be a contributing factor in the differential resistance of cardiomyocytes to AR.
RESUMO
It is well known that 5-lipoxygenase derivates of arachidonic acid play an important pathogenic role during myocardial infarction. Therefore, the gene encoding arachidonate 5-lipoxygenase (ALOX5) appears to be an attractive target for RNA interference (RNAi) application. In experiments on cultivated cardiomyocytes with anoxia-reoxygenation (AR) and in vivo using rat model of heart ischemia-reperfusion (IR) we determined influence of ALOX5 silencing on myocardial cell death. ALOX5 silencing was quantified using real-time PCR, semi-quantitative PCR, and evaluation of LTC(4) concentration in cardiac tissue. A 4.7-fold decrease of ALOX5 expression (P < 0.05) was observed in isolated cardiomyocytes together with a reduced number of necrotic cardiomyocytes (P < 0.05), increased number live (P < 0.05) and unchanged number of apoptotic cells during AR of cardiomyocytes. Downregulation of ALOX5 expression in myocardial tissue by 19% (P < 0.05) resulted in a 3.8-fold reduction of infarct size in an open chest rat model of heart IR (P < 0.05). Thus, RNAi targeting of ALOX5 protects heart cells against IR injury both in culture and in vivo.