RESUMEN
TNF-related apoptosis-inducing ligand (TRAIL) is a promising cytokine for killing tumor cells. However, a number of studies have demonstrated that different cancer cells resist TRAIL treatment and, moreover, TRAIL can promote invasion and metastasis in resistant cells. Here we report that TRAIL rapidly activates caspase-8 in a panel of non-small-cell lung carcinomas (NSCLCs). Adenocarcinomas derived from the lung in addition to high caspase-8 expression are characterized by increased expression of DR4 compared with adjacent non-neoplastic tissues. Blocking DR4 or lowering caspase-8 expression significantly reduced apoptosis in NSCLC cell lines, indicating the importance of DR4 and signifying that higher levels of caspase-8 in lung adenocarcinomas make them more susceptible to TRAIL treatment. Despite rapid and robust initial responsiveness to TRAIL, surviving cells quickly acquired resistance to the additional TRAIL treatment. The expression of cellular-FLIP-short (c-FLIPS) was significantly increased in surviving cells. Such upregulation of c-FLIPS was rapidly reduced and TRAIL sensitivity was restored by treatment with cycloheximide. Silencing of c-FLIPS, but not c-FLIP-long (c-FLIPL), resulted in a remarkable increase in apoptosis and significant reduction of clonogenic survival. Furthermore, chelation of intracellular Ca(2+) or inhibition of calmodulin caused a rapid proteasomal degradation of c-FLIPS, a significant increase of the two-step processing of procaspase-8, and reduced clonogenicity in response to TRAIL. Thus, our results revealed that the upregulation of DR4 and caspase-8 expression in NSCLC cells make them more susceptible to TRAIL. However, these cells could survive TRAIL treatment via upregulation of c-FLIPS, and it is suggested that blocking c-FLIPS expression by inhibition of Ca(2+)/calmodulin signaling significantly overcomes the acquired resistance of NSCLC cells to TRAIL.
Asunto(s)
Apoptosis/efectos de los fármacos , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/metabolismo , Calcio/metabolismo , Calmodulina/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNF/farmacología , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/antagonistas & inhibidores , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/genética , Calmodulina/antagonistas & inhibidores , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Caspasa 8/metabolismo , Línea Celular Tumoral , Cicloheximida/farmacología , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Inhibidores de la Síntesis de la Proteína/farmacología , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacología , Transducción de Señal/efectos de los fármacos , Ligando Inductor de Apoptosis Relacionado con TNF/genética , Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
In experiments on the primary culture of isolated neonatal rat cardiomyocytes it was determined that cardiomyocytes express ALOX5 gene encoding enzyme 5-lipoxygenase. Anoxia-reoxygenation does not affect significantly the expression of 5-lipoxygenase mRNA in cardiomyocytes. Transfection of 5-lipoxygenase-specific small interfering RNA's (siRNA) into cardiomyocytes lead to a significant reduction of 5-lipoxygenase mRNA expression in cardiomyocytes 24 hours after transfection. ALOX5 gene silencing resulted in improved viability of cell population (by 13.3% P < 0.001) due to decreased number of necrotic (by 14.6%, P < 0.001), but not apoptotic, cells during anoxia-reoxygenation. Our results indicate that siRNA against ALOX5 effectively protects cardiomyocytes against anoxia-reoxygenation injury.
Asunto(s)
Araquidonato 5-Lipooxigenasa/genética , Silenciador del Gen , Miocitos Cardíacos/enzimología , Miocitos Cardíacos/patología , Oxígeno/metabolismo , ARN Interferente Pequeño/farmacología , Animales , Animales Recién Nacidos , Apoptosis , Hipoxia de la Célula , Células Cultivadas , Miocitos Cardíacos/metabolismo , Necrosis , Interferencia de ARN , RatasRESUMEN
The cells death and genes expression in neonatal cardiomyocytes culture at two anoxia-reoxygenation modeling were investigated. The primary culture of neonatal cardiomyocytes was undergone 30 min of anoxia followed by 24 h (A-R1) and the second anoxia-reoxygenation--30 min and 60 min respectively (A-R2). The percentages of living, necrotic, apoptotic and autophagic cells were determined by staining with bis-benzimide, propidium iodide and monodansylcadaverine. Anoxia-reoxygenation significantly influenced the ratio of living, necrotic, apoptotic and autophagic cells both at its first A-RI and second A-R2 episodes. It was shown that the main mechanism of cell death after the both periods of anoxia-reoxygenation is necrosis. The changes of mRNA levels of genes of heat shock proteins HSP70 and HSP90, antiapoptotic protein Bcl2 and key regulator of autophagy FRAP in cardiomyocytes culture were established. The data obtained allow to make suggestion that in 24 h after the first episode of anoxia-reoxygenation A-R1 the overexpression of heat shock proteins starts the cascade of reactions that causes the necrotic cell death prevalent and the blocking of apoptotic program at second anoxia-reoxygenation A-R2.