RESUMEN
DNA repair has been suggested to be a major cause of spontaneous drug resistance in patients with lung adenocarcinomas (LADC). Among the DNA repair-related proteins, excision repair cross-complementation group 1 (ERCC1) has been shown to be essential for repairing cisplatin-induced interstrand cross-linkage. However, the role of other DNA repair-related proteins in drug resistance has not been clearly elucidated. In this study, we used suppression subtractive hybridization and microarray analysis to identify the DNA repair-related genes associated with cisplatin resistance. We focused on the association of XPC protein expression, which plays a pivotal role in the earliest response to global genomic repair, with the survival of LADC patients. Using suppression subtractive hybridization and a microarray analysis to identify drug resistance-associated DNA repair-related genes, we found that the mRNA levels of ERCC1, MSH-3, MSH-6 and XPC were significantly increased in LADC patients. Since the results of ERCC1 mRNA expression corresponded well with those in previous reports, in this study we focused on the clinical correlation between XPC expression and patient survival. The level of XPC protein was determined by immunohistochemical and immunoblotting analyses. We detected the XPC protein in 46 (43%) of 107 pathological LADC samples. XPC protein expression correlated with tumor stage, cigarette smoking and poor survival. In the in vitro experiments with LADC cell lines, increased XPC expression was associated with elevated drug resistance, and silencing of XPC expression reduced cisplatin resistance. Our results suggest that XPC expression predicts drug resistance in LADC.
Asunto(s)
Antineoplásicos/farmacología , Cisplatino/farmacología , Proteínas de Unión al ADN/genética , Resistencia a Antineoplásicos/genética , Endonucleasas/genética , Neoplasias Pulmonares/genética , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Adenocarcinoma del Pulmón , Animales , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Cisplatino/uso terapéutico , Reparación del ADN/efectos de los fármacos , Reparación del ADN/genética , Proteínas de Unión al ADN/metabolismo , Femenino , Perfilación de la Expresión Génica , Células HEK293 , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Masculino , Ratones , Ratones Endogámicos BALB C , Estadificación de Neoplasias , ARN Mensajero/genética , ARN Mensajero/metabolismo , Análisis de SupervivenciaRESUMEN
Using short hairpin RNA against p53, transient ectopic expression of wild-type p53 or mutant p53 (R248W or R175H), and a p53- and p21-dependent luciferase reporter assay, we demonstrated that growth arrest and apoptosis of FaDu (human pharyngeal squamous cell carcinoma), Hep3B (hepatoma), and MG-63 (osteosarcoma) cells induced by aloe-emodin (AE) are p53-independent. Co-immunoprecipitation and small interfering RNA (siRNA) studies demonstrated that AE caused S-phase cell cycle arrest by inducing the formation of cyclin A-Cdk2-p21 complexes through extracellular signal-regulated kinase (ERK) activation. Ectopic expression of Bcl-X(L) and siRNA-mediated Bax attenuation significantly inhibited apoptosis induced by AE. Cyclosporin A or the caspase-8 inhibitor Z-IETD-FMK blocked AE-induced loss of mitochondrial membrane potential and prevented increases in reactive oxygen species and Ca(++). Z-IETD-FMK inhibited AE-induced apoptosis, Bax expression, Bid cleavage, translocation of tBid to mitochondria, ERK phosphorylation, caspase-9 activation, and the release of cytochrome c, apoptosis-inducing factor (AIF), and endonuclease G from mitochondria. The stability of the mRNAs encoding caspase-8 and -10-associated RING proteins (CARPs) 1 and 2 was affected by AE, whereas CARP1 or 2 overexpression inhibited caspase-8 activation and apoptosis induced by AE. Collectively, our data indicate AE induces caspase-8-mediated activation of mitochondrial death pathways by decreasing the stability of CARP mRNAs in a p53-independent manner.
Asunto(s)
Apoptosis/efectos de los fármacos , Proteínas Portadoras/metabolismo , Caspasa 8/metabolismo , Emodina/farmacología , Proteína p53 Supresora de Tumor/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis , Western Blotting , Proteínas Portadoras/genética , Caspasa 8/genética , Ciclo Celular/efectos de los fármacos , Proteínas de Ciclo Celular , Línea Celular , Línea Celular Tumoral , Ciclina A/genética , Ciclina A/metabolismo , Quinasa 2 Dependiente de la Ciclina/genética , Quinasa 2 Dependiente de la Ciclina/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Células HEK293 , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patología , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Fosforilación/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Interferencia de ARN , Estabilidad del ARN/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteína p53 Supresora de Tumor/genéticaRESUMEN
DNA repair is one of the major causes of spontaneous drug and radiation resistance in patients with lung adenocarcinomas (LADC). 53BP1 is a mediator that relays signals from DNA damage sensors and activates various effectors for the DNA repair and cell survival. In this study we investigated the clinical and biological significance of 53BP1. Expression of 53BP1 was detected by immunoblotting and immunohistochemistry. Our data showed that 53BP1 was detected in 166 (75.8%) of 219 LADC patients. Expression of 53BP1 correlated with tumor stage, cigarette smoking, lymphovascular invasion and poor clinical outcome. In vitro, increased 53BP1 expression elevated drug resistance, and silencing of 53BP1 expression reduced cisplatin resistance. Our results suggest that 53BP1 expression plays an important role in cisplatin resistance and predicts the prognosis for LADC.
Asunto(s)
Adenocarcinoma/genética , Cisplatino/uso terapéutico , Resistencia a Antineoplásicos/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Neoplasias Pulmonares/genética , Adenocarcinoma/tratamiento farmacológico , Anciano , Animales , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Biomarcadores Farmacológicos/metabolismo , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Cisplatino/farmacología , Femenino , Regulación Neoplásica de la Expresión Génica/fisiología , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Péptidos y Proteínas de Señalización Intracelular/fisiología , Neoplasias Pulmonares/tratamiento farmacológico , Masculino , Ratones , Ratones Endogámicos BALB C , Persona de Mediana Edad , Células Tumorales Cultivadas , Proteína 1 de Unión al Supresor Tumoral P53RESUMEN
Enrofloxacin (ER) resistant Actinobacillus pleuropneumoniae strains emerged in Taiwan in 2002. The mechanism of ER resistance in A. pleuropneumoniae has not yet been reported. A total of 48 A. pleuropneumoniae isolates were obtained from the lungs of pigs with pleuropneumonia in Taiwan between September 2007 and April 2008. Twenty-nine isolates were found to be resistant to enrofloxacin. To understand the mechanisms of A. pleuropneumoniae's resistance to ER, enrofloxacin susceptibility of the isolates along with the mutations of the quinolone resistance-determining region (QRDR) of DNA gyrase and topoisomerase IV, qnr genes were analyzed. Enrofloxacin resistant isolates were found to carry at least one mutation in the QRDR of gyrA, leading to amino acid changes at codon 83 or 87. Efflux pump inhibitor (Phe-Arg-beta-naphthylamide) decreased enrofloxacin minimum inhibitory concentration 2-16-fold, suggesting participation of efflux in ER resistance. Plasmid mediated quinolone resistance genes qnr were not detected in these isolates. In conclusion, enrofloxacin resistance of A. pleuropneumoniae may be linked to multiple target gene mutations and active effluxs.
