RESUMO
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.
Assuntos
Apoptose/efeitos dos fármacos , Proteínas de Transporte/metabolismo , Caspase 8/metabolismo , Emodina/farmacologia , Proteína Supressora de Tumor p53/metabolismo , Animais , Proteínas Reguladoras de Apoptose , Western Blotting , Proteínas de Transporte/genética , Caspase 8/genética , Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular , Linhagem Celular , Linhagem Celular Tumoral , Ciclina A/genética , Ciclina A/metabolismo , Quinase 2 Dependente de Ciclina/genética , Quinase 2 Dependente de Ciclina/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Células HEK293 , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Interferência de RNA , Estabilidade de RNA/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína Supressora de Tumor p53/genéticaRESUMO
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.
Assuntos
Antineoplásicos/farmacologia , Cisplatino/farmacologia , Proteínas de Ligação a DNA/genética , Resistencia a Medicamentos Antineoplásicos/genética , Endonucleases/genética , Neoplasias Pulmonares/genética , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Adenocarcinoma de Pulmão , Animais , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Cisplatino/uso terapêutico , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/genética , Proteínas de Ligação a DNA/metabolismo , Feminino , Perfilação da Expressão Gênica , Células HEK293 , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Estadiamento de Neoplasias , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Análise de SobrevidaRESUMO
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.
Assuntos
Adenocarcinoma/genética , Cisplatino/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Neoplasias Pulmonares/genética , Adenocarcinoma/tratamento farmacológico , Idoso , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Biomarcadores Farmacológicos/metabolismo , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Cisplatino/farmacologia , Feminino , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Neoplasias Pulmonares/tratamento farmacológico , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , Células Tumorais Cultivadas , Proteína 1 de Ligação à Proteína Supressora de Tumor p53RESUMO
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.