RESUMEN
In this study, we investigated possible engagement of NF-kappaB and Ku autoantigen (Ku) activation in development of multidrug resistance (MDR) and circumvention of MDR by modulation of NF-kappaB and Ku. The NF-kappaB activity and NF-kappaB p65 subunit level were constitutively higher in MDR cells than in drug-sensitive parental cells. Interestingly, a faster running NF-kappaB DNA binding complex was identified as Ku, a DNA damage sensor and a key double strand break repair protein, and was positively correlated with the NF-kappaB activity in MDR cells and Ku- or both subunits of NF-kappaB-transfected cells. Also both NF-kappaB and Ku activities were activated or inhibited by treatment with etoposide (VP-16) or MG-132 (a proteasome inhibitor), respectively. Furthermore, PKA inhibitor suppressed markedly the constitutive and drug-induced activities of NF-kappaB and Ku in MDR cells and subsequently potentiated the cytotoxic activity of anticancer drugs. Our results proposed that the NF-kappaB and Ku activation could be one of multi-factorial MDR mechanism, and PKA inhibitor, likely via inhibition of NF-kappaB and Ku activities, could enhance the effectiveness of anticancer drugs against MDR cells with high activities of NF-kappaB and Ku.
Asunto(s)
Antígenos Nucleares , Carbazoles , ADN Helicasas , Proteínas de Unión al ADN/metabolismo , Resistencia a Múltiples Medicamentos , Resistencia a Antineoplásicos , FN-kappa B/metabolismo , Proteínas Nucleares/metabolismo , Sulfonamidas , Antineoplásicos/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Proteínas de Unión al ADN/antagonistas & inhibidores , Sinergismo Farmacológico , Inhibidores Enzimáticos/farmacología , Humanos , Indoles/farmacología , Isoquinolinas/farmacología , Autoantígeno Ku , FN-kappa B/antagonistas & inhibidores , Proteínas Nucleares/antagonistas & inhibidores , Pirroles/farmacología , Células Tumorales CultivadasRESUMEN
Current concepts of the pathogenesis of Parkinson's disease (PD) center on the formation of reactive oxygen species (ROS), and dopamine has been considered to be a major source of ROS. Recently, it has been shown in a postmortem study that nuclear translocation of nuclear factor-kappa B (NF-kappaB) was observed in dopaminergic neurons of patient with PD. However, its role is not known. The present study examined the possible role of NF-kappaB in ODA (auto-oxidized dopamine)-induced apoptosis to understand the process of PD. Using the electrophoretic mobility shift assay, it was found that ODA activated the DNA binding activity of NF-kappaB. Suppression of the transcriptional activity of NF-kappaB in PC12 cells by overexpression of a wild-type and a dominant negative mutant form (S32A/S36A) of inhibitor kappa B (IkappaB)-alpha led to increase of apoptotic cell death induced by treatment of ODA. In addition, overexpression of NF-kappaB in PC12 cells blocked ODA-induced cell death. However, JNK/SAPK activities, which mediate various stress signals, were similar among the parental, NF-kappaB- or dominant negative mutant IkappaB alpha-transfected cells. Therefore, these results suggest that activation of NF-kappaB during ODA-induced apoptosis may have a counteracting activity against the signals mediating apoptotic cell death and thereby delay the process of Parkinson's disease.
Asunto(s)
Apoptosis/fisiología , Dopamina/análogos & derivados , Dopamina/toxicidad , Proteínas I-kappa B , FN-kappa B/fisiología , Enfermedad de Parkinson/metabolismo , Feocromocitoma/metabolismo , Animales , Apoptosis/efectos de los fármacos , Muerte Celular/efectos de los fármacos , ADN/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Dopamina/metabolismo , Genes Dominantes , Proteínas Quinasas JNK Activadas por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Inhibidor NF-kappaB alfa , FN-kappa B/genética , FN-kappa B/metabolismo , FN-kappa B/farmacología , Oxidación-Reducción , Células PC12 , Enfermedad de Parkinson/etiología , Ratas , Factor de Transcripción ReIA , TransfecciónRESUMEN
DNA-dependent protein kinase (DNA-PK) is activated by DNA strand breaks and participates in DNA repair. Its regulatory subunit, Ku autoantigen, binds to DNA and recruits the catalytic subunit (DNA-PKcs). We show here a new role of DNA-PK in the development of multidrug resistance (MDR). The Ku-DNA binding activity, the levels of Ku70/Ku80 and DNA-PKcs in MDR variants, CEM/VLB(10-2), CEM/VLB(55-8) and CEM/VLB100 were higher than those in their parental drug-sensitive CEM cells in a drug resistance-dependent fashion. Also, CEM/VLB100 cells showed about 3-fold increase of DNA-PK enzyme activity as compared with CEM cells. Similar results were observed in another MDR cell line, FM3A/M mouse mammary carcinoma cells. Moreover, we observed that CEM/VLB100 cells were about 11-fold sensitive to wortmannin, which inhibits DNA-PK, compared with the CEM cells, and sensitized the MDR cells when combined with either bleomycin or vincristine, but have a little effect on CEM cells. Wortmannin was shown to inhibit DNA-PK and Ku-DNA binding activity in CEM/VLB100 cells dose dependently but had a little or no effect on their parental cells. Our results suggested that enhanced expression of DNA-PK participates in the development of MDR, and the use of DNA-PK inhibitors such as wortmannin is likely to improve the effectiveness of anticancer drugs and thus could partially overcome drug resistance in MDR cells, through its ability to inhibit Ku/DNA-PK activity.
Asunto(s)
Androstadienos/farmacología , Antígenos Nucleares , Antineoplásicos/toxicidad , ADN Helicasas , Resistencia a Múltiples Medicamentos , Inhibidores Enzimáticos/farmacología , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Bleomicina/toxicidad , Línea Celular , Proteína Quinasa Activada por ADN , Proteínas de Unión al ADN/genética , Femenino , Humanos , Autoantígeno Ku , Leucemia , Neoplasias Mamarias Experimentales , Ratones , Proteínas Nucleares/genética , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Ratas , Células Tumorales Cultivadas , Vincristina/toxicidad , WortmaninaRESUMEN
In the present study, we examined the modulation of heat shock factor 1 (HSF1) activity and expression of heat shock proteins (HSPs) after exposure to anticancer drugs. Anticancer drugs induced HSF1 DNA-binding activity, and this was followed by an increase of mitochondrial HSP75 and HSP60 levels and concurrent decrease of cytoplasmic HSP70 levels. Unlike heat shock-induced full phosphorylation, HSF1 was partially phosphorylated after exposure to vincristine, and this result was tightly correlated with the kinetics of JNK/SAPK activation, and up-regulation of mitochondrial HSP75 level and concurrent down-regulation of HSP70. Furthermore, the dominant-negative mutant of SEK1 blocked the phosphorylation of HSF1 and up-regulation of mitochondrial HSP75 in response to vincristine or vinblastine. These data suggest that anticancer drugs regulate the HSF1 transcriptional activity differently from heat shock, and JNK/SAPK pathway appears to be involved in anticancer drug-induced HSF1 phosphorylation and consequently differential regulation of mitochondrial HSP75 and HSP60 and cytoplasmic HSP70.