ABSTRACT
Objective: To investigate the effect of CrkL silence on hypoxia/reoxygenation (H/R)-induced apoptosis and survival inhibition in cardiomyocytes and the underlying mechanisms. Methods: H9C2 cardiomyocytes were infected with blank, negative lentivirus and CrkL RNAi lentivirus (CrkL mRNA silence), and then treated with H/R separately. The cardiomyocytes were divided into blank group, negative lentivirus group, CrkL silence group, blank + H/R group, negative lentivirus + H/R group and CrkL silence + H/R group. The expression of CrkL mRNA was detected by RT-PCR, and the expression of CrkL and p-ERK1/2 protein was detected by Western blotting analysis. The apoptosis rate of cardiomyocytes was analyzed by flow cytometry, and the cell proliferation rate was analyzed by MTT. Results: Compared with blank and negative lentivirus groups, CrkL silence group had significantly decreased CrkL mRNA and protein, p-ERK1 protein, p-ERK1/2 protein and proliferation rate (P<0.05 or P<0.01), and significantly increased apoptosis rate (P<0.01). The same was true for CrkL silence + H/R group when compared with the blank + H/R and negative lentivirus + H/R groups. Conclusion: CrkL silence can aggravate H/R-induced apoptosis and survival inhibition in cardiomyocytes, which might be mediated by the decrease of p-ERK1/2 protein expression.
ABSTRACT
"A leucemia mielóide crônica (LMC) é caracterizada pela presença da translocação t(9:22) que codifica a proteína quimérica oncogênica BCR-ABL. Imatinibe é uma droga alvo-específica que inibe a atividade tirosina quinase (TK) da proteína BCR-ABL. Entretanto, os níveis intracelulares do imatinibe podem ser alterados pelas proteínas transportadoras de efluxo de drogas: glicoproteína P (Pgp), proteína da resistência em câncer de mama (BCRP) e proteína relacionada à resistência à múltiplas drogas (MRP1), assim como a proteína transportadora de influxo de drogas (OCT1). O objetivo do presente estudo foi analisar a participação dessas proteínas, isoladamente ou em associação, na resistência ao imatinibe em linhagens celulares e células de pacientes com LMC. Para análise da atividade das proteínas transportadoras de efluxo, foi utilizado o fluorocromo rodamina-123 associado ao modulador ciclosporina-A (Rho+CSA) e o pheophorbide A associado ao fumitremorgin C (PhA+FTC), ambos através de citometria de fluxo. A análise dos RNAm dos genes ABCB1, ABCG2 e SLC22A1 que codificam as proteínas Pgp, BCRP e OCT1, respectivamente, foi realizada por PCR em tempo real. A inibição da TK BCR-ABL foi mensurada através dos níveis de fosforilação de CrkL (pCrkL), seu principal alvo de ativação. Observamos uma maior positividade para o ensaio Rho+CSA nas amostras que expressavam Pgp comparada com as que expressavam MRP1, sugerindo menor atividade dessa proteína em pacientes com LMC, ou ainda que tal ensaio possa ser menos específico para a atividade da MRP1. O ensaio PhA+FTC foi capaz de identificar a atividade da proteína BCRP em linhagens celulares e células de pacientes. Níveis reduzidos dos RNAm ABCB1 e SLC22A1, mas não do RNAm ABCG2, foram observados quando comparados com as amostras de indivíduos saudáveis. Não houve correlação entre os níveis da proteína Pgp e do RNAm ABCB1. A expressão de Pgp foi detectada na maioria das amostras de LMC, independente da fase da doença, e não foi associada com o prognóstico desfavorável. Variações nos níveis de expressão da Pgp foram observadas durante a evolução da LMC e relacionadas com o tratamento prévio. O imatinibe foi capaz de aumentar a expressão da Pgp, assim como os níveis do RNAm ABCB1 na linhagem K562-Lucena 1, Pgp+. Além disso, observamos uma maior redução de pCrkL e um maior percentual de morte celular nas células K562, Pgp-, quando comparadas à K562-Lucena 1, evidenciando um possível papel do imatinibe como substrato para a Pgp. Este fármaco também demonstrou ter potencial para funcionar como agente modulador da bomba de efluxo, uma vez que impediu o exporte de Rho das células K562-Lucena 1. Amostras de pacientes resistentes ao imatinibe exibiram altos níveis de atividade das proteínas transportadoras de efluxo de drogas (ensaio Rho+CSA). Nossos dados mostram que a atividade e/ou expressão dos transportadores de efluxo e influxo de drogas encontram-se alterados na maioria dos pacientes com LMC, porém não há correlação com a resposta ao imatinibe e o prognóstico na LMC. Entretanto, o conjunto dos resultados sugere um papel para a Pgp na resistência in vitro ao imatinibe"(AU)
"Chronic myeloid leukemia (CML) is characterized by the presence of the t(9:22) encoding the BCR-ABL chimeric oncogenic protein. Imatinib is a target specific drug that inhibits the activity of the tyrosine kinase (TK) protein BCR-ABL. However, imatinib intracellular concentration may be altered by transporter proteins. It was described that efflux proteins, P-glycoprotein (Pgp), breast cancer resistance protein (BCRP) and multidrug resistance related protein (MRP1), and the influx protein, the organic cation transporter protein (OCT1) may contribute for imatinib clinical resistance. The aim of this study was to evaluate the role of these proteins in imatinib resistance in cell lines and leukemic cells from CML patients. To analyze the activity of the efflux transporter proteins, fluorochrome rhodamine-123 associated with the modulator cyclosporin A (Rho+CSA) and pheophorbide A associated with fumitremorgin C (PhA+FTC) were used by flow cytometry. Analysis of ABCG1, ABCG2 and SLC22A1 genes, that encode the Pgp, BCRP and OCT1 proteins, respectively, was performed by real time PCR. The inhibition of BCR-ABL TK was measured by the levels of CrkL phosphorylation (pCrkL), its main target of activation. We observed a higher positivity for Rho+CSA assay in samples expressing Pgp, when compared with the ones expressing MRP1. These results suggest that patients with CML have lower activity of this protein, or this assay might be less specific to indicate the activity of MRP1. The PhA+FTC assay was able to identify BCRP activity in cell lines and cells from patients. Reduced levels of ABCB1 and SLC22A1, but not ABCG2 mRNA were observed when compared with samples from healthy individuals. There was no correlation between the levels of Pgp protein and ABCB1 mRNA. Pgp expression was detected in most samples of CML, regardless of disease stage and was not associated with poor prognosis. Changes in Pgp expression levels have been observed during the development of CML and were related to pretreatment. Imatinib was able to increase Pgp expression as well as ABCB1 mRNA levels in Pgp+ K562Lucena 1 cells. Moreover, we observed a greater pCrkL reduction and a higher percentage of cell death in Pgp- K562 cells compared to K562-Lucena 1, indicating a possible role of imatinib as a Pgp substrate. This drug has also been shown to have potential as an efflux pump modulating agent, once efflux of Rho was prevented in K562-Lucena 1. Imatinib resistant patient samples exhibited high levels of efflux transporter proteins activity (Rho+CSA assay). Our data show that the activity and / or expression of influx and efflux transporters of drugs are altered in most patients with CML, but no correlation with prognosis and response to imatinib in CML was observed. However, our results suggest a role for Pgp in imatinib resistance"(AU)