RESUMEN
In this study, we examined the mechanism of action of the novel epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor 5-benzylidene-hydantoin UPR1024, whose structure was designed to interact at the ATP-binding site of EGFR. The compound had antiproliferative and proapoptotic effects when tested on the non-small cell lung cancer cell line A549. The growth inhibitory effect was associated with an accumulation of the cells in the S phase of the cell cycle. Moreover, UPR1024 induced significant level of DNA strand breaks associated with increased expression of p53 and p21(WAF1) proteins, suggesting an additive mechanism of action. The presence of wild-type p53 improved the drug efficacy, although the effect was also detectable in p53 null cells. We also noted apoptotic cell death after treatment with UPR1024 at concentrations above 10 mumol/L for >24 h, with involvement of both the extrinsic and intrinsic pathways. The present data show that UPR1024 may be considered a combi-molecule capable of both blocking EGFR tyrosine kinase activity and inducing genomic DNA damage. UPR1024 or its derivatives might serve as a basis for development of drugs for the treatment of lung cancer in patients resistant to classic tyrosine kinase inhibitors.
Asunto(s)
Compuestos de Bencilideno/farmacología , Carcinoma de Pulmón de Células no Pequeñas/patología , Receptores ErbB/antagonistas & inhibidores , Hidantoínas/farmacología , Neoplasias Pulmonares/patología , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Daño del ADN/efectos de los fármacos , Evaluación Preclínica de Medicamentos , Resistencia a Antineoplásicos/efectos de los fármacos , Sinergismo Farmacológico , Receptores ErbB/metabolismo , Gefitinib , Humanos , Modelos Biológicos , Fosforilación , Quinazolinas/farmacología , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Exposure of C2C12 muscle cells to hypertonic stress induced an increase in cell content of creatine transporter mRNA and of creatine transport activity, which peaked after about 24 h incubation at 0.45 osmol (kg H(2)O)(-1). This induction of transport activity was prevented by addition of either cycloheximide, to inhibit protein synthesis, or of actinomycin D, to inhibit RNA synthesis. Creatine uptake by these cells is largely Na(+) dependent and kinetic analysis revealed that its increase under hypertonic conditions resulted from an increase in V(max) of the Na(+)-dependent component, with no significant change in the K(m) value of about 75 mumol l(-1). Quantitative real-time PCR revealed a more than threefold increase in the expression of creatine transporter mRNA in cells exposed to hypertonicity. Creatine supplementation significantly enhanced survival of C2C12 cells incubated under hypertonic conditions and its effect was similar to that obtained with the well known compatible osmolytes, betaine, taurine and myo-inositol. This effect seemed not to be linked to the energy status of the C2C12 cells because hypertonic incubation caused a decrease in their ATP content, with or without the addition of creatine at 20 mmol l(-1) to the medium. This induction of creatine transport activity by hypertonicity is not confined to muscle cells: a similar induction was shown in porcine endothelial cells.