ToxSci Advance Access published online on July 3, 2009
Toxicological Sciences, doi:10.1093/toxsci/kfp145
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Inhibition of p38-MAPK potentiates cisplatin-induced apoptosis via GSH depletion and increases intracellular drug accumulation in growth-arrested kidney tubular epithelial cells
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Departamento de Bioquímica y Biología Molecular I, Facultad de Biología, Universidad Complutense de Madrid, 28040 Madrid, Spain. E-mail: merodrig{at}bio.ucm.es; jcastro{at}solea.quim.ucm.es
# Departamento de Química Inorgánica, Universidad Autónoma de Madrid, 28049 Madrid, Spain. E-mail: adoracion.gomez{at}uam.es
Departamento de Nefrología, Fundación Jiménez Díaz, Avenida Reyes Católicos 2, 28040 Madrid, Spain. E-mail: aortiz{at}fjd.es
¶ Centro de Investigaciones Biológicas, CSIC, 28040 Madrid, Spain. E-mail: Aller{at}cib.csic.es
* Corresponding author: Felicísima Mata, Departamento de Bioquímica y Biología Molecular I, Facultad de Biología, Universidad Complutense de Madrid. 28040-Madrid, Spain. Phone: 34-913944156. Fax: +34-913944672. E-mail: fmata{at}bio.ucm.es
Received February 12, 2009; revision received June 17, 2009; accepted July 1, 2009
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Abstract |
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We were interested in analysing the regulation by mitogen-activated protein kinases (MAPKs) of cisplatin-provoked toxicity in epithelial renal tubule cell lines, when assayed under culture conditions (cell confluence plus serum deprivation), which mimic the characteristics of a non-proliferating epithelium. Under these restrictive growth conditions, cisplatin induced apoptosis with lower efficacy than in exponentially growing cells, and decreased p38-MAPK phosphorylation in NRK-52E and other (LLC-PK1, MDCK, HK2) cell lines. Moreover, cisplatin-provoked apoptosis was potentiated by co-treatment with p38-MAPK-specific inhibitors (SB203580, SB220025) or transfection with a kinase-negative mutant of MKK6, while JNK or MEK/ERK inhibitors were ineffective. By contrast, when applied to exponentially growing cells, cisplatin stimulated p38-MAPK phosphorylation and apoptosis, was attenuated by kinase inhibitors. Treatment of confluent/serum-deprived cells with cisplatin caused mitochondrial transmembrane potential disruption and activated the mitochondrial apoptotic pathway, as indicated by the decrease in Bcl-XL expression, increase in Bax expression and cytochrome c release, and these effects were potentiated by co-treatment with SB203580. Treatment of confluent/serum-deprived cells with cisplatin plus SB203580 decreased the intracellular GSH content, and increased intracellular cisplatin accumulation as well as cisplatin binding to DNA. Co-treatment with the GSH-depleting agent D,L-buthionine-R,S-sulfoximine also potentiated cisplatin-provoked apoptosis. In summary, p38-MAPK inhibition potentiates cisplatin-provoked apoptosis in growth- arrested epithelial renal tubule cells, a result that may be explained at least in part by GSH depletion and drug transport alteration.
Key Words: Cisplatin; Apoptosis; MAPK kinases; Cisplatin uptake; Intracellular glutathione; Renal tubule cells.