ToxSci Advance Access originally published online on July 3, 2009
Toxicological Sciences 2009 111(2):413-423; doi:10.1093/toxsci/kfp145
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Inhibition of p38-MAPK Potentiates Cisplatin-Induced Apoptosis via GSH Depletion and Increases Intracellular Drug Accumulation in Growth-Arrested Kidney Tubular Epithelial Cells
* Departamento de Bioquímica y Biología Molecular I, Facultad de Biología, Universidad Complutense de Madrid, 28040 Madrid, Spain
Departamento de Química Inorgánica, Universidad Autónoma de Madrid, 28049 Madrid, Spain
Departamento de Nefrología, Fundación Jiménez Díaz, 28040 Madrid, Spain
Centro de Investigaciones Biológicas, CSIC, 28040 Madrid, Spain
1 To whom correspondence should be addressed at Departamento de Bioquímica y Biología Molecular I, Facultad de Biología, Universidad Complutense de Madrid, 28040-Madrid, Spain. Fax: +34-913944672. E-mail: fmata{at}bio.ucm.es.
Received February 12, 2009; accepted July 1, 2009
 |
Abstract |
|---|
We were interested in analyzing 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 nonproliferating 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 cotreatment with p38-MAPK–specific inhibitors (SB203580, SB220025) or transfection with a kinase-negative mutant of MKK6, whereas c-Jun NH2-terminal kinase or extracellular signal-regulated kinase/MAPK and ERK Kinase 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 cotreatment with SB203580. Treatment of confluent/serum-deprived cells with cisplatin plus SB203580 decreased the intracellular reduced glutathione (GSH) content, and increased intracellular cisplatin accumulation as well as cisplatin binding to DNA. Cotreatment 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.