Original Paper
Cell Death and Differentiation (2006) 13, 1968–1981. doi:10.1038/sj.cdd.4401898; published online 17 March 2006
Aplidin® induces JNK-dependent apoptosis in human breast cancer cells via alteration of glutathione homeostasis, Rac1 GTPase activation, and MKP-1 phosphatase downregulation
Edited by G Nunez
L González-Santiago1,2,7, Y Suárez1,2,7, N Zarich3,7, M J Muñoz-Alonso1,2, A Cuadrado1,2, T Martínez1, L Goya4, A Iradi5, G Sáez-Tormo5, J V Maier6, A Moorthy6, A C B Cato6, J M Rojas3 and A Muñoz1
- 1Instituto de Investigaciones Biomédicas 'Alberto Sols', Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, Arturo Duperier, 4, Madrid E-28029, Spain
- 2Pharma Mar S.A., E-28770 Colmenar Viejo, Madrid, Spain
- 3Centro Nacional de Microbiología, Instituto de Salud Carlos III, Carr. Majadahonda-Pozuelo Km 2, E-28220 Majadahonda, Madrid, Spain
- 4Instituto del Frío, Consejo Superior de Investigaciones Científicas, José Antonio Novais 10, Madrid E-28040, Spain
- 5Facultad de Medicina, Universidad de Valencia, Avda. Blasco Ibáñez 17, Valencia E-46001, Spain
- 6Forschungszentrum Karlsruhe, Institute of Toxicology and Genetics, Karlsruhe D-76021, Germany
- 7These authors contributed equally to this work
Correspondence: A Muñoz, Molecular Endocrinology, Instituto de Investigaciones Biomédicas 'Alberto Sols', Arturo Duperier, 4, E-28029 Madrid, Spain. Tel: +34 91 585 4451; Fax: +34 91 585 4401; E-mail: amunoz@iib.uam.es
Received 11 April 2005; Revised 20 January 2006; Accepted 13 February 2006; Published online 17 March 2006.
Abstract
Aplidin® is an antitumor agent in phase II clinical trials that induces apoptosis through the sustained activation of Jun N-terminal kinase (JNK). We report that Aplidin® alters glutathione homeostasis increasing the ratio of oxidized to reduced forms (GSSG/GSH). Aplidin® generates reactive oxygen species and disrupts the mitochondrial membrane potential. Exogenous GSH inhibits these effects and also JNK activation and cell death. We found two mechanisms by which Aplidin® activates JNK: rapid activation of Rac1 small GTPase and downregulation of MKP-1 phosphatase. Rac1 activation was diminished by GSH and enhanced by L-buthionine (SR)-sulfoximine, which inhibits GSH synthesis. Downregulation of Rac1 by transfection of small interfering RNA (siRNA) duplexes or the use of a specific Rac1 inhibitor decreased Aplidin®-induced JNK activation and cytotoxicity. Our results show that Aplidin® induces apoptosis by increasing the GSSG/GSH ratio, a necessary step for induction of oxidative stress and sustained JNK activation through Rac1 activation and MKP-1 downregulation.
Keywords:
Aplidin®, apoptosis, JNK, Rac1 GTPase, MKP-1 phosphatase, glutathione
Abbreviations:
APL, Aplidin®; BCPS, bathocuproinedisulfonic acid; BHA, butylated hydroxyanisole; BSP, bromosulfophthalein; BSO, L-buthionine,S,R-sulfoximine; ERK, extracellular signal-regulated kinases; GSH, glutathione; GST, glutathione-S-transferase; JNK, Jun N-terminal kinase; LPO, lipoxygenases; MAPK, mitogen-activated protein kinase; MEF, mouse embryo fibroblasts; PLA2, phospholipase A2; ROS, reactive oxygen species; siRNA, small interfering RNA
