Aplidin TM induces the mitochondrial apoptotic pathway via oxidative stress-mediated JNK and p38 activation and protein kinase C d Luis F Garcı´a-Ferna´ndez 1,6 , Alejandro Losada 1,6 , Victoria Alcaide 1 , Alberto M A ´ lvarez 2 , Ana Cuadrado 3 , Laura Gonza´lez 3 , Keiko Nakayama 4 , Keiichi I Nakayama 5 , Jose´ Marı´a Ferna´ndez-Sousa 1 , Alberto Mun˜oz 3 and Jose´ Marı´a Sa´nchez-Puelles* ,1 1 Drug Discovery Department, PharmaMar, S.A., E-28760 Tres Cantos, Madrid, Spain; 2 Centro de Citometrı´a de Flujo, Facultad de Farmacia, Universidad Complutense, E-28040 Madrid, Spain; 3 Instituto de Investigaciones Biome´dicas ‘Alberto Sols’ (UAM- CSIC), E-28029 Madrid, Spain; 4 Department of Molecular Genetics, Medical Institute of Bioregulation, Kyushu University, Fukuoka 812-8582, Japan; 5 Department of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University, Fukuoka 812-8582, Japan Aplidin TM , a new antitumoural drug presently in phase II clinical trials, has shown both in vitro and in vivo activity against human cancer cells. Aplidin TM effectively inhibits cell viability by triggering a canonical apoptotic program resulting in alterations in cell morphology, caspase activation, and chromatin fragmentation. Pro-apoptotic concentrations of Aplidin TM induce early oxidative stress, which results in a rapid and persistent activation of both JNK and p38 MAPK and a biphasic activation of ERK. Inhibition of JNK and p38 MAPK blocks the apoptotic program induced by Aplidin TM , demonstrating its central role in the integration of the cellular stress induced by the drug. JNK and p38 MAPK activation results in downstream cytochrome c release and activation of caspases -9 and -3 and PARP cleavage, demonstrating the mediation of the mitochondrial apoptotic pathway in this process. We also demonstrate that protein kinase C delta (PKC-d) mediates the cytotoxic effect of Aplidin TM and that it is concomitantly processed and activated late in the apoptotic process by a caspase mediated mechanism. Remarkably, cells deficient in PKC-d show enhanced survival upon drug treatment as compared to its wild type counterpart. PKC-d thus appears as an important component necessary for full caspase cascade activation and execution of apoptosis, which most probably initiates a positive feedback loop further amplifying the apoptotic process. Oncogene (2002) 21, 7533 – 7544. doi:10.1038/sj.onc. 1205972 Keywords: Aplidin TM ; oxidative damage; JNK; p38; caspases; PKC-delta Introduction Aplidin TM (Figure 1), a marine cyclic depsipeptide derived from the Mediterranean tunicate Aplidium albicans (Rinehart, 2000), is a new anticancer agent with potent anti-neoplastic activity in vitro against a wide variety of human tumours. Aplidin TM was chosen for development as an antineoplastic agent because of its antitumour activity against subcutaneous implanted MRI-H254 gastric, PC-3 prostate and Burkitts lymphoma human xenografts as well as HTB-9 bladder carcinoma in the hollow fiber model (Faircloth et al., 1998, 1999). Clinical studies with Aplidin were initiated in early 1999 in different locations of Europe and Canada. Previous results from phase I clinical trials, including more than 200 treated patients, have shown hints of activity, in terms of stabilization of the disease during long-term intervals, in renal, medullary thyroid carcinoma and other neuroendocrine tumours, lung (NSC), head and neck and colorectal cancers. Anti- tumour activity in advanced resistant tumours has also been observed. On this basis, phase II disease oriented clinical studies are ongoing (Anthoney et al., 2000; Armand et al., 2001; Bowman et al., 2001; Ciruelos et al., 2002; Mauroun et al., 2001). Up to date, 17 patients have been already recruited for these studies (unpublished data). Little is known concerning the mechanism of action of Aplidin TM . In leukaemia cells, it has been suggested that Aplidin TM causes a G1 blockade and apoptosis, and inhibition of ornithine decarboxylase (ODC) (Erba et al., 1999). Also in leukaemia cells, Aplidin TM seems to cause a reduction in vascular endothelial growth factor (VEGF) secretion and downregulation of its receptor, VEGFR-1 (flt-1), involved in the process of vascularization and growth of certain tumours (Broggini et al., 2000). In addition, it has been described that its homologue didemnin B may interact with elongation factor 1a (Crews et al., 1994) and palmitoyl thioesterase (Crews et al., 1996). Apoptosis, or programmed cell death, is important for the destruction of undesired cells during develop- ment as well as tumour cells and other damaged cells. Received 26 March 2002; revised 12 August 2002; accepted 13 August 2002 *Correspondence: JM Sa´nchez-Puelles; E-mail: jmspuelles@pharmamar.com 6 These two authors contributed equally to this work Oncogene (2002) 21, 7533 – 7544 ª 2002 Nature Publishing Group All rights reserved 0950 – 9232/02 $25.00 www.nature.com/onc