Naturally Occurring Asteriscunolide A Induces Apoptosis and Activation of Mitogen-Activated Protein Kinase Pathway in Human Tumor Cell Lines GledyNegrı´n, 1 Jose ´ L. Eiroa, 2 Manuel Morales, 2 Jorge Triana, 2 Jose ´ Quintana, 1 and Francisco Este ´ vez 1 * 1 Department of Biochemistry and Molecular Biology, Instituto Canario de Investigacio ´n del Ca ´ncer (I.C.I.C.), University of Las Palmas de Gran Canaria, Plaza Dr. Pasteur s/n, Las Palmas de Gran Canaria, Spain 2 Department of Chemistry, University of Las Palmas de Gran Canaria, Las Palmas de Gran Canaria, Spain Sesquiterpene lactones have attracted much attention because they display a wide range of biological activities, including antitumor properties. Here, we show the effects of the naturally occurring sesquiterpene lactone asteriscunolide A (AS) on viability of human melanoma, leukemia and cells that overexpress antiapoptotic proteins, namely Bcl-2 and Bcl-x L . All cell lines were sensitive to this compound, with IC 50 values of 5 mM. The cytotoxic effects of AS were accompanied by a G 2 -M phase arrest of the cell cycle and a concentration- and time-dependent appearance of apoptosis as determined by DNA fragmentation, translocation of phosphatidylserine to the cell surface and sub-G 1 ratio. Apoptosis was associated with caspase-3 activity and poly(ADP-ribose) polymerase cleavage and was prevented by the nonspecific caspase inhibitor z-VAD-fmk, indicating that caspases are essential components in this pathway. The apoptotic effect of AS was also associated with (i) the release of cytochrome c from mitochondria which was accompanied by dissipation of the mitochondrial membrane potential (DC m ) and (ii) the activation of the mitogen-activated protein kinases (MAPKs) pathway. AS-induced cell death was potentiated by inhibition of extracellular signal-regulated kinases (ERK) 1/2 signaling with U0126 and PD98059. Intracellular reactive oxygen species (ROS) seem to play a pivotal role in this process since high levels of ROS were produced early (1 h) and apoptosis was completely blocked by the free radical scavenger N-acetyl-L-cysteine (NAC). The present study demonstrates that AS-induced cell death is mediated by an intrinsic-dependent apoptotic event involving mitochondria and MAPKs, and through a mechanism dependent on ROS generation. ß 2010 Wiley-Liss, Inc. Key words: sesquiterpene lactones; apoptosis; caspases; DNA fragmentation; reactive oxygen species; cell-cycle arrest; mitogen-activated protein kinases; c-Jun N-terminal kinases; extracellular signal-regulated kinases INTRODUCTION Many anticancer compounds have been shown to cause the death of sensitive cells through the induction of apoptosis. Essential executioners of apoptosis are the caspases, a family of conserved cysteine aspartate-specific proteases [1] that are generally synthesized as zymogens and activated via proteolytic cleavage. In general, two major pathways for apoptosis have been described [2]. In the extrinsic (or death receptor) pathway, apoptosis is mediated by death receptors (such as Fas or tumor necrosis factor receptors) [3] and involves caspase-8 activation, while in the intrinsic (or mitochondrial) pathway diverse proapoptotic signals stimulate the translocation of cytochrome c from mitochondria to cytoplasm that promotes caspase-9 activation. Since caspase-3 is responsible for cleaving specific cellular proteins during apoptosis [4] both pathways con- verge to this level and therefore caspases-8 and -9 cleave and activate the proenzyme. A great number of studies indicate that apoptosis can be mediated by the endoplasmic reticulum signaling pathway [5] and it may also occur independently of caspase activation [6]. Mitogen-activated protein kinases (MAPKs) are a family of serine/threonine protein kinases that play an essential role in a diverse array of cellular functions, including proliferation, differentiation, MOLECULAR CARCINOGENESIS 49:488–499 (2010) ß 2010 WILEY-LISS, INC. Abbreviations: MAPKs, mitogen-activated protein kinases; ERK, extracellular signal-regulated kinases; JNK, c-Jun N-terminal kinases; p38 MAPK , p38 mitogen-activated protein kinases; SAPK, stress- activated protein kinase; AS, asteriscunolide A; PARP, poly(ADP- ribose) polymerase; PBMCs, peripheral blood mononuclear cells; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; IC 50 , 50% inhibition of cell growth; TEM, transmission electron microscopy; JC-1, 5,5 0 ,6,6 0 -tetrachloro-1,1 0 ,3,3 0 -tetraethylbenzimi- dazolylcarbocyanine iodide; DCF, 2 0 ,7 0 -dichlorofluorescein; H 2 -DCF- DA, 2 0 ,7 0 -dichlorodihydrofluorescein diacetate; ROS, reactive oxy- gen species; MEK, mitogen-activated extracellular kinases; NAC, N-acetyl-L-cysteine; DCm, mitochondrial membrane potential. *Correspondence to: Plaza Dr. Pasteur s/n, Las Palmas de Gran Canaria 35016, Spain. Received 4 November 2009; Revised 19 January 2010; Accepted 26 January 2010 DOI 10.1002/mc.20629 Published online 15 March 2010 in Wiley InterScience (www.interscience.wiley.com)