Arsenic trioxide triggers a regulated form of caspase-independent necrotic cell death via the mitochondrial death pathway Christian Scholz 1 , Thomas Wieder 1 , Lilian Sta¨rck 1 , Frank Essmann 1 , Klaus Schulze-Osthoff 2 , Bernd Do¨rken 1 and Peter T Daniel* ,1 1 Department of Hematology, Oncology and Tumor Immunology, Charite´, Campus Berlin-Buch, Robert-Ro¨ssle-Klinik, Humboldt Universita¨t, Berlin, Germany; 2 Institute of Molecular Medicine, University of Du¨sseldorf, Du¨sseldorf, Germany Cell death is generally believed to occur either by accidental, lytic necrosis or by programmed cell death, that is, apoptosis. The initiation and execution of cell death, however, is far more complex and includes path- ways like caspase-independent apoptosis or actively triggered necrosis. In this study, we investigated the mechanisms of cell death induced by arsenic trioxide (arsenite, As 2 O 3 ), a clinically efficient agent in anticancer therapy. As 2 O 3 -induced cell death coincides with cyto- chrome c release, facilitates mitochondrial permeability transition and is sensitive to inhibition by Bcl-x L , indicating that cell demise is regulated through the mitochondrial apoptosis pathway. Nevertheless, only little caspase-3 activation was observed and As 2 O 3 -induced cell death was only weakly obstructed by the broad spectrum caspase inhibitor z-VAD-fmk. Moreover, disruption of caspase-9 or -2 failed to decrease the amount of As 2 O 3 - mediated cell death. Interestingly, As 2 O 3 -induced cell death had a predominantly necrosis-like phenotype as assessed by Annexin-V/propidium iodide staining and LDH release. Finally, blocking glutathione synthetase by buthionine sulfoximine enhanced the As 2 O 3 -mediated necrosis-like cell death without increasing caspase-3 cleavage. As 2 O 3 does, however, not directly inhibit caspases, but appears to interfere with caspase activation. Altogether, our data clearly delineate a mode of As 2 O 3 - triggered cell death that differs considerably from that induced by conventional anticancer drugs. These findings may explain the capability of As 2 O 3 to efficiently kill even chemoresistant tumor cells with disturbed apoptosis signaling and caspase activation, a frequent finding in malignancy. Oncogene (2005) 24, 1904–1913. doi:10.1038/sj.onc.1208233 Published online 24 January 2005 Keywords: arsenic trioxide; caspase-independence; ATP; mitochondria; necrosis Introduction Apoptosis and necrosis are two forms of cell death with distinct biochemical and morphological features. While apoptotic cells are defined in most cases by fragmented nuclei with condensed chromatin and formation of apoptotic bodies, necrotic cells are characterized by mitochondrial swelling and loss of plasma membrane integrity without severe nuclear damage (Wyllie et al., 1980). Mechanisms involved in necrosis have hardly been elucidated. In contrast, events leading to apoptotic cell death have been described to a greater extent (Kroemer and Reed, 2000). While apoptosis results in rapid phagocytosis and elimination of the affected cell, necrosis leads to spilling of cellular contents resulting in inflammation and tissue damage. In contrast to necrosis, apoptosis is mediated by caspases that are activated either by the extrinsic pathway or the intrinsic apoptosis pathway that is mediatedeitherviathemitochondriaortheendoplasmic reticulum. The extrinsic pathway is initiated by the ligation of a death receptor, for example, CD95 (Fas, APO-1) or the TRAIL receptors, and the subsequent assembly of the death-inducing signaling complex (DISC) that recruits and activates caspase-8 or -10 (Daniel et al., 2001; Peter and Krammer, 2003). In contrast, death-receptor-independent stimuli like growth-factor deprivation or anticancer drugs induce apoptosis through the mitochondrial pathway. Here, loss of mitochondrial transmembrane potential (DC m ) accompanies the release of cytochrome c into the cytosol. This triggers apoptosome formation and activation of the caspase cascade (Daniel et al., 2003) that is enhanced by Smac/Diablo and Omi/HtrA2, which are also released from mitochondria during apoptosis (van Loo et al., 2002). Arsenic trioxide (As 2 O 3 ), a common environmental toxin, has successfully been employed in patients with acute promyelocytic leukemia (APL) (Shen et al., 1997; Soignet et al., 1998; Niu et al., 1999). APL cells typically express the PML-RARa (promyelocytic leukemia gene product-retinoic acid receptor a) fusion protein. In line with this, degradation and targeting of PML to nuclear bodies was initially suggested to be the key event in As 2 O 3 -inducedcelldeath(Zhu et al.,1997).Nevertheless, Received 12 June 2004; revised 6 August 2004; accepted 20 September 2004; published online 24 January 2005 *Correspondence: P Daniel, Clinical and Molecular Oncology, Charite´, Campus Virchow Klinikum, Humboldt University, Augustenburger Platz 1, 13353 Berlin, Germany; E-mail: pdaniel@mdc-berlin.de Oncogene (2005) 24, 1904–1913 & 2005 Nature Publishing Group All rights reserved 0950-9232/05 $30.00 www.nature.com/onc