Mitochondria play a pivotal role in apoptosis: permeabilization of the outer mitochondrial membrane and the release of pro-apoptotic proteins from the intermembrane space of mitochondria are regarded as the key event in apoptosis induction. Here we demonstrate how non-toxic doses of the mitochondrial Complex II inhibitor thenoyltrifluoroacetone (TTFA), which specifically inhibits the ubiquinone-binding site of succinate dehydrogenase (SDH), synergistically stimulated cell death, induced by harmless doses of cisplatin in a panel of chemoresistant neuroblastoma cell lines. Apoptotic cell death was confirmed by cytochrome c release from the mitochondria, cleavage of poly ADP-ribose polymerase, processing of caspase-3, which is an important executive enzyme in apoptosis, and caspase-3-like activity. Methyl malonate, an inhibitor of the SDHA subunit partially reversed apoptosis stimulated by TTFA in SK-N-BE(2) neuroblastoma cells (NB), indicating that sensitization requires oxidation of succinate. In contrast, in IMR-32 NB cells, the same concentrations of TTFA markedly suppressed cisplatin-induced apoptosis. Comparison of oxygen consumption in cisplatin-resistant SK-N-BE(2) and cisplatin-sensitive IMR-32 cells clearly demonstrated impaired Complex II activity in IMR-32 cells. We also found that in SK-N-BE(2) cells co-treatment with cisplatin and TTFA markedly stimulated formation of reactive oxygen species (ROS), whereas in IMR cells, cisplatin-mediated ROS production was attenuated by TTFA, which explains apoptosis suppression in these cells. Thus, functionally active SDH is a prerequisite for the ROS-mediated sensitization to treatment by TTFA.

Institute of Environmental Medicine Division of Toxicology Karolinska Institutet Box 210 Stockholm SE 171 77 Sweden

Institute of Environmental Medicine Division of Toxicology Karolinska Institutet Box 210 Stockholm SE 171 77 Sweden; Department of Cytokinetics Institute of Biophysics Academy of Sciences of the Czech Republic v v i Královopolská 135 612 65 Brno Czech Republic; Department of Animal Physiology and Immunology Institute of Experimental Biology Faculty of Science Masaryk University Terezy Novákové 64 621 00 Brno Czech Republic

Institute of Environmental Medicine Division of Toxicology Karolinska Institutet Box 210 Stockholm SE 171 77 Sweden; MV Lomonosov Moscow State University 119991 Moscow Russia

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