Synergistic induction of apoptosis and caspase-independent autophagic cell death by a combination of nitroxide Tempo and heat shock in human leukemia U937 cells

We have shown that heat stress or a superoxidedismutase mimic nitroxide, Tempo, induces apoptosis,while their combination causes nonapoptotic cell death;however, the underlying mechanism for this switchremains unclear. Here we identified for the first time that10 mM Tempo present during heating at 44 °C for 30 minrapidly induced autophagy in U937 leukemic cells in spiteof Bax activation and mitochondrial outer membrane(MOM) permeabilization. This co-treatment inhibited theprocessing of heat-activated procaspases-2, -8, -9 and -3into active small subunits, leading to the inhibition ofcaspase-dependent apoptosis, and instead caused theinduction of autophagy. The inactivation of caspases, a keyevent, could result from oxidation of active-site-CysSH ofall caspases by a prooxidant oxo-ammonium cation, anintermediate derived Tempo during dismutation of heatinducedsuperoxide anion. In addition, the co-treatmentcaused mitochondrial calcium overloads, the mitochondrialinner membrane permeabilization, profound mitochondrialdysfunction, and liberation of Beclin 1 from the Bcl-2/Beclin 1 complex, all of which contributed to induction ofautophagy. These autophagic cells underwent propidiumiodide-positive necrosis in a delayed fashion, leading to thecomplete proliferative inhibition. Remarkably, rutheniumred and BAPTA, which interfere with mitochondrial calciumuptake, facilitated autophagic necrotic death. CyclosporinA, which binds to cyclophilin D, had a similarnecrotic effect. 3-Methyladenine facilitated the necrosis ofautophagic cells. In contrast, 5 mM Tempo-44 °C/10 minor 44 °C/30 min induced Bax-mediated MOM permeabilizationand caspase-dependent apoptosis more potently thanTempo alone. Thus, Tempo is a unique thermosensitizer tosynergistically induce apoptosis and autophagic cell death