Mechanism of cell death induction by nitroxide and hyperthermia

Heat stress and nitroxides induce reactive oxygen species (ROS) and proapoptotic effects. The underlying mechanisms remain largely elusive. Here we report that Tempo (2,2,6,6-tetramethylpiperidine-N-oxyl) is a potent thermosensitizer for promoting cell death in human leukemia U937 cells. Treatment with Tempo (10 mM, 37°C/30 min) and hyperthermia (44°C/30 min) induced 30 and 70–80% apoptosis, respectively, through Bax-mediated cytochrome c release and DEVDase activation. The Tempo/heat combination also caused Bax-mediated cytochrome c release, but switched heat-induced apoptosis to the particular pyknotic cell death, resulting in the irreparable inhibition of proliferation. Tempo and heat stress, but not the combination, caused an early transient elevation of H2O2/O2− and a late induction of only O2−, respectively. Mitochondrial Ca2+ overloads were indistinguishable after any treatment. Heat stress induced the pan-caspase inhibitor zVAD-fmk-suppressible low-Δψ (mitochondrial membrane potential) in 75% of cells as a result of DEVDase activation. In contrast, Tempo yielded low-Δψ by deprivation of the mitochondrial H+ gradient. The combined treatment induced 97% zVAD-resistant low-Δψ cells through irreversible mitochondrial dysfunction. Together, thus, Tempo or heat stress induced Bax-mediated mitochondrial apoptosis with the possible help of ROS or mitochondrial Ca2+, and Tempo when combined with hyperthermia acts a sensitizer by inducing irreparable pyknotic cell death through irreversible mitochondrial dysfunction.