Host: The Japan Radiation Research Society
We investigated the mechanisms for nitroxide Tempo to potentiate heat stress-induced apoptosis in human leukemic U937 cells. We treated the cells with either single or combined regiments of 5-10 mM Tempo, 44C/10 min or 44C/30 min hyperthermia and assessed apoptosis induction. The 5 mM Tempo-44C/10 min combination induced ∼80% apoptosis as did 44C/30 min heat stress, while 5 mM Tempo or 44C/10 min heating alone caused little apoptosis. The mechanistic basis for such a synergistic apoptotic enhancement involved (1) the activation of Bax and its translocation to mitochondria, leading to cytochrome c release and activation of the caspase-9 to -3 cascade, (2) activation of initiator caspases-8 and -2 as revealed by the biotin-zVAD-fmk pulldown experiment, and (3) zVAD-fmk-suppressible loss of mitochondrial membrane potential, which led to mitochondrial dysfunction. A stronger combination of 10 mM Tempo and 44C/30 min heating induced the caspase-3-independent cell death (CICD), involving zVAD-resistant loss of the potential (irreversible mitochondrial dysfunction) due partly to increases in ROS and mitochondria-associated Bnip3. Furthermore, transcriptional up regulation of inducible HSP70 and HSP27 by geldanamycin suppressed completely the 44C/30 min heat stress-induced apoptosis. The results together delineated the important mechanisms of hyperthermia-induced apoptosis and Tempo-mediated enhancement.
