Abstract
We have reported that Tempo, a nitroxide that interacts with mitochondrial ETC complexes and produces ROS, sensitizes p53-defective human leukemic U937 cells to heat-induced cell death (Zhao et al, FRBM, 2006). Here we describe that in U937 cells, increasing hyperthermic intensity and Tempo concentration elicit two distinct modes of synergistic cell death: caspase-dependent apoptosis (CDA) and caspase-independent cell death (CICD).
We treated U937 cells with 5-10 mM Tempo and/or 10-30 min exposure to 44C, and analyzed the cell death mechanisms. The results were as follows. (1) CDA: The 5 mM Tempo/44C-10 min combination induced the synergistic CDA to the similar extent as did the 44C-30 min heating. The common CDA mechanisms involved (i) activation of the initiator caspase-8-tBid signaling, (ii) activation of the initiator caspase-2, and (iii) Bax-induced cytochrome c release, followed by activation of the initiator caspase-9 and effector caspase-3/7. Both treatments also resulted in mitochondrial dysfunction, such as ROS increase, pan-caspase inhibitor zVAD-suppressible membrane-potential loss due to increased mitochondrial Ca2+ levels, and decreased ATP synthesis. (2) CICD: Notably, a stronger combination of 10 mM Tempo/44C-30 min switched CDA to the particular CICD without obvious sign of autophagy, leading to irreversible cell proliferation. This CICD was characterized by multiple molecular marks, including (i) Bnip upregulation in mitochondria, related in part to (ii) irreversible mitochondrial dysfunction, (iii) failure in processing conformationally activated, full-length procaspases to active caspases, and (iv) inhibition of Bid cleavage to tBid, despite the heat-induced Bax activation and cytochrome c release.
