A Bak-dependent mitochondrial amplification step contributes to Smac mimetic/glucocorticoid-induced necroptosis

Abstract

Necroptosis is a form of programmed cell death that critically depends on RIP3 and MLKL. However, the contribution of mitochondria to necroptosis is still poorly understood. In the present study, we discovered that mitochondrial perturbations play a critical role in Smac mimetic/Dexamethasone (Dexa)-induced necroptosis independently of death receptor ligands. We demonstrate that the Smac mimetic BV6 and Dexa cooperate to trigger necroptotic cell death in acute lymphoblastic leukemia (ALL) cells that are deficient in caspase activation due to absent caspase-8 expression or pharmacological inhibition by the caspase inhibitor zVAD.fmk, since genetic silencing or pharmacological inhibition of RIP3 or MLKL significantly rescue BV6/Dexa-induced necroptosis. In addition, RIP3 or MLKL knockout mouse embryonic fibroblasts (MEFs) are protected from BV6/Dexa/zVAD.fmk-induced cell death. In contrast, antagonistic antibodies against the death receptor ligands TNFα, TRAIL or CD95 ligand fail to rescue BV6/Dexa-triggered cell death. Kinetic studies revealed that prior to cell death BV6/Dexa treatment causes hyperpolarization of the mitochondrial membrane potential (MMP) followed by loss of MMP, reactive oxygen species (ROS) production, Bak activation and disruption of mitochondrial respiration. Importantly, knockdown of Bak significantly reduces BV6/Dexa-induced loss of MMP and delays cell death, but not ROS production, whereas ROS scavengers attenuate Bak activation, indicating that ROS production occurs upstream of BV6/Dexa-mediated Bak activation. Consistently, BV6/Dexa treatment causes oxidative thiol modifications of Bak protein. Intriguingly, knockdown or knockout of RIP3 or MLKL protect ALL cells or MEFs from BV6/Dexa-induced ROS production, Bak activation, drop of MMP and disruption of mitochondrial respiration, demonstrating that these mitochondrial events depend on RIP3 and MLKL. Thus, mitochondria might serve as an amplification step in BV6/Dexa-induced necroptosis. These findings provide new insights into the role of mitochondrial dysfunctions during necroptosis and have important implications for the development of novel treatment approaches to overcome apoptosis resistance in ALL.

Sodium hydrosulfide mitigates dexamethasone‐induced osteoblast dysfunction by interfering with mitochondrial function

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Mitochondrial and mitochondrial‐independent pathways of myocardial cell death during ischaemia and reperfusion injury

“…For instance, MLKL, the ultimate effector of necroptosis, is unlikely to directly assemble at mitochondrial membranes, as shown for the much higher efficiency of the MLKL N‐terminal domain to permeabilize liposomes with a composition resembling the plasma membrane, in comparison with cardiolipin‐rich liposomes . Nonetheless, at early stages of SMAC‐mimetic induced necroptosis in various cancer cell types, a disruption of mitochondrial membrane potential is observed that presumably depends on BAK‐BAX activity . In accordance with this, the pro‐apoptotic protein PUMA could act as an amplifier of necroptosis by exposing mitochondrial DNA to the cytosolic sensors, which further stimulates the necrosome formation .…”

Estrogen receptor variant ER‐α36 promotes tamoxifen agonist activity in glioblastoma cells

“…Change of mitochondrial membrane potential is considered an early apoptosis marker . To determine the effect of TAM on cell viability, we examined mitochondrial membrane potential in glioma cells treated with 5 μmol/L TAM.…”

“…Change of mitochondrial membrane potential is considered an early apoptosis marker. 29 To determine the effect of TAM on cell viability, Figure 3A, TAM at 5 μmol/L failed to change the mitochondrial membrane potential in U87 cells. However, when U251 cells were treated with 5 μmol/L TAM, the cells showed decreased mitochondrial membrane potential compared to the control cells treated with vehicle.…”