Mean and SD of three experiments performed in triplicate are shown; *< 0.05; **< 0.01. -Toc rescues both RSL3/BV6- and Erastin/BV6-induced ROS Capsazepine production, whereas Fer-1 prevents RSL3/BV6- but not Erastin/BV6-stimulated ROS generation Our results obtained so far demonstrate that inhibition of ROS accumulation by -Toc rescues both RSL3/BV6- and Erastin/BV6-induced cell death, while genetic or pharmacological inhibition of lipid peroxidation protect cells from RSL3/BV6- but not Erastin/BV6-stimulated cell death. Erastin/BV6. Of note, inhibition of lipid peroxidation by Fer-1 protects from RSL3/BV6-, but not from Erastin/BV6-stimulated ROS production, indicating that other forms of ROS besides lipophilic ROS occur during Erastin/BV6-induced cell death. Taken together, RSL3/BV6 and Erastin/BV6 differentially regulate redox signaling and cell death in ALL cells. While RSL3/BV6 cotreatment induces ferroptotic cell death, Erastin/BV6 stimulates oxidative cell death independently of iron. These findings have important implications for the therapeutic targeting of redox signaling to enhance Smac mimetic-induced cell death in ALL. < 0.05; **< 0.01; ***< 0.001. To explore the underlying mechanisms of the observed cooperative induction of cell death by the cotreatment with BV6 and RSL3 or Erastin, we decided cellular GSH levels. Erastin alone and in combination with BV6 caused a significant reduction of GSH levels, consistent with the described mode of action of Erastin as an inhibitor of the cystine/glutamate antiporter. In contrast, GSH levels remained largely unaffected upon treatment with BV6 [16] or RSL3 (Physique ?(Physique1B),1B), in line with the reported function of RSL3 as GPX4 inhibitor that triggers ferroptosis without changes in GSH levels [12]. Since inhibition of GPX4 or depletion of cellular GSH levels facilitate the accumulation of ROS [3], we hypothesized that RSL3 or Erastin act together with BV6 to cause alterations in ROS levels. To clarify this hypothesis, we analyzed cellular ROS levels in ALL cells before they succumb to cell death using Capsazepine the fluorescent ROS-sensitive dye CellROX. Importantly, both RSL3 and Erastin cooperated with BV6 to significantly increase ROS levels compared to either treatment alone (Physique ?(Physique1C1C). GPX4 inactivation has been shown to cause accumulation of lipid peroxides [12], and ROS accumulation in the vicinity of biomembranes has been described to favor lipid peroxidation [4]. Therefore, we next analyzed lipid peroxidation by staining viable (i.e. propidium iodide (PI)-unfavorable) cells with the membrane-targeted lipid ROS sensor BODIPY-C11, a fluorescent dye that detects lipid peroxides [17]. Of note, RSL3 and Erastin acted together with BV6 to significantly enhance lipid peroxidation compared to treatment with either agent alone (Physique ?(Figure1D1D). Collectively, these findings demonstrate that BV6 cooperates with RSL3 or Erastin to induce cell death, which is accompanied by accumulation of ROS and lipid peroxidation. RSL3/BV6 or Erastin/BV6 cotreatment triggers caspase-independent lipid peroxidation-induced cell death Next, we explored the involvement of caspases by monitoring caspase-3/7 activity. While cotreatment with RSL3/BV6 did not alter caspase-3/7 activity compared to untreated control cells, Erastin/BV6 caused a significant increase in caspase-3/7 activity (Physique ?(Figure2A).2A). To explore whether caspase activation is necessary for the induction of cell death, we used the pan-caspase inhibitor zVAD.fmk. Of note, the addition of zVAD.fmk failed to protect ALL cells from RSL3/BV6- or Erastin/BV6- stimulated cell death (Physique ?(Figure2B).2B). Control experiments showed that zVAD.fmk prevented Erastin/BV6-stimulated caspase-3/7 activation as well as low constitutive caspase-3/7 activity (Physique ?(Figure2A),2A), confirming that zVAD.fmk indeed blocks caspase activity Capsazepine in this setting. In addition, zVAD.fmk was unable to prevent RSL3/BV6- or Erastin/BV6-induced lipid peroxidation (Physique ?(Figure2C).2C). These findings demonstrate that RSL3/BV6 or Erastin/BV6 induce cell death in a caspase-independent lipid peroxidation manner. Open in a separate window Physique 2 RSL3/BV6- or Erastin/BV6- cotreatment triggers caspase-independent lipid peroxidation-induced cell death(ACC) ALL cells were treated with BV6 (Jurkat: 5 M, Rabbit polyclonal to PABPC3 Molt-4: 4 M), RSL3 (Jurkat: 0.1 M; Molt-4: RSL3 0.075 M) and/or Erastin (Era) (Jurkat: 5 M; Molt-4: 7.5 M) in the presence or absence of 20 M zVAD.fmk, which was added 2 hours before treatment. Caspase-3/7 activity was decided after 48 hours by Cell Event Caspase-3/7 Green Detection Reagent and ImageXpress Micro XLS system (A). Cell death was decided after 12 hours (Jurkat cells: Erastin/BV6) or 24 hours (Jurkat cells: RSL3/BV6, Molt-4 cells: RSL3/BV6, Erastin/BV6) by FSC/SSC analysis and flow cytometry (B). Lipid peroxidation was assessed after 18 hours (Erastin/BV6) or 24 hours (RSL3/BV6) by flow cytometry in PI-negative cells using the fluorescent dye BODIPY-C11 and is shown as fold increase compared to untreated cells (C). Mean and SD of at least three experiments performed in triplicate are shown; *< 0.05; **< 0.01; ***< 0.001. RSL3/BV6 but not Erastin/BV6 cotreatment triggers iron-dependent cell death To explore the involvement of ferroptotic cell death, which is characterized by its dependency on iron, we tested the effect of the iron chelator DFO. Importantly, DFO significantly reduced.