Targeting ferroptosis in rhabdomyosarcoma cells.

Recent data suggest that rhabdomyosarcoma (RMS) cells might be vulnerable to oxidative stress-induced cell death. Here, we show that RMS are susceptible to cell death induced by Erastin, an inhibitor of the glutamate/cystine antiporter xc- that can increase reactive oxygen species (ROS) production via glutathione (GSH) depletion. Prior to cell death, Erastin caused GSH depletion, ROS production and lipid peroxidation. Importantly, pharmacological inhibitors of lipid peroxidation (i.e., Ferrostatin-1, Liproxstatin-1), ROS scavengers (i.e., α-Tocopherol, GSH) and the iron chelator Deferoxamine inhibited ROS accumulation, lipid peroxidation and cell death, consistent with ferroptosis. Interestingly, the broad-spectrum protein kinase C (PKC) inhibitor Bisindolylmaleimide I as well as the PKCα- and ß-selective inhibitor Gö6976 significantly reduced Erastin-induced cell death. Similarly, genetic knockdown of PKCα significantly protected RMS cells from Erastin-induced cell death. Furthermore, the broad-spectrum nicotinamide adenine dinucleotide phosphate-oxidase (NOX) inhibitor Diphenyleneiodonium and the selective NOX1/4 isoform inhibitor GKT137831 significantly decreased Erastin-stimulated ROS, lipid ROS and cell death. These data provide new insights into the molecular mechanisms of ferroptosis in RMS, contributing to the development of new redox-based treatment strategies.

Lipoxygenase inhibitors protect acute lymphoblastic leukemia cells from ferroptotic cell death.

Ferroptosis has recently been identified as a mode of programmed cell death. However, little is yet known about the signaling mechanism. Here, we report that lipoxygenases (LOX) contribute to the regulation of RSL3-induced ferroptosis in acute lymphoblastic leukemia (ALL) cells. We show that the glutathione (GSH) peroxidase 4 (GPX4) inhibitor RSL3 triggers lipid peroxidation, production of reactive oxygen species (ROS) and cell death in ALL cells. All these events are impeded in the presence of Ferrostatin-1 (Fer-1), a small-molecule inhibitor of lipid peroxidation. Also, lipid peroxidation and ROS production precede the induction of cell death, underscoring their contribution to cell death upon exposure to RSL3. Importantly, LOX inhibitors, including the selective 12/15-LOX inhibitor Baicalein and the pan-LOX inhibitor nordihydroguaiaretic acid (NDGA), protect ALL cells from RSL3-stimulated lipid peroxidation, ROS generation and cell death, indicating that LOX contribute to ferroptosis. RSL3 triggers lipid peroxidation and cell death also in FAS-associated Death Domain (FADD)-deficient cells which are resistant to death receptor-induced apoptosis indicating that the induction of ferroptosis may bypass apoptosis resistance. By providing new insights into the molecular regulation of ferroptosis, our study contributes to the development of novel treatment strategies to reactivate programmed cell death in ALL.

RSL3 and Erastin differentially regulate redox signaling to promote Smac mimetic-induced cell death.

Redox mechanisms play an important role in the control of various signaling pathways. Here, we report that Second mitochondrial activator of caspases (Smac) mimetic-induced cell death is regulated by redox signaling. We show that RSL3, a glutathione (GSH) peroxidase (GPX) 4 inhibitor, or Erastin, an inhibitor of the cystine/glutamate antiporter, cooperate with the Smac mimetic BV6 to induce reactive oxygen species (ROS)-dependent cell death in acute lymphoblastic leukemia (ALL) cells. Addition of the caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD.fmk) fails to rescue ROS-induced cell death, demonstrating that RSL3/BV6- or Erastin/BV6-induced cell death occurs in a caspase-independent manner. Interestingly, the iron chelator Deferoxamine (DFO) significantly inhibits RSL3/BV6-induced cell death, whereas it is unable to rescue cell death by Erastin/BV6, showing that RSL3/BV6-, but not Erastin/BV6-mediated cell death depends on iron. ROS production is required for both RSL3/BV6- and Erastin/BV6-induced cell death, since the ROS scavenger α-tocopherol (α-Toc) rescues RSL3/BV6- and Erastin/BV6-induced cell death. By comparison, genetic or pharmacological inhibition of lipid peroxidation by GPX4 overexpression or ferrostatin (Fer)-1 significantly decreases RSL3/BV6-, but not Erastin/BV6-induced cell death, despite inhibition of lipid peroxidation upon exposure to RSL3/BV6 or 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.