Open Access

Jasmin Dächert

• Reactive oxygen species (ROS) are involved in various signalling mechanisms. Redox homeostasis is important in cancer cells, since they are dependent on upregulated antioxidant defence pathways to cope with elevated ROS levels. Therefore, targeting the antioxidant defence system and/ or increasing ROS to a lethal level may be a feasible strategy to counteract cancer cell progression. Acute lymphoblastic leukaemia (ALL) is the most frequent malignant childhood cancer, displaying on one side resistance to cell death induction and on the other side elevated ROS levels. Therefore, inducing ferroptosis, a ROS- and iron-dependent cell death pathway might be useful to trigger cell death in ALL as a novel treatment strategy. In the first study of this thesis we observed that RSL3, a glutathione (GSH) peroxidase 4 (GPX4) inhibitor, triggered ROS accumulation and lipid peroxidation which contributed to ferroptotic cell death. These observations were based on suppression of RSL3 stimulated cell death using different ferroptosis inhibitors like Ferrostatin-1 (Fer-1), Liproxstatin-1 (Lip-1), as well as iron chelator Deferoxamine (DFO) and the vitamin E derivate α-Tocopherol (α-Toc). RSL3-triggered ROS and lipid peroxide production were also inhibited through Fer-1 and α-Toc. Furthermore, lipoxygenases (LOX) were activated upon RSL3 stimulation and contributed to ferroptotic cell death in ALL as well. Selective inhibition of LOX with the 12/15-LOX inhibitor Baicalein and the pan-LOX inhibitor nordihydroguaiaretic acid (NDGA) abolished RSL3-induced ROS production, lipid peroxidation and cell death. In addition, RSL3 induced lipid peroxide-dependent ferroptotic cell death in FAS-associated Death Domain (FADD)-deficient, death receptor-induced apoptosis resistant cells, demonstrating that ferroptosis might circumvent apoptosis resistance. The second part of the study revealed that RSL3 and Erastin (Era), a GSH-depleting agent, inhibiting the cystine/glutamate antiporter system xc- and ferroptosis inducer, cooperated with the Smac mimetic BV6 to trigger cell death in ALL cells. RSL3/BV6 and Era/BV6 combination-induced cell death was dependent on ROS accumulation, but independent of caspases and key modulators of necroptosis. RSL3/BV6-treated ALL cells exhibited classical features of ferroptotic cell death with iron-dependency, ROS accumulation and lipid peroxidation which was diminished through either pharmacological inhibition (Fer-1, DFO, α-Toc) or genetic inhibition by overexpressing GPX4. Interestingly, Era/BV6-induced cell death in ALL cells was independent of iron but dependent on ROS accumulation, since α-Toc rescued from Era/BV6-triggered ROS production, lipid peroxidation and cell death. Moreover, inhibition of lipid peroxide formation through the addition of Fer-1 or by overexpressing GPX4 failed to rescue from Era/BV6-triggered cell death, even if Era/BV6-stimulated lipid peroxidation was diminished. Likewise, Fer-1 protected from RSL3/BV6-, but not from Era/BV6-generated ROS production, leading to the assumption that other ROS besides lipid-based ROS contributed to cell death in Era/BV6-treated cells. In summary, while RSL3/BV6 induced ferroptosis in ALL, Era/BV6 stimulated a ROS dependent cell death, which was neither dependent on iron nor caspases or receptor-interacting protein (RIP) kinase 1 nor 3. Additionally, using Erastin alone did not trigger ferroptotic cell death in ALL. Finally, with these two studies we tried to unravel the molecular pathway of ferroptosis by using RSL3 and Erastin as well described ferroptosis stimulators. Here, we demonstrate the possibility of a novel treatment strategy to reactivate programmed cell death by impeding redox homeostasis in ALL. Since ALL failed to induce ferroptosis upon Erastin treatment, we investigated in the third part of this thesis a new model system to induce ferroptosis upon Erastin and RSL3 exposure. Previous studies revealed that rhabdomyosarcoma (RMS) cells might be susceptible to oxidative stress-induced compounds. To this end, we used Erastin as a prototypic ferroptosis stimulus and GSH-depleting agent and demonstrated that GSH depletion, ROS and lipid ROS accumulation contributed to cell death. Additionally, Fer-1, Lip-1, DFO, lipophilic vitamin E derivate α-Toc and GSH, a cofactor of GPX4, protected from Erastin stimulated ROS accumulation, lipid peroxidation and cell death. Also, the use of a broad spectrum protein kinase C (PKC) inhibitor Bisindolylmaleimide I (Bim1), a PKCα and ß selective inhibitor Gö6976 and siRNA-mediated knockdown of PKCα suppressed Erastin-mediated cell death in RMS. Moreover broad spectrum nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase (NOX) inhibitor Diphenyleneiodonium (DPI) and a more selective NOX1/4 isoform inhibitor GKT137831 abrogated Erastin-generated ROS formation, lipid peroxidation and cell death. With this, we demonstrate that RMS are vulnerable to ferroptotic cell death and investigated the molecular mechanism of ferroptosis by unravelling that PKC and NOX could have a pivotal role in ROS-mediated ferroptosis signalling in RMS. In this regard, ferroptosis inducers may act as a possible novel treatment strategy for RMS, especially those with poor clinical outcome.