Inhibition of glutathione peroxidase 4 primes mouse C2C12 myoblasts and rhabdomyosarcoma cell lines to ferroptosis

Ferroptosis is a recently discovered form of cell death causally linked to the ability of iron to induce oxidative damage by peroxidation of polyunsaturated fatty acids (PUFAs). Misregulated ferroptosis has been implicated in a number of pathological processes and there is a growing interest in the pre-clinical use of ferroptosis inducers against tumors. Cells to prevent ferroptosis mostly engage in the activity of glutathione peroxidase 4 (GPx4), a selenoenzyme that uses glutathione for neutralizing lipid hydroperoxides. Two major ferroptosis inducers mediating GPx4 inhibition have been identified, namely Erastin (eradicator of RAS and ST-expressing cells) and RSL3 (RAS selective Lethal 3). In this work we have investigated their effect on mouse skeletal C2C12 myoblasts and cell lines of rhabdomyosarcoma (RMS), the most frequent soft-tissue tumor affecting children and adolescents. As evaluated by using specific fluorescent probes, treatment with Erastin or RSL3 agents resulted in a marked production of both cytoplasmic/mitochondrial ROS and lipid ROS, which correlated in a dose-dependent manner with a decreased cell viability, as evaluated by means of Neutral Red assays after 48 hours. In Erastin-treated cell lines ferroptosis was enhanced in the presence of iron supplementation (through ferric ammonium citrate), while it was prevented by pre-treatment with agents sequestering iron (bathophenanthrolinedisulfonic acid), antioxidant scavengers (glutathione and N-acetylcysteine) and lipid ROS scavengers (ferrostatin-1). We observed Erastin to be more effective to promote ferroptosis in the cell lines showing a higher proliferation rate. Indeed, inhibition of ERK signaling, as observed during differentiation or upon pharmacological treatment with PD090859 agent, prevented ferroptosis in Erastin-treated human RMS embryonal RD and C2C12 cell lines. Furthermore, we found Erastin and RSL3 to be more effective in inducing ferroptosis in RD subclones characterized by higher ERK1/2 phosphorylation and proliferation rate. Taken together, our data suggest that iron metabolism could play a key role in the cell fate of muscle cells; in addition, the use of ferroptotic inducers could offer a novel alternative to improve the efficacy of conventional antineoplastic cocktails utilized against RMS.