Cell growth potential drives ferroptosis susceptibility in rhabdomyosarcoma and myoblast cell lines

Purpose
Ferroptosis is a programmed form of iron-dependent cell death caused by lipid hydroperoxide accumulation, which can
be prevented by glutathione peroxidase 4 (GPx4) activity. Here we investigated the effects of ferroptosis inducers called
erastin and RSL3, which act by glutathione depletion and GPx4 inactivation, respectively, on muscle-derived cell lines
of embryonal and alveolar rhabdomyosarcoma (RMS), and mouse normal skeletal C2C12 myoblasts.
Methods
Myogenic lines were exposed to stepwise increasing concentrations of ferroptosis inducers either alone or in
combination with iron supplementation, iron chelating agents (Bathophenanthrolinedisulfonic acid, BPS), antioxidant
molecules (glutathione, N-acetylcysteine), lipid peroxidation inhibitors (Ferrostatin-1), and chemotherapeutic agents
(doxorubicin and actinomycin D). Drug susceptibility was quantified by measuring cell viability, proliferation and
differentiation via neutral red assay, crystal violet assay and Giemsa staining, respectively. The detection of lipid
hydroperoxide and protein levels was performed by immunofluorescence and Western blot analysis, respectively.
Results
Erastin and RSL3 increased lipid hydroperoxide levels preferentially in the embryonal U57810 and myoblast C2C12
lines, leading to ferroptosis that was accentuated by iron supplementation or prevented by co-treatment with BPS,
glutathione, N-acetylcysteine and Ferrostatin-1. The inhibition of extracellular regulated kinases (ERK) pathway
prevented ferroptosis in U57810 and C2C12 cells, whereas its increased activation in the embryonal RD cells mediated
by Caveolin-1 (Cav-1) overexpression led to augmented ferroptosis susceptibility. Finally, we observed the combination
of erastin or RSL3 with chemotherapeutic doxorubicin and actinomycin D agents to be effective in increasing cell death
in all RMS lines.
Conclusions
Erastin and RSL3 prime to ferroptosis rapidly dividing myogenic lines through a ERK pathway-dependent fashion.