erastin and Rhabdomyosarcoma

Topics: alpha-Tocopherol; Apoptosis; Cell Death; Cell Line, Tumor; Cyclohexylamines; Ferroptosis; Glutathione; Humans; Lipid Peroxidation; Oxidation-Reduction; Oxidative Stress; Phenylenediamines; Piperazines; Pyrazoles; Pyrazolones; Pyridines; Pyridones; Reactive Oxygen Species; Rhabdomyosarcoma

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.. 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.. 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.. Erastin and RSL3 trigger ferroptosis in highly proliferating myogenic lines through a ERK pathway-dependent fashion.

Topics: Animals; Carbolines; Cell Death; Cell Line; Cell Line, Tumor; Cell Proliferation; Cyclohexylamines; Dactinomycin; Doxorubicin; Glutathione; Glutathione Peroxidase; Humans; Lipid Peroxidation; Mice; Myoblasts; Phenylenediamines; Piperazines; Rhabdomyosarcoma

Rhabdomyosarcoma (RMS) cells have recently been reported to be sensitive to oxidative stress. Therefore, we investigated whether concomitant inhibition of the two main antioxidant defense pathways, that is, the thioredoxin (TRX) and the glutathione (GSH) systems, presents a new strategy to trigger cell death in RMS. In this study, we discover that GSH-depleting agents, i.e. γ-glutamylcysteine synthetase inhibitor, buthionine sulfoximine (BSO) or the cystine/glutamate antiporter inhibitor erastin (ERA), synergize with thioredoxin reductase (TrxR) inhibitor auranofin (AUR) to induce cell death in RMS cells. Interestingly, AUR causes accumulation of ubiquitinated proteins when combined with BSO or ERA, in line with recent reports showing that AUR inhibits the proteasome besides TrxR. Consistently, AUR/BSO or AUR/ERA cotreatment increases ubiquitination and expression of the short-lived proteins NOXA and MCL-1, accompanied by increased binding of NOXA to MCL-1. Notably, NOXA knockdown significantly rescues RMS cells from AUR/BSO- or AUR/ERA-induced cell death. In addition, AUR acts together with BSO or ERA to stimulate BAX/BAK and caspase activation. Of note, BSO or ERA abolish the AUR-stimulated increase in GSH levels, leading to reduced GSH levels upon cotreatment. Although AUR/BSO or AUR/ERA cotreatment enhances reactive oxygen species (ROS) production, only thiol-containing antioxidants (i.e., N-acetylcysteine (NAC), GSH), but not the non-thiol-containing ROS scavenger α-Tocopherol consistently suppress AUR/BSO- and AUR/ERA-stimulated cell death in both cell lines. Importantly, re-supply of GSH or its precursor NAC completely prevents AUR/ERA- and AUR/BSO-induced accumulation of ubiquitinated proteins, NOXA upregulation and cell death, indicating that GSH depletion rather than ROS production is critical for AUR/BSO- or AUR/ERA-mediated cell death. Thus, by demonstrating that GSH-depleting agents enhance the antitumor activity of AUR, we highlight new treatment options for RMS by targeting the redox homeostasis.

Topics: Antioxidants; Apoptosis; Auranofin; Buthionine Sulfoximine; Cell Line, Tumor; gamma-Glutamylcyclotransferase; Glutathione; Humans; Oxidation-Reduction; Oxidative Stress; Piperazines; Reactive Oxygen Species; Rhabdomyosarcoma; Thioredoxin-Disulfide Reductase; Thioredoxins