Erastin is a strong inducer of ferroptosis in human rhabdomyosarcoma cell lines

Rhabdomyosarcoma (RMS) is the most common soft tissue tumor affecting childhood and is characterized by cells showing morphological features and biochemical traits of the myogenic lineage (1). RMS tumors include two major histotypes, namely embryonal and alveolar, which are mainly characterized by RAS activating mutations and expression of the chimeric Pax3-Foxo1 transcription factor, respectively. Here we show that treatment of the human embryonal RD and alveolar RH30 cell lines with erastin, a molecule targeting the cystine/glutamate antiporter termed system xc – involved in the synthesis of intracellular glutathione, led to a pronounced ferroptosis, a newly discovered oxidative, nonapoptotic form of cell death closely dependent on iron (2). Erastin treatment at low doses (0.5 and 1 M) was already sufficient for inhibiting cell proliferation and triggering cell death independent from caspase-3 cleavage, as observed during a time-course of 48 hours by means of Crystal Violet, Neutral Red and Immunoblotting assays. Co-treatment of the cell lines with erastin and increasing doses of ammonium iron citrate was able to increase the ferroptotic-cell death, while co-treatment with the iron chelator deferoxamine completely prevented the cell-death induced by erastin. In addition, actively proliferating human RMS cell lines treated with erastin showed a marked production of intracellular ROS species, as evaluated by means of fluorescent cytosolic, mitochondrial and nuclear probes. Remarkably, unlike observed during cell proliferation, the erastin treatment of differentiated RMS cells did not produce any considerable effect on the cell viability, suggesting that the effects of erastin are strictly cell-cycle-dependent. On the whole, these preliminary data suggest that targeting iron metabolism may represent an important avenue to counteract the tumor progression in RMS.