Statin-Sensitive Akt1/Src/Caveolin-1 Signaling Enhances Oxidative Stress Resistance in Rhabdomyosarcoma.

Identifying the molecular mechanisms underlying radioresistance is a priority for the treatment of RMS, a myogenic tumor accounting for approximately 50% of all pediatric soft tissue sarcomas. We found that irradiation (IR) transiently increased phosphorylation of Akt1, Src, and Cav1 in human RD and RH30 lines. Synthetic inhibition of Akt1 and Src phosphorylation increased ROS levels in all RMS lines, promoting cellular radiosensitization. Accordingly, the elevated activation of the Akt1/Src/Cav1 pathway, as detected in two RD lines characterized by overexpression of a myristoylated Akt1 form (myrAkt1) or Cav1 (RDCav1), was correlated with reduced levels of ROS, higher expression of catalase, and increased radioresistance. We found that treatment with cholesterol-lowering drugs such as lovastatin and simvastatin promoted cell apoptosis in all RMS lines by reducing Akt1 and Cav1 levels and increasing intracellular ROS levels. Combining statins with IR significantly increased DNA damage and cell apoptosis as assessed by γ histone 2AX (γH2AX) staining and FACS analysis. Furthermore, in combination with the chemotherapeutic agent actinomycin D, statins were effective in reducing cell survival through increased apoptosis. Taken together, our findings suggest that the molecularly linked signature formed by Akt1, Src, Cav1, and catalase may represent a prognostic determinant for identifying subgroups of RMS patients with higher probability of recurrence after radiotherapy. Furthermore, statin-induced oxidative stress could represent a treatment option to improve the success of radiotherapy.

Iron supplementation enhances RSL3-induced ferroptosis to treat naïve and prevent castration-resistant prostate cancer.

Prostate cancer (PCa) is a leading cause of death in the male population commonly treated with androgen deprivation therapy that often relapses as androgen-independent and aggressive castration-resistant prostate cancer (CRPC). Ferroptosis is a recently described form of cell death that requires abundant cytosolic labile iron to promote membrane lipid peroxidation and which can be induced by agents that inhibit the glutathione peroxidase-4 activity such as RSL3. Exploiting in vitro and in vivo human and murine PCa models and the multistage transgenic TRAMP model of PCa we show that RSL3 induces ferroptosis in PCa cells and demonstrate for the first time that iron supplementation significantly increases the effect of RSL3 triggering lipid peroxidation, enhanced intracellular stress and leading to cancer cell death. Moreover, the combination with the second generation anti-androgen drug enzalutamide potentiates the effect of the RSL3 + iron combination leading to superior inhibition of PCa and preventing the onset of CRPC in the TRAMP mouse model. These data open new perspectives in the use of pro-ferroptotic approaches alone or in combination with enzalutamide for the treatment of PCa.

The modulation of iron metabolism affects the Rhabdomyosarcoma tumor growth in vitro and in vivo.

Rhabdomyosarcoma (RMS) is an aggressive rare neoplasm that derives from mesenchymal cells, which frequently develops resistance to the current therapies and the formation of metastases. Thus, new therapies are needed. The alteration of iron metabolism in cancer cells was effective in reducing the progression of many tumors but not yet investigated in RMS. Here we investigated the effect of iron modulation in RMS both in vitro and in vivo. We first characterized the most used RMS cell lines representing the most common subtypes, embryonal (ERMS, RD cells) and alveolar (ARMS, RH30 cells), for their iron metabolism, in basal condition and in response to its modulation. Then we investigated the effects of both iron overload and chelation strategies in vitro and in vivo. RMS cell lines expressed iron-related proteins, even if at lower levels compared to hepatic cell lines and they are correctly modulated in response to iron increase and deprivation. Interestingly, the treatment with different doses of ferric ammonium citrate (FAC, as iron source) and with deferiprone (DFP, as iron chelator), significantly affected the cell viability of RD and RH30. Moreover, iron supplementation (in the form of iron dextran) or iron chelation (in the form of DFP) were also effective in vivo in inhibiting the tumor mass growth both derived from RD and RH30 with iron chelation treatment the most effective one. All the data suggest that the iron modulation could be a promising approach to overcome the RMS tumor growth. The mechanism of action seems to involve the apoptotic cell death for both iron supplementation and chelation with the concomitant induction of ferroptosis in the case of iron supplementation.

Hyperactive Akt1 Signaling Increases Tumor Progression and DNA Repair in Embryonal Rhabdomyosarcoma RD Line and Confers Susceptibility to Glycolysis and Mevalonate Pathway Inhibitors.

In pediatric rhabdomyosarcoma (RMS), elevated Akt signaling is associated with increased malignancy. Here, we report that expression of a constitutively active, myristoylated form of Akt1 (myrAkt1) in human RMS RD cells led to hyperactivation of the mammalian target of rapamycin (mTOR)/70-kDa ribosomal protein S6 kinase (p70S6K) pathway, resulting in the loss of both MyoD and myogenic capacity, and an increase of Ki67 expression due to high cell mitosis. MyrAkt1 signaling increased migratory and invasive cell traits, as detected by wound healing, zymography, and xenograft zebrafish assays, and promoted repair of DNA damage after radiotherapy and doxorubicin treatments, as revealed by nuclear detection of phosphorylated H2A histone family member X (γH2AX) through activation of DNA-dependent protein kinase (DNA-PK). Treatment with synthetic inhibitors of phosphatidylinositol-3-kinase (PI3K) and Akt was sufficient to completely revert the aggressive cell phenotype, while the mTOR inhibitor rapamycin failed to block cell dissemination. Furthermore, we found that pronounced Akt1 signaling increased the susceptibility to cell apoptosis after treatments with 2-deoxy-D-glucose (2-DG) and lovastatin, enzymatic inhibitors of hexokinase, and 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGCR), especially in combination with radiotherapy and doxorubicin. In conclusion, these data suggest that restriction of glucose metabolism and the mevalonate pathway, in combination with standard therapy, may increase therapy success in RMS tumors characterized by a dysregulated Akt signaling.

Iron distribution in different tissues of homozygous Mask (msk/msk) mice and the effects of oral iron treatments.

Iron-refractory iron deficiency anemia (IRIDA) is an autosomal recessive disorder caused by genetic mutations on TMPRSS6 gene which encodes Matriptase2 (MT2). An altered MT2 cannot appropriately suppress hepatic BMP6/SMAD signaling in case of low iron, hence hepcidin excess blocks dietary iron absorption, leading to a form of anemia resistant to oral iron supplementation. In this study, using the IRIDA mouse model Mask, we characterized homozygous (msk/msk) compared to asymptomatic heterozygous (msk/wt) mice, assessing the major parameters of iron status in different organs, at different ages in both sexes. The effect of carbonyl iron diet was analyzed as control iron supplementation being used for many studies in mice. It resulted effective in both anemic control and msk/msk mice, as expected, even if there is no information about its mechanism of absorption. Then, we mainly compared two forms of oral iron supplement, largely used for humans: ferrous sulfate and Sucrosomial iron. In anemic control mice, the two oral formulations corrected hemoglobin levels from 11.40 ± 0.60 to 15.38 ± 1.71 g/dl in 2-4 weeks. Interestingly, in msk/msk mice, ferrous sulfate did not increase hemoglobin likely due to ferroportin/hepcidin-dependent absorption, whereas Sucrosomial iron increased it from 11.50 ± 0.60 to 13.53 ± 0.64 g/dl mainly in the first week followed by a minor increase at 4 weeks with a stable level of 13.30 ± 0.80 g/dl, probably because of alternative absorption. Thus, Sucrosomial iron, already used in other conditions of iron deficiency, may represent a promising option for oral iron supplementation in IRIDA patients.

H-ferritin suppression and pronounced mitochondrial respiration make Hepatocellular Carcinoma cells sensitive to RSL3-induced ferroptosis.

Ferroptosis is a form of regulated cell death dependent on iron, reactive oxygen species and characterized by the accumulation of lipid peroxides. It can be experimentally initiated by chemicals, such as erastin and RSL3, that modulate GPX4 activity, the cellular antioxidant machinery that avert lipid peroxidation. The study aimed to investigate mitochondrial respiration and ferritin function as biomarkers of ferroptosis sensitivity of HepG2 and HA22T/VGH, two Hepatocellular Carcinoma (HCC) cell line models. Cell viability was determined by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assay, labile iron levels were determined using Calcein-AM fluorescence microscopy, ferritin, glutathione and lipid peroxidation were assayed with commercially available kits. The Seahorse assay was used to investigate mitochondrial function in the cells. The study shows that highly differentiated HepG2 cells were more sensitive to RSL3-induced ferroptosis than the poorly differentiated HA22T/VGH (HCC) cell line (RSL3 IC50 0.07 µM in HepG2 vs 0.3 µM in HA22T/VGH). Interestingly, HepG2 exhibited higher mitochondrial respiration and lower glycolytic activity than HA22T/VGH and were more sensitive to RSL3-induced ferroptosis, indicating a mitochondrial-specific mechanism of action of RSL3. Interestingly, iron metabolism seems to be involved in this different sensitivity, specifically, the downregulation of H-ferritin (but not of L-subunit), makes HA22T/VGH more sensitive toward both RSL3-and iron-induced ferroptosis. Hence only the H-ferritin seems involved in the protection from this cell death process.

Caveolin-1 promotes radioresistance in rhabdomyosarcoma through increased oxidative stress protection and DNA repair.

The aim of this work was to investigate whether Caveolin-1 (Cav-1), a membrane scaffolding protein widely implicated in cancer, may play a role in radiation response in rhabdomyosarcoma (RMS), a pediatric soft tissue tumor. For this purpose, we employed human RD cells in which Cav-1 expression was stably increased via gene transfection. After radiation treatment, we observed that Cav-1 limited cell cycle arrest in the G2/M phase and enhanced resistance to cell senescence and apoptosis via reduction of p21Cip1/Waf1, p16INK4a and Caspase-3 cleavage. After radiotherapy, Cav-1-mediated cell radioresistance was characterized by low accumulation of H2AX foci, as confirmed by Comet assay, marked neutralization of reactive oxygen species (ROS) and enhanced DNA repair via activation of ATM, Ku70/80 complex and DNA-PK. We found that Cav-1-overexpressing RD cells, already under basal conditions, had higher glutathione (GSH) content and greater catalase expression, which conferred protection against acute treatment with hydrogen peroxide. Furthermore, pre-treatment of Cav-1-overexpressing cells with PP2 or LY294002 compounds restored the sensitivity to radiation treatment, indicating a role for Src-kinases and Akt pathways in Cav-1-mediated radioresistance. These findings were confirmed using radioresistant RD and RH30 lines generated by hypofractionated radiotherapy protocol, which showed marked increase of Cav-1, catalase and Akt, and sensitivity to PP2 and LY294002 treatment. In conclusion, these data suggest that concerted activity of Cav-1 and catalase, in cooperation with activation of Src-kinase and Akt pathways, may represent a network of vital mechanisms that allow irradiated RMS cells to evade cell death induced by oxidative stress and DNA damage.

Biochemical, Biophysical and Functional Characterization of an Insoluble Iron Containing Hepcidin-Ferritin Chimeric Monomer Assembled Together with Human Ferritin H/L Chains at Different Molar Ratios.

Hepcidin and ferritin are key proteins of iron homeostasis in mammals. In this study, we characterize a chimera by fusing camel hepcidin to a human ferritin H-chain to verify if it retained the properties of the two proteins. The construct (HepcH) is expressed in E. coli in an insoluble and iron-containing form. To characterize it, the product was incubated with ascorbic acid and TCEP to reduce and solubilize the iron, which was quantified with ferrozine. HepcH bound approximately five times more iron than the wild type human ferritin, due to the presence of the hepcidin moiety. To obtain a soluble and stable product, the chimera was denatured and renatured together with different amounts of L-ferritin of the H-chain in order to produce 24-shell heteropolymers with different subunit proportions. They were analyzed by denaturing and non-denaturing PAGE and by mass spectroscopy. At the 1:5 ratio of HepcH to H- or L-ferritin, a stable and soluble molecule was obtained. Its biological activity was verified by its ability to both bind specifically cell lines that express ferroportin and to promote ferroportin degradation. This chimeric molecule showed the ability to bind both mouse J774 macrophage cells, as well as human HepG2 cells, via the hepcidin-ferroportin axis. We conclude that the chimera retains the properties of both hepcidin and ferritin and might be exploited for drug delivery.

NCOA4-mediated ferritinophagy promotes ferroptosis induced by erastin, but not by RSL3 in HeLa cells.

Ferroptosis is a regulated cell death characterized by a lethal accumulation of lipid peroxides due to an increase of intracellular iron and a decrease of antioxidant capacity. The reduction of antioxidant activity is obtained by using chemical agents, such as erastin and RSL3, the first one inhibiting the transmembrane cystine-glutamate antiporter causing a cysteine and glutathione depletion and the second one inactivating directly the glutathione peroxidase 4 (GPX4) respectively. The role of iron and its related proteins in supporting the formation of lipid peroxides, is not completely understood hence to try to shed light on it we generated HeLa clones with altered ferritinophagy, the ferritin degradation process, by knocking-out or overexpressing Nuclear Receptor Coactivator 4 (NCOA4), the ferritin autophagic cargo-receptor. NCOA4 deficiency abolished ferritinophagy increasing ferritin level and making the cells more resistant to erastin, but unexpectedly more sensitive to RSL3. Interestingly, we found that erastin promoted ferritinophagy in HeLa cells expressing NCOA4, increasing the free iron, lipid peroxidation and the sensitivity to ferroptosis. In contrast, RSL3 did not modulate ferritinophagy, while NCOA4 overexpression delayed RSL3-induced cell death suggesting that RSL3 mechanism of action is independent of ferritin degradation process. Therefore, the ferritin-iron release in the execution of ferroptosis seems to depend on the inducing compound, its target and downstream pathway of cell death activation.

BMP6 binding to heparin and heparan sulfate is mediated by N-terminal and C-terminal clustered basic residues.

BACKGROUND: The bone morphogenetic protein 6 (BMP6) is a crucial inducer of hepcidin, the peptide hormone that regulates the iron availability in our body. Hepcidin expression is influenced by hepatic heparan sulfate (HS) and by heparin administration, suggesting BMP6 interaction with heparin/HS. The BMP2/4 subfamily has been deeply characterized to have a N-terminal heparin/HS binding domain (HBD), whose basic residues contact the sulfate groups on heparin and HS. Such detailed characterization is still required for other, structurally different BMPs, including BMP6. METHODS: BMP6 peptides encompassing potential HBDs were analysed on heparin-functionalized plates and microcantilevers, and on membrane HS expressing CHO-K1 cells. Monomeric wild-type BMP6 and mutants were produced, substituting the basic residues with non-charged ones, and their affinity to the heparin-column was measured. The BMP6-heparin interaction was also predicted at atomic level by in silico molecular dynamics. RESULTS: N-terminal and C-terminal BMP6 peptides showed high heparin affinity in solid-phase assays. The mutation of the two sites (R5L, R6S, R7L and K126N, K127N, R129S) abolished the heparin-binding activity of the recombinant monomeric BMP6. Monomeric BMP6 and peptides specifically bound to membrane HS of CHO-K1 cells through the same domains. Molecular dynamic studies supported the role of the two HBDs, suggesting a cooperative behaviour. CONCLUSIONS: In BMP6, N-terminal (R5, R6, R7) and C-terminal (K126, K127, R129) domains mediate the interaction with heparin and HS. GENERAL SIGNIFICANCE: This study provides the molecular mechanism supporting the use of heparin to sequester BMP6 and inhibit hepcidin expression, a novel clinical approach for high-hepcidin iron disorders.

Pentosan polysulfate to control hepcidin expression in vitro and in vivo.

Hepcidin peptide is crucial in the regulation of systemic iron availability controlling its uptake from the diet and its release from the body storage tissues. Hepcidin dysregulation causes different human disorders ranging from iron overload (e.g. hemochromatosis) to iron deficiency (e.g. anemia). Hepcidin excess is common in the Anemia of Chronic Diseases or Anemia of Inflammation and in the genetic form of anemia named IRIDA; the pharmacological downregulation of hepcidin in these disorders could improve the anemia. Commercial heparins were shown to be strong inhibitors of hepcidin expression, by interfering with BMP6/SMAD pathway. The non-anti-coagulant heparins, modified to abolish the anti-thrombin binding site, were equally potent and could be used to improve iron status. To perform its anti-hepcidin activity heparin needs 2O- and 6O-sulfation and an average molecular weight (MW) up to 4000-8000 Dalton, depending on the sulfation level. The pentosane polysulfate (PPS), which shares with heparin a high degree of sulfation, is a compound with low anti-coagulant activity that is already in use for pharmaceutical treatment. In the present work we analyzed the anti-hepcidin activity of PPS in vitro and in vivo. We found that it acts as a strong inhibitor of hepcidin expression in HepG2 cells with an effect already visible after 2-3 h of treatment. It also suppressed hepcidin in mice in a dose dependent manner after 3 h and with a significant redistribution of systemic iron without evident side effects. PPS is also able to abolish the LPS dependent hepcidin upregulation similarly to that showed for heparin derivatives. These results suggest PPS as an interesting compound to control hepcidin in vivo.

Cellular binding analysis of recombinant hybrid heteropolymer of camel hepcidin and human ferritin H chain. The unexpected human H-ferritin binding to J774 murine macrophage cells.

Ferritin is a molecule with enormous potentiality in biotechnology that have been already used to encapsulate molecules, as contrast in magnetic resonance imaging and to carry epitopes. We proposed to use it to carry another key protein of iron metabolism, hepcidin that is a small hormone peptide that control systemic iron homeostasis. In this work, we purified the previously produced camel hepcidin and human H-ferritin heteropolymer (HepcH-FTH) and to monitor its binding capability toward J744 cell line in presence or absence of ferric ammonium citrate. Fused camel hepcidin and human H-ferritin monomer (HepcH) as well as the assembled HepcH-FTH heteropolymer (ratio 1:5) was easily purified by a one-step purification using size exclusion chromatography. SDS-PAGE electrophoresis of HepcH, purified from soluble and insoluble fractions, showed a single band of 24 kDa with an estimated purity of at least 90%. The purification yields of HepcH from the soluble and insoluble fractions was, respectively, of about 6.80 and 2 mg/L of bacterial culture. Time curse cellular binding assays of HepcH-FTH revealed its great potential to bind the J774 cells after 15 min of incubation. Furthermore, HepcH-FTH was able to degrade ferroportin, the unique hepcidin receptor, even after 30 min of incubation with J774 cells treated with 100 µM ferric ammonium citrate. In conclusion, we proposed ferritin as a peptide carrier to promote the association of the hybrid HepcH-FTH nanoparticle with a particular type of cell for therapeutic or diagnostic.

The Antitumor Didox Acts as an Iron Chelator in Hepatocellular Carcinoma Cells.

Ribonucleotide reductase (RR) is the rate-limiting enzyme that controls the deoxynucleotide triphosphate synthesis and it is an important target of cancer treatment, since it is expressed in tumor cells in proportion to their proliferation rate, their invasiveness and poor prognosis. Didox, a derivative of hydroxyurea (HU), is one of the most potent pharmaceutical inhibitors of this enzyme, with low in vivo side effects. It inhibits the activity of the subunit RRM2 and deoxyribonucleotides (dNTPs) synthesis, and it seems to show iron-chelating activity. In the present work, we mainly investigated the iron-chelating properties of didox using the HA22T/VGH cell line, as a model of hepatocellular carcinoma (HCC). We confirmed that didox induced cell death and that this effect was suppressed by iron supplementation. Interestingly, cell treatments with didox caused changes of cellular iron content, TfR1 and ferritin levels comparable to those caused by the iron chelators, deferoxamine (DFO) and deferiprone (DFP). Chemical studies showed that didox has an affinity binding to Fe3+ comparable to that of DFO and DFP, although with slower kinetic. Structural modeling indicated that didox is a bidentated iron chelator with two theoretical possible positions for the binding and among them that with the two hydroxyls of the catechol group acting as ligands is the more likely one. The iron chelating property of didox may contribute to its antitumor activity not only blocking the formation of the tyrosil radical on Tyr122 (such as HU) on RRM2 (essential for its activity) but also sequestering the iron needed by this enzyme and to the cell proliferation.

Hepatic heparan sulfate is a master regulator of hepcidin expression and iron homeostasis in human hepatocytes and mice.

Hepcidin is a liver-derived peptide hormone that controls systemic iron homeostasis. Its expression is regulated by the bone morphogenetic protein 6 (BMP6)/SMAD1/5/8 pathway and by the proinflammatory cytokine interleukin 6 (IL6). Proteoglycans that function as receptors of these signaling proteins in the liver are commonly decorated by heparan sulfate, but the potential role of hepatic heparan sulfate in hepcidin expression and iron homeostasis is unclear. Here, we show that modulation of hepatic heparan sulfate significantly alters hepcidin expression and iron metabolism both in vitro and in vivo Specifically, enzymatic removal of heparan sulfate from primary human hepatocytes, CRISPR/Cas9 manipulation of heparan sulfate biosynthesis in human hepatoma cells, or pharmacological manipulation of heparan sulfate-protein interactions using sodium chlorate or surfen dramatically reduced baseline and BMP6/SMAD1/5/8-dependent hepcidin expression. Moreover inactivation of the heparan sulfate biosynthetic gene N-deacetylase and N-sulfotransferase 1 (Ndst1) in murine hepatocytes (Ndst1f/fAlbCre+) reduced hepatic hepcidin expression and caused a redistribution of systemic iron, leading to iron accumulation in the liver and serum of mice. Manipulation of heparan sulfate had a similar effect on IL6-dependent hepcidin expression in vitro and suppressed IL6-mediated iron redistribution induced by lipopolysaccharide in vivo These results provide compelling evidence that hepatocyte heparan sulfate plays a key role in regulating hepcidin expression and iron homeostasis in mice and in human hepatocytes.

The role of heparin, heparanase and heparan sulfates in hepcidin regulation.

Hepcidin is considered the major regulator of systemic iron homeostasis in human and mice, and its expression in the liver is mainly regulated at a transcriptional level. Central to its regulation are the bone morphogenetic proteins, particularly BMP6, that are heparin binding proteins. Heparin was found to inhibit hepcidin expression and BMP6 activity in hepatic cell lines and in mice, suggesting that endogenous heparan sulfates are involved in the pathway of hepcidin expression. This was confirmed by the study of cells and mice overexpressing heparanase, the enzyme that hydrolyzes heparan sulfates, and by cellular models with altered heparan sulfates. The evidences supporting the role of heparan sulfate in hepcidin expression are summarized in this chapter and open the way for new understanding in hepcidin expression and its control in pathological condition.

Sucrosomial® Iron Supplementation in Mice: Effects on Blood Parameters, Hepcidin, and Inflammation.

Sucrosomial® Iron is a recently developed formulation to treat iron deficiency based on ferric pyrophosphate covered by a matrix of phospholipids plus sucrose esters of fatty acids. Previous data indicated that Sucrosomial® Iron is efficiently absorbed by iron-deficient subjects, even at low dosage, and without side effects. Its structural properties may suggest that it is absorbed by an intestinal pathway which is different to the one used by ionic iron. Although, studies in vitro showed that Sucrosomial® Iron is readily absorbed, no animal models have been established to study this important aspect. To this aim, we induced iron deficient anemia in mice by feeding them with a low-iron diet, and then we treated them with either Sucrosomial® Iron or sulfate iron by gavage for up to two weeks. Both iron formulations corrected anemia and restored iron stores in a two-week period, but with different kinetics. Ferrous Sulfate was more efficient during the first week and Sucrosomial® Iron in the second week. Of note, when given at the same concentrations, Ferrous Sulfate induced the expression of hepcidin and four different inflammatory markers (Socs3, Saa1, IL6 and CRP), while Sucrosomial® Iron did not. We conclude that anemic mice are interesting models to study the absorption of oral iron, and that Sucrosomial® Iron is to be preferred over Ferrous Sulfate because of similar absorption but without inducing an inflammatory response.

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