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Inhibition of glutathione peroxidase 4 primes mouse C2C12 myoblasts and rhabdomyosarcoma cell lines to ferroptosis
Full text linkFerroptosis 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
Non-Anticoagulant Heparins Are Hepcidin Antagonists for the Treatment of Anemia
No full textThe peptide hormone hepcidin is a key controller of systemic iron homeostasis, and its expression in the liver is mainly regulated by bone morphogenetic proteins (BMPs), which are heparin binding proteins. In fact, heparins are strong suppressors of hepcidin expression in hepatic cell lines that act by inhibiting the phosphorylation of SMAD1/5/8 proteins elicited by the BMPs. The inhibitory effect of heparins has been demonstrated in cells and in mice, where subcutaneous injections of non-anticoagulant heparins inhibited liver hepcidin expression and increased iron bioavailability. The chemical characteristics for high anti-hepcidin activity in vitro and in vivo include the 2O-and 6O-sulfation and a molecular weight above 7 kDa. The most potent heparins have been found to be the super-sulfated ones, active in hepcidin suppression with a molecular weight as low as 4 kDa. Moreover, the alteration of endogenous heparan sulfates has been found to cause a reduction in hepcidin expression in vitro and in vivo, indicating that heparins act by interfering with the interaction between BMPs and components of the complex involved in the activation of the BMP/SMAD1/5/8 pathway. This review summarizes recent findings on the anti-hepcidin activity of heparins and their possible use for the treatment of anemia caused by hepcidin excess, including the anemia of chronic diseases
Hepcidin antagonists for potential treatments of disorders with hepcidin excess
No full textThe discovery of hepcidin clarified the basic mechanism of the control of systemic iron homeostasis. Hepcidin is mainly produced by the liver as a propeptide and processed by furin into the mature active peptide. Hepcidin binds ferroportin, the only cellular iron exporter, causing the internalization and degradation of both. Thus hepcidin blocks iron export from the key cells for dietary iron absorption (enterocytes), recycling of hemoglobin iron (the macrophages) and the release of storage iron from hepatocytes, resulting in the reduction of systemic iron availability. The BMP/HJV/SMAD pathway is the major regulator of hepcidin expression that responds to iron status. Also inflammation stimulates hepcidin via the IL6/STAT3 pathway with a support of an active BMP/HJV/SMAD pathway. In some pathological conditions hepcidin level is inadequately elevated and reduces iron availability in the body, resulting in anemia. These conditions occur in the genetic iron refractory iron deficiency anemia and the common anemia of chronic disease (ACD) or anemia of inflammation. Currently, there is no definite treatment for ACD. Erythropoiesis-stimulating agents and intravenous iron have been proposed in some cases but they are scarcely effective and may have adverse effects. Alternative approaches aimed to a pharmacological control of hepcidin expression have been attempted, targeting different regulatory steps. They include hepcidin sequestering agents (antibodies, anticalins, and aptamers), inhibitors of BMP/SMAD or of IL6/STAT3 pathway or of hepcidin transduction (siRNA/shRNA) or ferroportin stabilizers. In this review we summarized the biochemical interactions of the proteins involved in the BMP/HJV/SMAD pathway and its natural inhibitors, the murine and rat models with high hepcidin levels currently available and finally the progresses in the development of hepcidin antagonists, with particular attention to the role of heparins and heparin sulfate proteoglycans in hepcidin expression and modulation of the BMP6/SMAD pathway
Statin-Sensitive Akt1/Src/Caveolin-1 Signaling Enhances Oxidative Stress Resistance in Rhabdomyosarcoma
Full text linkObjective: The aim of the present study was to investigate the effects of targeting the mevalonate pathway (MVP) in rhabdomyosarcoma (RMS), a soft tissue tumor with a prevalence in young people. Methods: In silico analyses of RNA datasets were performed to correlate MVP with RMS patient survival. The sensitivity of RMS cell lines to MVP inhibitors was assessed in vitro by analysis of cell growth (crystal violet and clonogenic assays), cell migration (wound healing assay), cell survival (neutral red assay), and oxidative stress (ROS assay). The effects of MVP inhibitors were tested in vivo by analyzing RMS xenografts grown in NOD/SCID mice. Quantification of protein targets was performed using immunoblotting or immunohistochemistry analyses. Results: By analyzing RNA datasets from rhabdomyosarcoma (RMS), a soft tissue tumor with a prevalence in young people, we found In silico analysis showed upregulation of sterol regulatory element-binding protein 2 (SREBP2) and mevalonate pathway (MVP) genes, including 3-Hydroxy-3-Methylglutaryl-CoA Reductase (HMGCR), farnesyl-diphosphate synthase (FDPS), squalene epoxidase (SQLE), which correlated with worse overall patient survival and predicted statin sensitivity. Targeting of MVP in human RD and RH30 lines by inhibitors of SREBP2 (fatostatin), HMGCR (lovastatin and simvastatin), and FDPS (zoledronic acid) resulted in In impaired cell growth, migration, and viability, and increased oxidative cell death in combination with actinomycin D. Conversely, cholesterol (CHO) supplementation enhanced cell growth and migration. human RD and RH30 lines, treatment with 0.01–1 μM doses of fatostatin (SREBP2 inhibitor), lovastatin and simvastatin (HMGCR inhibitors), and zoledronic acid (FDPS inhibitor) impaired cell growth and migration, which were conversely stimulated by 50–100 μM cholesterol (CHO) supplementation. Treatment of RMS lines with higher doses of SREBP2 and MVP inhibitors (5–50 μM) promoted oxidative cell death and chemosensitization in combination with actinomycin D. Administration of lFatostatin and lovastatin or fatostatin to RD and RH30 cells produced produced a rapid attenuation of Erk1/2 and Akt1 phosphorylation signaling in RMS lines, and oral administration of lovastatin reduced tumor mass detectable after 4 h of treatment. Furthermore, tumor mass growth of xenografted RD cells in NOD/SCID mice was reduced by oral administration of lovastatin. LastlyFinally, we found we found that the forced Akt1 activation in RD cells was sufficient to drive SREBP2, HMGCR and SQLE protein expression, and enhance cell death promoting increased susceptibility to MVP inhibitors. Conclusions: Taken together, these data suggest that the axis formed by Akt1, SREBP2 and MVP axis is critical for RMS tumor growth, migration, and oxidative stress protection mainly primarily through the maintenancemaintaining adequate CHO levels that enable of proper intracellular signaling CHO levels that ensure proper intracellular signaling. Therefore, targeting stimulating CHO levels depletion by via SREBP2 and MVP inhibition may represent a viable option to improve the combination therapy protocol, especially in pAkt1-positive RMS
Erastin is a strong inducer of ferroptosis in human rhabdomyosarcoma cell lines
No full textRhabdomyosarcoma (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
Pentosan polysulfate to control hepcidin expression in vitro and in vivo
Full text linkHepcidin 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-anticoagulant 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
Genetic iron overload aggravates, and pharmacological iron restriction improves, MDS pathophysiology in a preclinical study
Full text link: Although iron overload is a common feature in myelodysplastic syndromes (MDS), it remains unclear how iron excess is detrimental for disease pathophysiology. Taking advantage of complementary approaches, we analyzed the impact of iron overload and restriction achieved through genetic activation of ferroportin (FPN) via the C326S mutation (FPNC326S) and pharmacologic inhibition (vamifeport) of the iron exporter FPN, respectively, in a MDS mouse model. Although FPNC326S-induced iron overload did not significantly improve the late stages of erythroid maturation, vamifeport-mediated iron restriction ameliorated anemia and red blood cell maturation in MDS mice, through the reduction of oxidative stress and apoptosis in erythroid progenitors. Iron overload aggravated, and restriction alleviated, reactive oxygen species formation, DNA damage, and cell death in hematopoietic stem and progenitor cells (HSPCs), resulting in altered cell survival and quality. Finally, myeloid bias, indicated by expanded bone marrow myeloid progenitors and circulating immature myeloid blasts, was exacerbated by iron excess and attenuated by iron restriction. Overall, vamifeport treatment resulted in improved anemia and significant survival increment in MDS mice. Interestingly, the combined therapy with vamifeport and the erythroid maturation agent luspatercept has superior effect in improving anemia and myeloid bias as compared with single treatments and offers additive beneficial effects in MDS. Our results prove, to our knowledge, for the first time in a preclinical model, that iron plays a pathologic role in transfusion-independent MDS. This is likely aggravated by transfusional iron overload, as suggested by observations in the FPNC326SMDS model. Ultimately, the beneficial effects of pharmacologic FPN inhibition uncovers the therapeutic potential of early prevention of iron toxicity in transfusion-independent MDS
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
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