1,720,996 research outputs found
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
Focus on the role of Caveolin and Cavin protein families in liposarcoma
No full textThe identification of ancillary biomarkers useful to improve diagnosis is a major challenge for adipocytic liposarcoma (LPS), the most common type among soft tissue sarcomas affecting adulthood. Recent findings have reported the expression of some proteins belonging to Caveolin and Cavin families as a critical hallmark distinctive of the least aggressive, well-differentiated LPS tumors. These proteins are involved in the biogenesis, morphology and function of caveolae, minute bulb-shaped domains of the plasma membrane that play a crucial role in the adipose tissue by controlling hormone-dependent uptake of nutrients and contributing to the maintenance of tissue integrity. In light of this, in this paper we covered different topics, including metabolism, hypoxia and cell mechanoprotection, to outline the rationale for considering a deeper investigation of Caveolin and Cavin protein members in LPS neoplasms as an opportunity to identify pro-differentiating mechanisms that could counteract tumor growth
Caveolin-1 overexpression accelerates tumor growth and metastasis of embryonal rhabdomyosarcoma
Full text linkCaveolin-1 (Cav-1) is a plasma membrane scaffolding protein that was shown to control the ERK pathway in muscle satellite cells. Oncogenic transformation of satellite cells is responsible of the generation of rhabdomyosarcoma (RMS), a soft tissue tumor affecting childhood and adolescence. We previously reported that Cav-1 is a marker of proliferating RMS cell lines and that its overexpression promotes increased malignancy of RMS cells in vitro and in vivo. Here we show that tail vein injection of the human embryonal RD cells with Cav-1 overexpression (RD Cav-1) into NOD/SCID mice resulted in formation of lung metastasis in about 9 weeks as compared to control cells that did not form metastasis. After performing ex vivo transplantation of lung metastases we isolated one cell population, termed lung metastatic RD1, which injected in mice again gave rise to lung metastases in 5 weeks; from these disseminated lungs we were able to isolate the lung metastatic RD2 cell population. All the distinct cell populations, including RD Cav-1 and lung metastatic RD1 and RD2 clones, retained high Cav-1 expression and showed high phosphorylation levels of ERK1/2, which completely prevented their ability to undergo myogenic differentiation. In addition, lung metastatic RD1 and RD2 clones exhibited an increased migration, adhesion and production of angiogenic stimuli in comparison to non-metastatic control RD and RD Cav-1 lines. Taken together, these data suggest a key role of Cav-1 in promoting both local tumor growth and metastasis of RMS through cooperation of the ERK signalling pathway
Caveolin-3, MURC and Trim72 predict myogenic differentiation in the human embryonal rhabdomyosarcoma RD cell line
Full text linkCaveolin-3 (Cav-3), muscle-restricted coiled coil MURC (also referred to as Cavin-4) and the recently identified large tripartite motif Trim72 are proteins controlling a number of important processes in skeletal muscle, ranging from myogenesis and insulin signaling up to sarcolemma repair. In this study we investigated their expression in rhabdomyosarcoma (RMS), a soft tissue tumor showing morphological and biochemical traits of the skeletal muscle lineage. Immunohistochemical analysis showed a similar pattern of expression of the three proteins in different human RMS samples. Immunoblotting analysis carried out using different RMS cell lines showed an increased expression of these proteins during myogenic differentiation as compared to proliferating cells. In addition, confocal microscopy analysis revealed that Cav-3, MURC and Trim72 all co-localize at the plasmalemma of differentiated embryonal RD cells. Ectopic expression of a point mutated Cav-3P104L form in RD cells was sufficient to mislocalize both MURC and Trim72 into perinuclear Golgi-like compartments, leading to a cell phenotype characterized by increased ERK phosphorylation and severe impairment of myogenic differentiation. Overall, these data suggest that a concurrent expression of Cav-3, MURC and Trim72 configures as a specific signature predicting and regulating cell differentiation in RMS
Caveolin-1 overexpression in the human embryonal rhabdomyosarcoma RD cells increases tumor growth and metastasis in vivo through a phospho-ERK-dependent signaling pathway
Full text linkCaveolin-1 (Cav-1) represents the principal component of caveolae, flask-shaped invaginations of the plasma membrane controlling different processes like endocytosis, mechanoprotection and signal transduction.
Cav-1 expression and/or function is often dysregulated in cancer, being associated to an increased or decreased tumor cell behavior depending on the cell context. We proposed Cav-1 as a marker of poorly differentiated cells in rhabdomyosarcoma (RMS), a class of childhood soft tissue sarcomas characterized by expression of myogenic markers. In addition, we have further shown that increasing or depleting Cav-1 expression levels in the human embryonal RD cells correlates with an augmented or decreased tumor cell behavior, respectively. In the attempt to further characterize the role of Cav-1 in RMS in vivo, here we demonstrate that injecting subcutaneously Cav-1-overexpressing RD cells in SCID nude mice led to rapid growth of huge RMS tumors in comparison to control mice; in addition, tail vein injection was followed by rapid lung metastasis formation. Ex vitro transplantation of lung metastatic cells followed by re-injection in the tail vein of SCID mice allowed us to isolate two clonal metastatic RD populations (named F1 and F2) characterized by increased ability to disseminate in a shorter time as compared to controls. FACS analyses revealed that both Cav-1 overexpressing RD cells and the metastatic F1-F2 populations exhibited a rapid G1 phase transition, characterized by massive activation of the phosphorylated ERK1/2 pathway. As a result, treatment of these cells with a pharmacological inhibitor of ERK phosphorylation (PD098059) was sufficient to prevent in vitro the increased proliferation and migration associated to the high Cav-1 levels. Overall, these data establish an important connection between the levels of Cav-1 and the activation of the ERK1/2 pathway underlying the augmented cell aggressiveness in RMS cells
Melatonin action in tumor skeletal muscle cells: an ultrastructural study
No full textMelatonin (Mel), or N-acetyl-5-methoxytryptamine, is a circadian hormone that can diffuse through all the biological membranes thanks to its amphiphilic structure, also overcoming the blood-brain barrier and placenta. Although Mel has been reported to exhibit strong antioxidant properties in healthy tissues, studies carried out on tumor cultures gave a different picture of its action, often describing Mel as effective to trigger the cell death of tumor cells by enhancing oxidative stress. Based on this premise, here Mel effect was investigated using a tumor cell line representative of the human alveolar rhabdomyosarcoma (ARMS), the most frequent soft tissue sarcoma affecting childhood. For this purpose, Mel was given either dissolved in ethanol (EtOH) or dimethyl sulfoxide (DMSO) at different concentrations and time exposures. Cell viability assays and ultrastructural observations demonstrated that Mel was able to induce a dose- and time-dependent cell death independently on the dissolution solvent. Microscopy analyses highlighted the presence of various apoptotic and necrotic patterns correlating with the increasing Mel dose and time of exposure. These findings suggest that Mel, triggering apoptosis in ARMS cells, could be considered as a promising drug for future multitargeted therapies
Synthetic inhibition of the SUMO pathway by targeting the SAE1 component via TAK-981 compound impairs growth and chemosensitizes embryonal and alveolar rhabdomyosarcoma cell lines
Full text link: Rhabdomyosarcoma (RMS) is a highly aggressive pediatric soft tissue sarcoma with limited therapeutic options, particularly for cases resistant to conventional treatments. The SUMOylation pathway, which plays a key role in regulating the cell cycle, apoptosis, and transcription, has emerged as a potential therapeutic target in RMS. Elevated levels of SUMO1 and SUMO2/3 conjugates in RMS cell lines, compared to normal human skeletal muscle cells, underscore the association between upregulated SUMOylation and aggressive cancer phenotypes. Understanding these molecular underpinnings is critical for the development of innovative and effective treatments. The investigation encompassed transcriptomic and protein analyses to profile SUMOylation pathway components across alveolar and embryonal RMS subtypes, aiming to identify heterogeneity that could guide personalized therapy approaches. TAK-981, a small molecule that selectively inhibits the SUMOylation of target proteins, was evaluated in combination with chemotherapeutic agents for additive or synergistic effects. Additionally, its impact on radiosensitivity and key signaling pathways, such as AKT, ERK and CAV1 phosphorylation, was assessed to elucidate its mechanism of action. Transcriptomic and proteomic analyses revealed distinct expression profiles of SUMOylation pathway components across RMS subtypes, highlighting heterogeneity that could guide personalized therapeutic strategies. Notably, SAE1 protein was overexpressed in RMS tissues and cells, positioning it as a potential biomarker for this cancer. Its activity was effectively counteracted by TAK-981, a SUMO inhibitor that demonstrated significant therapeutic potential by suppressing RMS cell proliferation and migration, and enhancing the cytotoxic effects of chemotherapeutic agents actinomycin D and doxorubicin. However, TAK-981 did not increase radiosensitivity, suggesting its selective action through chemical inhibition mechanisms. Mechanistically, TAK-981 reduced phosphorylation of key signaling proteins, including AKT, ERK and CAV1, which are critical for RMS cell survival. The findings of this study establish TAK-981 as a promising therapeutic agent for RMS. The results also provide foundational insights into the role of SUMOylation associated with the new biomarker SAE1 in RMS and its subtypes, paving the way for the development of personalized treatment strategies that leverage SUMO pathway inhibition
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
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
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