RESUMEN
BACKGROUND/AIM: Doxorubicin is first-line therapy for soft-tissue sarcoma, but patients can develop resistance which is usually fatal. As a novel therapeutic strategy, the present study aimed to determine the synergy of recombinant methioninase (rMETase) and doxorubicin against HT1080 fibrosarcoma cells compared to Hs27 normal fibroblasts, and rMETase efficacy against doxorubicin-resistant HT1080 cells in vitro. MATERIALS AND METHODS: The 50% inhibitory concentrations (IC50) of doxorubicin and rMETase, as well as their combination efficacy, against HT1080 human fibrosarcoma cells, Hs27 normal human fibroblasts and doxorubicin-resistant HT1080 (DR-HT1080) cells were determined. Dual-color HT1080 cells which expressed red fluorescent protein (RFP) in the cytoplasm and green fluorescent protein (GFP) in the nuclei were used to visualize nuclear fragmentation during treatment. Nuclear fragmentation was observed with an IX71 fluorescence microscope. RESULTS: The IC50 for doxorubicin was 3.3 µM for HT1080 cells, 12.4 µM for DR-HT1080 cells, and 7.25 µM for Hs27 cells. The IC50 for rMETase was 0.75 U/ml for HT1080 cells, 0.42 U/ml for DR-HT1080 cells, and 0.93 U/ml for Hs27 cells. The combination of rMETase and doxorubicin was synergistic against fibrosarcoma cells but not against normal fibroblasts. The combination of doxorubicin plus rMETase also caused more fragmented nuclei than either treatment alone in HT1080 cells. rMETase alone was highly effective against the DR-HT1080 cells as well as the parental HT1080 cells. CONCLUSION: The present results indicate the future clinical potential of rMETase in combination with doxorubicin for fibrosarcoma, including doxorubicin-resistant fibrosarcoma.
Asunto(s)
Liasas de Carbono-Azufre , Doxorrubicina , Resistencia a Antineoplásicos , Sinergismo Farmacológico , Fibrosarcoma , Proteínas Recombinantes , Humanos , Doxorrubicina/farmacología , Fibrosarcoma/tratamiento farmacológico , Fibrosarcoma/patología , Fibrosarcoma/metabolismo , Liasas de Carbono-Azufre/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Línea Celular Tumoral , Proteínas Recombinantes/farmacología , Antibióticos Antineoplásicos/farmacología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismoRESUMEN
This research aims to explore the mechanism by which microRNAs may regulate the biological behavior of tumor cells in ALDH1+ fibrosarcoma. We identified differentially expressed miRNAs in ALDH + NMFH-1 cells, screened genes related to sarcoma metastasis in the TCGA database, and finally obtained key genes regulated by miRNAs that are involved in metastasis. The function and mechanism of these key genes were then validated at the cellular level. Using the ULCAN database, a significant correlation was found between hsa-mir-206 and mortality in sarcoma patients. WGCNA analysis identified 352 genes related to tumor metastasis. Through Venn diagrams, we obtained 15 metastasis-related genes regulated by hsa-mir-206. Survival analysis showed that SYNPO2 expression is significantly correlated with survival rate and is significantly underexpressed in multiple tumors. SYNPO2 showed a negative correlation with macrophages and a positive correlation with CD8+ T cells. After inhibiting the expression of hsa-mir-206 with siRNA plasmids, the mRNA expression of SYNPO2 was significantly upregulated. The results of CCK8 assay, scratch assay, and transwell assay showed that the proliferation and migration ability of NFMH-1 cells were promoted after SYNPO2 was inhibited. ALDH1+ tumor stem cells promote the proliferation and invasion of malignant fibrous histiocytoma cells by inhibiting SYNPO2 through hsa-mir-206.
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Familia de Aldehído Deshidrogenasa 1 , Movimiento Celular , Proliferación Celular , Regulación Neoplásica de la Expresión Génica , MicroARNs , Células Madre Neoplásicas , Retinal-Deshidrogenasa , MicroARNs/genética , MicroARNs/metabolismo , Humanos , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Familia de Aldehído Deshidrogenasa 1/genética , Familia de Aldehído Deshidrogenasa 1/metabolismo , Proliferación Celular/genética , Retinal-Deshidrogenasa/genética , Retinal-Deshidrogenasa/metabolismo , Movimiento Celular/genética , Línea Celular Tumoral , Fibrosarcoma/patología , Fibrosarcoma/genética , Fibrosarcoma/metabolismo , Progresión de la Enfermedad , Ratones , AnimalesAsunto(s)
Proteína de la Leucemia Mieloide-Linfoide , Sarcoma , Factores de Transcripción , Proteínas Señalizadoras YAP , Humanos , Femenino , Adulto , Sarcoma/genética , Sarcoma/metabolismo , Sarcoma/patología , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Proteína de la Leucemia Mieloide-Linfoide/genética , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Fibrosarcoma/genética , Fibrosarcoma/metabolismo , Fibrosarcoma/patología , Translocación Genética , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/metabolismo , Hibridación Fluorescente in Situ , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Neoplasias de los Tejidos Blandos/genética , Neoplasias de los Tejidos Blandos/patología , Neoplasias de los Tejidos Blandos/metabolismoRESUMEN
Aerobic glycolysis is a metabolic reprogramming of tumor cells that is essential for sustaining their phenotype of fast multiplication by continuously supplying energy and mass. Pyruvate kinase M2 (PKM2) has a vital role in this process, which has given it high interest as a target for anticancer drug development. With potent toxicity to many types of cancer cells, polyphyllin II (PP2), a steroidal saponin isolated from the herbaceous plant Rhizoma paridis, brought to our attention that it might interfere with the PKM2 activity. In this study, we discovered that PP2 was a novel agonist of PKM2. PP2 activated recombinant PKM2 and changed the protein's oligomeric state to activate intracellular PKM2. At the same time, PP2 suppressed its protein kinase function by decreasing the content of nuclear PKM2. The mRNA levels of its downstream genes, such as Glut1, LDHA, and MYC, were inhibited. In addition, PP2 induced oxidative stress by downregulating the expression and activity of antioxidant proteins such as NQO1, TrxR, and Trx in HT-1080 cells, which in turn led to mitochondrial dysfunction and ultimately induced apoptosis. Moreover, PP2 reduced the proliferation and migration of HT-1080 cells. Thus, targeting the glycolysis pathway offers an unprecedented mode of action for comprehending PP2's pharmacological impacts and advances PP2's further development in fibrosarcoma therapy.
Asunto(s)
Apoptosis , Proliferación Celular , Fibrosarcoma , Saponinas , Humanos , Apoptosis/efectos de los fármacos , Fibrosarcoma/tratamiento farmacológico , Fibrosarcoma/patología , Fibrosarcoma/metabolismo , Proliferación Celular/efectos de los fármacos , Saponinas/farmacología , Saponinas/química , Piruvato Quinasa/metabolismo , Línea Celular Tumoral , Antineoplásicos/farmacología , Antineoplásicos/químicaRESUMEN
Autophagy plays a key role in removing protein aggregates and damaged organelles. In addition to its conventional degradative functions, autophagy machinery contributes to the release of cytosolic proteins through an unconventional secretion pathway. In this research, we analyzed autophagy-induced extracellular vesicles (EVs) in HT1080-derived human fibrosarcoma 2FTGH cells using transmission electron microscopy and atomic force microscopy (AFM). We preliminary observed that autophagy induces the formation of a subset of large heterogeneous intracellular vesicular structures. Moreover, AFM showed that autophagy triggering led to a more visible smooth cell surface with a reduced amount of plasma membrane protrusions. Next, we characterized EVs secreted by cells following autophagy induction, demonstrating that cells release both plasma membrane-derived microvesicles and exosomes. A self-forming iodixanol gradient was performed for cell subfractionation. Western blot analysis showed that endogenous LC3-II co-fractionated with CD63 and CD81. Then, we analyzed whether raft components are enriched within EV cargoes following autophagy triggering. We observed that the raft marker GD3 and ER marker ERLIN1 co-fractionated with LC3-II; dual staining by immunogold electron microscopy and coimmunoprecipitation revealed GD3-LC3-II association, indicating that autophagy promotes enrichment of raft components within EVs. Introducing a new brick in the crosstalk between autophagy and the endolysosomal system may have important implications for the knowledge of pathogenic mechanisms, suggesting alternative raft target therapies in diseases in which the generation of EV is active.
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Autofagia , Vesículas Extracelulares , Humanos , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/ultraestructura , Línea Celular Tumoral , Microdominios de Membrana/metabolismo , Exosomas/metabolismo , Exosomas/ultraestructura , Tetraspanina 30/metabolismo , Fibrosarcoma/metabolismo , Fibrosarcoma/patología , Proteínas Asociadas a Microtúbulos/metabolismoRESUMEN
Myxofibrosarcoma (MFS) is a common adult soft tissue sarcoma characterized by high-local recurrence rate, poorly understood molecular pathogenesis, lack of specific prognostic markers, and effective targeted therapies. To gain further insights into the disease, we analyzed a well-defined group of 133 primary MFS cases. Immunohistochemical (IHC) staining for p53, MET, RET, and RB was performed. Twenty-five cases were analyzed by targeted resequencing of known cancer driver hotspot mutations, whereas 66 and 64 MFSs were examined for the presence of genetic variants in TP53 and MET gene, respectively. All clinical, histologic, immunostaining, and genetic variables were analyzed for their impact on 5-years overall survival (OS) and 5-years event-free survival (EFS). In our series, no grade I tumors relapsed and high grade are related to a positive MET immunostaining (P = .034). Both local recurrence (P = .038) and distal metastases (P = .016) correlated to the presence of "single nucleotide variant (SNV) plus copy number variation (CNV)" in TP53. Multivariate analysis revealed that age (>60 years), metastasis at presentation, and positive IHC-p53 signal are risk factors for a poor OS (P = .003, P = .000, and P = .002), whereas age (>60 years), synchronous metastasis, and tumor size (>10 cm) predict an unfavorable 5-years EFS (P = .011, P = .000, and P = .023). Considering the smaller series (n = 66) that underwent molecular screening, the presence of "SNV+CNV" in TP53 represents a risk factor for a worse 5-years EFS (hazard ratio, 2.5; P = .017). The present series confirms that TP53 is frequently altered in MFS (86.4% of cases), appearing to play an important role in MFS tumorigenesis and being a potentially drugable target. A positive p53 immunostainings is related to a poor diagnosis, and it is the presence of a single nucleotide genetic alterations in TP53 that is essential in conferring MFS an aggressive phenotype, thus supporting the use of molecular profiling in MFS to better define the role of p53 as a prognostic factor.
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Fibrosarcoma , Proteína p53 Supresora de Tumor , Humanos , Proteína p53 Supresora de Tumor/metabolismo , Proteína p53 Supresora de Tumor/genética , Fibrosarcoma/genética , Fibrosarcoma/metabolismo , Fibrosarcoma/patología , Persona de Mediana Edad , Femenino , Masculino , Anciano , Adulto , Anciano de 80 o más Años , Mutación , Variaciones en el Número de Copia de ADN , Adulto Joven , Biomarcadores de Tumor/metabolismo , Biomarcadores de Tumor/genética , Proteínas Proto-Oncogénicas c-met/metabolismo , Proteínas Proto-Oncogénicas c-met/genética , Neoplasias de los Tejidos Blandos/genética , Neoplasias de los Tejidos Blandos/metabolismo , Neoplasias de los Tejidos Blandos/patologíaRESUMEN
This study demonstrated the anticancer efficacy of chalcones with indole moiety (MIPP, MOMIPP) in fibrosarcoma cells for the first time. The results showed that MIPP and MOMIPP reduced the viability of HT-1080 cells in a concentration-dependent manner. MOMIPP was more active than MIPP in HT-1080 cells, showing lower IC50 values (3.67 vs. 29.90 µM). Both compounds at a concentration of 1 µM induced apoptosis in HT-1080 cells, causing death strictly related to caspase activation, as cell viability was restored when the caspase inhibitor Z-VAD was added. Reactive oxygen species production was approximately 3-fold higher than in control cells, and cotreatment with the inhibitor of mitochondrial ATPase oligomycin diminished this effect. Such effects were also reflected in mitochondrial dysfunction, including decreased membrane potential. Interestingly, the compounds that were studied caused massive vacuolization in HT-1080 cells. Immunocytochemical staining and TEM analysis showed that HT-1080 cells exhibited increased expression of the LC3-II protein and the presence of autophagosomes with a double membrane, respectively. Both compounds induced apoptosis, highlighting a promising link between autophagy and apoptosis. This connection could be a new target for therapeutic strategies to overcome chemoresistance, which is a significant cause of treatment failure and tumour recurrence in fibrosarcoma following traditional chemotherapy.
Asunto(s)
Apoptosis , Autofagia , Chalconas , Fibrosarcoma , Indoles , Especies Reactivas de Oxígeno , Humanos , Apoptosis/efectos de los fármacos , Fibrosarcoma/tratamiento farmacológico , Fibrosarcoma/metabolismo , Fibrosarcoma/patología , Autofagia/efectos de los fármacos , Indoles/farmacología , Línea Celular Tumoral , Especies Reactivas de Oxígeno/metabolismo , Chalconas/farmacología , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Antineoplásicos/farmacología , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismoRESUMEN
BACKGROUND/AIM: The alkylating agent trabectedin, which binds the minor groove of DNA, is second-line therapy for soft-tissue sarcoma but has only moderate efficacy. The aim of the present study was to determine the synergistic efficacy of recombinant methioninase (rMETase) and trabectedin on fibrosarcoma cells in vitro, compared with normal fibroblasts. MATERIALS AND METHODS: HT1080 human fibrosarcoma cells expressing green fluorescent protein (GFP) in the nucleus and red fluorescent protein (RFP) in the cytoplasm and Hs27 normal human fibroblasts, were used. Each cell line was cultured in vitro and divided into four groups: no-treatment control; trabectedin treated; rMETase treated; and trabectedin plus rMETase treated. The dual-color HT1080 cells were used to quantitate nuclear fragmentation in each treatment group. RESULTS: The combination of rMETase and trabectedin was highly synergistic to decrease HT1080 cell viability. In contrast, there was no synergy on Hs27 cells. Moreover, nuclear fragmentation occurred synergistically with the combination of trabectedin and rMETase on dual-color HT1080 cells. CONCLUSION: The combination treatment of trabectedin plus rMETase was highly synergistic on fibrosarcoma cells in vitro suggesting that the combination can improve the outcome of trabectedin alone in future clinical studies. The lack of synergy of rMETase and trabectedin on normal fibroblasts suggests the combination is not toxic to normal cells. Synergy of the two drugs may be due to the high rate of nuclear fragmentation on treated HT1080 cells, and the late-S/G2 cell-cycle block of cancer cells by rMETase, which is a target for trabectedin. The results of the present study suggest the future clinical potential of the combination of rMETase and trabectedin for soft-tissue sarcoma.
Asunto(s)
Liasas de Carbono-Azufre , Supervivencia Celular , Dioxoles , Sinergismo Farmacológico , Fibroblastos , Fibrosarcoma , Tetrahidroisoquinolinas , Trabectedina , Humanos , Fibrosarcoma/tratamiento farmacológico , Fibrosarcoma/patología , Fibrosarcoma/metabolismo , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Trabectedina/farmacología , Liasas de Carbono-Azufre/farmacología , Liasas de Carbono-Azufre/administración & dosificación , Tetrahidroisoquinolinas/farmacología , Dioxoles/farmacología , Supervivencia Celular/efectos de los fármacos , Proteínas Recombinantes/farmacología , Línea Celular Tumoral , Antineoplásicos Alquilantes/farmacología , Núcleo Celular/metabolismo , Núcleo Celular/efectos de los fármacosRESUMEN
Cellular myxoma is a benign soft tissue tumor frequently associated with GNAS mutation that may morphologically resemble low-grade myxofibrosarcoma. This study aimed to identify the undescribed methylation profile of cellular myxoma and compare it to myxofibrosarcoma. We performed molecular analysis on twenty cellular myxomas and nine myxofibrosarcomas and analyzed the results using the methylation-based DKFZ sarcoma classifier. A total of 90% of the cellular myxomas had GNAS mutations (four loci had not been previously described). Copy number variations were found in all myxofibrosarcomas but in none of the cellular myxomas. In the classifier, none of the cellular myxomas reached the 0.9 threshold. Unsupervised t-SNE analysis demonstrated that cellular myxomas form their own clusters, distinct from myxofibrosarcomas. Our study shows the diagnostic potential and the limitations of molecular analysis in cases where morphology and immunohistochemistry are not sufficient to distinguish cellular myxoma from myxofibrosarcoma, particularly regarding GNAS wild-type tumors. The DKFZ sarcoma classifier only provided a valid prediction for one myxofibrosarcoma case; this limitation could be improved by training the tool with a more considerable number of cases. Additionally, the classifier should be introduced to a broader spectrum of mesenchymal neoplasms, including benign tumors like cellular myxoma, whose distinct methylation pattern we demonstrated.
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Variaciones en el Número de Copia de ADN , Metilación de ADN , Fibrosarcoma , Mixoma , Humanos , Mixoma/genética , Mixoma/diagnóstico , Mixoma/patología , Fibrosarcoma/genética , Fibrosarcoma/patología , Fibrosarcoma/diagnóstico , Fibrosarcoma/metabolismo , Persona de Mediana Edad , Femenino , Anciano , Masculino , Adulto , Mutación , Diagnóstico Diferencial , Subunidades alfa de la Proteína de Unión al GTP Gs/genética , Cromograninas/genética , Anciano de 80 o más Años , Neoplasias de los Tejidos Blandos/genética , Neoplasias de los Tejidos Blandos/diagnóstico , Neoplasias de los Tejidos Blandos/patologíaRESUMEN
In the tumor environment, hypoxia promotes tumor progression, such as cancer cell growth, migration and chemoresistance. This study aimed to evaluate the roles of free fatty acid receptors (FFARs) in the regulation of cancer cell functions under hypoxic conditions, using fibrosarcoma HT1080 cells. HT1080 cells expressed FFAR1, FFAR2 and FFAR3 genes, but not FFAR4 gene. FFAR1, FFAR2 and FFAR3 expression levels in HT1080 cells cultured at 1 % O2 were elevated, compared with 21 % O2. The cell growth activities of HT1080 cells cultured at 21 % O2 were inhibited by acetic acid (AA) and propanoic acid (PA), but not 1 % O2. HT1080 cell motility was markedly reduced by culturing at 1 % O2. The cell growth and motility of HT1080 cells were enhanced by FFAR2 knockdown. The cell viability to cisplatin (CDDP) of HT1080 cells cultured at 1 % O2 was increased, compared with 21 % O2. FFAR2 knockdown suppressed the cell viability to CDDP of HT1080 cells. On the other hand, the cell motility and viability to CDDP of HT1080 cells cultured at 21 % O2 were suppressed by TUG-770. When HT1080 cells were cultured at 1 % O2, the cell motility and viability to CDDP were decreased, correlating with FFAR1 expression level. Moreover, FFAR1 knockdown increased the cell viability to CDDP of HT1080 cells cultured at 1 % O2. These results suggest that FFAR-mediated signaling plays an important role in the modulation of cellular functions of HT1080 cells under hypoxic conditions.
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Ácidos Grasos no Esterificados , Fibrosarcoma , Humanos , Receptores Acoplados a Proteínas G/metabolismo , Cisplatino/farmacología , Transducción de Señal , Fibrosarcoma/metabolismo , Movimiento CelularRESUMEN
CREB3 subfamily belongs to the bZIP transcription factor family and comprises five members. Normally they are located on the endoplasmic reticulum (ER) membranes and proteolytically activated through RIP (regulated intramembrane proteolysis) on Golgi apparatus to liberate the N-terminus to serve as transcription factors. CREB3L1 acting as one of them transcriptionally regulates the expressions of target genes and exhibits distinct functions from the other members of CREB3 family in eukaryotes. Physiologically, CREB3L1 involves in the regulation of bone morphogenesis, neurogenesis, neuroendocrine, secretory cell differentiation, and angiogenesis. Pathologically, CREB3L1 implicates in the modulation of osteogenesis imperfecta, low grade fibro myxoid sarcoma (LGFMS), sclerosing epithelioid fibrosarcoma (SEF), glioma, breast cancer, thyroid cancer, and tissue fibrosis. This review summarizes the upstream and downstream regulatory network of CREB3L1 and thoroughly presents our current understanding of CREB3L1 research progress in both physiological and pathological conditions with special focus on the novel findings of CREB3L1 in cancers.
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Fibrosarcoma , Humanos , Fibrosarcoma/genética , Fibrosarcoma/metabolismo , Fibrosarcoma/patología , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico , Biomarcadores de Tumor/genética , Proteínas del Tejido Nervioso , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genéticaRESUMEN
Los tumores sólidos para crecer más de 2 mm, necesitan desarrollar nuevos vasos sanguíneos. Las células neoplásicas secretan factores de crecimiento que estimulan la angiogénesis y el crecimiento tumoral. La Carragenina bloquea un vitro la unión de los factores de crecimiento a sus receptores. Ensayamos in vivo su acción con el objetivo de analizar si inhibe el desarrollo de un fibrosarcoma murino. Para neutralizar la acción inflamatoria de la Carragenina usamos la Indometacina, que es un antiinflamatorio no esteroide. El tumor usado fue en fibrosarcoma inducido con metilcolanterno en ratones Balb/c y mantenido por pasaje seriado de células tumorales, en ratones de la misma cepa. El volumen de los tumores fue evaluado midiendo dos dimensiones y aplicando la fórmula V = 0.4 x d(2) x D. Los ratones con tumores fueron separados en grupos, uno de los cuales se usó como testigo y los otros tratados en forma separada con indometacina, Carragenina y Carragenina-Indometacina. Se comparó el volumen de los tumores de los ratones tratados con respecto al testigo y el de los tratados entre sí, utilizando el Test t de Student. Se demostró que la Carregenina y la indometacina, inhiben el desarrollo del fibrosarcoma. La acción inhibitoria de la Carragenina sobre el crecimiento tumoral es significativamente mayor que el efecto antitumoral de la Indometacina y el de la Corragenina-Indometacina.