RESUMEN
Ewing Sarcoma (ES) is the second most common primary malignant bone tumor in children and adolescents. microRNAs (miRNAs) are involved in cancer as tumor suppressors or oncogenes. We studied the involvement of miRNAs located on chromosomes 11q and 22q that participate in the most common translocation in ES. Of these, we focused on 3 that belong to the let-7 family.We studied the expression levels of let-7a, and let-7b and detected a significant correlation between low expression of let-7b and increased risk of relapse. let-7 is known to be a negative regulator of the RAS oncogene. Indeed, we detected an inverse association between the expression of let-7 and RAS protein levels and its downstream target p-ERK, following transfection of let-7 mimics and inhibitors. Furthermore, we identified let-7 as a negative regulator of HIF-1α and EWS-FLI-1. Moreover, we were able to show that HIF-1α directly binds to the EWS-FLI-1 promoter. Salirasib treatment in-vitro resulted in the reduction of cell viability, migration ability, and in the decrease of cells in S-phase. A significant reduction in tumor burden and in the expression levels of both HIF-1α and EWS-FLI-1 proteins were observed in mice after treatment.Our results support the hypothesis that let-7 is a tumor suppressor that negatively regulates RAS, also in ES, and that HIF-1α may contribute to the aggressive metastatic behavior of ES. Moreover, the reduction in the tumor burden in a mouse model of ES following Salirasib treatment, suggests therapeutic potential for this RAS inhibitor in ES.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , MicroARNs/metabolismo , Sarcoma de Ewing/metabolismo , Proteínas ras/metabolismo , Adolescente , Adulto , Animales , Antineoplásicos/uso terapéutico , Ciclo Celular , Movimiento Celular , Supervivencia Celular , Niño , Preescolar , Cromosomas Humanos Par 11/genética , Cromosomas Humanos Par 22/genética , Supervivencia sin Enfermedad , Farnesol/análogos & derivados , Farnesol/uso terapéutico , Femenino , Silenciador del Gen , Genes Supresores de Tumor , Humanos , Lactante , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Trasplante de Neoplasias , Proteínas de Fusión Oncogénica/metabolismo , Proteína Proto-Oncogénica c-fli-1/metabolismo , Proteína EWS de Unión a ARN/metabolismo , Distribución Aleatoria , Salicilatos/uso terapéutico , Sarcoma de Ewing/patología , Transducción de Señal , Adulto JovenRESUMEN
Histone deacetylase (HDAC) inhibitors, such as valproic acid (VPA), constitute a novel class of anticancer agents that cause an increase in acetylated histones and thus restore the expression of dormant tumor-suppressor and other genes related to cell differentiation, cell-cycle arrest or apoptosis of tumor cells. The Ras inhibitor farnesylthiosalicylic acid (FTS, salirasib) attenuates cancer cell proliferation in vitro and in vivo and, under certain circumstances, induces cell death. FTS by itself does not induce differentiation or complete growth arrest. The abovementioned activity of VPA as a differentiation agent suggested that it might be worth investigating its possible therapeutic potential in synergistic combination with FTS. Here, we examined whether the combined application of VPA and FTS could synergistically inhibit the proliferation of cancer cells that express oncogenic K-Ras (A549 nonsmall-cell lung carcinoma cells), DLD1 (colon carcinoma cells) or chronically active wild-type K-Ras and constitutively active B-Raf (ARO, thyroid carcinoma cells). The results showed that combined treatment with VPA and FTS synergistically reduces proliferation in all of these cancer cell lines by downregulating Ras and blocking the expression of Survivin and Aurora A. These alterations, which were most pronounced following the combined treatment, led to a mitotic crisis, as reflected by mislocalization of the chromosomal passenger complex. Our findings thus demonstrate that combination therapy with VPA and FTS might offer a promising therapeutic approach to the treatment of epithelial tumors.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Proteínas Asociadas a Microtúbulos/genética , Proteínas Serina-Treonina Quinasas/genética , Antineoplásicos/uso terapéutico , Aurora Quinasas , Western Blotting , Carcinoma de Pulmón de Células no Pequeñas/patología , Carcinoma de Células Pequeñas/patología , División Celular , Línea Celular Tumoral , Neoplasias del Colon/patología , Regulación hacia Abajo/efectos de los fármacos , Farnesol/análogos & derivados , Farnesol/uso terapéutico , Citometría de Flujo , Inhibidores de Histona Desacetilasas/uso terapéutico , Histona Desacetilasas/genética , Histona Desacetilasas/metabolismo , Humanos , Proteínas Inhibidoras de la Apoptosis , Neoplasias Pulmonares/patología , ARN Neoplásico/genética , ARN Neoplásico/aislamiento & purificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Salicilatos/uso terapéutico , Survivin , Ácido Valproico/uso terapéuticoRESUMEN
BACKGROUND: Myocarditis is an inflammatory disorder of the heart in which T lymphocytes have a central role. No effective treatment is currently at hand for management of the myocarditis. Lymphocyte function requires the active signal transducer Ras. We thus hypothesized that S-farnesylthiosalicylic acid (FTS), a synthetic small molecule that detaches Ras from the inner cell membrane and induces its rapid degradation, will attenuate experimental autoimmune myocarditis (EAM). METHODS AND RESULTS: Two groups of Lewis rats were induced to develop EAM by immunization with porcine cardiac myosin. Group A received 5 mg/kg of FTS, and group B received phosphate-buffered saline (PBS) according to two protocols: FTS or PBS was given 2 days before myosin immunization in protocol 1 and FTS or PBS was given 14 days after myosin immunization in protocol 2. FTS significantly suppressed myocarditis, and this effect was accompanied by a reduction in myosin-specific cellular and humoral immune responses. In the longer regimen, FTS treatment for 6 weeks was associated with preservation of myocardial function made evident by echocardiography. In vitro, FTS significantly attenuated the proliferation of lymphocytes from untreated myocarditic rats to myosin. CONCLUSIONS: FTS is effective in suppressing the progression of EAM and its consequent functional myocardial dysfunction. The effect may be mediated by suppression of the cellular and humoral responses to myosin.
Asunto(s)
Enfermedades Autoinmunes/tratamiento farmacológico , Inhibidores Enzimáticos/farmacología , Farnesol/análogos & derivados , Miocarditis/tratamiento farmacológico , Salicilatos/farmacología , Proteínas ras/antagonistas & inhibidores , Animales , Enfermedades Autoinmunes/inmunología , Enfermedades Autoinmunes/patología , Proliferación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Farnesol/farmacología , Corazón/efectos de los fármacos , Ganglios Linfáticos/efectos de los fármacos , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/patología , Activación de Linfocitos/efectos de los fármacos , Miocarditis/inmunología , Miocarditis/patología , Miocardio/inmunología , Miocardio/patología , Miosinas/inmunología , Ratas , Ratas Endogámicas Lew , Transducción de Señal/efectos de los fármacos , Porcinos/inmunología , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Linfocitos T/patologíaRESUMEN
Myocardial injury, developed after a period of ischemia/reperfusion (I/R) results in the destruction of functional heart tissue, this being replaced by scar tissue. Intracellular signaling pathways mediating cardiomyocyte death are partially understood and involve the activation of Ras. p38-MAPK, JNK and Mst-1 are downstream effectors of Ras protein. We hypothesized that S-farnesylthiosalicylic acid (FTS), a synthetic small molecule that detaches Ras from the inner cell membrane, consequently inhibiting Ras activity, reduces I/R myocardial injury in vitro and in vivo. Wistar rat hearts were isolated, mounted on the Langendorff apparatus and subjected to ischemia (30 min, 37 degrees C) and reperfusion. During the reperfusion period, the hearts were perfused with FTS (1 microM) solution or control buffer. Left anterior descending (LAD) ligation and subsequent reperfusion was performed in two groups of Wistar rats. Rats received 5mg/kg FTS or PBS according to two protocols: (A) FTS or PBS were administered daily 7 days prior, immediately before and 14 days (every other day) after LAD occlusion or (B) every other day for 14 days post-I/R. Hearts from FTS-treated rats (Langendorff) and FTS-treated rats (protocol A) showed a significant improvement in myocardial performance and smaller scar tissue compared with the PBS group. Infarct size in the FTS-treated group was 12.7+/-2% vs. 23.7+/-4% in the PBS-treated (in vitro) group and 17.3+/-2.5% vs. 36+/-7% compared with control I/R rats (in vivo) p<0.05. These effects may be associated with the down regulation of JNK as a short-term effector and with Mst-1 in the long-term remodeling process.
Asunto(s)
Farnesol/análogos & derivados , Daño por Reperfusión Miocárdica/prevención & control , Miocardio/metabolismo , Salicilatos/farmacología , Proteínas ras/antagonistas & inhibidores , Animales , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Esquema de Medicación , Farnesol/administración & dosificación , Farnesol/farmacología , Pruebas de Función Cardíaca , Técnicas In Vitro , Masculino , Daño por Reperfusión Miocárdica/metabolismo , Ratas , Ratas Endogámicas Lew , Ratas Wistar , Salicilatos/administración & dosificaciónRESUMEN
The removal of degenerated myelin is essential for repair in Wallerian degeneration that follows traumatic injury to axons and in autoimmune demyelinating diseases (e.g., multiple sclerosis). Microglia can remove degenerated myelin through phosphatidylinositol-3-kinase (PI3K)-dependent phagocytosis mediated by complement receptor-3 (CR3/MAC-1) and scavenger receptor-AI/II (SRAI/II). Paradoxically, these receptors are expressed in microglia after injury but myelin is not phagocytosed. Additionally, Galectin-3/MAC-2 is expressed in microglia that phagocytose but not in microglia that do not phagocytose, suggesting that Galectin-3/MAC-2 is instrumental in activating phagocytosis. S-trans, trans-farnesylthiosalicylic (FTS), which inhibits Galectin-3/MAC-2 dependent activation of PI3K through Ras, inhibited phagocytosis. K-Ras-GTP levels and PI3K activity increased during normal phagocytosis and decreased during FTS-inhibited phagocytosis. Galectin-3/MAC-2, which binds and stabilizes active Ras, coimmunoprecipitated with Ras and levels of the coimmunoprecipitate increased during normal phagocytosis. A role for Galectin-3/MAC-2 dependent activation of PI3K through Ras, mostly K-Ras, is thus suggested. An explanation may thus be offered for deficient phagocytosis by microglia that express CR3/MAC-1 and SRAI/II without Galectin-3/MAC-2 and efficient phagocytosis when CR3/MAC-1 and SRAI/II are co-expressed with Galectin-3/MAC-2.
Asunto(s)
Galectina 3/metabolismo , Antígeno de Macrófago-1/metabolismo , Microglía/inmunología , Vaina de Mielina/metabolismo , Fagocitosis/inmunología , Receptores Depuradores de Clase A/metabolismo , Animales , Animales Recién Nacidos , Células Cultivadas , Enfermedades Desmielinizantes/inmunología , Enfermedades Desmielinizantes/metabolismo , Enfermedades Desmielinizantes/fisiopatología , Gliosis/inmunología , Gliosis/metabolismo , Gliosis/fisiopatología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Regeneración Nerviosa/inmunología , Fosfatidilinositol 3-Quinasas/metabolismo , Transducción de Señal/inmunología , Degeneración Walleriana/inmunología , Degeneración Walleriana/metabolismo , Degeneración Walleriana/fisiopatología , Proteínas ras/metabolismoRESUMEN
Aberrant Ras pathway functions contribute to the malignant phenotype of lung cancers. Inhibitors of Ras might therefore be considered as potential drugs for lung cancer therapy. Here, we show that the Ras inhibitor farnesylthiosalicylic acid (salirasib) inhibits proliferation of human lung cancer cells harboring a mutated K-ras gene (A549, H23, or HTB54) or overexpressing a growth factor receptor (H1299 or HTB58) and enhances the cytotoxic effect of the chemotherapeutic drug gemcitabine. Salirasib inhibited active K-Ras in A549 cells, reversed their transformed morphology, and inhibited their anchorage-independent growth in vitro. Tumor growth in A549 and HTB58 cell nude mouse models was inhibited by i.p. administration of salirasib. P.o. formulated salirasib also inhibited A549 cell tumor growth. Our results suggest that p.o. salirasib may be considered as a potential treatment for lung cancer therapy.
Asunto(s)
Antineoplásicos/farmacología , División Celular/efectos de los fármacos , Farnesol/análogos & derivados , Neoplasias Pulmonares/patología , Salicilatos/farmacología , Proteínas ras/antagonistas & inhibidores , Animales , Línea Celular Tumoral , Farnesol/farmacología , Humanos , Ratones , Ratones DesnudosRESUMEN
PURPOSE: Our aim was to examine whether active Ras and MycN cooperation contributes to the malignant phenotype of human neuroblastoma with amplified MycN gene, an aggressive incurable tumor. EXPERIMENTAL DESIGN: Human neuroblastoma LAN-1 cells, in which the MycN gene is amplified, were used to examine the impact of the Ras inhibitor farnesylthiosalicylic acid on cell growth, on the levels Ras and MycN proteins, and on profiles of gene expression. RESULTS: We show that LAN-1 cells express relatively large amounts of MycN and active Ras-GTP. Inhibition of active Ras by farnesylthiosalicylic acid led to attenuation of the Raf-MEK-ERK and phosphoinositide 3-kinase-Akt-glycogen synthase-3 (GSK-3) pathways, to reduction in cyclin D1, phospho-retinoblastoma, and E2F, and to increase in the cyclin-dependent kinase inhibitor p27 and in retinoblastoma-binding protein-1, an inhibitor of E2F transcriptional activity. Ras inhibition by farnesylthiosalicylic acid or by a dominant-negative Ras also led to complete disappearance of MycN protein from the nuclei of LAN-1 cells. This was a result of blocking of Akt inactivation of GSK-3, leading to GSK-3-dependent phosphorylation with consequent proteosomal degradation of MycN. Loss of active Ras and of MycN in LAN-1 cells was manifested in profiles of gene expression that could be expected from the loss of MycN transcriptional activity and of Ras signaling. These changes explain the farnesylthiosalicylic acid-induced inhibition of LAN-1 cell growth. CONCLUSIONS: Active Ras is needed to block MycN degradation, promoting cooperative Ras- and MycN-dependent cell cycle progression in LAN-1 cells. Ras inhibitors are therefore likely candidates for the treatment of advanced neuroblastoma characterized by high expression of MycN.
Asunto(s)
Farnesol/análogos & derivados , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas ras/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Regulación hacia Abajo/efectos de los fármacos , Farnesol/farmacología , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Glucógeno Sintasa Quinasa 3/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Immunoblotting , Hibridación Fluorescente in Situ , Microscopía Confocal , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Neuroblastoma/genética , Neuroblastoma/metabolismo , Neuroblastoma/patología , Análisis de Secuencia por Matrices de Oligonucleótidos , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt , Proteínas Proto-Oncogénicas c-myc/genética , Proteína 1 de Unión a Retinoblastoma , Salicilatos/farmacología , Transducción de Señal/efectos de los fármacos , Transfección , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Quinasas raf/metabolismo , Proteínas ras/genéticaRESUMEN
Ras transformation requires Ras membrane anchorage, which is promoted by a farnesylcysteine carboxymethyl ester and by additional sequences specific to each Ras isoform. We showed previously that S-trans,trans-farnesylthiosalicylic acid (FTS) disrupts Ras membrane anchorage and that this disturbance contributes to inhibition of cell transformation and tumor growth. Most tumor cells develop resistance to anticancer agents. Here we examined whether tumor cells develop resistance to FTS and evaluated the therapeutic potential of FTS combined with cytotoxic drugs, because oncogenic Ras promotes antiapoptotic signals in tumors of epithelial origin. We showed that Panc-1 pancreatic cancer cells, SW480 colon cancer cells, and H-ras (EJ)-transformed Rat-1 fibroblasts exposed to FTS for prolonged periods (>6 months) do not escape FTS-induced growth inhibition and do not develop drug resistance. These cells continued to express reduced amounts of Ras, exhibit a reversed phenotype, and show an altered response to the cytotoxic drugs doxorubicin and gemcitabine. FTS-treated Panc-1 or SW480 cells acquired sensitivity to the cytotoxic drugs, whereas FTS-treated EJ cells lost sensitivity to doxorubicin, reflecting the opposite effects of oncogenic Ras on the survival of epithelial cells and fibroblasts. Treatment with FTS led to a marked increase in sensitivity to gemcitabine of the formerly resistant SW480 cells and a 100-fold increase in sensitivity to gemcitabine of Panc-1 cells. Such treatment in mice with preexisting Panc-1 tumors provided a synergistic effect of FTS and gemcitabine, leading to enhanced inhibition of tumor growth and a 65% increase in survival rate.