RESUMEN
The inhibitor-of-apoptosis family member survivin has been reported to inhibit apoptosis and regulate mitosis and cytokinesis. In multiple myeloma, survivin has been described to be involved in downstream sequelae of various therapeutic agents. We assessed 1093 samples from previously untreated patients, including two independent cohorts of 392 and 701 patients, respectively. Survivin expression was associated with cell proliferation, adverse prognostic markers, and inferior event-free and overall survival, supporting the evaluation of survivin as a therapeutic target in myeloma. The small molecule suppressant of survivin--YM155--is in clinical development for the treatment of solid tumors. YM155 potently inhibited proliferation and induced apoptosis in primary myeloma cells and cell lines. Gene expression and protein profiling revealed the critical roles of IL6/STAT3-signaling and the unfolded protein response in the efficacy of YM155. Both pathways converged to down regulate anti-apoptotic Mcl-1 in myeloma cells. Conversely, growth inhibition and apoptotic cell death by YM155 was rescued by ectopic expression of Mcl-1 but not survivin, identifying Mcl-1 as the pivotal downstream target of YM155 in multiple myeloma. Mcl-1 expression was likewise associated with adverse prognostic markers, and inferior survival. Our results strongly support the clinical evaluation of YM155 in patients with multiple myeloma.
Asunto(s)
Antineoplásicos/farmacología , Imidazoles/farmacología , Proteínas Inhibidoras de la Apoptosis/metabolismo , Mieloma Múltiple/diagnóstico , Mieloma Múltiple/tratamiento farmacológico , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Naftoquinonas/farmacología , Respuesta de Proteína Desplegada , Apoptosis/efectos de los fármacos , Apoptosis/genética , Procesos de Crecimiento Celular/efectos de los fármacos , Procesos de Crecimiento Celular/genética , Línea Celular Tumoral , Estudios de Cohortes , Regulación hacia Abajo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Proteínas Inhibidoras de la Apoptosis/genética , Interleucina-6/metabolismo , Mieloma Múltiple/mortalidad , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/genética , Pronóstico , Factor de Transcripción STAT3/metabolismo , Análisis de Supervivencia , Survivin , Transcriptoma , Respuesta de Proteína Desplegada/efectos de los fármacos , Respuesta de Proteína Desplegada/genéticaRESUMEN
Circulating microRNAs (miRNAs) have been recently detected in extracellular body fluids and proved themselves as promising biomarkers for a broad spectrum of diseases. The techniques to isolate, detect, and characterize extracellular miRNAs vary significantly from report to report. In this chapter we describe a consistent, efficient and highly reproducible protocol for isolation and detection of microvesicles-free circulating miRNA from human blood plasma or cell culture media. Furthermore, since exosomes-free circulating miRNAs are associated with Argonaut proteins (the same proteins to which miRNAs are bound inside the cell), we provide a protocol for immunoprecipitation of AGO2 associated miRNAs.
Asunto(s)
Proteínas Argonautas/aislamiento & purificación , Leucocitos Mononucleares/química , MicroARNs/aislamiento & purificación , Anticuerpos Monoclonales/química , Proteínas Argonautas/metabolismo , Medios de Cultivo Condicionados/química , Humanos , Inmunoprecipitación , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/metabolismo , MicroARNs/clasificación , Análisis de Secuencia por Matrices de Oligonucleótidos , Unión Proteica , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Mature miRNAs are 19-24 nucleotide noncoding RNAs that post-transcriptionally regulate gene expression in living cells by mediating targeted hydrolysis and translation inhibition of mRNAs. In recent years, miRNAs have been detected in a variety of biological fluids as extracellular nuclease-resistant entities. Importantly, extracellular circulating miRNAs are aberrantly expressed in blood plasma or serum during the course of many diseases, including cancer, and are promising noninvasive biomarkers. However, the biological function of extracellular miRNAs remains questionable. In this article, we summarise the current theories regarding extracellular miRNA origin and function, and suggest that these miRNAs are mostly byproducts of cellular activity. Nevertheless, some extracellular miRNA species might also carry cell-cell signaling function.
Asunto(s)
Sangre/metabolismo , Líquido Extracelular/metabolismo , MicroARNs/metabolismo , Animales , Apoptosis , Biomarcadores/sangre , Biomarcadores/metabolismo , Comunicación Celular , Humanos , MicroARNs/sangre , Estabilidad del ARN , Vesículas Secretoras/metabolismoRESUMEN
In contrast to normal cells, malignant cells are frequently aneuploid and contain multiple centrosomes. To allow for bipolar mitotic division, supernumerary centrosomes are clustered into two functional spindle poles in many cancer cells. Recently, we have shown that griseofulvin forces tumor cells with supernumerary centrosomes to undergo multipolar mitoses resulting in apoptotic cell death. Here, we describe the characterization of the novel small molecule GF-15, a derivative of griseofulvin, as a potent inhibitor of centrosomal clustering in malignant cells. At concentrations where GF-15 had no significant impact on tubulin polymerization, spindle tension was markedly reduced in mitotic cells upon exposure to GF-15. Moreover, isogenic cells with conditional centrosome amplification were more sensitive to GF-15 than parental controls. In a wide array of tumor cell lines, mean inhibitory concentrations (IC(50)) for proliferation and survival were in the range of 1 to 5 µmol/L and were associated with apoptotic cell death. Importantly, treatment of mouse xenograft models of human colon cancer and multiple myeloma resulted in tumor growth inhibition and significantly prolonged survival. These results show the in vitro and in vivo antitumor efficacy of a prototype small molecule inhibitor of centrosomal clustering and strongly support the further evaluation of this new class of molecules.
Asunto(s)
Centrosoma/efectos de los fármacos , Griseofulvina/análogos & derivados , Línea Celular Tumoral , Griseofulvina/farmacología , Humanos , Técnicas In Vitro , Distribución TisularRESUMEN
MicroRNAs (miRNAs), a class of post-transcriptional gene expression regulators, have recently been detected in human body fluids, including peripheral blood plasma as extracellular nuclease resistant entities. However, the origin and function of extracellular circulating miRNA remain essentially unknown. Here, we confirmed that circulating mature miRNA in contrast to mRNA or snRNA is strikingly stable in blood plasma and cell culture media. Furthermore, we found that most miRNA in plasma and cell culture media completely passed through 0.22 µm filters but remained in the supernatant after ultracentrifugation at 110 000g indicating the non-vesicular origin of the extracellular miRNA. Furthermore, western blot immunoassay revealed that extracellular miRNA ultrafiltrated together with the 96 kDa Ago2 protein, a part of RNA-induced silencing complex. Moreover, miRNAs in both blood plasma and cell culture media co-immunoprecipited with anti-Ago2 antibody in a detergent free environment. This is the first study to show that extracellular miRNAs are predominantly exosomes/microvesicles free and are associated with Ago proteins. We hypothesize that extracellular miRNAs are in the most part by-products of dead cells that remain in extracellular space due to the high stability of the Ago2 protein and Ago2-miRNA complex. Nevertheless, our data does not reject the possibility that some miRNAs can be associated with exosomes.