RESUMEN
Somatic mutations in isocitrate dehydrogenase (IDH)-1 and -2 have recently been described in glioma. This mutation leads to a neomorphic enzymatic activity as the conversion of isocitrate to alpha ketoglutarate (αKG) is replaced by the conversion of αKG to D-2-hydroxyglutarate (D-2HG) with NADPH oxidation. It has been suggested that this oncometabolite D-2HG via inhibition of αKG-dioxygenases is involved in multiple functions such as epigenetic modifications or hypoxia responses. The present study is aimed at deciphering how the mutant IDH can affect cancer pathogenesis, in particular with respect to its associated oncometabolite D-2HG. We show that the overexpression of mutant IDH in glioma cells or treatment with D-2HG triggered an increase in cell proliferation. However, although mutant IDH reduced cell sensitivity to the apoptotic inducer etoposide, D-2HG exhibited no effect on apoptosis. Instead, we found that the apoptotic effect was mediated through the mitochondrial NADH pool reduction and could be inhibited by oxamate. These data show that besides D-2HG production, mutant IDH affects other crucial metabolite pools. These observations lead to a better understanding of the biology of IDH mutations in gliomas and their response to therapy.
Asunto(s)
Proliferación Celular/genética , Glioma/genética , Isocitrato Deshidrogenasa/genética , Apoptosis/efectos de los fármacos , Apoptosis/genética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Etopósido/administración & dosificación , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Glioma/patología , Glutaratos/metabolismo , Humanos , Isocitrato Deshidrogenasa/biosíntesis , Complejo Cetoglutarato Deshidrogenasa , Mitocondrias/metabolismo , Mitocondrias/patología , Mutación , NAD/metabolismoRESUMEN
Lipids are key regulators of cell physiology through the control of many aspects of cellular life and survival. In particular, lipids have been implicated at different levels and through many different mechanisms in the cell death program called apoptosis. Here, we discuss the action of lipids in the regulation of the activation and the integration of Bax into the mitochondrial outer membrane, a key pro-apoptotic member of the BCL-2 family. We describe how, during apoptosis, lipids can act simultaneously or in parallel as receptors or ligands for Bax to stimulate or inhibit its pro-death activity.
Asunto(s)
Apoptosis/fisiología , Lípidos de la Membrana/metabolismo , Membranas Mitocondriales/metabolismo , Proteína X Asociada a bcl-2/metabolismo , Animales , HumanosRESUMEN
Glioma stem cells are highly resistant to cell death and as such are supposed to contribute to tumor recurrence by eluding anticancer treatments. Here, we show that spheroids that contain rat neural stem cells (NSCs) or rat glioma stem cells (cancer stem cells, CSCs) express isoforms 1 and 2 of pyruvate kinase (PKM1 and PKM2); however, the expression of PKM2 is considerably higher in glioma spheroids. Silencing of PKM2 enhances both apoptosis and differentiation of rat and human glioma spheroids. We establish that PKM2 was implicated in glioma spheroid differentiation through its interaction with Oct4, a major regulator of self-renewal and differentiation in stem cells. The small molecule Dichloroacetate (DCA), a pyruvate dehydrogenase kinase inhibitor, increases the amount of PKM2/Oct4 complexes and thus inhibited Oct4-dependent gene expression. Taken together, our results highlight a new molecular pathway through which PKM2 can manage gliomagenesis via the control of glioma stemness by Oct4.
Asunto(s)
Apoptosis , Diferenciación Celular , Glioma/metabolismo , Células Madre Neoplásicas/citología , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Piruvato Quinasa/metabolismo , Animales , Muerte Celular , Línea Celular Tumoral , Células Cultivadas , Glioma/genética , Glioma/fisiopatología , Humanos , Masculino , Ratones , Ratones Desnudos , Células Madre Neoplásicas/metabolismo , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/genética , Piruvato Quinasa/genética , Ratas , Ratas Sprague-DawleyRESUMEN
According to our previous study suggesting that antioxidant properties of phytochemicals in the diet decrease glioma aggressiveness, we used a SUVIMAX-like diet ("Supplementation en VItamines et Minéraux AntioXydants") (enriched with alpha-tocopherol, beta carotene, vitamin C, zinc, and sodium selenite), adapted to rats. The present results showed that each of the antioxidants inhibited growth of glioma cells in vitro. When used in combination for in vivo studies, we showed a highly significant delay in the clinical signs of the disease, but not a statistical significant difference in the incidence of glioma in an Ethyl-nitrosourea (ENU)-model. The SUVIMAX-like diet decreased candidate markers of tumoral aggressiveness and gliomagenesis progression. The mRNA expressions of 2 common markers in human glioma: Mn-SOD (Manganese Superoxide Dismutase) and IGFBP5 (insulin growth factor binding protein) were reduced in the tumors of rats fed the antioxidant diet. In addition, the transcripts of two markers linked to brain tumor proliferation, PDGFRb (platelet-derived growth factor receptor beta) and Ki-67, were also significantly decreased. On the whole, our results suggest a protective role for antioxidants to limit aggressiveness and to some extent, progression of gliomas, in a rat model.
Asunto(s)
Antioxidantes/farmacología , Neoplasias Encefálicas/prevención & control , Etilnitrosourea/toxicidad , Animales , Ácido Ascórbico/farmacología , Neoplasias Encefálicas/inducido químicamente , Proliferación Celular/efectos de los fármacos , Femenino , Glioma/inducido químicamente , Glioma/prevención & control , Proteína 5 de Unión a Factor de Crecimiento Similar a la Insulina/genética , Proteína 5 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Antígeno Ki-67/sangre , Masculino , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Selenito de Sodio/farmacología , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismo , Zinc/farmacología , alfa-Tocoferol/farmacología , beta Caroteno/farmacologíaRESUMEN
Apoptosis has a crucial role in anti-cancer treatment. The proteins of the BCL-2 family are core members of the apoptotic program. Thus, we postulated that alterations in the expression of BCL-2 protein family, and in particular in that of the Bcl-2 homology domain 3 (BH3)-only proteins (which can neutralized anti-apoptotic proteins or activate pro-apoptotic proteins) could account for differences in the overall survival (OS) of patients. To test this hypothesis, we analyzed the expression of 15 members of the BCL-2 protein family (Bax, Bak, Bok, Bcl-2, Bcl-xl, Bcl-w, Mcl-1, Bad, Bid, Bim, Bik, Bmf, Hrk, Noxa and Puma) in glioblastoma multiforme (GBM) tumors, the most frequent brain tumor in adults. We found that none of the individual expression of these proteins is associated with a significant variation in OS of the patients. However, when all BH3 proteins were pooled to determine a BH3(score), this score was significantly correlated with OS of GBM patients. We also noted that patients with a have high level of phospho-Bad and phospho-Bim displayed a lower OS. Thus, BH3 scoring/profiling could be used as an independent prognostic factor in GBM when globally analyzed.
Asunto(s)
Proteína Proapoptótica que Interacciona Mediante Dominios BH3/genética , Proteína Proapoptótica que Interacciona Mediante Dominios BH3/metabolismo , Glioblastoma/metabolismo , Anciano , Femenino , Glioblastoma/diagnóstico , Glioblastoma/genética , Glioblastoma/mortalidad , Humanos , Masculino , Persona de Mediana Edad , Familia de Multigenes , Fosforilación , Pronóstico , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Análisis de SupervivenciaRESUMEN
The Bax protein (Bcl-2-associated X protein) is pivotal for the apoptotic process. Bax, which resides in an inactive form in the cytosol of healthy cells, is activated during the early stages of apoptosis and becomes associated with mitochondria through poorly understood mechanisms. In this study, we show that a family of bioactive lipids, namely prostaglandins, regulates Bax-dependent apoptosis. The prostaglandin E(2) (PGE(2)) or its derivative PGA(2) binds to Bax, induces its change of conformation, and thereby triggers apoptosis. A cysteine present in the loop between the two transmembrane α-helices of Bax, Cys126 is critical for its activation. PGD(2) inhibits PGE(2) binding to Bax and PGE(2)-induced apoptosis, as well as cell death induced by staurosporine and UV-B in various cell lines. This result is consistent with the fact that apoptosis is accompanied during these treatments by an increase in PGE(2). This process is distinct, yet cooperative, from that involving the BH3-only protein Bid. Our results establish that the PGE(2)/PGD(2) balance is involved in a new early mechanism of control in the activation of Bax during apoptosis.
Asunto(s)
Apoptosis , Prostaglandinas/metabolismo , Proteína X Asociada a bcl-2/metabolismo , Animales , Apoptosis/efectos de los fármacos , Proteína Proapoptótica que Interacciona Mediante Dominios BH3/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Cisteína/metabolismo , Dinoprostona/farmacología , Humanos , Modelos Biológicos , Modelos Moleculares , Prostaglandina D2/farmacología , Unión Proteica/efectos de los fármacos , Estructura Secundaria de Proteína , Ratas , Transducción de Señal/efectos de los fármacos , Proteína X Asociada a bcl-2/químicaRESUMEN
Bcl-2 homologues (such as Bcl-x(L)) promote survival in part through sequestration of "activator" BH3-only proteins (such as Puma), preventing them from directly activating Bax. It is thus assumed that inhibition of interactions between activators and Bcl-x(L) is a prerequisite for small molecules to antagonize Bcl-x(L) and induce cell death. The biological properties, described here of a terphenyl-based alpha-helical peptidomimetic inhibitor of Bcl-x(L) attest that displacement of Bax from Bcl-x(L) is also critical. Terphenyl 14 triggers Bax-dependent but Puma-independent cell death, disrupting Bax/Bcl-x(L) interactions without affecting Puma/Bcl-x(L) interactions. In cell-free assays, binding of inactive Bax to Bcl-x(L), followed by its displacement from Bcl-x(L) by terphenyl 14, produces mitochondrially permeabilizing Bax molecules. Moreover, the peptidomimetic kills yeast cells that express Bax and Bcl-x(L), and it uses Bax-binding Bcl-x(L) to induce mammalian cell death. Likewise, ectopic expression of Bax in yeast and mammalian cells enhances sensitivity to another Bcl-x(L) inhibitor, ABT-737, when Bcl-x(L) is present. Thus, the interaction of Bcl-x(L) with Bax paradoxically primes Bax at the same time it keeps Bax activity in check, and displacement of Bax from Bcl-x(L) triggers an apoptotic signal by itself. This mechanism might contribute to the clinical efficiency of Bcl-x(L) inhibitors.
Asunto(s)
Proteína X Asociada a bcl-2/metabolismo , Proteína bcl-X/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Sitios de Unión , Compuestos de Bifenilo/farmacología , Muerte Celular/efectos de los fármacos , Muerte Celular/genética , Muerte Celular/fisiología , Línea Celular Tumoral , Sistema Libre de Células , Células Cultivadas , Técnicas de Silenciamiento del Gen , Células HeLa , Humanos , Técnicas In Vitro , Ratones , Ratones Noqueados , Mitocondrias/metabolismo , Modelos Biológicos , Imitación Molecular , Datos de Secuencia Molecular , Mutación , Nitrofenoles/farmacología , Piperazinas/farmacología , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , ARN Interferente Pequeño/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Sulfonamidas/farmacología , Compuestos de Terfenilo/farmacología , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Técnicas del Sistema de Dos Híbridos , Proteína X Asociada a bcl-2/deficiencia , Proteína X Asociada a bcl-2/genética , Proteína bcl-X/deficiencia , Proteína bcl-X/genéticaRESUMEN
Disruption of apoptosis is considered as an important factor aiding tumorigenesis, and aberrant DNA methylation of apoptosis-associated genes could be an important and significant mechanism through which tumor cells avoid apoptosis. However, little is known about (1) the impact of methylation status of apoptosis-associated genes on the presence of apoptosis evasion phenotype in glioma; and (2) the molecular mechanism governing the aberrant methylation of apoptosis-associated genes in glioma. By analyzing human glioma biopsies, we first show that low level of apoptosis in tumor is correlated with aberrant methylation of the bcl-2, bax and XAF-1 genes, but not with the aberrant methylation of the bcl-w, survivin, TMS1, caspase-8 and HRK genes. Our work also indicates that the expression levels of DNA methyltransferase 1 (Dnmt1), Dnmt3b and Dnmt1/Dnmt3a coregulate the methylation status of survivin, TMS1 and caspase-8, whereas no correlation was observed between the expression level of Dnmts and the methylation status of the bcl-w, bcl-2, bax, XAF-1 and HRK genes. Thus, these results indicate that the epigenetic regulation of some apoptosis-regulated genes could dictate whether glioma harbors the apoptosis evasion phenotype, and provide some bases to the identification of the methylation machineries of apoptosis-associated genes for which the Dnmt expression acts as a limiting factor.
Asunto(s)
Apoptosis/genética , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Glioblastoma/enzimología , Proteínas Adaptadoras Transductoras de Señales , Proteínas Reguladoras de la Apoptosis , Proteínas Adaptadoras de Señalización CARD , Caspasa 8/metabolismo , Proteínas del Citoesqueleto/metabolismo , ADN (Citosina-5-)-Metiltransferasas/genética , Metilación de ADN , Epigénesis Genética , Glioblastoma/genética , Humanos , Proteínas Inhibidoras de la Apoptosis/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Survivin , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Glioblastoma Multiforme (GBM) tumors are the most common type of brain tumors. These tumors are in general very malignant and can be characterized as rapidly progressive astrocytomas. The pathological characteristics of these tumors are exemplified by an active invasiveness, necrosis and a specialized form of angiogenesis, known as microvascular hyperplasia. These pathological features are thought to be due to tissue hypoxia. Cells that are under hypoxic stress can either develop an adaptive response that includes increasing the rate of glycolysis and angiogenesis or undergo cell death by promoting apoptosis and/or necrosis. The ability of tumor cells to maintain a balance between an adaptation to hypoxia and cell death is regulated by a family of transcription factors called hypoxia-inducing factors (HIF), which are essential for the regulation of the expression of a large number of hypoxia-responsive genes. The hypothesis that tumor hypoxia would facilitate the likelihood of metastases, tumor recurrence, resistance to chemotherapy and radiotherapy and the invasive potential; all of which culminate in a decrease in patient survival. In this review we will summarize the role of hypoxia in GBM with regard to drug therapy and toxicity and attempt to describe the possible interactions between hypoxia and apoptosis.
Asunto(s)
Apoptosis , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Hipoxia de la Célula , Glioblastoma/metabolismo , Glioblastoma/patología , Factor 1 Inducible por Hipoxia/genética , Animales , Neoplasias Encefálicas/terapia , Resistencia a Antineoplásicos , Quimioterapia , Regulación Neoplásica de la Expresión Génica , Glioblastoma/terapia , Glucólisis , Humanos , Factor 1 Inducible por Hipoxia/metabolismo , Necrosis , Invasividad Neoplásica , Recurrencia Local de Neoplasia , Neovascularización Patológica , Radioterapia , Transducción de SeñalRESUMEN
Prostaglandin E(2) plays multiple roles both in the physiology and the physiopathology of human brain, which are not completely understood. We have identified in a subset of human glioblastoma multiforme (GBM) tumors, the most common form of adult brain cancer, an increased expression of mPGES-1, the enzyme which catalyses the isomerization of PGH(2) into PGE(2) downstream of cyclooxygenase 2 (COX-2). The sensitivity of primary cultures of GBM to apoptosis was augmented by the overexpression of mPGES-1, whereas the knockdown of its expression by shRNA decreased the apoptotic threshold in vitro and stimulated tumor growth in vivo. Adding extracellular PGE(2) in the culture medium failed to reproduce mPGES-1 effect on the cell viability in vitro. However, the intracellular injection of PGE(2) induced a dose-dependent apoptosis in GBM cultures, which was dependent on the presence of Bax, a pro-apoptotic protein. We show that PGE(2) physically associates with Bax, triggering its apoptotic-like change in conformation and its subsequent association with mitochondria. Our results raise questions about the role of PGE(2) in the control of apoptosis and in its potential impact in central nervous system pathologies.
Asunto(s)
Apoptosis , Neoplasias Encefálicas/metabolismo , Dinoprostona/biosíntesis , Glioblastoma/metabolismo , Proteína X Asociada a bcl-2/metabolismo , Animales , Neoplasias Encefálicas/mortalidad , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Dinoprostona/farmacología , Glioblastoma/mortalidad , Glioblastoma/patología , Humanos , Oxidorreductasas Intramoleculares/metabolismo , Masculino , Ratones , Ratones Desnudos , Prostaglandina-E Sintasas , Tasa de Supervivencia , Proteína X Asociada a bcl-2/químicaRESUMEN
The association of Bax with mitochondria is an essential step in the implementation of apoptosis. By using a bacterial two-hybrid assay and crosslinking strategies, we have identified TOM22, a component of the translocase of the outer mitochondrial membrane (TOM), as a mitochondrial receptor of Bax. Peptide mapping showed that the interaction of Bax with TOM22 involved the first alpha helix of Bax and possibly two central alpha helices, which are homologous to the pore forming domains of some toxins. Antibodies directed against TOM22 or an antisense knockdown of the expression of TOM22 specifically inhibited the association of Bax with mitochondria and prevented Bax-dependent apoptosis. In yeast, a haploid strain for TOM22 exhibited a decreased expression of TOM22 and mitochondrial association of ectopically expressed human Bax. Our data provide a new perspective on the mechanism of association of Bax with mitochondria as it involves a classical import pathway.
Asunto(s)
Proteínas de Transporte de Membrana/metabolismo , Mitocondrias Hepáticas/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/genética , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Membranas Mitocondriales/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Canales Aniónicos Dependientes del Voltaje/metabolismo , Proteína X Asociada a bcl-2/metabolismo , Animales , Apoptosis/fisiología , Silenciador del Gen , Glioblastoma , Glioma , Humanos , Microscopía Confocal , Proteínas de Transporte de Membrana Mitocondrial/química , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales , Proteínas Mitocondriales/metabolismo , Unión Proteica/fisiología , Transporte de Proteínas/fisiología , Ratas , Receptores de Superficie Celular , Receptores Citoplasmáticos y Nucleares/metabolismo , Células Tumorales Cultivadas , Proteína X Asociada a bcl-2/deficienciaRESUMEN
To analyze the involvement of structured water (bound to macromolecules) in apoptosis-induced mitochondrial outer-membrane permeability, we compared the dynamics of water protons from nuclear magnetic resonance (NMR) data in apoptotic liver mitochondria with that of control mitochondria incubated in vitro with free Ca(2+) (opening of the permeability transition pore, PTP) or with Bax alpha. Our results demonstrate that water molecules in apoptotic mitochondria exhibit an accelerated translational motion of structured water common with that induced by the opening of the PTP, but limited in amplitude. On the other hand, no significant quantitative change in structured water was observed in apoptotic mitochondria, a phenomenon also observed with Bax alpha-induced permeability. We conclude that the changes observed in the different water phases differ both quantitatively and qualitatively during the opening of the PTP and the Bax alpha-induced permeability, and that the apoptotic mitochondria exhibit mixed properties between these model situations.
Asunto(s)
Apoptosis/fisiología , Calcio/farmacología , Membranas Mitocondriales/fisiología , Proteína X Asociada a bcl-2/farmacología , Animales , Western Blotting , Espectroscopía de Resonancia Magnética , Microscopía Electrónica , Mitocondrias Hepáticas/fisiología , Mitocondrias Hepáticas/ultraestructura , Membranas Mitocondriales/efectos de los fármacos , Membranas Mitocondriales/ultraestructura , Dilatación Mitocondrial , Permeabilidad/efectos de los fármacos , Ratas , Temperatura , Canales Aniónicos Dependientes del Voltaje/efectos de los fármacos , Canales Aniónicos Dependientes del Voltaje/farmacología , Canales Aniónicos Dependientes del Voltaje/fisiología , Agua/fisiologíaRESUMEN
Autophagy, a highly regulated programme found in almost all eukaryotes, is mainly viewed as a catabolic process that degrades nonessential cellular components into molecular building blocks, subsequently available for biosynthesis at a lesser expense than de novo synthesis. Autophagy is largely known to be regulated by nutritional conditions. Here we show that, in yeast cells grown under nonstarving conditions, autophagy can be induced by mitochondrial dysfunction. Electron micrographs and biochemical studies show that an autophagic activity can result from impairing the mitochondrial electrochemical transmembrane potential. Furthermore, mitochondrial damage-induced autophagy results in the preferential degradation of impaired mitochondria (mitophagy), before leading to cell death. Mitophagy appears to rely on classical macroautophagy machinery while being independent of cellular ATP collapse. These results suggest that in this case, autophagy can be envisioned either as a process of mitochondrial quality control, or as an ultimate cellular response triggered when cells are overwhelmed with damaged mitochondria.
Asunto(s)
Adenosina Trifosfato/metabolismo , Mitocondrias/fisiología , ATPasas de Translocación de Protón Mitocondriales/metabolismo , Saccharomyces cerevisiae/fisiología , Autofagia/fisiología , Muerte Celular/fisiología , Potencial de la Membrana Mitocondrial/fisiología , Proteínas Mitocondriales/metabolismo , Mutación , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
The release of holocytochrome c (cyt c) from mitochondria into the cytosol is reportedly a landmark of the execution phase of apoptosis. As shown here, the P-glycoprotein- (P-gp) expressing K562/ADR cell line (but not the parental K562 cell line) exhibits both cytosolic and mitochondrial cyt c in the absence of any signs of apoptosis. K562/ADR cells were found to be relatively resistant to a variety of different inducers of apoptosis, and blocking the P-gp did not reverse this resistance. The release of cyt c in non-apoptotic K562/ADR cells was not accompanied by that of any other mitochondrial apoptogenic protein, such as AIF or Smac/DIABLO, and was inhibited by Bcl-2 over expression. In addition, using a cell-free system, we show that mitochondria isolated from K562/ADR cells spontaneously released cyt c. These data suggest that cyt c release may be compatible with the preservation of mitochondrial integrity and function, as well as cell proliferation.
Asunto(s)
Citocromos c/metabolismo , Citosol/metabolismo , Resistencia a Antineoplásicos , Regulación Neoplásica de la Expresión Génica , Leucemia/metabolismo , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Apoptosis , Proteínas Reguladoras de la Apoptosis , Western Blotting , Línea Celular , Línea Celular Tumoral , Proliferación Celular , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Poliacrilamida , Activación Enzimática , Ensayo de Inmunoadsorción Enzimática , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Células K562 , Microscopía Confocal , Microscopía Electrónica , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Reacción en Cadena de la Polimerasa , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Factores de Tiempo , TransfecciónRESUMEN
Apoptosis dysfunction in metastases has been suggested to participate in their poor response to conventional anticancer treatments. To address this question, we have analyzed the sensitivity to cell death induced by non-steroid anti-inflammatory drug, Sulindac, the most common drug used in colon cancer chemotherapy, 5-fluorouracil (5-FU) and the short chain fatty acid, butyrate (Bu) in cell lines derived from a primary colorectal tumor (ALT-I) as well as the liver (ALT-F) and the lymph-node (ALT-G) metastases. We have previously shown both in vitro by analyzing anchorage-independent cell proliferation and in vivo by subcutaneous injection into athymic nude mice that the ALT-F and ALT-G cells were more tumorigenic than the primary ALT-I cells. All these cell lines, derived from an untreated patient, were highly resistant to apoptosis induced by 5-FU and Sulindac but were sensitive to Bu-induced apoptosis. The resistance to apoptosis was, as quantified by the induction of caspase activity and the relative percentage of apoptotic cells, higher in the metastatic cell lines, than in the ALT cell line. When compared to the primary tumor, more anti-apoptotic bcl-2 and less pro-apoptotic bax were expressed in the liver and lymph node metastatic cell lines. Quite remarkably, the expression of bax was up-regulated during Bu-treatment, a feature that could explain its powerful pro-apoptotic activity.
Asunto(s)
Antimetabolitos Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Butiratos/farmacología , Neoplasias del Colon/patología , Fluorouracilo/farmacología , Neoplasias Hepáticas/secundario , Metástasis Linfática/patología , Anexina A5/metabolismo , Antiinflamatorios no Esteroideos/farmacología , Western Blotting , Neoplasias del Colon/metabolismo , Resistencia a Antineoplásicos , Células HT29/efectos de los fármacos , Células HT29/metabolismo , Humanos , Neoplasias Hepáticas/metabolismo , Fenotipo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Sulindac/farmacología , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Proteína X Asociada a bcl-2 , Proteína bcl-XRESUMEN
We have identified, using site-directed mutagenesis, a proline located at position 13 of Baxalpha (Bax) as crucial for the maintenance of its cytosolic conformation. The substitution of this proline by a valine results in a strong binding of Bax to mitochondria and to conformational changes monitored by a decreased sensitivity of Bax to mild proteolysis and the enhancement of its oligomerization state. Deletion of the C-terminus of Bax does not modify its intracellular localization. On the other hand, the pro-apoptotic activity of Bax is enhanced by a deletion of the C-terminus in the absence of the N-terminus but is decreased in its presence. These results suggest that both extremities functionally interact to control the activity but not the subcellular localization of Bax.
Asunto(s)
Compartimento Celular , Proteínas Proto-Oncogénicas c-bcl-2 , Proteínas Proto-Oncogénicas/metabolismo , Secuencia de Aminoácidos , Proteína Proapoptótica que Interacciona Mediante Dominios BH3 , Proteínas Portadoras/metabolismo , Citosol/metabolismo , Membranas Intracelulares/metabolismo , Mitocondrias Hepáticas/metabolismo , Datos de Secuencia Molecular , Mutación , Prolina/genética , Prolina/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Transporte de Proteínas , Proteínas Proto-Oncogénicas/genética , Proteína X Asociada a bcl-2RESUMEN
We have analyzed the expression of the anti-apoptotic proteins bcl-2, bcl-xl and that of bax, a pro-apoptotic protein, in human WHO grade II astrocytomas (LGA) and WHO grade IV glioblastoma multiforme (GBM). Tumors were obtained immediately after surgical resection and were analyzed by immunohistochemistry (IHC), laser confocal microscopy (LCM) and immunoblots. Both IHC and immunoblot analysis indicated that the expression of bcl-xl was not significantly different between LGA and GBM. IHC indicated that the expression of bcl-2 was inversely correlated to the grade of the tumors (i.e more cells were bcl-2 positive in LGA than in GBM) while the expression of bax was unaffected by the grade of the tumor. In contrast, immunoblots revealed a parallel increase in the expression of bcl-2 and bax from the low to high grade tumor, suggesting a co-regulation of the expression of these two proteins during tumoral progression. Confocal analyses provide us with another possible level of complexicity in the regulation of apoptosis in these tumors, as these markers exhibited different subcellular localizations: bcl-2 was strictly associated with mitochondria and bcl-xl was present in both cytosolic and mitochondrial compartments while bax was found essentially in the cytosol of the tumoral cells. Taken together, our data suggest that the role of bcl-2 related proteins could be regulated at different levels in human astrocytomas (expression, subcellular localization, antigen exposure ...) which should be studied by different techniques.
Asunto(s)
Neoplasias Encefálicas/patología , Glioblastoma/patología , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas/genética , Adulto , Anciano , Apoptosis/genética , Astrocitoma/química , Astrocitoma/mortalidad , Astrocitoma/patología , Neoplasias Encefálicas/química , Neoplasias Encefálicas/mortalidad , Femenino , Regulación Neoplásica de la Expresión Génica , Proteína Ácida Fibrilar de la Glía/análisis , Glioblastoma/química , Glioblastoma/mortalidad , Humanos , Immunoblotting , Inmunohistoquímica , Masculino , Microscopía Confocal , Persona de Mediana Edad , Valor Predictivo de las Pruebas , Proteínas Proto-Oncogénicas/análisis , Proteínas Proto-Oncogénicas c-bcl-2/análisis , Análisis de Supervivencia , Proteína X Asociada a bcl-2 , Proteína bcl-XRESUMEN
The appearance of multidrug-resistant (MDR) proteins or the acquisition of a defective apoptotic programme are major drawbacks in the treatment of cancers since both induce a resistance to classical chemotherapy. However, a link between the two mechanisms has not, as yet, been clearly established. In this study, HL-60 cells cultured in the continual presence of a sub-lethal dose of doxorubicin (dox; HL-60/Dox) were used as a model to study acquired chemoresistance. During the induction of chemoresistance, the appearance of a functional P-glycoprotein (P-gp), in addition to the expression of anti-apoptotic Bcl-2, Bcl-XL and pro-apoptotic Bax proteins was assessed. Parental cells which are sensitive to dox, have no P-gp activity and express Bcl-2 and Bax. After 4 weeks of treatment, a functional P-gp was detected in HL-60/Dox cells. In addition, the synthesis of Bcl-2 appeared to be replaced by Bcl-XL while that of Bax remained unchanged. These cells were also resistant to apoptosis induced by both P-gp and non-P-gp substrates. This inability to induce apoptosis could have resulted from the induction of the expression of the inhibitor of apoptosis protein (XIAP). Our data show that acquired chemoresistance could involve a parallel induction of P-gp and an impairment of the apoptotic pathway.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/biosíntesis , Apoptosis , Células HL-60/efectos de los fármacos , Proteínas , Caspasa 3 , Caspasas/metabolismo , Citarabina/farmacología , Doxorrubicina/farmacología , Resistencia a Múltiples Medicamentos , Activación Enzimática , Citometría de Flujo , Fluoresceínas/farmacología , Humanos , Péptido Hidrolasas/metabolismo , Fenotipo , Probenecid/farmacología , Biosíntesis de Proteínas , Proteínas Proto-Oncogénicas/biosíntesis , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteína Inhibidora de la Apoptosis Ligada a X , Proteína X Asociada a bcl-2 , Proteína bcl-XRESUMEN
The expression of bcl-2-related proteins has been shown to be a key element in tumoral malignancy. The degradation of the extracellular matrix (ECM) by specialized matrix metalloproteinases (MMPs) is another major step in tumor invasion and metastasis. We have examined, in a rat glioma cell line A15A5, the effect of the stable transfection of human bcl-2, bax and bcl-xl on MMPs expression. Using a zymographic assay, we found that all transfected cell lines expressed a gelatinase activity which is predominantly associated with MMP-9. In bcl-2 and bcl-xl transfected cells, the transcription of MMP-9 was decreased compared to that of control or bax transfected cells. In addition, in bax transfected A15A5, we observed a down regulation of TIMP-1, the inhibitor of MMP-9. These results suggest that the ratio between MMP-9 and its inhibitor TIMP-1 is tightly controlled in cells overexpressing bcl-2 related proteins (i.e., high ratio in bax transfected A15A5 and low ratio in bcl-2 transfected A15A5). However, MMPs secreted by bcl-2 transfected cells were still capable of hydrolyzing FasL present on human lymphocytes. Our results suggest that the expression of bcl-2 related proteins could participate in the regulation of MMP-9/TIMP-1 in gliomas.
Asunto(s)
Metaloproteinasas de la Matriz/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Animales , Apoptosis , Proteína Ligando Fas , Glioma/genética , Glioma/metabolismo , Glioma/patología , Humanos , Metaloproteinasa 9 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/metabolismo , Metaloproteinasas de la Matriz/genética , Glicoproteínas de Membrana/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Inhibidor Tisular de Metaloproteinasa-1/genética , Inhibidor Tisular de Metaloproteinasa-1/metabolismo , Transfección , Células Tumorales CultivadasRESUMEN
The interaction of the anti-apoptotic members of the Bcl-2 family with mitochondria, through their hydrophobic C-terminus, has been proposed to play a crucial role in the execution phase of apoptosis. We report here that a substitution of the C-terminal end of pro-apoptotic bax by that of anti-apoptotic bcl-xL (baxCxL) does not modify its association with mitochondria in human and rat cells or in Saccharomyces cerevisiae. In addition, while bax sensitizes these cells to apoptotic stimuli, the construct baxCxL does not affect the apoptotic response in transfected cells. These results suggest that the C-terminus of bax plays an important role in apoptosis independently of its membrane addressing/targeting mechanism.