RESUMEN
Tumor cells, which undergo Epithelial-mesenchymal transition (EMT) acquire increased capacities of proliferation, invasion and have the ability to generate metastases by escaping the immune system during their systemic migration. To escape the immune system, cancer cells may induce tolerance or resist elimination by immune effectors via multiple mechanisms and we hypothesized that EMT may control the expression of immune checkpoint inhibitors, then promoting immune evasion. PD-L1 (programmed cell death ligand 1) but not PD-L2 nor Galectin 9 or Death receptor (DR4, DR5 and Fas) and ligands (FasL and TRAIL) expression was up-regulated during cytokine-driven EMT in a reversible manner. Moreover PD-L1 is overexpressed in VIMENTIN positive NSCLC tissues. We also demonstrated that the expression of PD-L1 required both TNFα and TGFß1. Indeed, TGFß1 decreased DNMT1 content and that resulted in PD-L1 promoter demethylation whereas TNFα induced the NF-κB pathway that promoted expression of demethylated PD-L1 promoter.
RESUMEN
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
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
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
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
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
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.