RESUMEN
RARA and RXRA contribute to myeloid maturation in both mice and humans, and deletion of Rxra and Rxrb augments leukemic growth in mice. While defining the domains of RXRA that are required for anti-leukemic effects in murine KMT2A-MLLT3 leukemia cells, we unexpectedly identified RXRA DT448/9PP as a constitutively active variant capable of inducing maturation and loss of their proliferative phenotype. RXRA DT448/9PP was associated with ligand-independent activity in reporter assays, with enhanced co-activator interactions, reduced engraftment in vivo, and activation of myeloid maturation transcriptional signatures that overlapped with those of cells treated with the potent RXRA agonist bexarotene, suggestive of constitutive activity that leads to leukemic maturation. Phenotypes of RXRA DT448/9PP appear to differ from those of two other RXRA mutations with forms of constitutive activity (F318A and S427F), in that DT448/9PP activity was resistant to mutations at critical ligand-interacting amino acids (R316A/L326A) and was resistant to pharmacological antagonists, suggesting it may be ligand-independent. These data provide further evidence that activated retinoid X receptors can regulate myeloid maturation and provide a novel constitutively active variant that may be germane for broader studies of retinoid X receptors in other settings.
Asunto(s)
Leucemia Mieloide Aguda , Leucemia Promielocítica Aguda , Receptor alfa X Retinoide , Animales , Proteínas de Unión al ADN , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Promielocítica Aguda/tratamiento farmacológico , Ratones , Receptor alfa X Retinoide/genética , Receptor alfa X Retinoide/metabolismoRESUMEN
Retinoid therapy transformed response and survival outcomes in acute promyelocytic leukemia (APL), but has demonstrated only modest activity in non-APL forms of acute myeloid leukemia (AML). The presence of natural retinoids in vivo could influence the efficacy of pharmacologic agonists and antagonists. We found that natural RXRA ligands, but not RARA ligands, were present in murine MLL-AF9-derived myelomonocytic leukemias in vivo and that the concurrent presence of receptors and ligands acted as tumor suppressors. Pharmacologic retinoid responses could be optimized by concurrent targeting RXR ligands (e.g. bexarotene) and RARA ligands (e.g. all-trans retinoic acid, ATRA), which induced either leukemic maturation or apoptosis depending on cell culture conditions. Co-repressor release from the RARA:RXRA heterodimer occurred with RARA activation, but not RXRA activation, providing an explanation for the combination synergy. Combination synergy could be replicated in additional, but not all, AML cell lines and primary samples, and was associated with improved survival in vivo, although tolerability of bexarotene administration in mice remained an issue. These data provide insight into the basal presence of natural retinoids in leukemias in vivo and a potential strategy for clinical retinoid combination regimens in leukemias beyond acute promyelocytic leukemia.
Asunto(s)
Leucemia Promielocítica Aguda , Retinoides , Animales , Diferenciación Celular , Ratones , Receptores de Ácido Retinoico/genética , Tretinoina/farmacologíaRESUMEN
The retinoid X receptor α (RXRA) has been implicated in diverse hematological processes. To identify natural ligands of RXRA that are present in hematopoietic cells, we adapted an upstream activation sequence-green fluorescent protein (UAS-GFP) reporter mouse to detect natural RXRA ligands in vivo. We observed reporter activity in diverse types of hematopoietic cells in vivo. Reporter activity increased during granulocyte colony-stimulating factor (G-CSF)-induced granulopoiesis and after phenylhydrazine (PHZ)-induced anemia, suggesting the presence of dynamically regulated natural RXRA ligands in hematopoietic cells. Mouse plasma activated Gal4-UAS reporter cells in vitro, and plasma from mice treated with G-CSF or PHZ recapitulated the patterns of reporter activation that we observed in vivo. Plasma from mice with dietary vitamin A deficiency only mildly reduced RXRA reporter activity, whereas plasma from mice on a fatty acid restriction diet reduced reporter activity, implicating fatty acids as plasma RXRA ligands. Through differential extraction coupled with mass spectrometry, we identified the long-chain fatty acid C24:5 as a natural RXRA ligand that was greatly increased in abundance in response to hematopoietic stress. Together, these data suggest that natural RXRA ligands are present and dynamically increased in abundance in mouse hematopoietic cells in vivo.
Asunto(s)
Células Madre Hematopoyéticas/metabolismo , Receptor alfa X Retinoide/metabolismo , Animales , Ácidos Grasos/sangre , Factor Estimulante de Colonias de Granulocitos/farmacología , Granulocitos/efectos de los fármacos , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Leucopoyesis/efectos de los fármacos , Ligandos , Ratones , Ratones Noqueados , Ratones Mutantes , Células Mieloides/metabolismo , Receptor alfa X Retinoide/genética , Vitamina A/sangreRESUMEN
Retinoid X receptors (RXRs) form a unique subclass within the nuclear receptor (NR) superfamily of ligand-dependent transcription factors. RXRs are obligatory partners for a number of other NRs, placing RXRs in a coordinating role at the crossroads of multiple signaling pathways. In addition, RXRs can function as self-sufficient homodimers. Recent advances have revealed RXRs as novel regulators of osteoclastogenesis and bone remodeling. This review outlines the versatility of RXR action in the control of transcription of bone-forming osteoblasts and bone-resorbing osteoclasts, both through heterodimerization with other NRs and through RXR homodimerization. RXR signaling is currently a major therapeutic target and, therefore, knowledge of how RXR signaling affects bone remodeling creates enormous potential for the translation of basic research findings into successful clinical therapies to increase bone mass and improve bone quality.
Asunto(s)
Remodelación Ósea , Receptores X Retinoide/metabolismo , Animales , Resorción Ósea/patología , Humanos , Modelos Biológicos , Multimerización de ProteínaRESUMEN
Osteoclasts are bone-resorbing cells that are important for maintenance of bone remodeling and mineral homeostasis. Regulation of osteoclast differentiation and activity is important for the pathogenesis and treatment of diseases associated with bone loss. Here, we demonstrate that retinoid X receptors (RXRs) are key elements of the transcriptional program of differentiating osteoclasts. Loss of RXR function in hematopoietic cells resulted in formation of giant, nonresorbing osteoclasts and increased bone mass in male mice and protected female mice from bone loss following ovariectomy, which induces osteoporosis in WT females. The increase in bone mass associated with RXR deficiency was due to lack of expression of the RXR-dependent transcription factor v-maf musculoaponeurotic fibrosarcoma oncogene family, protein B (MAFB) in osteoclast progenitors. Evaluation of osteoclast progenitor cells revealed that RXR homodimers directly target and bind to the Mafb promoter, and this interaction is required for proper osteoclast proliferation, differentiation, and activity. Pharmacological activation of RXRs inhibited osteoclast differentiation due to the formation of RXR/liver X receptor (LXR) heterodimers, which induced expression of sterol regulatory element binding protein-1c (SREBP-1c), resulting in indirect MAFB upregulation. Our study reveals that RXR signaling mediates bone homeostasis and suggests that RXRs have potential as targets for the treatment of bone pathologies such as osteoporosis.
Asunto(s)
Remodelación Ósea/fisiología , Diferenciación Celular/fisiología , Receptores Nucleares Huérfanos/metabolismo , Osteoclastos/metabolismo , Multimerización de Proteína/fisiología , Receptores X Retinoide/metabolismo , Animales , Femenino , Receptores X del Hígado , Factor de Transcripción MafB/biosíntesis , Factor de Transcripción MafB/genética , Masculino , Ratones , Ratones Noqueados , Receptores Nucleares Huérfanos/genética , Osteoclastos/citología , Osteoporosis/genética , Osteoporosis/metabolismo , Receptores X Retinoide/genética , Células Madre/citología , Células Madre/metabolismo , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/genética , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/metabolismo , Transcripción Genética/fisiología , Regulación hacia Arriba/fisiologíaRESUMEN
Retinoid X receptors (RXRs) form a distinct and unique subclass within the nuclear receptor (NR) superfamily of ligand-dependent transcription factors. RXRs regulate a plethora of genetic programs, including cell differentiation, the immune response, and lipid and glucose metabolism. Recent advances reveal that RXRs are important regulators of macrophages, key players in inflammatory and metabolic disorders. This review outlines the versatility of RXR action in the control of macrophage gene transcription through its heterodimerization with other NRs or through RXR homodimerization. We also highlight the potential of RXR-controlled transcriptional programs as targets for the treatment of pathologies associated with altered macrophage function, such as atherosclerosis, insulin resistance, autoimmunity, and neurodegeneration.
Asunto(s)
Macrófagos/inmunología , Receptores X Retinoide/inmunología , Animales , Humanos , Inmunidad Innata/inmunologíaRESUMEN
In this study, melittin, a well-characterized pore-forming lytic amphiphilic peptide susceptible to be vehiculized in lipid membranes, has been utilized to study their antiviral properties. For this purpose, an assay based on melittin loaded-immunoliposomes previously described by our group was adapted to antiviral purposes by means of monoclonal antibodies targeting the surface G glycoprotein of the fish viral haemorrhagic septicemia rhabdovirus (VHSV). We also studied the antiviral action of these immunoliposomes in vitro and the results showed that they are capable of inhibiting the VHSV infectivity by 95.2% via direct inactivation of the virus. Furthermore, the inhibition of the infectivity when treatments were added at different times post-infection and the analysis of the infection foci sizes suggested altogether that they also act by reducing the VHSV spread in cell culture and by killing the infected cells which express the G glycoprotein in their plasmatic membranes.
Asunto(s)
Antivirales/farmacología , Liposomas/metabolismo , Meliteno/farmacología , Proteínas de la Membrana/antagonistas & inhibidores , Novirhabdovirus/efectos de los fármacos , Proteínas Virales/antagonistas & inhibidores , Animales , Línea Celular , Peces , Pruebas de Sensibilidad Microbiana , Viabilidad Microbiana/efectos de los fármacos , Inactivación de VirusRESUMEN
Peroxisome proliferator-activated receptors (PPARs) are ligand dependent transcription factors. The three mammalian PPARs are key regulators of fatty acid and lipoprotein metabolism, glucose homeostasis, cellular proliferation/ differentiation and the immune response. PPARs are therefore important targets in the treatment of metabolic disorders such as insulin resistance and type 2 diabetes mellitus, and are also of interest in relation to chronic inflammatory diseases such as atherosclerosis, arthritis, chronic pulmonary inflammation, pancreatitis, inflammatory bowel disease, and psoriasis. Recent advances have attributed novel functions to PPARs in blood pressure regulation, neuroinflammation, nerve-cell protection, inflammatory pain reduction, and the hypothalamic control of metabolism. The abundant pleiotropic actions of PPARs suggest that PPAR agonists have enormous therapeutic potential. However, current PPAR-based therapies often have undesired side effects due to the concomitant activation of PPARs in non-target cells. There is therefore growing interest in the development of cell-specific PPAR agonists and improvement of the clinical use of PPAR ligands. This review gives an overview of PPAR functions and discusses the current and potential medical implications of PPAR ligands in various pathologies, ranging from metabolic disorders to cardiovascular disease, chronic inflammation, neurodegenerative disorders and cancer.
Asunto(s)
Terapia Molecular Dirigida , Receptores Activados del Proliferador del Peroxisoma/agonistas , Receptores Activados del Proliferador del Peroxisoma/metabolismo , Animales , Humanos , Ligandos , Receptores Activados del Proliferador del Peroxisoma/inmunologíaRESUMEN
Autoimmune glomerulonephritis is a common manifestation of systemic lupus erythematosus (SLE). In this study, we show that mice lacking macrophage expression of the heterodimeric nuclear receptors PPARγ or RXRα develop glomerulonephritis and autoantibodies to nuclear Ags, resembling the nephritis seen in SLE. These mice show deficiencies in phagocytosis and clearance of apoptotic cells, and they are unable to acquire an anti-inflammatory phenotype upon feeding of apoptotic cells, which is critical for the maintenance of self-tolerance. These results demonstrate that stimulation of PPARγ and RXRα in macrophages facilitates apoptotic cell engulfment, and they provide a potential strategy to avoid autoimmunity against dying cells and to attenuate SLE.
Asunto(s)
Apoptosis/inmunología , Nefritis Lúpica/inmunología , Nefritis Lúpica/patología , Macrófagos/inmunología , Macrófagos/patología , PPAR gamma/deficiencia , Fagocitosis/inmunología , Receptor alfa X Retinoide/deficiencia , Animales , Anticuerpos Antinucleares/biosíntesis , Anticuerpos Antinucleares/metabolismo , Anticuerpos Antinucleares/fisiología , Apoptosis/genética , Femenino , Nefritis Lúpica/genética , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , PPAR gamma/genética , PPAR gamma/fisiología , Fagocitosis/genética , Receptor alfa X Retinoide/genética , Receptor alfa X Retinoide/fisiología , Autotolerancia/genética , Autotolerancia/inmunologíaRESUMEN
In this work, high-performance liquid chromatography (HPLC) coupled to electrospray time-of-flight mass spectrometry (ESI-TOF-MS) and electrospray ion trap multiple-stage tandem mass spectrometry (ESI-IT-MS(2)) has been applied to screen phenolic compounds in olive leaf extracts. The use of a small particle size C18 column (1.8 micro) provided great resolution and made separation of a lot of isomers possible. The structural characterization was based on accurate mass data obtained by ESI-TOF-MS, and the nature of fragmentation ions were further confirmed by ESI-IT-MS(2) when possible. In addition, we employed tetrazolium salt (MTT)-based assays to assess the effects of olive leaf extracts on the growth of human tumor-derived cells. Upon this approach, we achieved an accurate profile of olive leaf phenolics along with the identification of several important isomers of secoiridoids and flavonoids. This will allow a better understanding of the complete composition of olive-leaf-bioactive compounds as well as their involvement in Olea europaea L. biochemical pathways. Importantly, olive leaf extracts exhibited dose-dependent inhibitory effects on the metabolic status (cell viability) of three breast cancer models in vitro. Since the tumoricidal activity of the extracts should be mainly attributed to the identified olive leaf phenolics, these findings warrant further investigation at the structure-function molecular level to definitely establish the anticancer value of these phytochemicals.
Asunto(s)
Antineoplásicos Fitogénicos/análisis , Antineoplásicos Fitogénicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Carcinoma/tratamiento farmacológico , Cromatografía Líquida de Alta Presión/métodos , Olea/química , Fenoles/análisis , Fenoles/farmacología , Antineoplásicos Fitogénicos/aislamiento & purificación , Supervivencia Celular/efectos de los fármacos , Cinamatos/análisis , Cinamatos/aislamiento & purificación , Cinamatos/farmacología , Femenino , Flavonoides/análisis , Flavonoides/aislamiento & purificación , Flavonoides/farmacología , Humanos , Glucósidos Iridoides , Iridoides/análisis , Iridoides/aislamiento & purificación , Iridoides/farmacología , Fenoles/aislamiento & purificación , Hojas de la Planta/química , Piranos/análisis , Piranos/aislamiento & purificación , Piranos/farmacología , Espectrometría de Masa por Ionización de Electrospray/métodos , Espectrometría de Masas en Tándem/métodosRESUMEN
Trastuzumab (Herceptin) targets the human epidermal growth factor receptor 2 (HER2), which is overexpressed in 20-30% of breast and ovarian cancers carrying a bad prognosis. Our purpose was to target HER2-overexpressing human breast cancer cells with pegylated immunoliposomes bearing trastuzumab and containing melittin, which has recently shown anticancer properties. Using a panel of human breast cancer cells with different HER2 expression levels, these immunoliposomes decreased cancer cells viability in a dose-response manner and in correlation to their level of HER2 expression. Specific binding of the immunoliposomes to SKBr3 breast cancer cells was shown by ImageStream-based analysis. The morphological changes observed in the treated cells suggested a cytolytic process. This preclinical approach may suppose an effective strategy for the treatment of HER2-overexpressing tumors, and can support the development of an early phases I-II clinical trial. Trastuzumab resistant breast cancer cells (JIMT-1), can also be targeted using this approach.
Asunto(s)
Anticuerpos Monoclonales/administración & dosificación , Antineoplásicos/administración & dosificación , Neoplasias de la Mama/metabolismo , Sistemas de Liberación de Medicamentos/métodos , Meliteno/administración & dosificación , Receptor ErbB-2/biosíntesis , Anticuerpos Monoclonales Humanizados , Western Blotting , Línea Celular Tumoral , Separación Celular , Supervivencia Celular/efectos de los fármacos , Cromatografía Líquida de Alta Presión , Femenino , Citometría de Flujo , Humanos , Procesamiento de Imagen Asistido por Computador , Liposomas , TrastuzumabRESUMEN
Previous studies have documented that while several drug-resistant cells enter apoptosis upon treatment with histone deacetylase inhibitors (iHDACs), their drug-sensitive counterparts do not. In the present study, we have investigated at the molecular level why parental drug-sensitive tumor cells do not respond to Trichostatin A and suberoylanilide hydroxamic acid, two iHDACs that promote apoptosis in drug-resistant leukaemia cells. Taking murine leukaemia L1210 cells as a model, we have determined that: (i) PKC-alpha expression is more elevated in parental L1210 than in drug-resistant L1210/R cells, (ii) activation of PKC neutralizes iHDACs-mediated apoptosis in L1210/R cells, (iii) depletion of PKC in parental L1210 cells results in a positive response to iHDACs-mediated apoptosis, and (iv) transfection of a mutant constitutively active PKC-alpha form in L1210/R cells makes the cells refractory to apoptosis induction by iHDACs. These results allow us to conclude that activation/high expression of PKC-alpha protects parental drug-sensitive L1210 cells from iHDACs-mediated apoptosis. Thus, determination of PKC-alpha levels/activity in leukaemia seems to be relevant when choosing efficient chemotherapy protocols based on the use of apoptosis-inducing anticancer drugs.
Asunto(s)
Apoptosis/efectos de los fármacos , Resistencia a Múltiples Medicamentos/genética , Inhibidores Enzimáticos/farmacología , Inhibidores de Histona Desacetilasas , Leucemia/genética , Proteína Quinasa C-alfa/fisiología , Animales , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Células HL-60 , Humanos , Ácidos Hidroxámicos/farmacología , Leucemia/tratamiento farmacológico , Ratones , Proteínas Mutantes/genética , Proteínas Mutantes/fisiología , Proteína Quinasa C-alfa/genética , Transfección , Células Tumorales Cultivadas , VorinostatRESUMEN
The antitumor activity of the histone deacetylase inhibitors was tested in three well-characterized pancreatic adenocarcinoma cell lines, IMIM-PC-1, IMIM-PC-2, and RWP-1. These cell lines have been previously characterized in terms of their origin, the status of relevant molecular markers for this kind of tumor, resistance to other antineoplastic drugs, and expression of differentiation markers. In this study, we report that histone deacetylase inhibitors induce apoptosis in pancreatic cancer cell lines, independently of their intrinsic resistance to conventional antineoplastic agents. The histone deacetylase inhibitor-induced apoptosis is due to a serine protease-dependent and caspase-independent mechanism. Initially, histone deacetylase inhibitors increase Bax protein levels without affecting Bcl-2 levels. Consequently, the apoptosis-inducing factor (AIF) and Omi/HtrA2 are released from the mitochondria, with the subsequent induction of the apoptotic program. These phenomena require AIF relocalization into the nuclei to induce DNA fragmentation and a serine protease activity of Omi/HtrA2. These data, together with previous results from other cellular models bearing the multidrug resistance phenotype, suggest a possible role of the histone deacetylase inhibitors as antineoplastic agents for the treatment of human pancreatic adenocarcinoma.