RESUMEN
Pyrazole analogues of the staurosporine aglycone K252c, in which the lactam ring was replaced by a pyrazole moiety, were synthesized. In this series, one or the other nitrogen atoms of the indolocarbazole scaffold was substituted by aminoalkyl chains, aiming at improving protein kinase inhibition as well as cellular potency toward acute myeloid leukemia (AML) cell lines. Compound 19a, substituted at the N12-position by a 3-(methylamino)propyl group, showed high cellular activity in the low micromolar range toward three AML cell lines (MOLM-13, OCI-AML3 and MV4-11) with selectivity over non-cancerous cells (NRK, H9c2). 19a is also a highly potent inhibitor of the three Pim kinase isoforms, Pim-3 being the most inhibited with an IC50 value in the nanomolar range. A selectivity screening toward a panel of 50 protein kinases showed that 19a also potently inhibited PRK2 and to a lower extent AMPK, MARK3, GSK3ß and JAK3. Our results enhance the understanding of the structural characteristics of indolopyrazolocarbazoles essential for potent protein kinase inhibition with therapeutic potential against AML.
Asunto(s)
Antineoplásicos , Leucemia Mieloide Aguda , Humanos , Inhibidores de Proteínas Quinasas/química , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/metabolismo , Pirazoles/química , Línea Celular Tumoral , Apoptosis , Proliferación Celular , Tirosina Quinasa 3 Similar a fms , Antineoplásicos/químicaRESUMEN
We synthesized new analogues of the anti-AML agent VS-II-173. We studied the effect of the substitution at the 1- and 5-positions of the pyrazolo[4,3-a]phenanthridine scaffold on Pim-1 kinase inhibition and cytotoxicity against AML MOLM-13 cells. We found that compounds 20 and 21, substituted at the 1-position exhibited stronger Pim-1 inhibition together with a high potency toward MOLM-13 cells, associated with apoptosis induction and selectivity over non-cancerous NRK cells.
Asunto(s)
Antineoplásicos , Leucemia Mieloide Aguda , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Apoptosis , Línea Celular Tumoral , Proliferación Celular , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/metabolismo , Fenantridinas/farmacología , Inhibidores de Proteínas Quinasas , Proteínas Proto-Oncogénicas c-pim-1RESUMEN
Garden chervil, Anthriscus cerefolium (L.) Hoffm. is an important herb commonly applied in Norwegian large-scale commercial kitchens. This species is a highly enriched source of phenolics, containing 1260 mg gallic acid equivalents (GAE) 100-1 g DM, however, the individual phenolic compounds have been scarcely characterized. Here we report on the qualitative and quantitative content of phenolics in garden chervil. The structure of the main phenolic compound was elucidated to be the previously undescribed compound 1,3-dicaffeoyl-5-malonyl-δ-quinide (1) by means of 1D- and 2D NMR and high-resolution mass spectrometry. The known flavones apigenin 7-O-ß-(2â³-apiofuranosylglucopyranoside) (= apiin) (2), apigenin 7-(2â³-apiosyl-6â³-malonylglucoside) (3) and luteolin 7-glucoside (4) were also identified. Compound 3 is reported for the first time from this plant species. The main phenolic compound, 1,3-dicaffeoyl-5-malonyl-δ-quinide, exhibited moderate cytotoxicity towards acute monocytic leukaemia cells (MOLM-13) and rat kidney epithelial cells (NRK) with EC50 between 400 and 600 µM.
Asunto(s)
Apiaceae , Polifenoles , Animales , Apiaceae/química , Apigenina , Cromatografía Líquida de Alta Presión , Lactonas , Fenoles/análisis , Extractos Vegetales/química , Extractos Vegetales/farmacología , Ratas , VerdurasRESUMEN
Novel chemotherapeutic strategies for acute myeloid leukemia (AML) treatment are called for. We have recently demonstrated that the phenazine 5,10-dioxide natural products iodinin (3) and myxin (4) exhibit potent and hypoxia-selective cell death on MOLM-13 human AML cells, and that the N-oxide functionalities are pivotal for the cytotoxic activity. Very few structure-activity relationship studies dedicated to phenazine 5,10-dioxides exist on mammalian cell lines and the present work describes our efforts regarding in vitro lead optimizations of the natural compounds iodinin (3) and myxin (4). Prodrug strategies reveal carbamate side chains to be the optimal phenol-attached group. Derivatives with no oxygen-based substituent (-OH or -OCH3) in the 6th position of the phenazine skeleton upheld potency if alkyl or carbamate side chains were attached to the phenol in position 1. 7,8-Dihalogenated- and 7,8-dimethylated analogs of 1-hydroxyphenazine 5,10-dioxide (21) displayed increased cytotoxic potency in MOLM-13 cells compared to all the other compounds studied. On the other hand, dihalogenated compounds displayed high toxicity towards the cardiomyoblast H9c2 cell line, while MOLM-13 selectivity of the 7,8-dimethylated analogs were less affected. Further, a parallel artificial membrane permeability assay (PAMPA) demonstrated the majority of the synthesized compounds to penetrate cell membranes efficiently, which corresponded to their cytotoxic potency. This work enhances the understanding of the structural characteristics essential for the activity of phenazine 5,10-dioxides, rendering them promising chemotherapeutic agents.
RESUMEN
Cyanobacteria produce a variety of chemically diverse cyclic lipopeptides with potent antifungal activities. These cyclic lipopeptides have an amphipathic structure comprised of a polar peptide cycle and hydrophobic fatty acid side chain. Many have antibiotic activity against a range of human and plant fungal pathogens. This review article aims to summarize the present knowledge on the chemical diversity and cellular effects of cyanobacterial cyclic lipopeptides that display antifungal activity. Cyclic antifungal lipopeptides from cyanobacteria commonly fall into four structural classes; hassallidins, puwainaphycins, laxaphycins, and anabaenolysins. Many of these antifungal cyclic lipopeptides act through cholesterol and ergosterol-dependent disruption of membranes. In many cases, the cyclic lipopeptides also exert cytotoxicity in human cells, and a more extensive examination of their biological activity and structure-activity relationship is warranted. The hassallidin, puwainaphycin, laxaphycin, and anabaenolysin structural classes are unified through shared complex biosynthetic pathways that encode a variety of unusual lipoinitiation mechanisms and branched biosynthesis that promote their chemical diversity. However, the biosynthetic origins of some cyanobacterial cyclic lipopeptides and the mechanisms, which drive their structural diversification in general, remain poorly understood. The strong functional convergence of differently organized chemical structures suggests that the production of lipopeptide confers benefits for their producer. Whether these benefits originate from their antifungal activity or some other physiological function remains to be answered in the future. However, it is clear that cyanobacteria encode a wealth of new cyclic lipopeptides with novel biotechnological and therapeutic applications.
Asunto(s)
Antifúngicos , Cianobacterias , Antibacterianos , Antifúngicos/farmacología , Lipopéptidos/farmacología , Péptidos Cíclicos/farmacologíaRESUMEN
In addition to the rapidly expanding field of using microalgae for food and feed, microalgae represent a tremendous potential for new bioactive compounds with health-promoting effects. One field where new therapeutics is needed is cancer therapy. As cancer therapy often cause severe side effects and loose effect due to development of drug resistance, new therapeutic agents are needed. Treating cancer by modulating the immune response using peptides has led to unprecedented responses in patients. In this review, we want to elucidate the potential for microalgae as a source of new peptides for possible use in cancer management. Among the limited studies on anti-cancer effects of peptides, positive results were found in a total of six different forms of cancer. The majority of studies have been performed with different strains of Chlorella, but effects have also been found using peptides from other species. This is also the case for peptides with immunomodulating effects and peptides with other health-promoting effects (e.g., role in cardiovascular diseases). However, the active peptide sequence has been determined in only half of the studies. In many cases, the microalga strain and the cultivation conditions used for producing the algae have not been reported. The low number of species that have been explored, as opposed to the large number of species available, is a clear indication that the potential for new discoveries is large. Additionally, the availability and cost-effectiveness of microalgae make them attractive in the search for bioactive peptides to prevent cancer.
Asunto(s)
Chlorella , Microalgas , Secuencia de Aminoácidos , Humanos , PéptidosRESUMEN
Cyclin-dependent kinase 8 (CDK8) plays a vital role in regulating cell transcription either through its association with the mediator complex or by the phosphorylation of transcription factors. CDK8-mediated activation of oncogenes has proved to be important in a variety of cancer types including hematological malignancies. We have designed and synthesized a series of new synthetic steroids. The compounds were evaluated as CDK8 inhibitors in vitro. The three most potent compounds exhibit Kd-values towards CDK8 in the low nanomolar range (3.5-18 nM). Furthermore, the compounds displayed selectivity for CDK8 in a panel of 465 different kinases. The cell studies indicated a selectivity to kill AML-cancer cell lines compared to normal cell lines.
Asunto(s)
Antineoplásicos/farmacología , Quinasa 8 Dependiente de Ciclina/antagonistas & inhibidores , Diseño de Fármacos , Inhibidores de Proteínas Quinasas/farmacología , Esteroides/farmacología , Antineoplásicos/síntesis química , Antineoplásicos/química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Quinasa 8 Dependiente de Ciclina/metabolismo , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Estructura Molecular , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Esteroides/síntesis química , Esteroides/química , Relación Estructura-ActividadRESUMEN
The exchange proteins directly activated by cAMP 1 and 2 (Epac1 and Epac2) are expressed in a cell specific manner in the liver, but their biological functions in this tissue are poorly understood. The current study was undertaken to begin to determine the potential roles of Epac1 and Epac2 in liver physiology and disease. Male C57BL/6J mice in which expression of Epac1 and/or Epac2 are deleted, were subjected to partial hepatectomy and the regenerating liver was analyzed with regard to lipid accumulation, cell replication and protein expression. In response to partial hepatectomy, deletion of Epac1 and/or Epac2 led to increased hepatocyte proliferation 36 h post surgery, and the transient steatosis observed in wild type mice was virtually absent in mice lacking both Epac1 and Epac2. The expression of the protein cytochrome P4504a14, which is implicated in hepatic steatosis and fibrosis, was substantially reduced upon deletion of Epac1/2, while a number of factors involved in lipid metabolism were significantly decreased. Moreover, the number of Küpffer cells was affected, and Epac2 expression was increased in the liver of wild type mice in response to partial hepatectomy, further supporting a role for these proteins in liver function. This study establishes hepatic phenotypic abnormalities in mice deleted for Epac1/2 for the first time, and introduces Epac1/2 as regulators of hepatocyte proliferation and lipid accumulation in the regenerative process.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Regeneración Hepática/fisiología , Animales , Proliferación Celular/fisiología , Hígado Graso/metabolismo , Fibrosis/metabolismo , Hepatectomía/métodos , Metabolismo de los Lípidos/fisiología , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
More than 40 years ago, the present standard induction therapy for acute myeloid leukemia (AML) was developed. This consists of the metabolic inhibitor cytarabine (AraC) and the cytostatic topoisomerase 2 inhibitor daunorubucin (DNR). In light of the high chance for relapse, as well as the large heterogeneity, novel therapies are needed to improve patient outcome. We have tested the anti-AML activity of 15 novel compounds based on the scaffolds pyrrolo[2,3-a]carbazole-3-carbaldehyde, pyrazolo[3,4-c]carbazole, pyrazolo[4,3-a]phenanthridine, or pyrrolo[2,3-g]indazole. The compounds were inhibitors of Pim kinases, but could also have inhibitory activity against other protein kinases. Ser/Thr kinases like the Pim kinases have been identified as potential drug targets for AML therapy. The compound VS-II-173 induced AML cell death with EC50 below 5 µmol/L, and was 10 times less potent against nonmalignant cells. It perturbed Pim-kinase-mediated AML cell signaling, such as attenuation of Stat5 or MDM2 phosphorylation, and synergized with DNR to induce AML cell death. VS-II-173 induced cell death also in patients with AML blasts, including blast carrying high-risk FLT3-ITD mutations. Mutation of nucleophosmin-1 was associated with good response to VS-II-173. In conclusion new scaffolds for potential AML drugs have been explored. The selective activity toward patient AML blasts and AML cell lines of the pyrazolo-analogue VS-II-173 make it a promising drug candidate to be further tested in preclinical animal models for AML.
Asunto(s)
Carbazoles/química , Indazoles/química , Leucemia Mieloide Aguda/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Apoptosis/efectos de los fármacos , Carbazoles/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Citarabina/química , Citarabina/farmacología , Humanos , Indazoles/farmacología , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Mutación/efectos de los fármacos , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/química , Proteínas Proto-Oncogénicas c-pim-1/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-pim-1/genética , Transducción de Señal/efectos de los fármacos , Inhibidores de Topoisomerasa II/química , Inhibidores de Topoisomerasa II/farmacologíaRESUMEN
Previous studies demonstrate essential roles for the exchange proteins directly activated by cAMP 1 and 2 (Epac1 and Epac2; here collectively referred to as Epac) in the brain. In the hippocampus, Epac contributes to the control of neuronal growth and differentiation and has been implicated in memory and learning as well as in anxiety and depression. In the present study we address the hypothesis that Epac affects hippocampal cellular responses to acute restraint stress. Stress causes activation of the hypothalamus-pituitary-adrenal (HPA)-axis, and glucocorticoid receptor (GR) signaling is essential for proper feedback regulation of the stress response, both in the brain and along the HPA axis. In the hippocampus, GR expression is regulated by cAMP and the brain enriched micro RNA miR-124. Epac has been associated with miR-124 expression in hippocampal neurons, but not in regulation of GR. We report that hippocampal expression of Epac1 and Epac2 increased in response to acute stress in female wild type mice. In female mice genetically deleted for Epac, nuclear translocation of GR in response to restraint stress was significantly delayed, and moreover, miR-124 expression was decreased in these mice. Male mice lacking Epac also showed abnormalities in miR-124 expression, but the phenotype was less profound than in females. Serum corticosterone levels were slightly altered immediately after stress in both male and female mice deleted for Epac. The presented data indicate that Epac1 and Epac2 are involved in controlling cellular responses to acute stress in the mouse hippocampus and provide novel insights into the underlying transcriptional and signaling networks. Interestingly, we observe sex specific differences when Epac is deleted. As the incidence and prevalence of stress-related diseases are higher in women than in men, the Epac knockout models might serve as genetic tools to further elucidate the cellular mechanisms underlying differences between male and female with regard to regulation of stress.
Asunto(s)
Eliminación de Gen , Factores de Intercambio de Guanina Nucleótido/deficiencia , Factores de Intercambio de Guanina Nucleótido/genética , Hipocampo/citología , Transducción de Señal/genética , Animales , Corticosterona/sangre , Femenino , Hipocampo/metabolismo , Hipocampo/patología , Masculino , Ratones , Ratones Endogámicos C57BL , MicroARNs/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Estrés Psicológico/genética , Estrés Psicológico/patologíaRESUMEN
A new efficient total synthesis of the phenazine 5,10-dioxide natural products iodinin and myxin and new compounds derived from them was achieved in few steps, a key-step being 1,6-dihydroxyphenazine di-N-oxidation. Analogues prepared from iodinin, including myxin and 2-ethoxy-2-oxoethoxy derivatives, had fully retained cytotoxic effect against human cancer cells (MOLM-13 leukemia) at atmospheric and low oxygen level. Moreover, iodinin was for the first time shown to be hypoxia selective. The structure-activity relationship for leukemia cell death induction revealed that the level of N-oxide functionality was essential for cytotoxicity. It also revealed that only one of the two phenolic functions is required for activity, allowing the other one to be modified without loss of potency.
Asunto(s)
Productos Biológicos/síntesis química , Productos Biológicos/farmacología , Línea Celular Tumoral , Humanos , Fenazinas/síntesis química , Fenazinas/química , Fenazinas/farmacología , Relación Estructura-ActividadRESUMEN
Adrenocorticotropic hormone regulates adrenal steroidogenesis mainly via the intracellular signaling molecule cAMP. The effects of cAMP are principally relayed by activating protein kinase A (PKA) and the more recently discovered exchange proteins directly activated by cAMP 1 and 2 (EPAC1 and EPAC2). While the intracellular roles of PKA have been extensively studied in steroidogenic tissues, those of EPACs are only emerging. EPAC1 and EPAC2 are encoded by the genes RAPGEF3 and RAPGEF4, respectively. Whereas EPAC1 is ubiquitously expressed, the expression of EPAC2 is more restricted, and typically found in endocrine tissues. Alternative promoter usage of RAPGEF4 gives rise to three different isoforms of EPAC2 that vary in their N-termini (EPAC2A, EPAC2B, and EPAC2C) and that exhibit distinct expression patterns. EPAC2A is expressed in the brain and pancreas, EPAC2B in steroidogenic cells of the adrenal gland and testis, and EPAC2C has until now only been found in the liver. In this review, we discuss current knowledge on EPAC expression and function with focus on the known roles of EPAC in adrenal gland physiology.
RESUMEN
There is emerging evidence asserting the importance of orphan nuclear receptors (ONRs) in cancer initiation and progression. In breast cancer, there is a lot unknown about ONRs in terms of their expression profile and their transcriptional targets in the various stages of tumor progression. With the classification of breast tumors into distinct molecular subtypes, we assess ONR expression in the different breast cancer subtypes and with patient outcomes. Complementing this, we review evidence implicating ONR-dependent molecular pathways in breast cancer progression to identify candidate ONRs as potential prognostic markers and/or as therapeutic targets.
RESUMEN
BACKGROUND: Hypoxia- and Myc-dependent transcriptional regulatory pathways are frequently deregulated in cancer cells. These pathways converge in many cellular responses, but the underlying molecular mechanisms are unclear. METHODS: The ability of Miz-1 and Arnt to interact was identified in a yeast two-hybrid screen. The mode of interaction and the functional consequences of complex formation were analyzed by diverse molecular biology methods, in vitro. Statistical analyses were performed by Student's t-test and ANOVA. RESULTS: In the present study we demonstrate that the aryl hydrocarbon receptor nuclear translocator (Arnt), which is central in hypoxia-induced signaling, forms a complex with Miz-1, an important transcriptional regulator in Myc-mediated transcriptional repression. Overexpression of Arnt induced reporter gene activity driven by the proximal promoter of the cyclin-dependent kinase inhibitor 2B gene (CDKN2B), which is an established target for the Myc/Miz-1 complex. In contrast, mutated forms of Arnt, that were unable to interact with Miz-1, had reduced capability to activate transcription. Moreover, repression of Arnt reduced endogenous CDKN2B expression, and chromatin immunoprecipitation demonstrated that Arnt interacts with the CDKN2B promoter. The transcriptional activity of Arnt was counteracted by Myc, but not by a mutated variant of Myc that is unable to interact with Miz-1, suggesting mutually exclusive interaction of Arnt and Myc with Miz-1. Our results also establish CDKN2B as a hypoxia regulated gene, as endogenous CDKN2B mRNA and protein levels were reduced by hypoxic treatment of U2OS cells. CONCLUSIONS: Our data reveal a novel mode of regulation by protein-protein interaction that directly ties together, at the transcriptional level, the Myc- and hypoxia-dependent signaling pathways and expands our understanding of the roles of hypoxia and cell cycle alterations during tumorigenesis.
Asunto(s)
Translocador Nuclear del Receptor de Aril Hidrocarburo/metabolismo , Carcinogénesis/genética , Inhibidor p15 de las Quinasas Dependientes de la Ciclina/biosíntesis , Genes myc/genética , Factor 1 Inducible por Hipoxia/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Línea Celular Tumoral , Inmunoprecipitación de Cromatina , Inhibidor p15 de las Quinasas Dependientes de la Ciclina/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Immunoblotting , Inmunoprecipitación , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/fisiología , Transfección , Técnicas del Sistema de Dos HíbridosRESUMEN
The alkylating prodrug of melphalan, J1 (melphalanyl-L-p-fluorophenylalanyl ethyl ester) is currently in early clinical trials. Preclinical studies have shown that J1-mediated cytotoxicity is dependent on hydrolytic activity of tumor cells. In this report we have analyzed potential peptidases and esterases of importance for release of free melphalan from J1. Exposure of tumor cell lines to J1 resulted in a significant increased level of free intracellular melphalan, at least tenfold at C(max), compared to exposure to melphalan at the same molar concentration. This efficient intracellular delivery could be inhibited in both magnitude and in time by bestatin, a broad spectrum inhibitor of the aminopeptidases, including the metalloproteinase aminopeptidase N (APN, EC 3.4.11.2.), and ebelactone A, an esterase inhibitor. These effects resulted, as expected, in decreased cytotoxic effects of J1. A specific role of APN in hydrolyzing J1 releasing free melphalan was demonstrated in vitro with pure APN enzyme. By using plasmid-based overexpression of APN or down regulation of endogenous APN with siRNA in different tumor cell lines we here confirm the involvement of APN in J1-mediated cytotoxic and apoptotic signaling. In conclusion, this study demonstrates a role of APN in the activation of the melphalan prodrug J1 and subsequently, its cytotoxicity. Given that APN is shown to be overexpressed in several solid tumors our data suggest that J1 may be activated in a tumor selective manner.
Asunto(s)
Antineoplásicos/metabolismo , Antígenos CD13/metabolismo , Dipéptidos/metabolismo , Melfalán/farmacología , Antineoplásicos/química , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Antígenos CD13/genética , Caspasa 3/metabolismo , Línea Celular Tumoral , Dipéptidos/química , Dipéptidos/farmacología , Sistemas de Liberación de Medicamentos , Activación Enzimática , Regulación Enzimológica de la Expresión Génica/fisiología , Regulación Neoplásica de la Expresión Génica/fisiología , Humanos , Estructura Molecular , Profármacos/química , Profármacos/metabolismo , Profármacos/farmacología , Interferencia de ARN , ARN Interferente Pequeño , Factores de TiempoRESUMEN
Tamoxifen, a partial estrogen receptor antagonist, is part of the standard treatment of both primary and advanced breast cancers. However, significant proportions of breast cancers are either de novo resistant or develop tamoxifen resistance during the course of treatment through mechanisms which have been only partly characterized. We have previously found that high vascular endothelial growth factor (VEGF) or VEGF receptor 2 (VEGFR2) expression and concomitant high p38 mitogen-activated protein kinase activity within breast cancers predict a poor outcome for tamoxifen-treated patients. Here, we have molecularly dissected how VEGF/VEGFR2 and p38 are linked, and contribute to tamoxifen resistance within breast cancer using a MCF-7 BC cell model with different 4-hydroxytamoxifen (4-OHT) responsiveness. We report that MCF-7 breast cancer cell lines with tamoxifen resistance have increased secretion of VEGF and increased signaling through VEGFR2 compared with parental MCF-7 cells. 4-OHT treatment caused the ablation of VEGF secretion in parental MCF-7 cells, whereas in the tamoxifen-resistant subline, a VEGF/VEGFR2 signaling loop was still evident upon treatment. Increased basal levels of total and phosphorylated p38 were observed in tamoxifen-resistant cells. Pharmacologic inhibition of p38 reduced the proliferation of both tamoxifen-responsive and tamoxifen-resistant cells and showed an additive growth-inhibitory effect in combination with 4-OHT. A connection between VEGF/VEGFR2 and p38 signaling was identified by VEGF and VEGFR2 knockdown, which equally reduced both the total and the active forms of p38 in tamoxifen-resistant cells. Taken together, our results suggest that decreased sensitivity to 4-OHT is caused by a death-protecting VEGF/VEGFR2 and p38 growth factor loop in breast cancer cells. Inhibition of these signaling pathways may be beneficial to overcome tamoxifen resistance.
Asunto(s)
Comunicación Autocrina/efectos de los fármacos , Neoplasias de la Mama/metabolismo , Resistencia a Antineoplásicos/efectos de los fármacos , Tamoxifeno/análogos & derivados , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Antineoplásicos Hormonales/farmacología , Neoplasias de la Mama/enzimología , Neoplasias de la Mama/patología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , ARN Interferente Pequeño/metabolismo , Transducción de Señal/efectos de los fármacos , Tamoxifeno/farmacología , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidoresRESUMEN
Steroidogenic factor 1 (SF1) is expressed in a time- and cell-specific manner in the endocrine system. In this study we present evidence to support that methylation of CpG sites located in the proximal promoter of the gene encoding SF1 contributes to the restricted expression pattern of this nuclear receptor. DNA methylation analyses revealed a nearly perfect correlation between the methylation status of the proximal promoter and protein expression, such that it was hypomethylated in cells that express SF1 but hypermethylated in nonexpressing cells. Moreover, in vitro methylation of this region completely repressed reporter gene activity in transfected steroidogenic cells. Bisulfite sequencing of DNA from embryonic tissue demonstrated that the proximal promoter was unmethylated in the developing testis and ovary, whereas it was hypermethylated in tissues that do not express SF1. Together these results indicate that the DNA methylation pattern is established early in the embryo and stably inherited thereafter throughout development to confine SF1 expression to the appropriate tissues. Chromatin immunoprecipitation analyses revealed that the transcriptional activator upstream stimulatory factor 2 and RNA polymerase II were specifically recruited to this DNA region in cells in which the proximal promoter is hypomethylated, providing functional support for the fact that lack of methylation corresponds to a transcriptionally active gene. In conclusion, we identified a region within the SF1/Sf1 gene that epigenetically directs cell-specific expression of SF1.
Asunto(s)
Metilación de ADN , Factor Esteroidogénico 1/genética , Animales , Secuencia de Bases , Tipificación del Cuerpo/genética , Islas de CpG , Regulación del Desarrollo de la Expresión Génica , Silenciador del Gen , Células HeLa , Humanos , Ratones , Ratones Endogámicos C57BL , Modelos Biológicos , Datos de Secuencia Molecular , Células 3T3 NIH , Especificidad de Órganos/genética , Regiones Promotoras Genéticas , Factor Esteroidogénico 1/metabolismo , Distribución Tisular , Células Tumorales CultivadasRESUMEN
The orphan nuclear receptor steroidogenic factor 1 (SF-1) is an essential regulator of endocrine organogenesis, sexual differentiation, and steroidogenesis. SF-1 is a transcriptional regulator of cAMP responsive genes, but the exact mechanisms by which cAMP-dependent PKA modulates SF-1 dependent transcription leading to increased steroidogenic output have not been determined. In this report the effects of PKA activation on SF-1 in living cells have been examined by the use of full-length SF-1 cDNA fused to the cDNA encoding green fluorescent protein (GFP). The GFP-SF-1 fusion protein localized to the nucleus of both steroidogenic Y1 cells and nonsteroidogenic COS-1 cells, and the functional properties of wild-type SF-1 were conserved. When the catalytic subunit of PKA was coexpressed with GFP-SF-1, we observed that the fluorescence emission was markedly elevated. These findings were confirmed by Western blot analysis, showing that stimulation of PKA increased SF-1 protein levels. The PKA- induced expression of SF-1 protein was not accompanied by an increase in SF-1 mRNA levels. However, pulse-chase studies showed a decrease in SF-1 degradation rate in response to activation of PKA, indicating that PKA elevates the level of SF-1 by increasing the stability of SF-1 protein.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas de Unión al ADN/metabolismo , Factores de Transcripción/metabolismo , Animales , Células COS , Proteínas de Unión al ADN/genética , Estabilidad de Medicamentos , Activación Enzimática/fisiología , Factores de Transcripción Fushi Tarazu , Proteínas Fluorescentes Verdes , Proteínas de Homeodominio , Proteínas Luminiscentes/genética , Ratones , Mutación , Fosforilación , ARN Mensajero/metabolismo , Receptores Citoplasmáticos y Nucleares , Proteínas Recombinantes de Fusión/metabolismo , Factor Esteroidogénico 1 , Factores de Transcripción/genética , Células Tumorales CultivadasRESUMEN
The orphan nuclear receptor steroidogenic factor-1 (SF-1) plays pivotal roles in the development and function of steroidogenic organs. Here we describe the differential effect of protein kinase A (PKA) on coregulation of SF-1 dependent transcription by two p160 family members, p300/CBP co-integrator-associated protein (p/CIP) and transcription intermediary factor-2 (TIF2). Thus, whereas p/CIP-stimulated SF-1 dependent transcription is further potentiated by PKA, we show that activation of PKA leads to selective downregulation of TIF2 protein and a subsequent repression of TIF2 coactivator function. Using a yeast two-hybrid screen we also identified a novel zinc finger containing protein, which interacts with SF-1 via the AF-2 domain.