RESUMEN
Androgen biosynthesis in the human fetus proceeds through the adrenal sex steroid precursor dehydroepiandrosterone, which is converted to testosterone in the gonads, followed by further activation to 5α-dihydrotestosterone in genital skin, thereby facilitating male external genital differentiation. Congenital adrenal hyperplasia due to P450 oxidoreductase deficiency results in disrupted dehydroepiandrosterone biosynthesis, explaining undervirilization in affected boys. However, many affected girls are born virilized, despite low circulating androgens. We hypothesized that this is due to a prenatally active, alternative androgen biosynthesis pathway from 17α-hydroxyprogesterone to 5α-dihydrotestosterone, which bypasses dehydroepiandrosterone and testosterone, with increased activity in congenital adrenal hyperplasia variants associated with 17α-hydroxyprogesterone accumulation. Here we employ explant cultures of human fetal organs (adrenals, gonads, genital skin) from the major period of sexual differentiation and show that alternative pathway androgen biosynthesis is active in the fetus, as assessed by liquid chromatography-tandem mass spectrometry. We found androgen receptor expression in male and female genital skin using immunohistochemistry and demonstrated that both 5α-dihydrotestosterone and adrenal explant culture supernatant induce nuclear translocation of the androgen receptor in female genital skin primary cultures. Analyzing urinary steroid excretion by gas chromatography-mass spectrometry, we show that neonates with P450 oxidoreductase deficiency produce androgens through the alternative androgen pathway during the first weeks of life. We provide quantitative in vitro evidence that the corresponding P450 oxidoreductase mutations predominantly support alternative pathway androgen biosynthesis. These results indicate a key role of alternative pathway androgen biosynthesis in the prenatal virilization of girls affected by congenital adrenal hyperplasia due to P450 oxidoreductase deficiency.
Asunto(s)
17-alfa-Hidroxiprogesterona/metabolismo , Andrógenos/biosíntesis , Fenotipo del Síndrome de Antley-Bixler/genética , Feto/metabolismo , Receptores Androgénicos/genética , Virilismo/metabolismo , Glándulas Suprarrenales/embriología , Glándulas Suprarrenales/metabolismo , Andrógenos/genética , Células Cultivadas , Femenino , Feto/embriología , Genitales/embriología , Genitales/metabolismo , Gónadas/embriología , Gónadas/metabolismo , Humanos , Masculino , Receptores Androgénicos/metabolismo , Diferenciación Sexual , Virilismo/genéticaRESUMEN
BACKGROUND: Diffuse intrinsic pontine glioma (DIPG) is a lethal type of pediatric brain tumor that is resistant to conventional chemotherapies. Palbociclib is a putative novel DIPG treatment that restricts the proliferation of rapidly dividing cancer cells via selective inhibition of cyclin-dependent kinase (CDK) 4 and CDK6. However, implementing palbociclib as a monotherapy for DIPG is unfeasible, as CDK4/6 inhibitor resistance is commonplace and palbociclib does not readily cross the blood-brain barrier (BBB) or persist in the central nervous system. To inhibit the growth of DIPG cells, we aimed to use palbociclib in combination with the rapamycin analog temsirolimus, which is known to ameliorate resistance to CDK4/6 inhibitors and inhibit BBB efflux. MATERIALS AND METHODS: We tested palbociclib and temsirolimus in three patient-derived DIPG cell lines. The expression profiles of key proteins in the CDK4/6 and mammalian target of rapamycin (mTOR) signaling pathways were assessed, respectively, to determine feasibility against DIPG. Moreover, we investigated effects on cell viability and examined in vivo drug toxicity. RESULTS: Immunoblot analyses revealed palbociclib and temsirolimus inhibited CDK4/6 and mTOR signaling through canonical perturbation of phosphorylation of the retinoblastoma (RB) and mTOR proteins, respectively; however, we observed noncanonical downregulation of mTOR by palbociclib. We demonstrated that palbociclib and temsirolimus inhibited cell proliferation in all three DIPG cell lines, acting synergistically in combination to further restrict cell growth. Flow cytometric analyses revealed both drugs caused G1 cell cycle arrest, and clonogenic assays showed irreversible effects on cell proliferation. Palbociclib did not elicit neurotoxicity in primary cultures of normal rat hippocampi or when infused into rat brains. CONCLUSION: These data illustrate the in vitro antiproliferative effects of CDK4/6 and mTOR inhibitors in DIPG cells. Direct infusion of palbociclib into the brain, in combination with systemic delivery of temsirolimus, represents a promising new approach to developing a much-needed treatment for DIPG.
RESUMEN
OBJECTIVE The pan-histone deacetylase inhibitor panobinostat has preclinical efficacy against diffuse intrinsic pontine glioma (DIPG), and the oral formulation has entered a Phase I clinical trial. However, panobinostat does not cross the blood-brain barrier in humans. Convection-enhanced delivery (CED) is a novel neurosurgical drug delivery technique that bypasses the blood-brain barrier and is of considerable clinical interest in the treatment of DIPG. METHODS The authors investigated the toxicity, distribution, and clearance of a water-soluble formulation of panobinostat (MTX110) in a small- and large-animal model of CED. Juvenile male Wistar rats (n = 24) received panobinostat administered to the pons by CED at increasing concentrations and findings were compared to those in animals that received vehicle alone (n = 12). Clinical observation continued for 2 weeks. Animals were sacrificed at 72 hours or 2 weeks following treatment, and the brains were subjected to neuropathological analysis. A further 8 animals received panobinostat by CED to the striatum and were sacrificed 0, 2, 6, or 24 hours after infusion, and their brains explanted and snap-frozen. Tissue-drug concentration was determined by liquid chromatography tandem mass spectrometry (LC-MS/MS). Large-animal toxicity was investigated using a clinically relevant MRI-guided translational porcine model of CED in which a drug delivery system designed for humans was used. Panobinostat was administered at 30 µM to the ventral pons of 2 juvenile Large White-Landrace cross pigs. The animals were subjected to clinical and neuropathological analysis, and findings were compared to those obtained in controls after either 1 or 2 weeks. Drug distribution was determined by LC-MS/MS in porcine white and gray matter immediately after CED. RESULTS There were no clinical or neuropathological signs of toxicity up to an infused concentration of 30 µM in both small- and large-animal models. The half-life of panobinostat in rat brain after CED was 2.9 hours, and the drug was observed to be distributed in porcine white and gray matter with a volume infusion/distribution ratio of 2 and 3, respectively. CONCLUSIONS CED of water-soluble panobinostat, up to a concentration of 30 µM, was not toxic and was distributed effectively in normal brain. CED of panobinostat warrants clinical investigation in patients with DIPG.
Asunto(s)
Antineoplásicos/administración & dosificación , Neoplasias del Tronco Encefálico/tratamiento farmacológico , Convección , Glioma/tratamiento farmacológico , Panobinostat/administración & dosificación , Animales , Antineoplásicos/farmacocinética , Neoplasias del Tronco Encefálico/diagnóstico por imagen , Proteínas de Unión al Calcio/metabolismo , Cromatografía Liquida , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Proteína Ácida Fibrilar de la Glía/metabolismo , Glioma/diagnóstico por imagen , Imagen por Resonancia Magnética , Masculino , Proteínas de Microfilamentos/metabolismo , Panobinostat/farmacocinética , Fosfopiruvato Hidratasa/metabolismo , Ratas , Ratas Wistar , Porcinos , Espectrometría de Masas en Tándem , Factores de Tiempo , Distribución Tisular/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Targeting epigenetic changes in diffuse intrinsic pontine glioma (DIPG) may provide a novel treatment option for patients. This report demonstrates that sodium valproate, a histone deacetylase inhibitor (HDACi), can increase the cytotoxicity of carboplatin in an additive and synergistic manner in DIPG cells in vitro. Sodium valproate causes a dose-dependent decrease in DIPG cell viability in three independent ex vivo cell lines. Furthermore, sodium valproate caused an increase in acetylation of histone H3. Changes in cell viability were consistent with an induction of apoptosis in DIPG cells in vitro, determined by flow cytometric analysis of Annexin V staining and assessment of apoptotic markers by western blotting. Subsequently, immunofluorescent staining of neuronal and glial markers was used to determine toxicity in normal rat hippocampal cells. Pre-treatment of cells with sodium valproate enhanced the cytotoxic effects of carboplatin, in three DIPG cell lines tested. These results demonstrate that sodium valproate causes increased histone H3 acetylation indicative of HDAC inhibition, which is inversely correlated with a reduction in cell viability. Cell viability is reduced through an induction of apoptosis in DIPG cells. Sodium valproate potentiates carboplatin cytotoxicity and prompts further work to define the mechanism responsible for the synergy between these two drugs and determine in vivo efficacy. These findings support the use of sodium valproate as an adjuvant treatment for DIPG.
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Adyuvantes Farmacéuticos/farmacología , Anticonvulsivantes/farmacología , Neoplasias del Tronco Encefálico/tratamiento farmacológico , Glioma/tratamiento farmacológico , Ácido Valproico/farmacología , Acetilación/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Neoplasias del Tronco Encefálico/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Reposicionamiento de Medicamentos/métodos , Epigénesis Genética/efectos de los fármacos , Glioma/metabolismo , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Inhibidores de Histona Desacetilasas/farmacología , Humanos , RatasRESUMEN
A new class of anion transporter named 'perenosins' consisting of a pyrrole linked through an imine to either an indole, benzimidazole or indazole is reported. The indole containing members of the perenosin family function as effective transmembrane Cl(-)/NO3(-) antiporters and HCl cotransporters in a manner similar to the prodigiosenes. The compounds reduce the viability of MDA-MB-231 and MCF-7.
Asunto(s)
Aniones/metabolismo , Antineoplásicos Fitogénicos/farmacología , Antiportadores/farmacología , Iminas/química , Indoles/farmacología , Pirroles/farmacología , Antineoplásicos Fitogénicos/síntesis química , Antineoplásicos Fitogénicos/química , Antiportadores/síntesis química , Antiportadores/química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Indoles/química , Transporte Iónico/efectos de los fármacos , Células MCF-7 , Modelos Moleculares , Estructura Molecular , Pirroles/síntesis química , Pirroles/química , Relación Estructura-ActividadRESUMEN
5-Aminoimidazole-4-carboxamide ribonucleotide (known as ZMP) is a metabolite produced in de novo purine biosynthesis and histidine biosynthesis, but only utilized in the cell by a homodimeric bifunctional enzyme (called ATIC) that catalyzes the last two steps of de novo purine biosynthesis. ZMP is known to act as an allosteric activator of the cellular energy sensor adenosine monophosphate-activated protein kinase (AMPK), when exogenously administered as the corresponding cell-permeable ribonucleoside. Here, we demonstrate that endogenous ZMP, produced by the aforementioned metabolic pathways, is also capable of activating AMPK. Using an inhibitor of ATIC homodimerization to block the ninth step of de novo purine biosynthesis, we demonstrate that the subsequent increase in endogenous ZMP activates AMPK and its downstream signaling pathways. We go on to illustrate the viability of using this approach to AMPK activation as a therapeutic strategy with an in vivo mouse model for metabolic disorders.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Aminoimidazol Carboxamida/análogos & derivados , Transferasas de Hidroximetilo y Formilo/antagonistas & inhibidores , Complejos Multienzimáticos/antagonistas & inhibidores , Nucleótido Desaminasas/antagonistas & inhibidores , Purinas/biosíntesis , Ribonucleótidos/farmacología , Aminoimidazol Carboxamida/farmacología , Animales , Activación Enzimática , Células HCT116 , Humanos , Transferasas de Hidroximetilo y Formilo/metabolismo , Células MCF-7 , Masculino , Ratones , Ratones Endogámicos C57BL , Modelos Animales , Complejos Multienzimáticos/metabolismo , Nucleótido Desaminasas/metabolismo , Multimerización de Proteína/efectos de los fármacosRESUMEN
Very high diastereoselectivity can be achieved by 1,3-chelation-controlled allylation of aldehydes that possess a non-chelating α-ether substituent, even if the α-position is a quaternary centre and/or a spiro-epoxide. This reaction was used as a key step in an enantioselective synthesis of the angiogenesis inhibitor luminacinâ D.
Asunto(s)
Aldehídos/química , Benzaldehídos/química , Quelantes/química , Compuestos Epoxi/química , Éter/química , Compuestos de Espiro/química , Catálisis , EstereoisomerismoRESUMEN
Human sexual differentiation is a critical process whereby a strict dimorphism is established that enables future reproductive success as phenotypic males and females. Significant components of this differentiation pathway unfold during the first three months of gestation when they are sensitive to disruption by abnormal hormonal influences. Excessive exposure of female development to androgens in conditions such as congenital adrenal hyperplasia causes virilization. However, recently we have suggested that female development normally takes place in the presence of low, yet significant, levels of androgen, implying a need for strict regulation to avoid virilization and the potential for a biological role of androgens in females that has not been fully elucidated. Here, we review androgen-dependent male differentiation of the external genitalia in humans, and link this to current understanding of female development and steroidogenesis in the developing adrenal cortex.