RESUMO
BACKGROUND: Current strategies to inhibit androgen receptor (AR) are circumvented in castration-resistant prostate cancer (CRPC). Cyclin-dependent kinase 7 (CDK7) promotes AR signalling, in addition to established roles in cell cycle and global transcription, providing a rationale for its therapeutic targeting in CRPC. METHODS: The antitumour activity of CT7001, an orally bioavailable CDK7 inhibitor, was investigated across CRPC models in vitro and in xenograft models in vivo. Cell-based assays and transcriptomic analyses of treated xenografts were employed to investigate the mechanisms driving CT7001 activity, alone and in combination with the antiandrogen enzalutamide. RESULTS: CT7001 selectively engages with CDK7 in prostate cancer cells, causing inhibition of proliferation and cell cycle arrest. Activation of p53, induction of apoptosis, and suppression of transcription mediated by full-length and constitutively active AR splice variants contribute to antitumour efficacy in vitro. Oral administration of CT7001 represses growth of CRPC xenografts and significantly augments growth inhibition achieved by enzalutamide. Transcriptome analyses of treated xenografts indicate cell cycle and AR inhibition as the mode of action of CT7001 in vivo. CONCLUSIONS: This study supports CDK7 inhibition as a strategy to target deregulated cell proliferation and demonstrates CT7001 is a promising CRPC therapeutic, alone or in combination with AR-targeting compounds.
Assuntos
Neoplasias de Próstata Resistentes à Castração , Masculino , Humanos , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Neoplasias de Próstata Resistentes à Castração/genética , Neoplasias de Próstata Resistentes à Castração/metabolismo , Linhagem Celular Tumoral , Ensaios Antitumorais Modelo de Xenoenxerto , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismo , Nitrilas/uso terapêutico , Quinases Ciclina-Dependentes/uso terapêutico , Inibidores Enzimáticos/uso terapêutico , Proliferação de CélulasRESUMO
Mitochondrial electron transport drives ATP synthesis but also generates reactive oxygen species, which are both cellular signals and damaging oxidants. Superoxide production by respiratory complex III is implicated in diverse signaling events and pathologies, but its role remains controversial. Using high-throughput screening, we identified compounds that selectively eliminate superoxide production by complex III without altering oxidative phosphorylation; they modulate retrograde signaling including cellular responses to hypoxic and oxidative stress.
Assuntos
Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Sequestradores de Radicais Livres/farmacologia , Mitocôndrias/efeitos dos fármacos , Pirazóis/farmacologia , Pirimidinas/farmacologia , Superóxidos/antagonistas & inibidores , Trifosfato de Adenosina/biossíntese , Animais , Antimicina A/análogos & derivados , Antimicina A/antagonistas & inibidores , Antimicina A/farmacologia , Relação Dose-Resposta a Droga , Feminino , Células HEK293 , Ensaios de Triagem em Larga Escala , Humanos , Peróxido de Hidrogênio/antagonistas & inibidores , Peróxido de Hidrogênio/metabolismo , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Masculino , Mitocôndrias/metabolismo , Fosforilação Oxidativa/efeitos dos fármacos , Estresse Oxidativo , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Transdução de Sinais , Superóxidos/metabolismoRESUMO
We studied acute changes of secretory vesicle pH in pancreatic beta-cells with a fluorescent pH indicator, lysosensor green DND-189. Fluorescence was decreased by 0.66 +/- 0.10% at 149 +/- 16 s with 22.2 mM glucose stimulation, indicating that vesicular pH was alkalinized by approximately 0.016 unit. Glucose-responsive pH increase was observed when cytosolic Ca2+ influx was blocked but disappeared when an inhibitor of glycolysis or mitochondrial ATP synthase was present. Glutamate dimethyl ester (GME), a plasma membrane-permeable analog of glutamate, potentiated glucose-stimulated insulin secretion at 5 mM without changing cellular ATP content or cytosolic Ca2+ concentration ([Ca2+]). Application of GME at basal glucose concentration decreased DND-189 fluorescence by 0.83 +/- 0.19% at 38 +/- 2 s. These results indicated that the acutely alkalinizing effect of glucose on beta-cell secretory vesicle pH was dependent on glucose metabolism but independent of modulations of cytosolic [Ca2+]. Moreover, glutamate derived from glucose may be one of the mediators of this alkalinizing effect of glucose, which may have potential relevance to the alteration of secretory function by glutamate.
Assuntos
Glucose/metabolismo , Glucose/farmacologia , Glutamatos/farmacologia , Insulina/metabolismo , Ilhotas Pancreáticas/ultraestrutura , Vesículas Secretórias/efeitos dos fármacos , Trifosfato de Adenosina/metabolismo , Animais , Corantes Fluorescentes , Concentração de Íons de Hidrogênio , Secreção de Insulina , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Oxirredução , Canais de Potássio/efeitos dos fármacos , Canais de Potássio/fisiologia , Vesículas Secretórias/metabolismoRESUMO
Glucose-responsive (GR) neurons from hypothalamic nuclei are implicated in the regulation of feeding and satiety. To determine the role of intracellular ATP in the closure of ATP-sensitive K(+) (K(ATP)) channels in these cells and associated glia, the cytosolic ATP concentration ([ATP](c)) was monitored in vivo using adenoviral-driven expression of recombinant targeted luciferases and bioluminescence imaging. Arguing against a role for ATP in the closure of K(ATP) channels in GR neurons, glucose (3 or 15 mM) caused no detectable increase in [ATP](c), monitored with cytosolic luciferase, and only a small decrease in the concentration of ATP immediately beneath the plasma membrane, monitored with a SNAP25-luciferase fusion protein. In contrast to hypothalamic neurons, hypothalamic glia responded to glucose (3 and 15 mM) with a significant increase in [ATP](c). Both neurons and glia from the cerebellum, a glucose-unresponsive region of the brain, responded robustly to 3 or 15 mM glucose with increases in [ATP](c). Further implicating an ATP-independent mechanism of K(ATP) channel closure in hypothalamic neurons, removal of extracellular glucose (10 mM) suppressed the electrical activity of GR neurons in the presence of a fixed, high concentration (3 mM) of intracellular ATP. Neurons from both brain regions responded to 5 mM lactate (but not pyruvate) with an oligomycin-sensitive increase in [ATP](c). High levels of the plasma membrane lactate-monocarboxylate transporter, MCT1, were found in both cell types, and exogenous lactate efficiently closed K(ATP) channels in GR neurons. These data suggest that (1) ATP-independent intracellular signalling mechanisms lead to the stimulation of hypothalamic neurons by glucose, and (2) these effects may be potentiated in vivo by the release of lactate from neighbouring glial cells.
Assuntos
Trifosfato de Adenosina/metabolismo , Células Quimiorreceptoras/fisiologia , Citosol/metabolismo , Glucose/metabolismo , Hipotálamo/fisiologia , Neurônios/fisiologia , Trifosfato de Adenosina/fisiologia , Animais , Membrana Celular/metabolismo , Células Cultivadas , Cerebelo/citologia , Cerebelo/metabolismo , Eletrofisiologia , Metabolismo Energético , Hipotálamo/citologia , Lactatos/metabolismo , Masculino , Transportadores de Ácidos Monocarboxílicos/metabolismo , NADP/metabolismo , Neuroglia/metabolismo , Concentração Osmolar , Canais de Potássio/metabolismo , Ratos , Ratos Sprague-Dawley , Ratos WistarRESUMO
Recruitment of secretory vesicles to the cell surface is essential for the sustained secretion of insulin in response to glucose. At present, the molecular motors involved in this movement, and the mechanisms whereby they may be regulated, are undefined. To investigate the role of kinesin family members, we labelled densecore vesicles in clonal beta-cells using an adenovirally expressed, vesicle-targeted green fluorescent protein (phogrin.EGFP), and employed immunoadsorption to obtain highly purified insulin-containing vesicles. Whereas several kinesin family members were expressed in this cell type, only conventional kinesin heavy chain (KHC) was detected in vesicle preparations. Expression of a dominant-negative KHC motor domain (KHC(mut)) blocked all vesicular movements with velocity >0.4 micro m second(-1), which demonstrates that kinesin activity was essential for vesicle motility in live beta-cells. Moreover, expression of KHC(mut) strongly inhibited the sustained, but not acute, stimulation of secretion by glucose. Finally, vesicle movement was stimulated by ATP dose-dependently in permeabilized cells, which suggests that glucose-induced increases in cytosolic [ATP] mediate the effects of the sugar in vivo, by enhancing kinesin activity. These data therefore provide evidence for a novel mechanism whereby glucose may enhance insulin release.
Assuntos
Células Clonais/metabolismo , Glucose/metabolismo , Ilhotas Pancreáticas/metabolismo , Cinesinas/metabolismo , Transporte Proteico/genética , Vesículas Secretórias/metabolismo , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/farmacologia , Animais , Células Cultivadas , Células Clonais/efeitos dos fármacos , Citosol/metabolismo , Relação Dose-Resposta a Droga , Exocitose/efeitos dos fármacos , Exocitose/genética , Glucose/farmacologia , Proteínas de Fluorescência Verde , Humanos , Insulina/metabolismo , Secreção de Insulina , Ilhotas Pancreáticas/efeitos dos fármacos , Cinesinas/genética , Proteínas Luminescentes , Proteínas de Membrana , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mutação/genética , Transporte Proteico/efeitos dos fármacos , Proteínas Tirosina Fosfatases , Proteínas Tirosina Fosfatases Classe 8 Semelhantes a Receptores , Proteínas Recombinantes de Fusão , Vesículas Secretórias/efeitos dos fármacosRESUMO
The mechanisms by which glucose may affect protein kinase C (PKC) activity in the pancreatic islet beta-cell are presently unclear. By developing adenovirally expressed chimeras encoding fusion proteins between green fluorescent protein and conventional (betaII), novel (delta), or atypical (zeta) PKCs, we show that glucose selectively alters the subcellular localization of these enzymes dynamically in primary islet and MIN6 beta-cells. Examined by laser scanning confocal or total internal reflection fluorescence microscopy, elevated glucose concentrations induced oscillatory translocations of PKCbetaII to spatially confined regions of the plasma membrane. Suggesting that increases in free cytosolic Ca(2+) concentrations ([Ca(2+)](c)) were primarily responsible, prevention of [Ca(2+)](c) increases with EGTA or diazoxide completely eliminated membrane recruitment, whereas elevation of cytosolic [Ca(2+)](c) with KCl or tolbutamide was highly effective in redistributing PKCbetaII both to the plasma membrane and to the surface of dense core secretory vesicles. By contrast, the distribution of PKCdelta.EGFP, which binds diacylglycerol but not Ca(2+), was unaffected by glucose. Measurement of [Ca(2+)](c) immediately beneath the plasma membrane with a ratiometric "pericam," fused to synaptic vesicle-associated protein-25, revealed that depolarization induced significantly larger increases in [Ca(2+)](c) in this domain. These data demonstrate that nutrient stimulation of beta-cells causes spatially and temporally complex changes in the subcellular localization of PKCbetaII, possibly resulting from the generation of Ca(2+) microdomains. Localized changes in PKCbetaII activity may thus have a role in the spatial control of insulin exocytosis.
Assuntos
Glucose/farmacologia , Ilhotas Pancreáticas/fisiologia , Isoenzimas/metabolismo , Proteína Quinase C/metabolismo , Animais , Linhagem Celular , Membrana Celular/enzimologia , Membrana Celular/fisiologia , Células Clonais , Citosol/enzimologia , Diazóxido/farmacologia , Ácido Egtázico/farmacologia , Proteínas de Fluorescência Verde , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/enzimologia , Cinética , Proteínas Luminescentes/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Camundongos , Potássio/farmacologia , Proteína Quinase C beta , Transporte Proteico , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Glucose-stimulated increases in mitochondrial metabolism are generally thought to be important for the activation of insulin secretion. Pyruvate dehydrogenase (PDH) is a key regulatory enzyme, believed to govern the rate of pyruvate entry into the citrate cycle. We show here that elevated glucose concentrations (16 or 30 vs 3 mM) cause an increase in PDH activity in both isolated rat islets, and in a clonal beta-cell line (MIN6). However, increases in PDH activity elicited with either dichloroacetate, or by adenoviral expression of the catalytic subunit of pyruvate dehydrogenase phosphatase, were without effect on glucose-induced increases in mitochondrial pyridine nucleotide levels, or cytosolic ATP concentration, in MIN6 cells, and insulin secretion from isolated rat islets. Similarly, the above parameters were unaffected by blockade of the glucose-induced increase in PDH activity by adenovirus-mediated over-expression of PDH kinase (PDK). Thus, activation of the PDH complex plays an unexpectedly minor role in stimulating glucose metabolism and in triggering insulin release.
Assuntos
Adenoviridae/genética , Glucose/farmacologia , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Piruvato Desidrogenase (Lipoamida)/metabolismo , Animais , Células Cultivadas , Células Clonais , Ácido Dicloroacético/farmacologia , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Vetores Genéticos , Glucose/metabolismo , Secreção de Insulina , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/enzimologia , Masculino , Microscopia de Fluorescência , Mitocôndrias/metabolismo , Inibidores de Proteínas Quinases , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases , Piruvato Desidrogenase (Lipoamida)-Fosfatase/genética , Piruvato Desidrogenase (Lipoamida)-Fosfatase/metabolismo , Piruvato Desidrogenase Quinase de Transferência de Acetil , Ratos , Ratos Wistar , TransfecçãoRESUMO
Normal glucose-stimulated insulin secretion is pulsatile, but the molecular mechanisms underlying this pulsatility are poorly understood. Oscillations in the intracellular free [ATP]/[ADP] ratio represent one possible mechanism because they would be expected to cause fluctuations in ATP-sensitive K(+) channel activity and hence oscillatory Ca(2+) influx. After imaging recombinant firefly luciferase, expressed via an adenoviral vector in single human or mouse islet beta-cells, we report here that cytosolic free ATP concentrations oscillate and that these oscillations are affected by glucose. In human beta-cells, oscillations were observed at both 3 and 15 mmol/l glucose, but the oscillations were of a longer wavelength at the higher glucose concentration (167 vs. 66 s). Mouse beta-cells displayed oscillations in both cytosolic free [Ca(2+)] and [ATP] only at elevated glucose concentrations, both with a period of 120 s. To explore the causal relationship between [Ca(2+)] and [ATP] oscillations, the regulation of each was further investigated in populations of MIN6 beta-cells. Incubation in Ca(2+)-free medium lowered cytosolic [Ca(2+)] but increased [ATP] in MIN6 cells at both 3 and 30 mmol/l glucose. Removal of external Ca(2+) increased [ATP], possibly by decreasing ATP consumption by endoplasmic reticulum Ca(2+)-ATPases. These results allow a model to be constructed of the beta-cell metabolic oscillator that drives nutrient-induced insulin secretion.