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1.
J Biol Chem ; 291(42): 22231-22243, 2016 Oct 14.
Artículo en Inglés | MEDLINE | ID: mdl-27576691

RESUMEN

Androgen receptor (AR) is a validated drug target for all stages of prostate cancer including metastatic castration-resistant prostate cancer (CRPC). All current hormone therapies for CRPC target the C-terminal ligand-binding domain of AR and ultimately all fail with resumed AR transcriptional activity. Within the AR N-terminal domain (NTD) is activation function-1 (AF-1) that is essential for AR transcriptional activity. Inhibitors of AR AF-1 would potentially block most AR mechanisms of resistance including constitutively active AR splice variants that lack the ligand-binding domain. Here we provide evidence that sintokamide A (SINT1) binds AR AF-1 region to specifically inhibit transactivation of AR NTD. Consistent with SINT1 targeting AR AF-1, it attenuated transcriptional activities of both full-length AR and constitutively active AR splice variants, which correlated with inhibition of growth of enzalutamide-resistant prostate cancer cells expressing AR splice variants. In vivo, SINT1 caused regression of CRPC xenografts and reduced expression of prostate-specific antigen, a gene transcriptionally regulated by AR. Inhibition of AR activity by SINT1 was additive to EPI-002, a known AR AF-1 inhibitor that is in clinical trials (NCT02606123). This implies that SINT1 binds to a site on AF-1 that is unique from EPI. Consistent with this suggestion, these two compounds showed differences in blocking AR interaction with STAT3. This work provides evidence that the intrinsically disordered NTD of AR is druggable and that SINT1 analogs may provide a novel scaffold for drug development for the treatment of prostate cancer or other diseases of the AR axis.


Asunto(s)
Antagonistas de Receptores Androgénicos/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Proteínas de Neoplasias , Neoplasias de la Próstata , Pirrolidinonas/farmacología , Receptores Androgénicos/biosíntesis , Activación Transcripcional/efectos de los fármacos , Animales , Línea Celular Tumoral , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/metabolismo , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/metabolismo , Dominios Proteicos , Pirrolidinonas/farmacocinética , Factor de Transcripción STAT3/metabolismo
2.
Clin Cancer Res ; 22(11): 2744-54, 2016 06 01.
Artículo en Inglés | MEDLINE | ID: mdl-26712685

RESUMEN

PURPOSE: The PI3K/Akt/mTOR pathway is activated in most castration-resistant prostate cancers (CRPC). Transcriptionally active androgen receptor (AR) plays a role in the majority of CRPCs. Therefore, cotargeting full-length (FL) AR and PI3K/Akt/mTOR signaling has been proposed as a possible, more effective therapeutic approach for CRPC. However, truncated AR-splice variants (AR-V) that are constitutively active and dominant over FL-AR are associated with tumor progression and resistance mechanisms in CRPC. It is currently unknown how blocking the PI3K/Akt/mTOR pathway impacts prostate cancer driven by AR-Vs. Here, we evaluated the efficacy and mechanism of combination therapy to block mTOR activity together with EPI-002, an AR N-terminal domain (NTD) antagonist that blocks the transcriptional activities of FL-AR and AR-Vs in models of CRPC. EXPERIMENTAL DESIGN: To determine the functional roles of FL-AR, AR-Vs, and PI3K/Akt/mTOR pathways, we employed EPI-002 or enzalutamide and BEZ235 (low dose) or everolimus in human prostate cancer cells that express FL-AR or FL-AR and AR-Vs (LNCaP95). Gene expression and efficacy were examined in vitro and in vivo RESULTS: EPI-002 had antitumor activity in enzalutamide-resistant LNCaP95 cells that was associated with decreased expression of AR-V target genes (e.g., UBE2C). Inhibition of mTOR provided additional blockade of UBE2C expression. A combination of EPI-002 and BEZ235 decreased the growth of LNCaP95 cells in vitro and in vivo CONCLUSIONS: Cotargeting mTOR and AR-NTD to block transcriptional activities of FL-AR and AR-Vs provided maximum antitumor efficacy in PTEN-null, enzalutamide-resistant CRPC. Clin Cancer Res; 22(11); 2744-54. ©2015 AACR.


Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Compuestos de Bencidrilo/farmacología , Glicerol/análogos & derivados , Neoplasias de la Próstata Resistentes a la Castración/tratamiento farmacológico , Receptores Androgénicos/genética , Empalme Alternativo , Animales , Antineoplásicos Hormonales/farmacología , Antineoplásicos Hormonales/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Sinergismo Farmacológico , Everolimus/administración & dosificación , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Glicerol/farmacología , Imidazoles/administración & dosificación , Masculino , Ratones Endogámicos NOD , Ratones SCID , Feniltiohidantoína/administración & dosificación , Feniltiohidantoína/análogos & derivados , Neoplasias de la Próstata Resistentes a la Castración/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Quinolinas/administración & dosificación , Receptores Androgénicos/metabolismo , Transducción de Señal , Serina-Treonina Quinasas TOR/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto
3.
Toxins (Basel) ; 3(6): 647-59, 2011 06.
Artículo en Inglés | MEDLINE | ID: mdl-22069731

RESUMEN

Aspergillus flavus and Aspergillus parasiticus are important pathogens of cotton, corn, peanuts and other oil-seed crops, producing toxins both in the field and during storage. We have designed three siRNA sequences (Nor-Ia, Nor-Ib, Nor-Ic) to target the mRNA sequence of the aflD gene to examine the potential for using RNA silencing technology to control aflatoxin production. Thus, the effect of siRNAs targeting of two key genes in the aflatoxin biosynthetic pathway, aflD (structural) and aflR (regulatory gene) and on aflatoxin B(1 )(AFB(1)), and aflatoxin G(1) (AFG(1)) production was examined. The study showed that Nor-Ib gave a significant decrease in aflD mRNA, aflR mRNA abundance, and AFB(1) production (98, 97 and 97% when compared to the controls) in A. flavus NRRL3357, respectively. Reduction in aflD and aflR mRNA abundance and AFB(1 )production increased with concentration of siRNA tested. There was a significant inhibition in aflD and AFB(1) production by A. flavus EGP9 and AFG(1 )production by A. parasiticus NRRL 13005. However, there was no significant decrease in AFG(1) production by A. parasiticus SSWT 2999. Changes in AFB(1) production in relation to mRNA levels of aflD showed a good correlation (R = 0.88; P = 0.00001); changes in aflR mRNA level in relation to mRNA level of aflD also showed good correlation (R = 0.82; P = 0.0001). The correlations between changes in aflR and aflD gene expression suggests a strong relationship between these structural and regulatory genes, and that aflD could be used as a target gene to develop efficient means for aflatoxin control using RNA silencing technology.


Asunto(s)
Aflatoxina B1/biosíntesis , Aflatoxinas/biosíntesis , Oxidorreductasas de Alcohol/genética , Aspergillus flavus/metabolismo , Proteínas Fúngicas/genética , Genes Fúngicos , Interferencia de ARN , Aflatoxina B1/análisis , Aflatoxinas/análisis , Aspergillus flavus/genética , Aspergillus flavus/crecimiento & desarrollo , Cromatografía Líquida de Alta Presión , Productos Agrícolas/microbiología , Regulación Fúngica de la Expresión Génica , Genes Reguladores , Oxidorreductasas de Alcohol Dependientes de NAD (+) y NADP (+) , Protoplastos/metabolismo , ARN Interferente Pequeño/genética , Reacción en Cadena en Tiempo Real de la Polimerasa
4.
PLoS One ; 5(9)2010 Sep 29.
Artículo en Inglés | MEDLINE | ID: mdl-20927371

RESUMEN

Higher order chromatin folding is critical to a number of developmental processes, including the regulation of gene expression. Recently developed biochemical techniques such as RNA TRAP and chromosome conformation capture (3C) have provided us with the tools to probe chromosomal structures. These techniques have been applied to the ß-globin locus, revealing a complex pattern of interactions with regions along the chromosome that the gene resides on. However, biochemical and microscopy data on the nature of ß-globin interactions with other chromosomes is contradictory. Therefore we developed a novel 4C variant, Complete-genome 3C by vectorette amplification (4Cv), which allows an unbiased and quantitative method to examine chromosomal structure. We have used 4Cv to study the microenvironment of the ß-globin locus in mice and show that a significant proportion of the interactions of ß-globin are inter-chromosomal. Furthermore, our data show that in the liver, where the gene is active, ß-globin is more likely to interact with other chromosomes, compared to the brain where the gene is silent and is more likely to interact with other regions along the same chromosome. Our data suggest that transcriptional activation of the ß-globin locus leads to a change in nuclear position relative to the chromosome territory.


Asunto(s)
Cromatina/metabolismo , Cromosomas de los Mamíferos/metabolismo , Técnicas Genéticas , Globinas beta/genética , Globinas beta/metabolismo , Animales , Encéfalo/embriología , Encéfalo/metabolismo , Cromatina/genética , Cromosomas de los Mamíferos/genética , Hígado/embriología , Hígado/metabolismo , Ratones , Ratones Endogámicos C57BL , Unión Proteica , Activación Transcripcional
5.
ScientificWorldJournal ; 10: 90-102, 2010 Jan 08.
Artículo en Inglés | MEDLINE | ID: mdl-20062956

RESUMEN

The way in which the genome of a multicellular organism can orchestrate the differentiation of trillions of cells and many organs, all from a single fertilized egg, is the subject of intense study. Different cell types can be defined by the networks of genes they express. This differential expression is regulated at the epigenetic level by chromatin modifications, such as DNA and histone methylation, which interact with structural and enzymatic proteins, resulting in the activation or silencing of any given gene. While detailed mechanisms are emerging on the role of different chromatin modifications and how these functions are effected at the molecular level, it is still unclear how their deposition across the epigenomic landscape is regulated in different cells. A raft of recent evidence is accumulating that implicates long noncoding RNAs (lncRNAs) in these processes. Most genomes studied to date undergo widespread transcription, the majority of which is not translated into proteins. In this review, we will describe recent work suggesting that lncRNAs are more than transcriptional "noise", but instead play a functional role by acting as tethers and guides to bind proteins responsible for modifying chromatin and mediating their deposition at specific genomic locations. We suggest that lncRNAs are at the heart of developmental regulation, determining the epigenetic status and transcriptional network in any given cell type, and that they provide a means to integrate external differentiation cues with dynamic nuclear responses through the regulation of a metastable epigenome. Better characterization of the lncRNA-protein "interactome" may eventually lead to a new molecular toolkit, allowing researchers and clinicians to modulate the genome at the epigenetic level to treat conditions such as cancer.


Asunto(s)
Cromatina/metabolismo , Epigénesis Genética , ARN no Traducido/metabolismo , Animales , Cromatina/genética , Impresión Genómica/genética , Histonas/metabolismo , Humanos , Metilación , Modelos Biológicos , ARN no Traducido/genética , Inactivación del Cromosoma X/genética
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