RESUMEN

The constitutive active/androstane receptor (CAR) controls genes involved in xenochemical metabolism. Although numerous cofactors have been reported to be involved in CAR-mediated transactivation, unknown and poorly defined proteins recruited by CAR have yet to be characterized. In this study, a novel CAR-interacting protein, cell cycle and apoptosis regulator 1 (CCAR1), was identified by coimmunoprecipitation analysis using human hepatocarcinoma HepG2 cells expressing FLAG epitope-tagged CAR. We demonstrated that CCAR1 can act as an enhancer-dependent coactivator of CAR. First, we showed that overexpression of CCAR1 enhanced CAR-induced reporter gene activity with triplicate consensus direct repeat 4 motif (DR4-Luc), xenobiotic-responsive enhancer module (XREM)-enhancer of CYP3A4 (XREM-Luc), and phenobarbital-responsive enhancer module of UDP-glucuronosyltransferases 1A1 (UGT1A1) (gtPBREM)-enhancer of UGT1A1 (gtPBREM-Luc)-driven reporter plasmids but not PBREM-enhancer of CYP2B6 (PBREM-Luc)-driven reporter activity. Furthermore, we showed that knockdown of CCAR1 suppressed CAR-induced UGT1A1 mRNA expression but did not affect CAR-induced CYP2B6 mRNA expression in HepTR/CAR and HepaRG cells. Moreover, CCAR1 could be recruited to the gtPBREM of the UGT1A1 enhancer by CAR but not to the PBREM of the CYP2B6 enhancer. Moreover, we showed that CCAR1 can act as a secondary coactivator by cooperating with the p160 family of steroid receptor coactivators (SRCs). These findings demonstrated CCAR1 to be a novel transcriptional cofactor for CAR and provided insight regarding the mechanism of CAR-mediated gene-selective transactivation.

Asunto(s)

Proteínas Reguladoras de la Apoptosis/genética , Apoptosis/genética , Proteínas de Ciclo Celular/genética , Ciclo Celular/genética , Elementos de Facilitación Genéticos/genética , Receptores Citoplasmáticos y Nucleares/genética , Activación Transcripcional/genética , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Receptor de Androstano Constitutivo , Citocromo P-450 CYP2B6/genética , Citocromo P-450 CYP3A/genética , Elementos de Facilitación Genéticos/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/genética , Genes Reporteros/genética , Glucuronosiltransferasa/genética , Células Hep G2 , Humanos , Reactores Nucleares , Fenobarbital/farmacología , ARN Mensajero/genética , Receptores de Esteroides/genética , Activación Transcripcional/efectos de los fármacos , Xenobióticos/farmacología

RESUMEN

Androgens are key regulators that play a critical role in the male reproductive system and have anabolic effects on bone mineral density and skeletal muscle mass. We have previously reported that YK11 is a novel selective androgen receptor modulator (SARM) and induces myogenic differentiation and selective gene regulation. In this study, we show that treatment of YK11 and dihydrotestosterone (DHT) accelerated cell proliferation and mineralization in MC3T3-E1 mouse osteoblast cells. Further, YK11-treated cells increased osteoblast specific differentiation markers, such as osteoprotegerin and osteocalcin, compared to untreated cells. These observations were attenuated by androgen receptor (AR) antagonist treatment. To clarify the effect of YK11, we investigated rapid non-genomic signaling by AR. The phosphorylated Akt protein level was increased by YK11 and DHT treatment, suggesting that YK11 activates Akt-signaling via non-genomic signaling of AR. Because it is known Akt-signaling is a key regulator of androgen-mediated osteoblast differentiation, YK11 has osteogenic activity as well as androgen.

Asunto(s)

Andrógenos/farmacología , Norpregnadienos/farmacología , Osteoblastos/efectos de los fármacos , Células 3T3 , Animales , Calcificación Fisiológica/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Ratones , Proteína Oncogénica v-akt/biosíntesis , Proteína Oncogénica v-akt/genética , Osteogénesis/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacos

RESUMEN

The constitutive androstane receptor (CAR) is a nuclear receptor that acts as a transcription factor for a variety of genes, including genes encoding xenobiotic, steroid, and drug-metabolizing enzymes and transporters. Transactivation of a target gene by a transcription factor is generally mediated through the concerted and stepwise recruitment of various proteins termed coregulators, including coactivators and corepressors. In this study, TRIM24 (also known as transcriptional intermediary factor 1 alpha) was found to interact with the CAR. TRIM24 enhanced the CAR-dependent transactivation in reporter assays using the direct repeat-4 motif, a binding site of the CAR. This enhancement was synergistically augmented in the presence of steroid receptor coactivator (SRC) 1 or SRC2, both of which are coactivators of the CAR. In addition, TRIM24 was recruited to the CAR-binding element of the CYP2B6 promoter together with the CAR. We also noted that knockdown of TRIM24 suppressed CAR-induced CYP2B6 mRNA expression in HepTR/CAR and HepaRG cells and suppressed CAR-induced CYP3A4 mRNA expression in HepaRG cells but not HepTR/CAR cells. From these results, we suggest that TRIM24 is a novel coactivator of the CAR that is involved in cell- and/or promoter- selective transactivation.

Asunto(s)

Proteínas Portadoras/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Activación Transcripcional , Proteínas Portadoras/genética , Receptor de Androstano Constitutivo , Citocromo P-450 CYP2B6/genética , Citocromo P-450 CYP2B6/metabolismo , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , Técnicas de Silenciamiento del Gen , Células Hep G2 , Humanos , Coactivador 1 de Receptor Nuclear/genética , Coactivador 1 de Receptor Nuclear/metabolismo , Coactivador 2 del Receptor Nuclear/genética , Coactivador 2 del Receptor Nuclear/metabolismo , Regiones Promotoras Genéticas/genética , Interferencia de ARN , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Receptores Citoplasmáticos y Nucleares/genética

RESUMEN

BACKGROUND: Pregnane X receptor (PXR) is a key regulator of the induction of drug metabolizing enzymes. PXR has been studied for its importance in drug-drug or herb-drug interactions, and it is also a molecular target for the treatment of inflammatory and metabolic diseases. PURPOSE: This study aims to determine new natural PXR-ligands from traditional plant medicines. METHODS: The PXR activation activity was measured by a mammalian one hybrid assay of PXR. Identification of the active compound from Alisma rhizome (the rhizomes of Alisma orientale) was carried out by bioassay-guided fractionation method. The transcriptional activity of the liver-enriched nuclear receptors was measured by the luciferase reporter assay. The interaction between the SRC-1 and PXR was measured by a mammalian 2-hybrid assay. The expression of endogenous CYP3A4 mRNA in both cultured hPXR-overexpressing hepatoma cells and human primary hepatocytes were measured by quantitative RT-PCR method. RESULTS: The extract of Alisma rhizome showed the most potent activation activity by screening of a library of medicinal plant extracts. Alisol B 23-acetate (ABA) was identified to be the active compound of Alisma rhizome. ABA caused a concentration-dependent increase on the PXR-dependent transactivation of a luciferase reporter gene, but did not affect the ligand binding activity of the liver-enriched nuclear receptors, such as CAR, LXR, FXR, PPARα, PPARδ and PPARγ, emphasizing that ABA is a potent and specific agonist of PXR. With ABA treatment, the direct interaction between the ligand-binding domain of PXR and the receptor interaction domain of SRC1 was observed. ABA also induced the expression of endogenous CYP3A4 mRNA in both cultured hPXR-overexpressing hepatoma cells and human primary hepatocytes. CONCLUSION: Since the rhizomes of Alisma orientale are used for a wide range of ailments in traditional Chinese medicine and Japanese Kampo medicine, this study could possibly extend into the clinical usage of these medicines via the mechanism of PXR activation.

Asunto(s)

Alisma/química , Colestenonas/farmacología , Extractos Vegetales/farmacología , Receptores de Esteroides/agonistas , Rizoma/química , Animales , China , Humanos , Medicina Tradicional China , Receptor X de Pregnano

RESUMEN

The constitutive androstane receptor (CAR) and pregnane X receptor (PXR) regulate various genes involved in xenobiotics and drug metabolism. In many cases, CAR/PXR share ligands termed dual ligands of CAR/PXR. It is difficult to investigate the effect of CAR/PXR dual ligands in cell lines because CAR and PXR expression is scarcely detected in cultured cell lines. Here, we established a tetracycline-inducible human CAR and stably human PXR-overexpressing HepG2 cell line (HepTR/hCAR/hPXR) to examine CAR/PXR dual ligands. In the present study, we investigated the regulation of CYP2B6, CYP2C9, CYP3A4, and UDP-glucuronosyl transferase, which are target genes of CAR/PXR, by dual ligands of CAR/PXR in two transfectants. Activation of CAR and PXR in cells treated with a high dose of CITCO [6-(4-chlorophenyl)-imidazo(2,1-b)thiazole-5-carbaldehyde] or cotreated with rifampicin and tetracycline resulted in synergistic enhancement of CYP3A4, but not CYP2B6, CYP2C9, or UGT1A1, mRNA expression in HepTR/hCAR/hPXR cells. In contrast, this synergistic effect was not observed in HepTR/hCAR cells. These observations were also demonstrated in human primary hepatocytes. Taken together, our results suggest that dual ligands of CAR/PXR show distinct gene regulation patterns by cross-talk between CAR and PXR. Furthermore, the two newly established cell lines are useful tools to investigate dual ligands of CAR/PXR.

Asunto(s)

Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Isoquinolinas/farmacología , Oximas/farmacología , Receptores Citoplasmáticos y Nucleares/agonistas , Receptores de Esteroides/agonistas , Rifampin/farmacología , Tiazoles/farmacología , Receptor de Androstano Constitutivo , Citocromo P-450 CYP2B6/genética , Citocromo P-450 CYP2B6/metabolismo , Citocromo P-450 CYP2C9/genética , Citocromo P-450 CYP2C9/metabolismo , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , Agonismo Parcial de Drogas , Glucuronosiltransferasa/genética , Glucuronosiltransferasa/metabolismo , Células Hep G2 , Humanos , Ligandos , Receptor X de Pregnano , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptor Cross-Talk , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores de Esteroides/genética , Receptores de Esteroides/metabolismo , Transfección

RESUMEN

Animal defense mechanisms against both endogenous and exogenous toxic compounds function mainly through receptor-type transcription factors, including the constitutive androstane receptor (CAR). Following xenobiotic stimulation, CAR translocates into the nucleus and transactivates its target genes including oxygenic and conjugative enzymes and transporters in hepatocytes. We identified subcellular localization signals in the rat CAR: two nuclear localization signals (NLS1 and 2); two nuclear export signals (NES1 and 2); and a cytoplasmic retention region. The nuclear import of CAR is regulated by the importin-Ran system and microtubule network. Five splice variants (SV1-5) were identified in rat liver in addition to wild-type CAR. When expressed in immortalized cells, their artificial transcripts were inactive as transcription factors. A CAR mutant with three consecutive alanine residues inserted into the ligand-binding domain of CAR showed ligand-dependent activation of target genes in immortalized cells, which is in marked contrast to the constitutive transactivating nature of wild-type CAR. Using this assay system, androstenol and clotrimazole, both of which are inverse agonists of CAR, were classified as an antagonist and weak agonist, respectively. A member of the DEAD box DNA/RNA helicase family (DP97) and protein arginine methyltransferase 5 (PRMT5) were found to be gene (or promotor)-specific coactivators of CAR. The expression of the CAR gene might be under the control of clock genes mediated by the nuclear receptor Rev-erb-α.

Asunto(s)

Receptores Citoplasmáticos y Nucleares/química , Receptores Citoplasmáticos y Nucleares/fisiología , Transporte Activo de Núcleo Celular , Androstenoles , Animales , Núcleo Celular/metabolismo , Ritmo Circadiano/genética , Clotrimazol , Receptor de Androstano Constitutivo , Expresión Génica , Humanos , Carioferinas/fisiología , Ratones , Microtúbulos/fisiología , Miembro 1 del Grupo D de la Subfamilia 1 de Receptores Nucleares/fisiología , Ratas , Receptores Citoplasmáticos y Nucleares/agonistas , Receptores Citoplasmáticos y Nucleares/metabolismo , Transducción de Señal/fisiología , Proteína de Unión al GTP ran/fisiología

RESUMEN

It has been noticed that crosstalk between androgen receptor (AR) and mammalian target of rapamycin (mTOR) signaling pathways plays a crucial role in the proliferation of prostate cancer cells. To clarify this mechanism, we focused on DEPTOR, a naturally occurring inhibitor of mTOR. The treatment of a human AR-positive prostate cancer cell line, LNCaP, with the AR-agonist dihydrotestosterone (DHT) repressed DEPTOR mRNA expression in a time-dependent manner. This repression was abrogated by treatment with the AR-antagonist bicalutamide. Knockdown of DEPTOR mRNA by siRNA resulted in the increased phosphorylation of 70 kDa ribosomal protein S6 kinase 1 (S6K), a substrate of mTORC1, accompanied by the elevated expression of cyclin D1, a positive regulator of cell proliferation. Furthermore, the ChIP assay demonstrated that AR could bind to AR-responsible element-like region within the 4th intron of the DEPTOR gene. The amount of acetylated histone H3 (Lys9, Lys14) was reduced by the DHT treatment in this region. Taken together, these results propose that AR-dependent prostate cancer cell proliferation requires decreased DEPTOR transcription directly controlled by AR.

Asunto(s)

Proliferación Celular/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Neoplasias de la Próstata/patología , Receptores Androgénicos/fisiología , Transducción de Señal/fisiología , Serina-Treonina Quinasas TOR/fisiología , Transcripción Genética/genética , Antagonistas de Andrógenos/farmacología , Anilidas/farmacología , Línea Celular Tumoral , Ciclina D1/metabolismo , Dihidrotestosterona/farmacología , Histonas/metabolismo , Humanos , Masculino , Nitrilos/farmacología , Fosforilación , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptor Cross-Talk , Receptores Androgénicos/metabolismo , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismo , Factores de Tiempo , Compuestos de Tosilo/farmacología , Células Tumorales Cultivadas

RESUMEN

The constitutive androstane receptor (CAR) plays a key role in the expression of xenobiotic/steroid and drug metabolizing enzymes and their transporters. In this study, we demonstrated that protein arginine methyltransferase 5 (PRMT5) is a novel CAR-interacting protein. Furthermore, the PRMT-dependent induction of a CAR reporter gene, which was independent of methyltransferase activity, was enhanced in the presence of steroid receptor coactivator 1 (SRC1), peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC-1α) or DEAD box DNA/RNA helicase DP97. Using tetracycline inducible-hCAR system in HepG2 cells, we showed that knockdown of PRMT5 with small interfering RNA suppressed tetracycline -induced mRNA expression of CYP2B6 but not of CYP2C9 or CYP3A4. PRMT5 enhanced phenobarbital-mediated transactivation of a phenobarbital-responsive enhancer module (PBREM)-driven reporter gene in co-operation with PGC-1α in rat primary hepatocytes. Based on these findings, we suggest PRMT5 to be a gene (or promoter)-selective coactivator of CAR by mediating the formation of complexes between hCAR and appropriate coactivators.

Asunto(s)

Proteína-Arginina N-Metiltransferasas/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Línea Celular , Receptor de Androstano Constitutivo , Citocromo P-450 CYP2B6/genética , Citocromo P-450 CYP2B6/metabolismo , Citocromo P-450 CYP2C9/genética , Citocromo P-450 CYP2C9/metabolismo , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Coactivador 1 de Receptor Nuclear/genética , Coactivador 1 de Receptor Nuclear/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , Proteína-Arginina N-Metiltransferasas/genética , Receptores Citoplasmáticos y Nucleares/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Activación Transcripcional

RESUMEN

The constitutive androstane receptor (CAR, NR1I3) is very important for drug development and for understanding pharmacokinetic drug-drug interactions. We screened by mammalian one hybrid assay among natural compounds to discover novel ligands of human constitutive androstane receptor (hCAR). hCAR transcriptional activity was measured by luciferase assay and mRNA levels of CYP2B6 and CYP3A4 in HepTR-hCAR cells and human primary hepatocytes were measured by real-time RT-PCR. Nigramide J (NJ) whose efficacy is comparable to those of hitherto known inverse agonists such as clotrimazole, PK11195, and ethinylestradiol. NJ is a naturally occurring cyclohexane-type amide alkaloid that was isolated from the roots of Piper nigrum. The suppressive effect of NJ on the CAR-dependent transcriptional activity was found to be species specific, in the descending order of hCAR, rat CAR, and mouse CAR. The unliganded hCAR-dependent transactivation of reporter and endogenous genes was suppressed by NJ at concentrations higher than 5 µmol/L. The ligand-binding cavity of hCAR was shared by NJ and CITCO, because they were competitive in the binding to hCAR. NJ interfered with the interaction of hCAR with coactivator SRC-1, but not with its interaction with the corepressor NCoR1. Furthermore, NJ is agonist of human pregnane X receptor (hPXR). NJ is a dual ligand of hCAR and hPXR, being an agonist of hPXR and an inverse agonist of hCAR.

RESUMEN

A chemical study of the roots of Euphorbia fischeriana resulted in the isolation of seven triterpenes (1-7), including two new compounds: (24R,S)-3ß-24,31-epoxy-24-methylcycloartane (1) and (24R,S)-3ß,31-dihydroxy-24-methoxy-24-methylcycloartane (2). Their structures were elucidated through extensive spectroscopic analyses. Cycloartanes 1-4 showed significant human CYP3A4 promoter activity through a series of luciferase reporter assays. Of these compounds, 3 and 4 activated the pregnane X receptor (PXR) and induced CYP3A4 mRNA expression in human primary hepatocytes. However, despite showing the most potent human CYP3A4 promoter activity via a PXR-independent pathway, 2 did not affect CYP3A4 mRNA expression in human primary hepatocytes. This difference is correlated to substitutions in C-24 and C-25 of the cycloartane structure.

Asunto(s)

Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , Euphorbia/química , Triterpenos/química , Humanos , Conformación Molecular , Estructura Molecular , Regiones Promotoras Genéticas/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Triterpenos/farmacología

RESUMEN

Cyclization-carbonylation of α,ß-alkynic hydrazones and (o-alkynylphenyl) (methoxymethyl) sulfides with Pd(tfa)2 in DMSO/MeOH afforded methyl pyrazole-4-carboxylates and benzo[b]thiophene-3-carboxylates, respectively, in good yields. A simple change of the ligand (solvent) allowed controlled, effective switching between cyclization-carbonylation-cyclization-coupling (CCC-coupling) reactions and cyclization-carbonylation reactions.

Asunto(s)

Ácidos Carboxílicos/síntesis química , Hidrazonas/química , Paladio/química , Pirazoles/síntesis química , Tiofenos/síntesis química , Catálisis , Cationes Bivalentes , Ciclización , Dimetilsulfóxido/química , Ligandos , Metanol/química

RESUMEN

Pregnane X receptor (PXR) is known as a xenosensor, playing a key role in response to xenochemical stimuli. Activation of PXR enhanced expression of various drug-metabolizing enzymes and transporters such as cytochrome P450 3A4 (CYP3A4). During a screening of a natural compounds library for novel ligands of human xenosensing receptors by the mammalian one-hybrid assay, two cyclohexene-type amide alkaloids were isolated, with nigramide C (NigC) showing the most potent activation of human PXR (hPXR). NigC-mediated hPXR activation was enhanced by overexpression of steroid receptor coactivator 1 (SRC1), peroxisome proliferator-activated receptor γ, coactivator 1α, and protein arginine methyltransferase 1. A direct interaction between the ligand-binding domain of hPXR and the receptor interaction domain of SRC1 was observed. NigC induced the expression of endogenous CYP3A4 mRNA and protein in both cultured hepatoma cells and primary hepatocytes. However, in primary hepatocytes, the relative agonist activity of NigC was not as potent as that of rifampicin, probably because of lower metabolic stability of NigC in these cells. In conclusion, NigC was found to be an effective agonist of hPXR. NigC is a useful tool for investigation of hPXR function.

Asunto(s)

Hepatocitos/efectos de los fármacos , Piper nigrum , Extractos Vegetales/farmacología , Raíces de Plantas , Receptores de Esteroides/agonistas , Femenino , Células Hep G2 , Hepatocitos/metabolismo , Humanos , Masculino , Extractos Vegetales/química , Extractos Vegetales/aislamiento & purificación , Receptor X de Pregnano , Receptores de Esteroides/fisiología

RESUMEN

The myogenic differentiation of C2C12 myoblast cells is induced by the novel androgen receptor (AR) partial agonist, (17α,20E)-17,20-[(1-methoxyethylidene)bis-(oxy)]-3-oxo-19-norpregna-4,20-diene-21-carboxylic acid methyl ester (YK11), as well as by dihydrotestosterone (DHT). YK11 is a selective androgen receptor modulator (SARM), which activates AR without the N/C interaction. In this study, we further investigated the mechanism by which YK11 induces myogenic differentiation of C2C12 cells. The induction of key myogenic regulatory factors (MRFs), such as myogenic differentiation factor (MyoD), myogenic factor 5 (Myf5) and myogenin, was more significant in the presence of YK11 than in the presence of DHT. YK11 treatment of C2C12 cells, but not DHT, induced the expression of follistatin (Fst), and the YK11-mediated myogenic differentiation was reversed by anti-Fst antibody. These results suggest that the induction of Fst is important for the anabolic effect of YK11.

Asunto(s)

Andrógenos/farmacología , Folistatina/genética , Mioblastos/efectos de los fármacos , Norpregnadienos/farmacología , Receptores Androgénicos/metabolismo , Antagonistas de Andrógenos/farmacología , Animales , Diferenciación Celular/efectos de los fármacos , Línea Celular , Flutamida/análogos & derivados , Flutamida/farmacología , Ratones , Proteína MioD/genética , Mioblastos/citología , Mioblastos/metabolismo , Factor 5 Regulador Miogénico/genética , Miogenina/genética , ARN Mensajero/metabolismo

RESUMEN

Palladium and CO: Carbonylation of 1 with [Pd(tfa)2(±)-L1] (tfa = trifluoroacetate) affords the spirofuranone 2 with inversion of the stereochemistry at C17 in 96 % yield. C17-epi-1 also gave the same product 2 with retention of the stereochemistry at C17. Labelling studies show that (13)CO was incorporated into the C5' position of the furanone ring. The first asymmetric version of this new reaction was achieved.

Asunto(s)

Carbamatos/química , Monóxido de Carbono/química , Furanos/síntesis química , Oxazoles/química , Paladio/química , Catálisis , Ciclización , Estructura Molecular

RESUMEN

The basal transcriptional activity of unliganded human constitutive androstane receptor (hCAR) was shown to be repressed by the potent liver X receptor (LXR) agonist, TO901317, in a concentration-dependent manner using a reporter assay in cultured cells. TO901317 also repressed the basal transcriptional activity of both mouse and rat CAR. The certified hCAR agonist, CITCO, partially reversed this repressive effect of TO901317 on hCAR basal activity. Unlike hCAR, a three alanine insertion mutant and the splice variant 2 of hCAR require agonists, such as CITCO, to become transcriptionally active and the CITCO-induced reporter activity was repressed by TO901317. As has been previously shown for the typical hCAR inverse agonist, PK11195, TO901317 blocked the interaction of hCAR with steroid receptor co-activator 1 (SRC1). In contrast, the interaction between hCAR and nuclear receptor corepressor 1 (NCoR1) was promoted by PK11195 and TO901317. Furthermore, the hCAR-mediated basal induction of endogenous cytochrome P450 2B6 (CYP2B6) mRNA was adversely affected by co-treatment with TO901317.

Asunto(s)

Hidrocarburos Fluorados/farmacología , Receptores Nucleares Huérfanos/agonistas , Receptores Citoplasmáticos y Nucleares/agonistas , Receptores Citoplasmáticos y Nucleares/genética , Sulfonamidas/farmacología , Animales , Hidrocarburo de Aril Hidroxilasas/metabolismo , Células Cultivadas , Receptor de Androstano Constitutivo , Citocromo P-450 CYP2B6 , Inducción Enzimática/efectos de los fármacos , Variación Genética , Humanos , Hidrocarburos Fluorados/efectos adversos , Receptores X del Hígado , Ratones , Coactivador 1 de Receptor Nuclear/fisiología , Oxidorreductasas N-Desmetilantes/metabolismo , Oximas/farmacología , Ratas , Receptor Cross-Talk/efectos de los fármacos , Receptores Citoplasmáticos y Nucleares/fisiología , Sulfonamidas/efectos adversos , Tiazoles/farmacología , Transcripción Genética/efectos de los fármacos

RESUMEN

Adenosine monophosphate (AMP)-activated protein kinase (AMPK) is a major cellular energy sensor and master regulator of metabolic homeostasis; thus, AMPK plays a central role in studies on diabetes and related metabolic diseases. From the rhizomes of Polygonatum odoratum (Mill.) Druce, six homoisoflavonoids (1-6) and one dihydrochalcone (7) were isolated, and the structures of polygonatones A-D (4-7) were elucidated by various spectroscopic analyses. Compounds 1-7 were evaluated for their effect on AMPK activation. The amount of active phosphorylated AMPK and acetyl-CoA carboxylase in rat liver epithelial IAR-20 cells increased when the cells were incubated with the aforementioned compounds. Specifically, (3R)-5,7-dihydroxyl-6-methyl-8-methoxyl-3-(4'-hydroxylbenzyl)-chroman-4-one (1), (3R)-5,7-dihydroxyl-6,8-dimethyl-3-(4'-hydroxylbenzyl)-chroman-4-one (2), (3R)-5,7-dihydroxyl-6-methyl-3-(4'-hydroxylbenzyl)-chroman-4-one (3), and polygonatone D (7) exhibited significant activation effects.

Asunto(s)

Proteínas Quinasas Activadas por AMP/metabolismo , Chalconas/química , Isoflavonas/química , Polygonatum/química , Células 3T3-L1 , Proteínas Quinasas Activadas por AMP/química , Acetil-CoA Carboxilasa/química , Acetil-CoA Carboxilasa/metabolismo , Animales , Línea Celular , Chalconas/aislamiento & purificación , Chalconas/farmacología , Isoflavonas/aislamiento & purificación , Isoflavonas/farmacología , Ratones , Fosforilación/efectos de los fármacos , Ratas , Rizoma/química

RESUMEN

The interaction between HER2 and aryl hydrocarbon receptor (AhR) signaling cascades during mammosphere formation of MCF-7 cells was studied. A newly established clonal MCF-7 cell line (HER2-5), stably overexpressing HER2, showed significantly enhanced levels of AhR mRNA and protein compared with MCF-7 cells. AhR was required for the HER2-mediated induction of interleukin-6 mRNA and for mammosphere formation in HER2-5 and MCF-7 cells. Mammosphere forming efficiency was suppressed by an AhR antagonist in a dose-dependent manner, as well as by knockdown of AhR. Taken together, these results indicate that AhR enhances mammosphere formation by human HER2-overexpressing breast cancer cells.

Asunto(s)

Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Receptor ErbB-2/biosíntesis , Receptores de Hidrocarburo de Aril/biosíntesis , Neoplasias de la Mama/enzimología , Neoplasias de la Mama/genética , Técnicas de Cultivo de Célula , Procesos de Crecimiento Celular/fisiología , Línea Celular Tumoral , Femenino , Humanos , Células MCF-7 , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Receptores de Hidrocarburo de Aril/genética , Receptores de Hidrocarburo de Aril/metabolismo , Transducción de Señal , Esferoides Celulares , Transcripción Genética , Transfección , Regulación hacia Arriba

RESUMEN

The constitutive androstane receptor (CAR) plays a key role in the expression of xenobiotic/steroid and drug metabolizing enzymes and their transporters. In this study, we demonstrated that DP97, a member of the DEAD box DNA/RNA helicase protein family, is a novel CAR-interacting protein. Using HepG2 cells expressing human CAR in the presence of tetracycline, we showed that knockdown of DP97 with small interfering RNAs suppressed tetracycline-inducible mRNA expression of CYP2B6 and UGT1A1 but not CYP3A4. Thus, DP97 was found to be a gene (or promoter)-selective co-activator for hCAR. DP97-mediated CAR transactivation was synergistically enhanced by the co-expression of SRC-1 or PGC1α, therefore it might act as mediator between hCAR and appropriate co-activators.

Asunto(s)

ARN Helicasas DEAD-box/metabolismo , Proteínas de Neoplasias/metabolismo , Receptores Citoplasmáticos y Nucleares/genética , Activación Transcripcional , Receptor de Androstano Constitutivo , ARN Helicasas DEAD-box/genética , Células HEK293 , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Proteínas de Neoplasias/genética , Coactivador 1 de Receptor Nuclear/genética , Coactivador 1 de Receptor Nuclear/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , Receptores Citoplasmáticos y Nucleares/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo

RESUMEN

Human bronchial mucins, such as MUC5AC, have traditionally been defined as a family of high-molecular weight glycoproteins. Changes in the contents of sugar chains on MUC5AC are among the fundamental features in inflammatory respiratory disease. The changes have been shown to lead to unfavorable alterations in the viscosity of mucus, resulting in impairment of mucociliary transport, vulnerability to viral/bacterial infection as sugar chains play an important role in adhesion of some viruses and bacteria to the epithelium, and finally inflammatory cell infiltration in the airway. Recently, we found that expression of some glycosyltransferases associated with the contents and structure of sugar chains is regulated by phosphatidylinositol-phospholipase (PI-PL) C signaling in cells. L-Carbocisteine, a mucoregulatory drug, normalized or balanced fucosylated and sialylated sugar chains, such as sialyl Lewis x through inhibition of PI-PL C signaling. We prepared MUC5AC fusion protein with tandem repeats associated with MUC5AC, and confirmed that L-carbocisteine inhibited the increases in viscosity associated with sialyl Lewis x expression levels. In addition, the clinical study (2008) noted that L-carbocisteine reduced the frequency of common colds and exacerbation of symptoms in patients with COPD. These favorable effects in patients may be due to normalization of sugar chain contents on mucins. We suggest that the inhibitory effect on infection of airway epithelial cells by rhinoviruses, respiratory syncytial virus, and influenza viruses by treatment with L-carbocisteine may also be based on the regulation of sugar chain contents or structures on mucins.

Asunto(s)

Antiinfecciosos Locales/farmacología , Carbocisteína/farmacología , Expectorantes/farmacología , Mucina 5AC/química , Moco/química , Fosfoinositido Fosfolipasa C/antagonistas & inhibidores , Infecciones del Sistema Respiratorio/etiología , Animales , Antiinfecciosos Locales/uso terapéutico , Carbocisteína/uso terapéutico , Conformación de Carbohidratos , Expectorantes/uso terapéutico , Humanos , Mucina 5AC/fisiología , Moco/metabolismo , Moco/fisiología , Oligosacáridos/metabolismo , Fosfoinositido Fosfolipasa C/fisiología , Ratas , Infecciones del Sistema Respiratorio/tratamiento farmacológico , Antígeno Sialil Lewis X , Transducción de Señal/fisiología , Viscosidad

RESUMEN

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor. Recent studies have reported the anti-tumor effects of the AhR in breast cancer. In this study, we investigated the anti-tumor effect of AhR activation based on the cancer stem cell hypothesis. We show that AhR activation suppressed mammosphere formation of MCF-7 cells and decreased the proportion of cells with high ALDH-1 (aldehyde dehydrogenase 1) activity. In addition, we also demonstrate that AhR activation regulates self-renewal signaling by down-regulating Wnt/ß-catenin and Notch.

Asunto(s)

Regulación Neoplásica de la Expresión Génica , Receptores de Hidrocarburo de Aril/genética , Receptores de Hidrocarburo de Aril/metabolismo , Esferoides Celulares/metabolismo , Familia de Aldehído Deshidrogenasa 1 , Western Blotting , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A1/metabolismo , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Isoenzimas/metabolismo , Metilcolantreno/farmacología , Microscopía Confocal , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Interferencia de ARN , Receptores de Hidrocarburo de Aril/agonistas , Receptores Notch/metabolismo , Retinal-Deshidrogenasa/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Esferoides Celulares/efectos de los fármacos , Esferoides Celulares/patología , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , beta-naftoflavona/farmacología