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1.
J Steroid Biochem Mol Biol ; 194: 105459, 2019 11.
Artículo en Inglés | MEDLINE | ID: mdl-31470108

RESUMEN

The effect of androgen on angiogenesis has been documented. However, its underlying molecular mechanisms have not been well illustrated. Here, we show that treatment with an androgen receptor (AR) agonist, metribolone (R1881; 0.05-5 nM), or dihydrotestosterone (DHT; 0.5-2 nM), concentration- and time-dependently inhibited proliferation in human umbilical venous endothelial cells (HUVEC). This inhibitory effect was confirmed in human microvascular endothelial cells (HMEC-1). Flow cytometric analysis demonstrated that R1881 induced G0/G1 phase cell cycle arrest in HUVEC. Blockade of the AR activity by pre-treatment with an AR antagonist, hydroxyflutamide (HF), or knockdown of AR expression using the shRNA technique abolished the R1881-induced HUVEC proliferation inhibition, suggesting that AR activation can inhibit endothelial cell proliferation. We further investigated the signaling pathway contributing to the proliferation inhibition induced by AR activation. Our data suggest that R1881 reduced the proliferation rate of HUVEC through activating the AR/cSrc/AKT/p38/ERK/NFκB pathway, subsequently up-regulating p53 expression, which in turn increased the levels of p21 and p27 protein, hence decreasing the activities of cyclin-dependent kinase 2 (CDK2) and CDK4, and finally reduced the cell proliferation rate. An extra-nuclear pathway involved in the proliferation inhibition induced by AR activation in vascular endothelial cells was confirmed by showing that membrane-impermeable testosterone-bovine serum albumin (BSA) treatment significantly increased the levels of p53, p27 and p21 protein and reduced cell proliferation. These data highlight the underlying molecular mechanisms by which AR activation induced proliferation inhibition in vascular endothelial cells.


Asunto(s)
Andrógenos/farmacología , Células Endoteliales/efectos de los fármacos , Metribolona/farmacología , Receptores Androgénicos/metabolismo , Línea Celular , Proliferación Celular/efectos de los fármacos , Células Endoteliales/metabolismo , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , FN-kappa B/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores Androgénicos/genética , Proteína p53 Supresora de Tumor/metabolismo
2.
Endocrinology ; 159(8): 2884-2890, 2018 08 01.
Artículo en Inglés | MEDLINE | ID: mdl-29850791

RESUMEN

3ßHSD1 enzymatic activity is essential for synthesis of potent androgens from adrenal precursor steroids in prostate cancer. A germline variant in HSD3B1, the gene that encodes 3ßHSD1, encodes for a stable enzyme, regulates adrenal androgen dependence, and is a predictive biomarker of poor clinical outcomes after gonadal testosterone deprivation therapy. However, little is known about HSD3B1 transcriptional regulation. Generally, it is thought that intratumoral androgen synthesis is upregulated after gonadal testosterone deprivation, enabling development of castration-resistant prostate cancer. Given its critical role in extragonadal androgen synthesis, we sought to directly interrogate the transcriptional regulation of HSD3B1 in multiple metastatic prostate cancer cell models. Surprisingly, we found that VCaP, CWR22Rv1, LNCaP, and LAPC4 models demonstrate induction of HSD3B1 upon androgen stimulation for approximately 72 hours, followed by attenuation around 120 hours. 3ßHSD1 protein levels mirrored transcriptional changes in models harboring variant (LNCaP) and wild-type (LAPC4) HSD3B1, and in these models androgen induction of HSD3B1 is abrogated via enzalutamide treatment. Androgen treatment increased flux from [3H]-dehydroepiandrosterone to androstenedione and other downstream metabolites. HSD3B1 expression was reduced 72 hours after castration in the VCaP xenograft mouse model, suggesting androgen receptor (AR) regulation of HSD3B1 also occurs in vivo. Overall, these data suggest that HSD3B1 is unexpectedly positively regulated by androgens and ARs. These data may have implications for the development of treatment strategies tailored to HSD3B1 genotype status.


Asunto(s)
Regulación Neoplásica de la Expresión Génica/genética , Complejos Multienzimáticos/genética , Progesterona Reductasa/genética , Neoplasias de la Próstata Resistentes a la Castración/genética , Receptores Androgénicos/metabolismo , Esteroide Isomerasas/genética , Andrógenos/metabolismo , Andrógenos/farmacología , Androstenodiona/metabolismo , Animales , Antineoplásicos Hormonales/farmacología , Línea Celular Tumoral , Deshidroepiandrosterona/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Masculino , Metribolona/farmacología , Ratones , Complejos Multienzimáticos/efectos de los fármacos , Trasplante de Neoplasias , Orquiectomía , Feniltiohidantoína/análogos & derivados , Feniltiohidantoína/farmacología , Progesterona Reductasa/efectos de los fármacos , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/terapia , Neoplasias de la Próstata Resistentes a la Castración/metabolismo , Neoplasias de la Próstata Resistentes a la Castración/terapia , Receptores Androgénicos/efectos de los fármacos , Esteroide Isomerasas/efectos de los fármacos , Testosterona , Congéneres de la Testosterona/farmacología , Regulación hacia Arriba
3.
J Cell Biochem ; 119(9): 7204-7211, 2018 09.
Artículo en Inglés | MEDLINE | ID: mdl-29749659

RESUMEN

AR-mediated androgen signaling plays a key role in female reproductive system. Granulosa-lutein cells (GCs) are the main sites for expression of androgen receptor (AR). There is also a close relation between AKT signaling and AR. Here, we assayed the role for a synthetic AR ligand methyltrienolone (R1881) in expressions of AKTs and AR. Controlled ovarian hyperstimulation (COH) was performed in 20 normal women. Mural GCs were isolated by filtration method, cultured, and passaged. Then, the cells were starved for 48 h with 10% charcoal stripped FBS. The cells were then treated with R1881, bicalutamide (AR blocker), LY294002 (PI3K/AKT pathway blocker), and combination of them for 48 h. Finally, GCs were evaluated for quantitative real-time PCR analysis of AKT1, AKT2, AKT3, and AR, and also Western blot assessment of total AKT and phosphorylated AKT (p-AKT) [Ser473 and Thr308]. Addition of R1881 to the GCs culture showed high expressions of AKT1, AKT2, and AKT3 (P ≤ 0.05 vs LY294002 group and bicalutamide group). Expressions of AKT1 and AKT2 were decreased in the GCs under exposure to bicalutamide or LY294002 (P ≤ 0.05 vs R1881). AKT1, AKT2, and AKT3 showed decreased rates of expressions in the LY294002 + bicalutamide group (P ≤ 0.05 vs R1881). AR, total AKT and p-AKT showed no significant differences between groups. Our findings indicate that 46 h exposure with R1881 could affect AKTs expressions in the GCs of pre-ovulatory phase, but it cannot promote AR expression and AKTs activation.


Asunto(s)
Células de la Granulosa/metabolismo , Células Lúteas/metabolismo , Metribolona/farmacología , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores Androgénicos/genética , Receptores Androgénicos/metabolismo , Análisis de Varianza , Células Cultivadas , Cromonas/farmacología , Femenino , Expresión Génica/efectos de los fármacos , Humanos , Morfolinas/farmacología , Inducción de la Ovulación/métodos , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilación , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Estadísticas no Paramétricas
4.
Endocr Relat Cancer ; 25(4): 453-469, 2018 04.
Artículo en Inglés | MEDLINE | ID: mdl-29431615

RESUMEN

Despite altered metabolism being an accepted hallmark of cancer, it is still not completely understood which signaling pathways regulate these processes. Given the central role of androgen receptor (AR) signaling in prostate cancer, we hypothesized that AR could promote prostate cancer cell growth in part through increasing glucose uptake via the expression of distinct glucose transporters. Here, we determined that AR directly increased the expression of SLC2A12, the gene that encodes the glucose transporter GLUT12. In support of these findings, gene signatures of AR activity correlated with SLC2A12 expression in multiple clinical cohorts. Functionally, GLUT12 was required for maximal androgen-mediated glucose uptake and cell growth in LNCaP and VCaP cells. Knockdown of GLUT12 also decreased the growth of C4-2, 22Rv1 and AR-negative PC-3 cells. This latter observation corresponded with a significant reduction in glucose uptake, indicating that additional signaling mechanisms could augment GLUT12 function in an AR-independent manner. Interestingly, GLUT12 trafficking to the plasma membrane was modulated by calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2)-5'-AMP-activated protein kinase (AMPK) signaling, a pathway we previously demonstrated to be a downstream effector of AR. Inhibition of CaMKK2-AMPK signaling decreased GLUT12 translocation to the plasma membrane by inhibiting the phosphorylation of TBC1D4, a known regulator of glucose transport. Further, AR increased TBC1D4 expression. Correspondingly, expression of TBC1D4 correlated with AR activity in prostate cancer patient samples. Taken together, these data demonstrate that prostate cancer cells can increase the functional levels of GLUT12 through multiple mechanisms to promote glucose uptake and subsequent cell growth.


Asunto(s)
Quinasa de la Proteína Quinasa Dependiente de Calcio-Calmodulina/metabolismo , Proteínas Facilitadoras del Transporte de la Glucosa/metabolismo , Próstata/metabolismo , Neoplasias de la Próstata/metabolismo , Receptores Androgénicos/metabolismo , Transducción de Señal/fisiología , Andrógenos/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proteínas Facilitadoras del Transporte de la Glucosa/genética , Humanos , Masculino , Metribolona/farmacología , Fosforilación/efectos de los fármacos , Próstata/efectos de los fármacos , Próstata/patología , Neoplasias de la Próstata/patología , ARN Interferente Pequeño , Transducción de Señal/efectos de los fármacos
5.
J Clin Invest ; 127(6): 2326-2338, 2017 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-28463227

RESUMEN

The clinical utility of inhibiting cytochrome P450 17A1 (CYP17), a cytochrome p450 enzyme that is required for the production of androgens, has been exemplified by the approval of abiraterone for the treatment of castration-resistant prostate cancer (CRPC). Recently, however, it has been reported that CYP17 inhibitors can interact directly with the androgen receptor (AR). A phase I study recently reported that seviteronel, a CYP17 lyase-selective inhibitor, ædemonstrated a sustained reduction in prostate-specific antigen in a patient with CRPC, and another study showed seviteronel's direct effects on AR function. This suggested that seviteronel may have therapeutically relevant activities in addition to its ability to inhibit androgen production. Here, we have demonstrated that CYP17 inhibitors, with the exception of orteronel, can function as competitive AR antagonists. Conformational profiling revealed that the CYP17 inhibitor-bound AR adopted a conformation that resembled the unliganded AR (apo-AR), precluding nuclear localization and DNA binding. Further, we observed that seviteronel and abiraterone inhibited the growth of tumor xenografts expressing the clinically relevant mutation AR-F876L and that this activity could be attributed entirely to competitive AR antagonism. The results of this study suggest that the ability of CYP17 inhibitors to directly antagonize the AR may contribute to their clinical efficacy in CRPC.


Asunto(s)
Antagonistas de Receptores Androgénicos/farmacología , Antineoplásicos Hormonales/farmacología , Neoplasias de la Próstata Resistentes a la Castración/tratamiento farmacológico , Esteroide 17-alfa-Hidroxilasa/antagonistas & inhibidores , Transporte Activo de Núcleo Celular , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Resistencia a Antineoplásicos , Sinergismo Farmacológico , Células HEK293 , Humanos , Concentración 50 Inhibidora , Masculino , Metribolona/farmacología , Ratones Endogámicos NOD , Ratones SCID , Feniltiohidantoína/análogos & derivados , Feniltiohidantoína/farmacología , Unión Proteica , Receptores Androgénicos/metabolismo , Esteroide 17-alfa-Hidroxilasa/metabolismo , Testosterona/farmacología , Activación Transcripcional/efectos de los fármacos , Ensayos Antitumor por Modelo de Xenoinjerto
6.
Sci Rep ; 7: 45300, 2017 03 24.
Artículo en Inglés | MEDLINE | ID: mdl-28338058

RESUMEN

The incidence of prostate cancer (PC) is growing rapidly throughout the world, in probable association with the adoption of western style diets. Thus, understanding the molecular pathways triggering the development of PC is crucial for both its prevention and treatment. Here, we investigated the role of the metabolism-associated protein, CREB3L4, in the proliferation of PC cells. CREB3L4 was upregulated by the synthetic androgen, R1881, in LNCaP PC cells (an androgen-dependent cell line). Knockdown of CREB3L4 resulted in decreased androgen-dependent PC cell growth. LNCaP cells transfected with siCREB3L4 underwent G2/M arrest, with upregulation of the proteins cyclin B1, phospho-CDK1, p21Waf1/Cip1, and INCA1, and downregulation of cyclin D1. Moreover, depletion of CREB3L4 resulted in significantly decreased expression of a subset of androgen-receptor (AR) target genes, including PSA, FKBP5, HPGD, KLK2, and KLK4. We also demonstrated that CREB3L4 directly interacts with the AR, and increases the binding of AR to androgen response elements (AREs). We also identified a role for the unfolded protein response (and its surrogate, IRE1α), in activating CREB3L4. Cumulatively, we postulate that CREB3L4 expression is mediated by an AR-IRE1α axis, but is also directly regulated by AR-to-ARE binding. Thus, our study demonstrates that CREB3L4 plays a key role in PC cell proliferation, which is promoted by both AR and IRE1α.


Asunto(s)
Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Proliferación Celular , Proteínas Nucleares/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proteína de Unión a Elemento de Respuesta al AMP Cíclico , Regulación hacia Abajo/efectos de los fármacos , Endorribonucleasas/metabolismo , Puntos de Control de la Fase G2 del Ciclo Celular/efectos de los fármacos , Humanos , Masculino , Metribolona/farmacología , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Unión Proteica , Proteínas Serina-Treonina Quinasas/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Receptores Androgénicos/metabolismo , Transducción de Señal/efectos de los fármacos , Respuesta de Proteína Desplegada/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacos
7.
Endocrinology ; 158(4): 1015-1021, 2017 04 01.
Artículo en Inglés | MEDLINE | ID: mdl-28324044

RESUMEN

5α-Reductase types 1 and 2, encoded by SRD5A1 and SRD5A2, are the two enzymes that can catalyze the conversion of testosterone to dihydrotestosterone, the most potent androgen receptor (AR) agonist in prostate cells. 5α-Reductase type 2 is the predominant isoform expressed in the normal prostate. However, its expression decreases during prostate cancer (PCa) progression, whereas SRD5A1 increases, and the mechanism underlying this transcriptional regulatory switch is still unknown. Interrogation of SRD5A messenger RNA expression in three publicly available data sets confirmed that SRD5A1 is increased in primary and metastatic PCa compared with nontumoral prostate tissues, whereas SRD5A2 is decreased. Activation of AR, a major oncogenic driver of PCa, induced the expression of SRD5A1 from twofold to fourfold in three androgen-responsive PCa cell lines. In contrast, AR repressed SRD5A2 expression in this context. Chromatin-immunoprecipitation studies established that AR is recruited to both SRD5A1 and SRD5A2 genes following androgen stimulation but initiates transcriptional activation only at SRD5A1 as monitored by recruitment of RNA polymerase II and the presence of the H3K27Ac histone mark. Furthermore, we showed that the antiandrogens bicalutamide and enzalutamide block the AR-mediated regulation of both SRD5A1 and SRD5A2, highlighting an additional mechanism explaining their beneficial effects in patients. In summary, we identified an AR-dependent transcriptional regulation that explains the differential expression of 5α-reductase types 1 and 2 during PCa progression. Our work thus defines a mechanism by which androgens control their own synthesis via differential regulatory control of the expression of SRD5A1 and SRD5A2.


Asunto(s)
3-Oxo-5-alfa-Esteroide 4-Deshidrogenasa/metabolismo , Proteínas de la Membrana/metabolismo , Próstata/metabolismo , Neoplasias de la Próstata/metabolismo , Receptores Androgénicos/metabolismo , Andrógenos/farmacología , Línea Celular Tumoral , Progresión de la Enfermedad , Humanos , Calicreínas/metabolismo , Masculino , Metribolona/farmacología , Próstata/efectos de los fármacos , Próstata/patología , Antígeno Prostático Específico/metabolismo , Neoplasias de la Próstata/patología
8.
Sci Rep ; 7: 40719, 2017 01 16.
Artículo en Inglés | MEDLINE | ID: mdl-28091582

RESUMEN

The endoplasmic reticulum (ER) comprises thirty percent of the newly translated proteins in eukaryotic cells. The quality control mechanism within the ER distinguishes between properly and improperly folded proteins and ensures that unwanted proteins are retained in the ER and subsequently degraded through ER-associated degradation (ERAD). Besides cleaning of misfolded proteins ERAD is also important for physiological processes by regulating the abundance of normal proteins of the ER. Thus it is important to unreveal the regulation patterns of ERAD. Here, we describe that ERAD pathway is regulated by androgen, where its inhibitor SVIP was downregulated, all other ERAD genes were upregulated. Consistently, androgen treatment increased the degradation rate of ERAD substrates. Using several independent techniques, we showed that this regulation is through androgen receptor transactivation. ERAD genes found to be upregulated in prostate cancer tissues and silencing expression of Hrd1, SVIP, and gp78 reduced the in vitro migration and malignant transformation of LNCaP cells. Our data suggests that expression levels of ERAD components are regulated by androgens, that promotes ERAD proteolytic activity, which is positively related with prostate tumorigenesis.


Asunto(s)
Andrógenos/metabolismo , Degradación Asociada con el Retículo Endoplásmico , Neoplasias de la Próstata/metabolismo , Andrógenos/farmacología , Biomarcadores , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Transformación Celular Neoplásica/efectos de los fármacos , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Relación Dosis-Respuesta a Droga , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Degradación Asociada con el Retículo Endoplásmico/efectos de los fármacos , Degradación Asociada con el Retículo Endoplásmico/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Masculino , Metribolona/farmacología , Neoplasias de la Próstata/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo
9.
Sci Rep ; 6: 37310, 2016 11 17.
Artículo en Inglés | MEDLINE | ID: mdl-27853318

RESUMEN

Androgen-Induced bZIP (AIbZIP) is structurally a bZIP transmembrane transcription factor belonging to the CREB/ATF family. This molecule is highly expressed in androgen-sensitive prostate cancer cells and is transcriptionally upregulated by androgen treatment. Here, we investigated molecular mechanism of androgen-dependent expression of AIbZIP and its physiological function in prostate cancer cells. Our data showed that SAM pointed domain-containing ETS transcription factor (SPDEF), which is upregulated by androgen treatment, directly activates transcription of AIbZIP. Knockdown of AIbZIP caused a significant reduction in the proliferation of androgen-sensitive prostate cancer cells with robust expression of p21. Mechanistically, we demonstrated that AIbZIP interacts with old astrocyte specifically induced substance (OASIS), which is a CREB/ATF family transcription factor, and prevents OASIS from promoting transcription of its target gene p21. These findings showed that AIbZIP induced by the androgen receptor (AR) axis plays a crucial role in the proliferation of androgen-sensitive prostate cancer cells, and could be a novel target of therapy for prostate cancer.


Asunto(s)
Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/fisiología , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Regulación Neoplásica de la Expresión Génica , Proteínas Nucleares/fisiología , Andrógenos/farmacología , Sitios de Unión , Línea Celular Tumoral , Proliferación Celular , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Regulación hacia Abajo , Expresión Génica , Aparato de Golgi/metabolismo , Células HEK293 , Humanos , Masculino , Metribolona/farmacología , Proteínas del Tejido Nervioso/metabolismo , Regiones Promotoras Genéticas , Neoplasias de la Próstata , Unión Proteica , Proteolisis , Proteínas Proto-Oncogénicas c-ets/metabolismo , Transducción de Señal , Activación Transcripcional
10.
J Biol Chem ; 291(11): 5512-26, 2016 Mar 11.
Artículo en Inglés | MEDLINE | ID: mdl-26786102

RESUMEN

The transcription factor c-Fos controls many important cellular processes, including cell growth and apoptosis. c-Fos expression is rapidly elevated in the prostate upon castration-mediated androgen withdrawal through an undefined mechanism. Here we show that androgens (5α-dihydrotestosterone and R1881) suppress c-Fos protein and mRNA expression induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) or EGF in human prostate cancer (PCa) cell lines. Such suppression transpires through a transcriptional mechanism, predominantly at the proximal serum response element of the c-fos promoter. We show that androgen signaling suppresses TPA-induced c-Fos expression through repressing a PKC/MEK/ERK/ELK-1 signaling pathway. Moreover, our results support the hypothesis that p38(MAPK), PI3K, and PKCδ are involved in the androgenic regulation of c-Fos through controlling MEK/ERK. Stable silencing of c-Fos and PKCδ with shRNAs suggests that R1881 promotes cell death induced by low-dose TPA through a mechanism that is dependent on both PKCδ and loss of c-Fos expression. Reciprocally, loss of either PKCδ or c-Fos activates p38(MAPK) while suppressing the activation of ERK1/2. We also provide the first demonstration that R1881 permits cell death induced by low-dose TPA in the LNCaP androgen-dependent PCa cell line and that TPA-induced cell death is independent of exogenous androgen in the castration-resistant variants of LNCaP, C4-2 and C4-2B. Acquisition of androgen-independent killing by TPA correlates with activation of p38(MAPK), suppression of ERK1/2, and loss of c-Fos. These results provide new insights into androgenic control of c-Fos and use of PKC inhibitors in PCa therapy.


Asunto(s)
Adenocarcinoma/tratamiento farmacológico , Andrógenos/farmacología , Dihidrotestosterona/farmacología , Metribolona/farmacología , Neoplasias de la Próstata/tratamiento farmacológico , Proteínas Proto-Oncogénicas c-fos/genética , Acetato de Tetradecanoilforbol/farmacología , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Regulación hacia Abajo/efectos de los fármacos , Factor de Crecimiento Epidérmico/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Masculino , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Próstata/efectos de los fármacos , Próstata/metabolismo , Próstata/patología , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Proteína Quinasa C/metabolismo , ARN Mensajero/genética , Transducción de Señal/efectos de los fármacos , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo
11.
Planta Med ; 81(10): 804-12, 2015 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-26039262

RESUMEN

Understanding the molecular mechanism of action of traditional medicines is an important step towards developing marketable drugs from them. Piperine, an active constituent present in the Piper species, is used extensively in Ayurvedic medicines (practiced on the Indian subcontinent). Among others, piperine is known to possess a male contraceptive effect; however, the molecular mechanism of action for this effect is not very clear. In this regard, detailed docking and molecular dynamics simulation studies of piperine with the androgen-binding protein and androgen receptors were carried out. Androgen receptors control male sexual behavior and fertility, while the androgen-binding protein binds testosterone and maintains its concentration at optimal levels to stimulate spermatogenesis in the testis. It was found that piperine docks to the androgen-binding protein, similar to dihydrotestosterone, and to androgen receptors, similar to cyproterone acetate (antagonist). Also, the piperine-androgen-binding protein and piperine-androgen receptors interactions were found to be stable throughout 30 ns of molecular dynamics simulation. Further, two independent simulations for 10 ns each also confirmed the stability of these interactions. Detailed analysis of the piperine-androgen-binding protein interactions shows that piperine interacts with Ser42 of the androgen-binding protein and could block the binding with its natural ligands dihydrotestosterone/testosterone. Moreover, piperine interacts with Thr577 of the androgen receptors in a manner similar to the antagonist cyproterone acetate. Based on the in silico results, piperine was tested in the MDA-kb2 cell line using the luciferase reporter gene assay and was found to antagonize the effect of dihydrotestosterone at nanomolar concentrations. Further detailed biochemical experiments could help to develop piperine as an effective male contraceptive agent in the future.


Asunto(s)
Alcaloides/química , Alcaloides/farmacología , Proteína de Unión a Andrógenos/metabolismo , Benzodioxoles/química , Benzodioxoles/farmacología , Anticonceptivos Masculinos/farmacología , Piperidinas/química , Piperidinas/farmacología , Alcamidas Poliinsaturadas/química , Alcamidas Poliinsaturadas/farmacología , Receptores Androgénicos/metabolismo , Alcaloides/metabolismo , Proteína de Unión a Andrógenos/química , Benzodioxoles/metabolismo , Dominio Catalítico , Línea Celular/efectos de los fármacos , Simulación por Computador , Anticonceptivos Masculinos/química , Dihidrotestosterona/farmacología , Humanos , Enlace de Hidrógeno , Masculino , Metribolona/química , Metribolona/metabolismo , Metribolona/farmacología , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Piperidinas/metabolismo , Alcamidas Poliinsaturadas/metabolismo , Conformación Proteica , Receptores Androgénicos/química , Serina/metabolismo
12.
Prostate ; 75(5): 527-38, 2015 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-25586052

RESUMEN

BACKGROUND: The central role of androgen receptor (AR) signaling is established in prostate cancer growth and progression. We propose CYP3A5 is part of a feedback loop that modulates the sensitivity of AR to androgen exposure. The purpose of this study is to elucidate the mechanism of regulation of AR expression by CYP3A5. METHODS: To identify the role of CYP3A5 in regulating AR signaling, CYP3A5 protein expression was inhibited using CYP3A5 siRNA and azamulin. Both cell fractionation and immunocytochemical approaches in combination with dihydrotestosterone (DHT) and R1881 treatment were used to evaluate changes in AR nuclear translocation. RESULTS: CYP3A5 siRNA blocked growth of LNCaP and C4-2 cells by 30-60% (P ≤ 0.005). Azamulin, a CYP3A pharmacologic inhibitor, reduced the growth of LNCaP, C4-2 and 22RV1 lines by ∼ 40% (P ≤ 0.005). CYP3A5 siRNA inhibited growth in response to DHT and R1881 treatment in LNCaP and C4-2 by decreasing nuclear AR localization and resulting in diminished PSA and TMPRSS2 expression. Decreased AR nuclear localization resulting from CYP3A5 inhibition resulted in growth inhibition comparable to IC60 and IC40 of bicalutamide in LNCaP and C4-2 cell lines. Conversely, the CYP3A inducer rifampicin enhanced AR nuclear localization. CONCLUSION: As CYP3A5 regulates the nuclear translocation of AR; co-targeting CYP3A5 may provide a novel strategy for enhancing the efficacy of androgen deprivation therapy. Consequentially, these data suggest that concomitant medications may impact androgen deprivation therapy's efficacy.


Asunto(s)
Citocromo P-450 CYP3A/fisiología , Neoplasias de la Próstata/enzimología , Receptores Androgénicos/metabolismo , Transducción de Señal/fisiología , Western Blotting , Hidrocarburos Aromáticos con Puentes/farmacología , Línea Celular Tumoral , Inhibidores del Citocromo P-450 CYP3A/farmacología , Dihidrotestosterona/farmacología , Técnica del Anticuerpo Fluorescente , Humanos , Masculino , Metribolona/farmacología , Neoplasias de la Próstata/patología , ARN Interferente Pequeño/farmacología , Reacción en Cadena en Tiempo Real de la Polimerasa , Triazoles/farmacología
13.
Cell Death Dis ; 5: e1548, 2014 Dec 04.
Artículo en Inglés | MEDLINE | ID: mdl-25476896

RESUMEN

The functions of androgen receptor (AR) in stromal cells are still debated in spite of the demonstrated importance of these cells in organ development and diseases. Here, we show that physiological androgen concentration (10 nM R1881 or DHT) fails to induce DNA synthesis, while it consistently stimulates cell migration in mesenchymal and transformed mesenchymal cells. Ten nanomolar R1881 triggers p27 Ser10 phosphorylation and its stabilization in NIH3T3 fibroblasts. Activation of Rac and its downstream effector DYRK 1B is responsible for p27 Ser10 phosphorylation and cell quiescence. Ten nanomolar androgen also inhibits transformation induced by oncogenic Ras in NIH3T3 fibroblasts. Overexpression of an AR mutant unable to interact with filamin A, use of a small peptide displacing AR/filamin A interaction, and filamin A knockdown indicate that the androgen-triggered AR/filamin A complex regulates the pathway leading to p27 Ser10 phosphorylation and cell cycle arrest. As the AR/filamin A complex is also responsible for migration stimulated by 10 nM androgen, our report shows that the androgen-triggered AR/filamin A complex controls, through Rac 1, the decision of cells to halt cell cycle and migration. This study reveals a new and unexpected role of androgen/AR signalling in coordinating stromal cell functions.


Asunto(s)
Dihidrotestosterona/farmacología , Filaminas/metabolismo , Células Madre Mesenquimatosas/metabolismo , Receptores Androgénicos/metabolismo , Transducción de Señal , Proteína de Unión al GTP rac1/metabolismo , Animales , Puntos de Control del Ciclo Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibrosarcoma/genética , Fibrosarcoma/metabolismo , Fibrosarcoma/patología , Filaminas/genética , Regulación de la Expresión Génica , Humanos , Masculino , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/efectos de los fármacos , Metribolona/farmacología , Ratones , Células 3T3 NIH , Fosforilación , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , Receptores Androgénicos/genética , Serina/metabolismo , Congéneres de la Testosterona/farmacología , Células Tumorales Cultivadas , Proteína de Unión al GTP rac1/genética , Proteínas ras/genética , Proteínas ras/metabolismo
14.
Oncotarget ; 5(20): 10017-33, 2014 Oct 30.
Artículo en Inglés | MEDLINE | ID: mdl-25294820

RESUMEN

Prostate cancer (PCA) kills thousands of men every year, demanding additional approaches to better understand and target this malignancy. Recently, critical role of aberrant lipogenesis is highlighted in prostate carcinogenesis, offering a unique opportunity to target it to reduce PCA. Here, we evaluated efficacy and associated mechanisms of silibinin in inhibiting lipid metabolism in PCA cells. At physiologically achievable levels in human, silibinin strongly reduced lipid and cholesterol accumulation specifically in human PCA cells but not in non-neoplastic prostate epithelial PWR-1E cells. Silibinin also decreased nuclear protein levels of sterol regulatory element binding protein 1 and 2 (SREBP1/2) and their target genes only in PCA cells. Mechanistically, silibinin activated AMPK, thereby increasing SREBP1 phosphorylation and inhibiting its nuclear translocation; AMPK inhibition reversed silibinin-mediated decrease in nuclear SREBP1 and lipid accumulation. Additionally, specific SREBP inhibitor fatostatin and stable overexpression of SREBP1 further confirmed the central role of SREBP1 in silibinin-mediated inhibition of PCA cell proliferation and lipid accumulation and cell cycle arrest. Importantly, silibinin also inhibited synthetic androgen R1881-induced lipid accumulation and completely abrogated the development of androgen-independent LNCaP cell clones via targeting SREBP1/2. Together, these mechanistic studies suggest that silibinin would be effective against PCA by targeting critical aberrant lipogenesis.


Asunto(s)
Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/metabolismo , Silimarina/farmacología , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/metabolismo , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Humanos , Metabolismo de los Lípidos/efectos de los fármacos , Masculino , Metribolona/antagonistas & inhibidores , Metribolona/farmacología , Terapia Molecular Dirigida , Fosforilación , Neoplasias de la Próstata/patología , Neoplasias de la Próstata Resistentes a la Castración/prevención & control , Piridinas/farmacología , Silibina , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/antagonistas & inhibidores , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/biosíntesis , Proteína 2 de Unión a Elementos Reguladores de Esteroles/antagonistas & inhibidores , Proteína 2 de Unión a Elementos Reguladores de Esteroles/biosíntesis , Proteína 2 de Unión a Elementos Reguladores de Esteroles/metabolismo , Tiazoles/farmacología , Transfección
15.
PLoS One ; 9(9): e107991, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-25268119

RESUMEN

Androgen ablation therapy causes a temporary reduction in tumor burden in patients with advanced prostate cancer. Unfortunately the malignancy will return to form lethal castration-recurrent prostate cancer (CRPC). The androgen receptor (AR) remains transcriptionally active in CRPC in spite of castrate levels of androgens in the blood. AR transcriptional activity resides in its N-terminal domain (NTD). Possible mechanisms of continued AR transcriptional activity may include, at least in part, expression of constitutively active splice variants of AR that lack the C-terminal ligand-binding domain (LBD). Current therapies that target the AR LBD, would not be effective against these AR variants. Currently no drugs are clinically available that target the AR NTD which should be effective against these AR variants as well as full-length AR. Niphatenones were originally isolated and identified in active extracts from Niphates digitalis marine sponge. Here we begin to characterize the mechanism of niphatenones in blocking AR transcriptional activity. Both enantiomers had similar IC50 values of 6 µM for inhibiting the full-length AR in a functional transcriptional assay. However, (S)-niphatenone had significantly better activity against the AR NTD compared to (R)-niphatenone. Consistent with niphatenones binding to and inhibiting transactivation of AR NTD, niphatenones inhibited AR splice variant. Niphatenone did not affect the transcriptional activity of the related progesterone receptor, but slightly decreased glucocorticoid receptor (GR) activity and covalently bound to GR activation function-1 (AF-1) region. Niphatenone blocked N/C interactions of AR without altering either AR protein levels or its intracellular localization in response to androgen. Alkylation with glutathione suggests that niphatenones are not a feasible scaffold for further drug development.


Asunto(s)
Antagonistas de Receptores Androgénicos/farmacología , Antineoplásicos Hormonales/farmacología , Éteres de Glicerilo/farmacología , Línea Celular Tumoral , Humanos , Concentración 50 Inhibidora , Masculino , Metribolona/farmacología , Neoplasias de la Próstata/tratamiento farmacológico , Estructura Terciaria de Proteína , Receptores Androgénicos/química , Receptores Androgénicos/fisiología , Estereoisomerismo , Activación Transcripcional/efectos de los fármacos
16.
Mol Cancer ; 13: 214, 2014 Sep 12.
Artículo en Inglés | MEDLINE | ID: mdl-25216853

RESUMEN

BACKGROUND: Prostate cancer (PCa) is the second leading cause of cancer mortality of men in Western countries. The androgen receptor (AR) and AR-agonists (androgens) are required for the development and progression of the normal prostate as well as PCa. However, it is discussed that in addition to their tumor promoting activity, androgens may also exhibit tumor suppressive effects. A biphasic growth response to androgens a growth-promoting and -inhibition has been observed that suggests that administration of supraphysiological androgen levels mediates growth reduction in AR expressing PCa cells. METHODS: Detection of senescence markers, three dimensional interphase fluorescence in situ hybridization (3D-iFISH), qRT-PCR, Western blotting, detection of GFP fusions, prostatectomy, ex vivo culturing. RESULTS: Here, we describe that supraphysiological levels of androgens induce cell cycle arrest and markers of cellular senescence in human PCa cells, which may in part explain the growth inhibitory role of androgens. The expression of the senescence associated beta galactosidase is observed by treatment with the natural androgen DHT or the less metabolized synthetic androgen R1881. The induction of senescence marker was detected in human PCa cell lines as well as in human primary PCa tissue derived from prostatectomy treated ex vivo. Using interphase FISH (iFISH) suggests that the androgen-induced cellular senescence is associated with localizing the genomic E2F1 locus to senescence associated heterochromatic foci. Analysis of different signaling pathways in LNCaP cells suggest that the p16-Rb-E2F1 pathway is essential for the induction of cellular senescence since treatment with siRNA directed against p16 reduces the level of androgen-induced cellular senescence. Based on the rapid induction of androgen-mediated cellular senescence we identified the Src-PI3K-Akt-signaling pathway and autophagy being in part involved in androgen regulation. CONCLUSIONS: Taken together, our data suggest that AR-agonists at supraphysiological levels mediate induction of cellular senescence in human PCa cells, which may have a protective anti-cancer role. These results provide also new insights for understanding androgen-mediated regulation of PCa growth.


Asunto(s)
Antineoplásicos/farmacología , Biomarcadores de Tumor/genética , Dihidrotestosterona/farmacología , Metribolona/farmacología , Neoplasias de la Próstata/patología , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Senescencia Celular , Factor de Transcripción E2F1/genética , Humanos , Imagenología Tridimensional , Hibridación Fluorescente in Situ , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Masculino , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/cirugía
17.
Endocr Relat Cancer ; 21(5): 723-37, 2014 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-24981110

RESUMEN

Chitinase 3-like 1 (CHI3L1 or YKL40) is a secreted glycoprotein highly expressed in tumours from patients with advanced stage cancers, including prostate cancer (PCa). The exact function of YKL40 is poorly understood, but it has been shown to play an important role in promoting tumour angiogenesis and metastasis. The therapeutic value and biological function of YKL40 are unknown in PCa. The objective of this study was to examine the expression and function of YKL40 in PCa. Gene expression analysis demonstrated that YKL40 was highly expressed in metastatic PCa cells when compared with less invasive and normal prostate epithelial cell lines. In addition, the expression was primarily limited to androgen receptor-positive cell lines. Evaluation of YKL40 tissue expression in PCa patients showed a progressive increase in patients with aggressive disease when compared with those with less aggressive cancers and normal controls. Treatment of LNCaP and C4-2B cells with androgens increased YKL40 expression, whereas treatment with an anti-androgen agent decreased the gene expression of YKL40 in androgen-sensitive LNCaP cells. Furthermore, knockdown of YKL40 significantly decreased invasion and migration of PCa cells, whereas overexpression rendered them more invasive and migratory, which was commensurate with an enhancement in the anchorage-independent growth of cells. To our knowledge, this study characterises the role of YKL40 for the first time in PCa. Together, these results suggest that YKL40 plays an important role in PCa progression and thus inhibition of YKL40 may be a potential therapeutic strategy for the treatment of PCa.


Asunto(s)
Adipoquinas/metabolismo , Lectinas/metabolismo , Neoplasias de la Próstata/metabolismo , Anciano , Anciano de 80 o más Años , Antagonistas de Andrógenos/farmacología , Andrógenos/farmacología , Línea Celular , Línea Celular Tumoral , Movimiento Celular , Proteína 1 Similar a Quitinasa-3 , Dihidrotestosterona/farmacología , Progresión de la Enfermedad , Humanos , Masculino , Metribolona/farmacología , Persona de Mediana Edad , Invasividad Neoplásica , Feniltiohidantoína/análogos & derivados , Feniltiohidantoína/farmacología , Neoplasias de la Próstata/patología
18.
Cancer Lett ; 351(2): 272-80, 2014 Sep 01.
Artículo en Inglés | MEDLINE | ID: mdl-24998678

RESUMEN

LuCaP serially transplantable xenografts are valuable preclinical models of locally advanced or metastatic prostate cancer. For the first time, we recently succeeded in establishing and serially passaging spheroid cultures of several LuCaP xenografts. Here, we characterized in depth the molecular and cellular phenotype of LuCaP 147 cultures and found faithful retention of the characteristics of the original xenograft, including immunophenotype, genetic fidelity, gene expression profile and responsiveness to androgen. Furthermore, we demonstrated capabilities for high-throughput drug screening and that anti-cancer agents induced cell cycle arrest and apoptosis in spheroid cultures. Finally, we showed that cells formed tumors when re-introduced into mice, providing an authentic in vitro-in vivo preclinical model of a subtype of prostate cancer with a hypermutator phenotype and an SPOP mutation.


Asunto(s)
Proteínas Nucleares/genética , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Proteínas Represoras/genética , Antagonistas de Andrógenos/farmacología , Anilidas/farmacología , Animales , Línea Celular Tumoral , Xenoinjertos , Humanos , Inmunofenotipificación , Masculino , Metribolona/farmacología , Ratones , Mutación , Nitrilos/farmacología , Fenotipo , Neoplasias de la Próstata/metabolismo , Esferoides Celulares , Compuestos de Tosilo/farmacología , Complejos de Ubiquitina-Proteína Ligasa
19.
J Biomed Opt ; 19(11): 111605, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-24933682

RESUMEN

Increasing interest in the role of lipids in cancer cell proliferation and resistance to drug therapies has motivated the need to develop better tools for cellular lipid analysis. Quantification of lipids in cells is typically done by destructive chromatography protocols that do not provide spatial information on lipid distribution and prevent dynamic live cell studies. Methods that allow the analysis of lipid content in live cells are therefore of great importance. Using micro-Raman spectroscopy and coherent anti-Stokes Raman scattering (CARS) microscopy, we generated a lipid profile for breast (T47D, MDA-MB-231) and prostate (LNCaP, PC3) cancer cells upon exposure to medroxyprogesterone acetate (MPA) and synthetic androgen R1881. Combining Raman spectra with CARS imaging, we can study the process of hormone-mediated lipogenesis. Our results show that hormone-treated cancer cells T47D and LNCaP have an increased number and size of intracellular lipid droplets and higher degree of saturation than untreated cells. MDA-MB-231 and PC3 cancer cells showed no significant changes upon treatment. Principal component analysis with linear discriminant analysis of the Raman spectra was able to differentiate between cancer cells that were treated with MPA, R1881, and untreated.


Asunto(s)
Neoplasias de la Mama/química , Lípidos/análisis , Microscopía/métodos , Neoplasias de la Próstata/química , Espectrometría Raman/métodos , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Femenino , Humanos , Gotas Lipídicas/química , Gotas Lipídicas/efectos de los fármacos , Gotas Lipídicas/metabolismo , Lípidos/química , Masculino , Acetato de Medroxiprogesterona/farmacología , Metribolona/farmacología , Imagen Molecular/métodos , Neoplasias de la Próstata/metabolismo
20.
Biochem J ; 462(2): 315-27, 2014 Sep 01.
Artículo en Inglés | MEDLINE | ID: mdl-24844355

RESUMEN

We show that HEXIM1 (hexamethylene bis-acetamide inducible 1) functions as an AR (androgen receptor) co-repressor as it physically interacts with the AR and is required for the ability of anti-androgens to inhibit androgen-induced target gene expression and cell proliferation. Oncomine™ database and IHC (immunohistochemistry) analyses of human prostate tissues revealed that expression of HEXIM1 mRNA and protein are down-regulated during the development and progression of prostate cancer. Enforced down-regulation of HEXIM1 in parental hormone-dependent LNCaP cells results in resistance to the inhibitory action of anti-androgens. Conversely, ectopic expression of HEXIM1 in the CRPC (castration-resistant prostate cancer) cell line, C4-2, enhances their sensitivity to the repressive effects of the anti-androgen bicalutamide. Novel insight into the mechanistic basis for HEXIM1 inhibition of AR activity is provided by the present studies showing that HEXIM1 induces expression of the histone demethylase KDM5B (lysine-specific demethylase 5B) and inhibits histone methylation, resulting in the inhibition of FOXA1 (forkhead box A1) licensing activity. This is a new mechanism of action attributed to HEXIM1, and distinct from what has been reported so far to be involved in HEXIM1 regulation of other nuclear hormone receptors, including the oestrogen receptor.


Asunto(s)
Antagonistas de Andrógenos/farmacología , Neoplasias de la Próstata/metabolismo , Proteínas de Unión al ARN/metabolismo , Receptores Androgénicos/metabolismo , Anilidas/farmacología , Línea Celular Tumoral , Elementos de Facilitación Genéticos , Células Epiteliales/metabolismo , Expresión Génica/efectos de los fármacos , Histonas/metabolismo , Humanos , Histona Demetilasas con Dominio de Jumonji/metabolismo , Masculino , Metribolona/farmacología , Nitrilos/farmacología , Proteínas Nucleares/metabolismo , Próstata/metabolismo , Neoplasias de la Próstata Resistentes a la Castración/metabolismo , Transporte de Proteínas , Proteínas Represoras/metabolismo , Compuestos de Tosilo/farmacología , Factores de Transcripción , Factores de Elongación Transcripcional/metabolismo
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