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1.
Anticancer Drugs ; 23(9): 959-69, 2012 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-22713594

RESUMEN

Gonadotropin-releasing hormone (GnRH) agonists are widely used for the treatment of advanced prostate cancer (PCa). Agonists activate the GnRH receptor (GnRH-R), triggering apoptosis in PCa cells. In gonadotropes, the amount of GnRH-R in the plasma membrane is regulated by protein folding and endoplasmic reticulum retention, mechanisms that can be overcome by the pharmacoperone IN3. Our aim was to describe the intracellular distribution of GnRH-R in PCa cells and its relation to response to GnRH analog treatments. The expressions of GnRH-R in PCa biopsies were evaluated by immunohistochemistry and the intracellular distribution was determined by immunofluorescence in primary cell cultures from human PCa samples. Cultured cells were pretreated with IN3 and then with leuprolide. Cell survival was evaluated by 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT) thiazolyl blue formazan and cell cycle and apoptosis by flow cytometry. We observed that the expression of GnRH-R decreased according to malignant progression. Most GnRH-R are located inside the cell, colocalizing with endoplasmic reticulum markers. The treatment with IN3 decreased cellular GnRH-R retention, increasing plasma membrane expression in approximately 60%. Pretreatment with IN3 decreased PCa cell survival compared with leuprolide-alone treatment, primarily because of an increase in apoptosis. We conclude that the response of PCa cells to leuprolide is related to the amount of GnRH-R in the plasma membrane. Therefore, pretreatment evaluation of the amount of these receptors may be a predictor of the outcome of leuprolide treatment in PCa patients. Assessment of systemic IN3 effect would be necessary to determine its utility as an adjuvant treatment in hormone-resistant tumors.


Asunto(s)
Apoptosis/efectos de los fármacos , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Membrana Celular/efectos de los fármacos , Indoles/farmacología , Leuprolida/farmacología , Neoplasias de la Próstata/patología , Piridinas/farmacología , Receptores LHRH/agonistas , Western Blotting , Técnicas de Cultivo de Célula , Ciclo Celular/efectos de los fármacos , Membrana Celular/metabolismo , Supervivencia Celular/efectos de los fármacos , Sinergismo Farmacológico , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Citometría de Flujo , Humanos , Inmunohistoquímica , Masculino , Neoplasias de la Próstata/metabolismo , Pliegue de Proteína , Receptores LHRH/biosíntesis , Células Tumorales Cultivadas
2.
Cancer Invest ; 24(3): 261-8, 2006.
Artículo en Inglés | MEDLINE | ID: mdl-16809153

RESUMEN

Contradictory data have been reported regarding the effect of GnRH agonists and antagonists on cell growth and survival, using prostate cancer-derived cell lines expressing either endogenous or exogenous GnRH receptors. We addressed the issue studying the effect of leuprolide (agonist) and cetrorelix (antagonist) on cell growth, apoptosis and GnRH receptor expression using a primary cell coculture system. Also, binding characteristics of prostate GnRH receptor in this culture system are described. Epithelial and stromal cells were obtained from prostate adenocarcinoma samples and cocultured in a bicameral system. Expression of GnRH receptors was evaluated by semiquantitative RT-PCR (transcript level) and Western blot (protein level). Cell growth was estimated by MTT method and apoptosis by DNA fragmentation using COMET assay. Saturation and competition binding studies were carried out using 125I-GnRH as radioligand. GnRH receptors from cell cultures of prostate cancer exhibited a single class of binding sites with a Kd of 1.11 +/- 0.28 nM and a Bmax of 2.81 +/- 0.37 pmol/mg of membrane protein for GnRH. Leuprolide and cetrorelix showed no effect on GnRH receptor expression. Both analogues showed a significant reduction in cell growth rate and an increase in DNA-fragmented cell number. These effects were dependent on the analogue concentrations (from 5-20 ng/mL). Considering that the culture system used in this work represents more closely the in vivo conditions of tumor cells than metastatic derived cell lines, we conclude that GnRH analogues have a significant inhibitory effect on cell viability of cells expressing GnRH receptors. In addition, GnRH receptors expressed in tumor prostatic cells seem not discriminate between agonist and antagonist, both analogues activating these receptors. Also, leuprolide and cetrorelix treatments did not influence GnRH receptor expression in our culture system. These differences with pituitary receptors may be explained by differences in affinity, transduction mechanism and molecular context in prostatic tissue.


Asunto(s)
Adenocarcinoma/tratamiento farmacológico , Antineoplásicos Hormonales/farmacología , Antagonistas de Hormonas/farmacología , Leuprolida/farmacología , Neoplasias de la Próstata/tratamiento farmacológico , Apoptosis/efectos de los fármacos , Western Blotting , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Técnicas de Cocultivo , Ensayo Cometa , Células Epiteliales/efectos de los fármacos , Humanos , Inmunohistoquímica , Masculino , Receptores LHRH/biosíntesis , Receptores LHRH/efectos de los fármacos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células del Estroma/efectos de los fármacos
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