title: Leuprorelin acetate long-lasting effects on GnRH receptors of prostate cancer cells: an atomic force microscopy study of agonist/receptor interaction.
abstract: High cell-surface GnRH receptor (GnRH-R) levels have been shown to have a major influence on the extent of GnRH agonist-mediated tumor growth inhibition. The ability of the GnRH agonist leuprorelin acetate (LA) to induce a post-transcriptional upregulation of GnRH-R at the plasma membrane of androgen-sensitive (LNCaP) and -insensitive (PC-3) prostate cancer (PCa) cells has been previously demonstrated by Western blotting. Here we performed single molecule force spectroscopy by using Atomic Force Microscopy (AFM), which has proven to be a powerful tool allowing for investigation of living cell surface biological features, such as the so far unclear GnRH agonist/receptor interaction. Thus, in the hormone-insensitive PC-3 cells, we characterized the strength of the LA-receptor binding, and the amount and distribution of the functional receptor molecules on the cell surface. The effect of a long and continuous treatment (up to 30 days) with the agonist (10(-11) and 10(-6) M) on the same parameters was also investigated. A GnRH-R increase was observed, reaching the maximum (∼80%) after 30 days of treatment with the highest dose of LA (10(-6) M). The analogue-induced increase in GnRH-R was also demonstrated by Western blotting. In addition, two different receptor bound strengths were detected by AFM, which suggests the existence of two GnRH-R classes. A homogeneous distribution of the unbinding events has been found on untreated and treated PC-3 cell surfaces. The persistence of high receptor levels at the membrane of these living cells may warrant the maintenance of the response to LA also in androgen-unresponsive PCa. Moreover, the determination of ligand/receptor bond strength could shed light on the poorly understood event of LA/GnRH-R interaction and/or address structural/chemical agonist optimizations.
authors: Istituto di Istologia ed Embriologia, Facoltà di Medicina e Chirurgia A. Gemelli, Università Cattolica del Sacro Cuore, Roma, Italy.
authors: Gina
authors: G
authors: Lama
authors: Massimiliano
authors: M
authors: Papi
authors: Cristiana
authors: C
authors: Angelucci
authors: Giuseppe
authors: G
authors: Maulucci
authors: Gigliola
authors: G
authors: Sica
authors: Marco
authors: M
authors: De Spirito
mesh_terms: D018931:Antineoplastic Agents, Hormonal / Q000378:metabolism / Q000494:pharmacology; D001665:Binding Sites; D001667:Binding, Competitive; D015153:Blotting, Western; D045744:Cell Line, Tumor; D007987:Gonadotropin-Releasing Hormone / Q000378:metabolism / Q000494:pharmacology; D057809:HEK293 Cells; D006801:Humans; D016729:Leuprolide / Q000378:metabolism* / Q000494:pharmacology; D008297:Male; D018625:Microscopy, Atomic Force / Q000379:methods*; D011471:Prostatic Neoplasms / Q000378:metabolism / Q000473:pathology; D011966:Receptors, LHRH / Q000819:agonists / Q000235:genetics / Q000378:metabolism*
chemical_list: D018931:Antineoplastic Agents, Hormonal; D011966:Receptors, LHRH; D007987:Gonadotropin-Releasing Hormone; D016729:Leuprolide