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. © 2013 Lama et al.

Lama, G., Papi, M., Angelucci, C., Maulucci, G., Sica, G., De Spirito, M., Leuprorelin Acetate Long-Lasting Effects on GnRH Receptors of Prostate Cancer Cells: An Atomic Force Microscopy Study of Agonist/Receptor Interaction, <<PLOS ONE>>, 2013; 8 (1): e52530-1-e52530-9. [doi:10.1371/journal.pone.0052530] [http://hdl.handle.net/10807/41083]

Leuprorelin Acetate Long-Lasting Effects on GnRH Receptors of Prostate Cancer Cells: An Atomic Force Microscopy Study of Agonist/Receptor Interaction

Lama, Gina;Papi, Massimiliano;Angelucci, Cristiana;Maulucci, Giuseppe;Sica, Gigliola;De Spirito, Marco
2013

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. © 2013 Lama et al.
Inglese
Lama, G., Papi, M., Angelucci, C., Maulucci, G., Sica, G., De Spirito, M., Leuprorelin Acetate Long-Lasting Effects on GnRH Receptors of Prostate Cancer Cells: An Atomic Force Microscopy Study of Agonist/Receptor Interaction, <<PLOS ONE>>, 2013; 8 (1): e52530-1-e52530-9. [doi:10.1371/journal.pone.0052530] [http://hdl.handle.net/10807/41083]
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