We previously demonstrated by Western blotting and immunocytochemistry that a GnRH agonist (leuprorelin acetate, LA) was able to induce a post-transcriptional increase in GnRH receptor (GnRH-R) expression at the plasma membrane of androgen-sensitive (LNCaP) and -insensitive (PC-3) prostate cancer (PCa) cells. In the present study, we used atomic force microscopy (AFM) to gain a deeper insight into the effects of LA on the behaviour of GnRH-R in highly invasive and poorly differentiated PC-3 cells. The use of this powerful, non-destructive technique allows to identify and study the biological features of the living cell surface, as ligand-receptor interactions. Here, we investigated for 6, 12, 18, 24 and 30 days, the effect of LA (10-11 and 10-6 M) in PC-3 cells on: i) amount of LA/GnRH-R binding events (i.e. GnRH-R quantification), ii) strength of the analogue-receptor binding, iii) receptor topography. Briefly, analogue molecules were immobilized onto conical AFM tips and the single agonist/receptor interactions were measured by force-distance cycles. In agreement with our previous results, the number of GnRH-R augmented during 30 days due to the effect of LA treatment. The increasing rate of GnRH-R was dose-dependent until the 24th day and reached the maximum (~70%) after 30 days of treatment with the highest dose of LA (10-6M). At least 2 different receptor bound strengths have been detected, probably due to the presence of two GnRH-R classes. The majority of the sites showed a relatively low bound strength (~37 piconewton). A LA/GnRH-R complex lifetime of ~ 9s and ~3.4 s for the higher and lower bound strength receptors, respectively, has been determined. Regarding GnRH-R topography, a homogeneous distribution of the binding events has been found on untreated and LA-treated PC-3 cell surfaces. The persistence of high receptor levels at the androgen-insensitive cell surface may warrant the maintenance of the response to the analogue in androgen-unresponsive PCa also, which might be useful in clinical practice. Moreover, the definition of parameters as ligand/receptor bond strength and lifetime could shed light on the poorly understood molecular basis of LA/GnRH-R interaction and might be used to address structural/chemical agonist optimizations

Lama, G., Angelucci, C., Cupelli, E., Sica, G., De Spirito, M., Papi, M., EFFECTS OF GnRH AGONIST TREATMENT ON GnRH RECEPTORS IN HUMAN PROSTATE CANCER CELLS: AN ATOMIC FORCE MICROSCOPY STUDY, Abstract de <<65° Congresso Società Italiana di Anatomia e Istologia>>, (Padova, 27-29 September 2011 ), <<ITALIAN JOURNAL OF ANATOMY AND EMBRYOLOGY>>, 2011; 116 (1): 97-97 [http://hdl.handle.net/10807/6915]

EFFECTS OF GnRH AGONIST TREATMENT ON GnRH RECEPTORS IN HUMAN PROSTATE CANCER CELLS: AN ATOMIC FORCE MICROSCOPY STUDY

Lama, Gina;Angelucci, Cristiana;Cupelli, Elisa;Sica, Gigliola;De Spirito, Marco;Papi, Massimiliano
2011

Abstract

We previously demonstrated by Western blotting and immunocytochemistry that a GnRH agonist (leuprorelin acetate, LA) was able to induce a post-transcriptional increase in GnRH receptor (GnRH-R) expression at the plasma membrane of androgen-sensitive (LNCaP) and -insensitive (PC-3) prostate cancer (PCa) cells. In the present study, we used atomic force microscopy (AFM) to gain a deeper insight into the effects of LA on the behaviour of GnRH-R in highly invasive and poorly differentiated PC-3 cells. The use of this powerful, non-destructive technique allows to identify and study the biological features of the living cell surface, as ligand-receptor interactions. Here, we investigated for 6, 12, 18, 24 and 30 days, the effect of LA (10-11 and 10-6 M) in PC-3 cells on: i) amount of LA/GnRH-R binding events (i.e. GnRH-R quantification), ii) strength of the analogue-receptor binding, iii) receptor topography. Briefly, analogue molecules were immobilized onto conical AFM tips and the single agonist/receptor interactions were measured by force-distance cycles. In agreement with our previous results, the number of GnRH-R augmented during 30 days due to the effect of LA treatment. The increasing rate of GnRH-R was dose-dependent until the 24th day and reached the maximum (~70%) after 30 days of treatment with the highest dose of LA (10-6M). At least 2 different receptor bound strengths have been detected, probably due to the presence of two GnRH-R classes. The majority of the sites showed a relatively low bound strength (~37 piconewton). A LA/GnRH-R complex lifetime of ~ 9s and ~3.4 s for the higher and lower bound strength receptors, respectively, has been determined. Regarding GnRH-R topography, a homogeneous distribution of the binding events has been found on untreated and LA-treated PC-3 cell surfaces. The persistence of high receptor levels at the androgen-insensitive cell surface may warrant the maintenance of the response to the analogue in androgen-unresponsive PCa also, which might be useful in clinical practice. Moreover, the definition of parameters as ligand/receptor bond strength and lifetime could shed light on the poorly understood molecular basis of LA/GnRH-R interaction and might be used to address structural/chemical agonist optimizations
2011
Inglese
Lama, G., Angelucci, C., Cupelli, E., Sica, G., De Spirito, M., Papi, M., EFFECTS OF GnRH AGONIST TREATMENT ON GnRH RECEPTORS IN HUMAN PROSTATE CANCER CELLS: AN ATOMIC FORCE MICROSCOPY STUDY, Abstract de <<65° Congresso Società Italiana di Anatomia e Istologia>>, (Padova, 27-29 September 2011 ), <<ITALIAN JOURNAL OF ANATOMY AND EMBRYOLOGY>>, 2011; 116 (1): 97-97 [http://hdl.handle.net/10807/6915]
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