TY - JOUR
T1 - EFFECTS OF GnRH AGONIST TREATMENT ON GnRH RECEPTORS IN HUMAN PROSTATE CANCER CELLS: AN ATOMIC FORCE MICROSCOPY STUDY
AU - Lama, Gina
AU - Angelucci, Cristiana
AU - Cupelli, Elisa
AU - Sica, Gigliola
AU - De Spirito, Marco
AU - Papi, Massimiliano
PY - 2011
Y1 - 2011
N2 - 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
AB - 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
KW - AFM
KW - GnRH ANALOGUE
KW - GnRH RECEPTOR
KW - PROSTATE CANCER CELLS
KW - AFM
KW - GnRH ANALOGUE
KW - GnRH RECEPTOR
KW - PROSTATE CANCER CELLS
UR - http://hdl.handle.net/10807/6915
M3 - Conference article
SN - 1122-6714
VL - 116
SP - 97
EP - 97
JO - Italian Journal of Anatomy and Embryology
JF - Italian Journal of Anatomy and Embryology
T2 - 65° Congresso Società Italiana di Anatomia e Istologia
Y2 - 27 September 2011 through 29 September 2011
ER -