ATOMIC FORCE MICROSCOPY OF GnRH MEMBRANE RECEPTORS IN HUMAN PROSTATE CANCER CELLS TREATED WITH A GnRH AGONIST

We previously demonstrated by Western blotting and immunocytochemistry that a GnRH agonist (leuprorelin acetate, LA) was able to induce an 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 6-30 days, the effect of LA (10-11 and 10-6M) 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 AFM tips and the single agonist/receptor interactions were measured by force-distance cycles. In agreement with our previous results, the 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 10-6M LA. Two 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. 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 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 LA 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