Characterization of high‐affinity receptors for bombesin/gastrin releasing peptide on the human prostate cancer cell lines PC‐3 and DU‐145: Internalization of receptor bound 125I‐(Tyr4) bombesin by tumor cells

Specific receptors for bombesin/gastrin releasing peptide (GRP) on the androgen‐independent human prostate cancer cell lines PC‐3 and DU‐145 were characterized. No specific binding of 125I‐[Tyr4]‐bombesin to the androgen‐dependent human prostate cancer cell line LNCaP was detectable. The binding of 125I‐[Tyr4]‐bombesin to PC‐3 and DU‐145 cells was found to be time‐ and temperature‐dependent, saturable, and reversible. Scatchard analysis revealed a single class of binding sites with high affinity (Kd 9.8 × 10−11 M for PC‐3, and 9.1 × 10‐11 M for DU‐145 cells at 25°C) and with a binding capacity of 44,000 binding sites/cell and 19,000 binding sites/cell, respectively. Bound 125I‐[Tyr4]‐bombesin was rapidly internalized by PC‐3 cells. The nonhydrolyzable GTP analog GTP‐gamma‐S caused a dose‐dependent inhibition of 125I‐[Tyr4]‐bombesin binding to PC‐3 and DU‐145 cells, indicating that a G‐protein (guanine nucleotide‐binding protein) couples the bombesin receptor to intracellular effector systems. Bombesin and GRP(14‐27) inhibited the binding of 125I‐[Tyr4]‐bombesin to both cell lines in a dose‐dependent manner with inhibition constants (Ki of 0.5 nM and 0.4 nM, respectively. Both cell lines express the bombesin/GRP preferring bombesin receptor subtype, since, in displacement studies, neuromedin B was more than 200 times less potent than bombesin and GRP(14‐27) in inhibiting the binding of 125I‐[Tyr4]‐bombesin. Two synthetic bombesin/GRP antagonists, RC‐3095 and RC‐3110, powerfully inhibited the specific binding of 125I‐[Tyr4]‐bombesin with Ki 0.92 nM and 0.26 nM on PC‐3 cells, and 3.3 nM and 0.89 nM on DU‐145 cells, respectively. These findings indicate that the PC‐3 and DU‐145 human prostate cancer cell lines possess specific high‐affinity receptors for bombesin/GRP, and are suitable models for the evaluation of the antineoplastic activity of new bombesin/GRP antagonists in the treatment of androgen‐independent prostate cancer. © 1994 Wiley‐Liss, Inc.