BOMBESIN ANTAGONISTS INHIBIT IN-VITRO AND IN-VIVO GROWTH OF HUMAN GASTRIC-CANCER AND BINDING OF BOMBESIN TO ITS RECEPTORS

We investigated the effect of bombesin/gastrin-releasing peptide (GRP) antagonist RC-3095 and other analogs on the growth of Hs746T human ga stric cancer cells implanted in nude mice or cultured in vitro and on the binding of bombesin to its receptors. Nude mice bearing xenografts of the Hs746T cell line received s.c. injections of RC-3095 (10 mug t wice daily) or the vehicle (control) for 21 days. Administration of an tagonist RC-3095 inhibited the growth of Hs746T tumors. Treatment with RC-3095 produced a significant decrease in tumor volume, prolonged th e tumor volume doubling time from 3.6 days to 5.1 days, and decreased the tumor growth rate by 76.9%. The tumor growth delay time in mice tr eated with RC-3095 was 2.8 days. Treatment with RC-3095 also decreased the final tumor weight by 88.3% and reduced DNA and protein contents in tumors by 91.5% and 89.5%, respectively, as compared to controls. T he presence of specific receptors for bombesin/GRP was investigated on the crude membranes of implanted tumors of Hs746T cells. Saturation b inding assays showed that the binding of [I-125-Tyr4]bombesin to the m embranes was saturable and reversible. Scatchard analysis indicated th e presence of a single class of binding sites with a high affinity (K( d) = 0.24 +/- 0.07 nM) and a low binding capacity (B(max) = 57.0 +/- 0 .9 fmol/mg protein). In displacement studies, the binding of [I-121-Ty r4]bombesin was inhibited in a dose-dependent manner by unlabelled bom besin(1-14), [Tyr4]-bombesin and GRP(14 27), but not by structurally u nrelated peptides. Synthetic bombesin/GRP antagonists RC-3095, RC-3110 , and RC-3950-II were all able to inhibit effectively the binding of [ I-125-Tyr4]bombesin to the membranes of Hs746T cells. RC-3950-II showe d a higher binding affinity for bombesin receptors than RC-3095 or RC- 3110. Addition of the non-hydrolyzable guanine-nucleotide analog GTP [ S] to the binding buffer caused a significant reduction in the amount of [I-125-Tyr4]bombesin bound to the cells, indicating that the bombes in receptor is coupled to a G-protein. In cell cultures, bombesin sign ificantly stimulated the growth of Hs746T cells in vitro as shown by a n increase in the uptake of [H-3]thymidine. Bombesin antagonist RC-309 5 could effectively inhibit the bombesin-stimulated growth of Hs746T c ells in cultures. These observations suggest that bombesin/GRP may act as growth factors through specific receptors present on the membranes of Hs746T cells. Bombesin/GRP antagonists appear to nullify the effec ts of bombesin/GRP and may be useful for the treatment of gastric canc ers.