Bombesin antagonists inhibit in vitro and in vivo growth of human gastric cancer and binding of bombesin to its receptors

Abstract

We investigated the effect of bombesin/gastrin-releasing peptide (GRP) antagonist RC-3095 and other analogs on the growth of Hs746T human gastric 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 micrograms twice daily) or the vehicle (control) for 21 days. Administration of antagonist RC-3095 inhibited the growth of Hs746T tumors. Treatment with RC-3095 produced a significant decrease in tumor volume, prolonged the 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 treated 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. The presence of specific receptors for bombesin/GRP was investigated on the crude membranes of implanted tumors of Hs746T cells. Saturation binding assays showed that the binding of [125I-Tyr4]bombesin to the membranes was saturable and reversible. Scatchard analysis indicated the presence of a single class of binding sites with a high affinity (Kd = 0.24 +/- 0.07 nM) and a low binding capacity (Bmax = 57.0 +/- 0.9 fmol/mg protein). In displacement studies, the binding of [125I-Tyr4]bombesin was inhibited in a dose-dependent manner by unlabelled bombesin(1-14), [Tyr4]-bombesin and GRP (14-27), but not by structurally unrelated peptides. Synthetic bombesin/GRP antagonists RC-3095, RC-3110, and RC-3950-II were all able to inhibit effectively the binding of [125I-Tyr4]bombesin to the membranes of Hs746T cells. RC-3950-II showed 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 [125I-Tyr4]bombesin bound to the cells, indicating that the bombesin receptor is coupled to a G-protein. In cell cultures, bombesin significantly stimulated the growth of Hs746T cells in vitro as shown by an increase in the uptake of [3H]thymidine. Bombesin antagonist RC-3095 could effectively inhibit the bombesin-stimulated growth of Hs746T cells 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 effects of bombesin/GRP and may be useful for the treatment of gastric cancers.

Inhibition of growth of human malignant glioblastoma in nude mice by antagonists of bombesin/gastrin-releasing peptide

“…Pinski et al (1994d) showed that RC-3095 at 10 77 M concentration inhibited the growth of U-87MG and U-373MG cells in vitro, by approximately 40 and 60% respectively, in the presence of 10 75 M GRP(14 ± 27). Similar experiments on cancer cell lines that respond to bombesin-like peptides, indicated that exposure to low concentrations of GRP(14 ± 27) results in stimulation of growth, while exposure of the same cells to higher concentrations of GRP(14 ± 27) may produce growth inhibition (Qin et al, 1994a;Wang et al, 1996;Kiaris and Schally, unpublished data).…”

International Union of Pharmacology. LXVIII. Mammalian Bombesin Receptors: Nomenclature, Distribution, Pharmacology, Signaling, and Functions in Normal and Disease States

“…BW2258U89 was reported to inhibit small cell lung cancer growth (Moody et al, 1995b) and to inhibit bombesin-stimulated gastrin release in vivo in dogs and rats and blocked the satiety effect of bombesin in rats (Kirkham et al, 1994). An additional series of substituted pseudopeptide analogs with position 14 substitutions in addition to the 13-14 bond have been described and widely used by Schally's group for inhibition of various tumor cell growth (Radulovic et al, 1991a;Cai et al, 1992Cai et al, , 1994Qin et al, 1994Qin et al, , 1995Jungwirth et al, 1998;Bajo et al, 2004). Two analogs with high potency in this group include [D-Phe 6 ,13-14, Tac 14 ]Bn 6 -14 (tac ϭ thiazolidine-4-carboxylic acid) (RC-3950-II) ) (K i 0.078 nM, murine BB 2 receptor) and [D-Tpi 6 ,13-14]bombesin 6 -14 (RC-3095) (K i 0.92 nM, murine BB2 receptor) Qin et al, 1994Qin et al, , 1995.…”

“…An additional series of substituted pseudopeptide analogs with position 14 substitutions in addition to the 13-14 bond have been described and widely used by Schally's group for inhibition of various tumor cell growth (Radulovic et al, 1991a;Cai et al, 1992Cai et al, , 1994Qin et al, 1994Qin et al, , 1995Jungwirth et al, 1998;Bajo et al, 2004). Two analogs with high potency in this group include [D-Phe 6 ,13-14, Tac 14 ]Bn 6 -14 (tac ϭ thiazolidine-4-carboxylic acid) (RC-3950-II) ) (K i 0.078 nM, murine BB 2 receptor) and [D-Tpi 6 ,13-14]bombesin 6 -14 (RC-3095) (K i 0.92 nM, murine BB2 receptor) Qin et al, 1994Qin et al, , 1995. A final group of potent antagonists in this class were synthesized by J. Martínez's group, with the most potent being JMV641 and JMV594 (Azay et al, 1996;Lamharzi et al, 1998).…”

Constitutive activation of the gastrin-releasing peptide receptor expressed by the nonmalignant human colon epithelial cell line NCM460.

“…Given the ability of the GRP-R to cause the proliferation of human stomach (33)(34)(35) and colon cancer (14-17) cell lines, we next determined whether ectopic receptor expression altered the growth characteristics of transfected NCM460 cells. Nontransfected NCM460 cells were similar to rabbit colonocytes maintained in primary culture insofar as they required a complex defined media for survival (25).…”