gastrins and benzotript

The 78 kDa gastrin-binding protein (GBP) is a likely target for the antiproliferative effects of gastrin receptor antagonists and non-steroidal anti-inflammatory drugs (NSAIDs) on colorectal carcinoma cells (Baldwin GS, Murphy VJ, Yang Z, and Hashimoto T. J Pharmacol Exp Ther 1998;286:1110-14). This study tested the hypotheses that the GBP bound actin, and that the interaction could be disrupted by gastrin receptor antagonists and NSAIDs. Binding of actin to the GBP was assessed by competition with (125)I-[Nle(15)]-gastrin(2,17) in a covalent cross-linking assay, and by comparison of (125)I-actin binding to the N- and C-terminal GBP domains, which had been expressed independently in E. coli as glutathione-S-transferase (GST) fusion proteins. The ability of gastrin receptor antagonists and NSAIDs to interfere with the actin-GBP interaction was measured by release of (125)I-actin from preformed complexes with the N- and C-terminal domain-GST fusion proteins. Actin purified from skeletal muscle or from gastric mucosal cytosol competed with (125)I-[Nle(15)]-gastrin(2,17) for binding to the GBP with IC(50) values of 2.6 +/- 0.7 microM, and 2.1 +/- 0.7 microM, respectively. The amount of (125)I-actin from either source bound to the N-terminal GBP domain was 8.2 times greater than the amount bound to the C-terminal domain. Binding of actin to both domains was inhibited by the gastrin receptor antagonists proglumide and benzotript, and by NSAIDs. We conclude that the GBP may associate with the cytoskeleton via an interaction between its N-terminal domain and actin, and that the association may be disrupted either by gastrin receptor antagonists or by NSAIDs.

Topics: Actins; Animals; Anti-Inflammatory Agents, Non-Steroidal; Benzamides; Binding, Competitive; Carrier Proteins; Gastrins; Mitochondrial Trifunctional Protein; Molecular Weight; Multienzyme Complexes; Proglumide; Protein Structure, Tertiary; Receptors, Cholecystokinin; Swine

A 78 kDa gastrin-binding protein is a likely target for the anti-proliferative effects of the cholecystokinin (CCK) receptor antagonists D,L-4-benzamido-N,N-dipropylglutaramic acid (proglumide) and N-4-chlorobenzoyl-L-tryptophan (benzotript) on colorectal carcinoma cell lines [Baldwin, G.S., 1994. Antiproliferative gastrin/cholecystokinin receptor antagonists target the 78-kDa gastrin-binding protein. Proc. Natl. Acad. Sci. USA 91, 7593-7597.]. Definition of the physiological role of the gastrin-binding protein has been hampered by the very low affinity of benzotript for the gastrin-binding protein. Benzotript analogues were therefore tested for their ability to inhibit the binding of iodinated gastrin to the gastrin-binding protein. The affinity of the most potent analogue (the D-isomer of benzotript, CR 665) was similar to the value reported previously for the L-isomer. In order to isolate more potent binding inhibitors, several selective CCK receptor antagonists were also tested as inhibitors of the binding of gastrin to the gastrin-binding protein. The affinity of the most potent binding inhibitor PD 149164 (benzenebutanoic acid, 4-fluoro-!b/-[[3-(1H-indol-3-yl)-2-methyl-1-oxo-2-[[(tricyclo-[3.3 .1. 1(3,7)]dec-2-yloxy)carbonyl]amino]propyl]amino]-, [R-(R*,S*)]-) was approximately 10-fold higher than the L-isomer of benzotript. PD 149164 may serve as the lead compound for the future development of more potent and selective gastrin-binding protein inhibitors.

Topics: Adamantane; Animals; Anti-Ulcer Agents; Benzamides; Carrier Proteins; Cells, Cultured; Fatty Acids; Fibroblasts; Gastrins; Humans; Indoles; Mitochondria; Mitochondrial Trifunctional Protein; Multienzyme Complexes; Oxidation-Reduction; Proglumide; Protein Binding; Receptors, Cholecystokinin; Swine

The mitochondrial enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase trifunctional protein (trifunctional protein) plays a major role in mitochondrial fatty acid oxidation. The enzyme complex consists of four molecules of alpha-subunit containing both hydratase and dehydrogenase domains and four molecules of beta-subunit containing the thiolase domain. The primary structure of a gastrin-binding protein (GBP) was highly homologous to that of the alpha-subunit of the trifunctional protein. Here, we report that gastrin inhibits the hydratase, dehydrogenase, and thiolase activities of the trifunctional protein. The gastrin/cholecystokinin receptor antagonist benzotript, which inhibited binding of gastrin to the GBP, also inhibited all three activities of the trifunctional protein. In addition, benzotript inhibits the activities of multifunctional enzymes having similar structures, such as the peroxisomal enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase bifunctional protein and the Pseudomonas fragi fatty acid oxidation enzyme complex. This reagent, however, hardly inhibited various monofunctional enzymes involved in fatty acid oxidation.

Topics: Acetyl-CoA C-Acyltransferase; Animals; Benzamides; Carnitine O-Palmitoyltransferase; Coenzyme A Ligases; Enoyl-CoA Hydratase; Enzyme Inhibitors; Fatty Acid Desaturases; Fibroblasts; Gastrins; Humans; Kinetics; Liver; Mitochondria; Mitochondrial Trifunctional Protein; Multienzyme Complexes; Palmitic Acid; Proglumide; Pseudomonas; Rats

A 78 kDa gastrin-binding protein (GBP) has previously been identified as the target of the anti-proliferative effects of non-selective gastrin/cholecystokinin receptor antagonists on colorectal carcinoma cell lines. The GBP was related in sequence to a family of fatty acid oxidation enzymes possessing enoyl CoA hydratase and 3-hydroxyacyl CoA dehydrogenase activity. This study aims to define the binding site for gastrin and gastrin antagonists in greater detail. The N- and C-terminal halves of the porcine GBP were expressed independently as glutathione S-transferase fusion proteins in E. coli. Affinities of gastrin and gastrin antagonists for the fusion proteins were measured by competition for 125I-[Nle15]-gastrin binding in a covalent cross-linking assay. The N- and C-terminal fusion proteins bound gastrin with affinities of 9.9 +/- 6.1 and 71 +/- 48 microM, respectively (n = 3). These values were 40-fold and 300-fold lower than the affinity of the full-length GBP for gastrin (0.23 +/- 0.15 microM). In contrast, the affinities of the N- and C-terminal halves for the antagonists proglumide (22 +/- 13 and 10 +/- 4 mM, respectively) and benzotript (350 +/- 90 and 400 +/- 160 micro M, respectively) were similar to each other and to the affinities of proglumide and benzotript for the full-length GBP (5.1 +/- 3.6 mM and 200 +/- 120 microM, respectively). It is concluded that proglumide and benzotript bind independently to both the hydratase and dehydrogenase active sites of the GBP, while a single molecule of gastrin may bind simultaneously to both active sites. A model is proposed which is consistent with these data, and which will assist in the development of more potent and selective GBP antagonists.

Topics: Animals; Base Sequence; Benzamides; Binding Sites; Carrier Proteins; DNA Primers; Escherichia coli; Gastrins; In Vitro Techniques; Mitochondrial Trifunctional Protein; Models, Biological; Molecular Structure; Molecular Weight; Multienzyme Complexes; Polymerase Chain Reaction; Proglumide; Protein Binding; Receptors, Cholecystokinin; Recombinant Fusion Proteins; Swine

The role of gastrin in the control of growth of renal G401 cells isolated from a human nephroblastoma (Wilms' tumour) was investigated. G401 cell growth was enhanced in the presence of exogenous gastrin. Addition of anti-gastrin antibodies to serum-free medium significantly inhibited the growth of G401 cells. G401 cells contained the equivalent of 4.3 pg/10(6) cells of gastrin, and serum-free medium collected over 48 hr from G401 cells contained the equivalent of 38 ng/10(6) cells of gastrin, as determined by radioimmunoassay. Growth of G401 cells was inhibited in a concentration-related way by a variety of gastrin/CCK receptor antagonists. Devazepide and proglumide were, respectively, the most and the least potent inhibitors of G401 cell growth (potency order devazepide > L-365,260 = lorglumide > loxiglumide > benzotript > proglumide). These gastrin/CCK receptor antagonists had similar growth-inhibitory activities in human colonic adenocarcinoma HCT-116 cells. Growth of HCT-116 cells was stimulated to a lesser extent, as compared with G401 cells, by exogenous gastrin, and endogenous gastrin was not detectable in HCT-116 cells. The results are consistent with a role for a gastrin-like peptide in the control of growth of a renal cell line. The data suggest that gastrin/CCK receptor antagonists warrant further investigation as therapeutic agents for the control of gastrin-responsive tumours derived from outside, as well as inside, the gastrointestinal tract, including tumours derived from the kidney.

Topics: Benzamides; Benzodiazepinones; Cell Division; Devazepide; Gastrins; Humans; Indoles; Kidney Neoplasms; Meglumine; Phenylurea Compounds; Proglumide; Receptors, Cholecystokinin; Tumor Cells, Cultured; Wilms Tumor

Gastrin has been postulated to be a physiological growth factor, but compelling in vitro evidence of this has been difficult to obtain. In the present study we investigated whether small cell lung carcinoma cell lines could provide a useful model system to study the effects of gastrin on signal transduction and cell proliferation in vitro. We found that the addition of gastrin to small cell lung cancer cells loaded with the fluorescent Ca2+ indicator fura 2-tetraacetoxymethylester causes a rapid and transient increase in the intracellular concentration of Ca2+ ([Ca2+]i) followed by homologous desensitization. The [Ca2+]i response was especially prominent in the small cell lung carcinoma cell line H510. In this cell line, gastrin I, gastrin II, cholecystokinin residues 26-33 (CCK-8), and unsulfated CCK-8 increased [Ca2+]i in a concentration-dependent fashion with half-maximum effects at 7, 2.5, 3, and 5 nM, respectively. The Ca(2+)-mobilizing effects of gastrin and CCK-8 were prevented by proglumide, benzotript, and the specific gastrin/CCKB receptor antagonist L365260. Gastrin stimulated the clonal growth of H510 cells in semisolid (agarose-containing) medium, increasing both the number and the size of the colonies. Gastrin and CCK agonists were equally effective in promoting clonal growth. The broad-spectrum neuropeptide antagonists [D-Arg1,D-Phe5,D-Trp7,9,Leu11] substance P and [Arg6,D-Trp7,9,MePhe8] substance P (6-11) markedly inhibited gastrin-stimulated Ca2+ mobilization and clonal growth. These results show that gastrin acts as a direct growth factor through gastrin/CCKB receptors and demonstrate, for the first time, that these peptides can stimulate the proliferation of cells outside the gastrointestinal tract.

Topics: Benzamides; Calcium; Carcinoma, Small Cell; Cell Division; Gastrins; Humans; Lung Neoplasms; Proglumide; Sincalide; Tumor Cells, Cultured

Polyamines are essential factors of cell growth and differentiation. Modulation of the cellular polyamine content by 2-difluoromethylornithine (DFMO) inhibiting ornithine decarboxylase (ODC), or by hormones inducing ODC, influences cell growth. Gastrin acts trophically on some colonic carcinomas and their growth is inhibited by gastrin receptor blockers. The mechanism of the trophic action of gastrin on colonic carcinomas is not known. In this study the effect of gastrin, gastrin receptor blockers, epidermal growth factor (EGF) and DFMO on growth and ODC activity of four human colon carcinoma cell lines (SW 403, SW 1116, LS 174 T and Lovo) was investigated. Growth and ODC activity of all cell lines were inhibited by DFMO. Growth of the SW 403 cell line was increased by gastrin and inhibited by the gastrin receptor blocker benzotrypte. The other cell lines did not respond to gastrin and the gastrin receptor blocker. In SW 403 cells ODC activity was increased by gastrin, and was also elevated after treatment with the gastrin receptor blocker. These in vitro results were confirmed by studies on tumours that developed from SW 403 cells in nude mice. Combination of benzotrypte and DFMO did not enhance the antiproliferative effect. EGF increased growth of SW 403 cells, but no induction of ODC activity was measured. LS 174 T cells were not stimulated by EGF. Medium replacement was the strongest stimulus of ODC activity in SW 403 cells already inducing ODC after 3 h. During cell culture ODC activity was high after seeding and decreased continuously with increasing cell density. These data suggest that gastrin induces ODC in gastrin-sensitive colonic carcinoma cells. DFMO appears to be a valuable antiproliferative agent in colonic carcinoma cells.

Topics: Animals; Anti-Ulcer Agents; Benzamides; Cell Division; Colonic Neoplasms; Eflornithine; Epidermal Growth Factor; Gastrins; Humans; Mice; Mice, Nude; Neoplasm Transplantation; Ornithine Decarboxylase; Ornithine Decarboxylase Inhibitors; Pentagastrin; Receptors, Cholecystokinin; Tumor Cells, Cultured

We have previously demonstrated trophic effects of gastrin on mouse colon cancer (MC-26) cells, in vivo, and demonstrated the presence of gastrin receptors (GR) on these cells. The cellular and intracellular mechanism by which gastrin expresses trophic effects on colon cancer cells is, however, as yet unknown. For us to start investigating the possible mechanisms involved, it was important that we first develop an in vitro model, in which gastrin expresses its trophic effects directly on the MC-26 cells. The growth-promoting effects of gastrin on the MC-26 cells were examined in various in vitro culture models, in terms of [3H]thymidine incorporation and cell number. A significant trophic effect of gastrin could be demonstrated on quiescent cells in culture, in the absence of serum. The optimal cell-culture conditions for observing trophic effects of gastrin were defined and included a 24-h period of rapid growth of MC-26 cells in serum-supplemented normal growth medium, followed by a 24-h period of culture in serum-free medium containing an optimal dose (1.0 mM) of thymidine, to achieve growth-arrest of the cells. Addition of gastrin (0.5 to 25 nM) to the quiescent, growth-arrested cells resulted in significant dose-dependent increases in both the incorporation of [3H]thymidine uptake by the cells, and a significant increase in cell number. The concentration of GR on the growth-arrested quiescent MC-26 cells in culture was significantly increased compared to the GR concentration on the control, asynchronized cells. The increased presence of GR on the growth-arrested, synchronized MC-26 cells may have allowed us to observe a significant trophic effect of gastrin on the MC-26 cells, in vitro itself. To determine if gastrin was functioning as an autocrine growth factor for MC-26 cells, we examined the effect of gastrin antibodies on the growth of MC-26 cells; no significant effect of the antigastrin IgG on the growth of MC-26 cells was observed.

Topics: Animals; Antibodies; Benzamides; Cell Cycle; Cell Division; Colonic Neoplasms; Gastrins; Growth Substances; Mice; Proglumide; Receptors, Cholecystokinin; Thymidine; Tritium; Tumor Cells, Cultured

The gastrointestinal hormone gastrin has been shown to stimulate the growth of normal colonic mucosa. To examine for a possible role of gastrin in the proliferation of cultured colon tumor cells, we have studied the effects of two gastrin receptor antagonists, proglumide and benzotript, and of antibodies to gastrin. We find that proglumide (50% effective concentration, 2 to 5 mM) and benzotript (50% effective concentration, 0.4 to 0.8 mM) inhibit the monolayer growth of six human colon cancer cell lines. Addition of exogenous gastrin abrogated the growth-inhibitory effect of proglumide. The anchorage-independent growth of colon carcinoma cells was also inhibited by the two gastrin antagonists. Also, a dose-dependent increase in carcinoembryonic antigen secretion was observed upon treatment with proglumide and benzotript in three cell lines examined. Half-maximal inhibition of labeled gastrin binding was observed at concentrations of 0.4 mM benzotript and 8.6 mM proglumide. In addition, antigastrin antiserum added to HCT 116 cells adapted to growth in serum-free medium resulted in a concentration-dependent inhibition of cellular proliferation. These data suggest that gastrin may function as an autocrine growth factor in colon carcinoma.

Topics: Antibodies; Benzamides; Carcinoembryonic Antigen; Cell Division; Cell Line; Colonic Neoplasms; Gastrins; Humans; Proglumide; Receptors, Cholecystokinin

Pancreatico-biliary diversion (PBD) stimulates pancreatic growth in the rat. The present experiment was designed to investigate the mechanism of this phenomenon. The potential roles of endogenous CCK, gastrin, and secretin were studied. Hormone measurements by specific RIA's show that PBD was associated with higher CCK plasma concentrations and, conversely, with lower gastrin circulating levels. Secretin and pancreatic polypeptide were unaffected by PBD. Seven days' subcutaneous administration of proglumide (1000 mg/kg/day), benzotript (100 mg/kg/day), two CCK and gastrin receptor antagonists, and Ranitidine (100 mg/kg/day) resulted in a significant inhibition of PBD-induced pancreatic growth, assessed by measurements of pancreatic weight, DNA, RNA and protein content. These results suggest, therefore, that CCK plays a central role in the development of the pancreatic adaptive response to PBD.

Topics: Adaptation, Physiological; Animals; Benzamides; Biliary Tract; Cholecystokinin; DNA; Gastrins; Glutamine; Male; Organ Size; Pancreas; Pancreatic Polypeptide; Proglumide; Proteins; Ranitidine; Rats; RNA; Secretin

Benzotript (N-p-chlorobenzoyl-L-tryptophan) has been shown to be a receptor-antagonist in vivo and in vitro for peptides from the gastrin family. In the present study, we examine tryptophan, and some of its N- and C-acylated derivatives, as well as some phenylalanine derivatives, to show their ability to inhibit gastrin-induced acid secretion in the rat in vivo and to compete for the binding of [125I]-(Leu-15)-HG-17 to its cellular receptor on rabbit isolated gastric mucosal cells. N- and C- derivatives of tryptophan and phenylalanine were found to inhibit gastrin-induced acid secretion and binding of [125I]-(Leu-15)-HG-17 to its mucosal cell receptors. By either criterion, the relative antagonistic potencies of the compounds tested were: tert-butyloxycarbonyl-L-tryphophan-p-nitrophenyl ester approximately equal to tert-butyloxycarbonyl-L-tryptophan-carbamoylmethyl ester greater than tert-butyloxycarbonyl-L-tryptophyl-L-methionyl-carbamoylmethyl ester approximately equal to tert-butyloxycarbonyl-L-phenylalanine-carbamoylmethyl ester approximately equal to tert-butyloxycarbonyl-L-tryptophyl-L-methionyl-amide greater than tert-butyloxycarbonyl-L-tryptophan greater than tert-butyloxycarbonyl-L-phenylalanine greater than benzyloxycarbonyl-L-tryptophan approximately equal to benzotript, with minor differences between the in vivo and the in vitro experiments. These results demonstrate that both the nature of the amino acid residue and the N- and C-substitutions are important in determining antagonist activity and affinity for gastrin receptors.

Topics: Animals; Benzamides; Esters; Gastric Acid; Gastric Mucosa; Gastrins; Male; Phenylalanine; Rabbits; Rats; Rats, Inbred Strains; Receptors, Cell Surface; Receptors, Cholecystokinin; Structure-Activity Relationship; Tryptophan

Proglumide has been shown to be an in vivo inhibitor of secretagogue-stimulated gastric acid secretion. In the present study, we have examined the ability of proglumide and benzotript, a new tryptophan derivative, to inhibit acid output from isolated gastric fundic parietal cells from rabbit. As measured with the [14C]aminopyrine (AP) accumulation method as an index of acid secretion, the two drugs inhibited basal AP with IC-50 values of 1 X 10(-2) M for proglumide and 1 X 10(-3) M for benzotript. In the case of secretagogue stimulation (1) benzotript slightly affected histamine-induced AP (15% inhibition at 5 X 10(-3) M), proglumide did not; (2) both proglumide and benzotript inhibited in a non-competitive manner acetylcholine-induced AP; (3) these isolated cells were sensitive to gastrin and the dose-response curve for the stimulant was biphasic (maximum for 1 X 10(-9) M), suggesting a desensitization mechanism. Proglumide and benzotript competitively inhibited both [125I]gastrin binding to its receptor sites and gastrin-induced AP, suggesting they are members of a class of gastrin-receptor antagonists. But, this suggestion cannot exclude other post-receptorial mechanisms involved in the acid output from parietal cells.

Topics: Aminopyrine; Animals; Benzamides; Gastric Juice; Gastrins; Glutamine; In Vitro Techniques; Proglumide; Rabbits; Receptors, Cell Surface; Receptors, Cholecystokinin

A method for studying the interaction of 125I-human gastrin with a rat gastric mucosal membrane fraction is described, and the saturability, high affinity and reversibility of the preparations, as well as the correlation of pharmacological responses to antigastrin-drugs with the observed binding are discussed. Proglumide and other antigastrin drugs inhibit gastrin binding in a dose-dependent way, and their activities in such a system are well correlated with the "in vivo" antisecretory activities of these drugs.

Topics: Animals; Anti-Ulcer Agents; Benzamides; Binding, Competitive; Cell Membrane; Dose-Response Relationship, Drug; Female; Gastric Mucosa; Gastrins; Humans; Proglumide; Pyridines; Rats; Receptors, Cell Surface; Thioacetamide