Bismuth ions inhibit the biological activity of non-amidated gastrins in vivo.

The peptide hormone gastrin binds two ferric ions with high affinity, and iron binding is essential for the biological activity of non-amidated gastrins in vitro and in vivo. Bi3+ ions also bind to glycine-extended gastrin17 (Ggly), but inhibit Ggly-induced cell proliferation and migration in gastrointestinal cell lines in vitro. The aims of the present study were firstly, to establish the mechanism by which Bi3+ ions inhibit the binding of Fe3+ ions to Ggly, and secondly, to test the effect of Bi3+ ions on the activity of non-amidated gastrins in vivo. The interaction between Bi3+ ions, Fe3+ ions and Ggly was investigated by ultraviolet spectroscopy. The effect of Bi3+ ions on colorectal mucosal proliferation was measured in three animal models. In vitro in the presence of Bi3+ ions the affinity of Fe3+ ions for Ggly was substantially reduced; the data was better fitted by a mixed, rather than a competitive, inhibition model. In rats treated with Ggly alone proliferation in the rectal mucosa was increased by 318%, but was reduced to control values (p < 0.001) in animals receiving oral bismuth plus Ggly. Proliferation in the colonic mucosa of mice overexpressing Ggly or progastrin was significantly greater than in wild-type mice, but was no greater than control (p < 0.01) in animals receiving oral bismuth. Thus a reduction in the binding of Fe3+ ions to Ggly and progastrin in the presence of Bi3+ ions is a likely explanation for the ability of oral bismuth to block the biological activity of non-amidated gastrins in vivo.

Stimulation of proliferation in the colorectal mucosa by gastrin precursors is blocked by desferrioxamine.

Precursors of the peptide hormone gastrin stimulate proliferation in the colorectal mucosa and promote the development of colorectal carcinoma. Gastrins bind two ferric ions selectively and with high affinity, and the biological activity of glycine-extended gastrin (Ggly) in vitro is dependent on the presence of ferric ions. The aim of the present study was to determine whether or not iron is required for biological activity of progastrin and Ggly in vivo. Rats that had undergone a colostomy were infused with Ggly, and proliferation was measured in the defunctioned rectal mucosa. Proliferation was also measured in the colonic mucosa of hGAS and MTI-Ggly mice, which, by definition, overexpress progastrin and Ggly, respectively. The requirement for iron was assessed by thrice-weekly injection of the chelating agent desferrioxamine (DFO). The proliferation index in the defunctioned rectal mucosa was significantly increased in the Ggly-infused rats, and the increase was significantly reduced after treatment with DFO. Treatment with DFO significantly reduced the crypt height and proliferation index in the colonic mucosa of hGAS and MTI-Ggly mice but had no effect on the same variables in wild-type mice. These observations are consistent with the hypothesis that the biological activity of progastrin and Ggly in vivo is dependent on the presence of ferric ions and further suggest that chelating agents may block the stimulatory effects of gastrin precursors in the development of colorectal carcinoma.

Production, secretion, and biological activity of the C-terminal flanking peptide of human progastrin.

BACKGROUND & AIMS: Processing of progastrin, the 80-amino acid precursor of the hormone gastrin, generates a variety of peptides with distinct distributions and biological activities. However, little is known regarding the expression, secretion, and biological activity of the 6-amino acid C-terminal flanking peptide (CTFP) of progastrin. The objectives were to determine the concentration of CTFP in normal subjects and patients with gastrointestinal diseases and to investigate the biological activity of CTFP. METHODS: CTFP, gastrin-amide (Gamide), glycine-extended gastrin (Ggly), and progastrin were measured using region-specific radioimmunoassay (RIA) in antral extracts and resected colorectal cancers (CRC) and in plasma from normal subjects (fasting and meal stimulated) and from patients with CRC, multiple endocrine neoplasia type 1 (MEN-1), or pernicious anemia. The effect of CTFP on proliferation, migration, and activation of the mitogen-activated protein kinase (MAPK) pathway in several types of gastrointestinal cell lines was determined. RESULTS: CTFP is by far the predominant progastrin-derived peptide found in the antrum (4-fold higher than Gamide), resected CRC, and circulation (60-fold higher than Gamide) and is released after meal stimulation. The hypergastrinemic patients (MEN-1, pernicious anemia) had elevated plasma Gamide but unaltered CTFP demonstrating differential secretion of these 2 progastrin-derived peptides. Finally, CTFP stimulated proliferation and migration and activated MAPK of cells in culture. CONCLUSIONS: The high and regulated expression of CTFP in healthy and diseased subjects combined with the evidence for biological activity of CTFP demonstrates that CTFP is not an inactive metabolite of progastrin processing but is a bioactive peptide with potential roles in the normal and diseased gastrointestinal tract.

Synthesis, expression and biological activity of the prohormone for gastrin releasing peptide (ProGRP).

Gastrin-releasing peptide (GRP) has a widespread distribution and multiple stimulating effects on endocrine and exocrine secretions and metabolism. The prohormone for GRP (ProGRP, 125 amino acids) is processed to the amidated, biologically active end products GRP(1-27) and GRP(18-27). Amidated forms of GRP are putative autocrine or paracrine growth factors in a number of cancers including colorectal cancer. However, the potential role and biological activity of proGRP has not been investigated. Using a newly developed antisera directed to the N terminus of human proGRP, proGRP immunoreactivity was detected in all of the endometrial, prostate, and colon cancer cell lines tested and in nine of 10 resected colorectal carcinomas. However, no amidated forms were detected, suggesting an attenuation of processing in tumors. Recombinant proGRP was expressed as a His-tag fusion protein and purified by metal affinity chromatography and HPLC. ProGRP stimulated proliferation of a colon cancer cell line and activated MAPK, but unlike GRP(18-27)amide had no effect on inositol phosphate production. ProGRP did not compete with iodinated bombesin in binding assays on Balb-3T3 cells transfected with the known GRP receptors, GRP-R or BRS-3. We conclude that proGRP is present in a number of cancer cell lines and in resected colorectal tumors and is biologically active. Our results suggest that antagonists to GRP precursors rather than the amidated end products should be developed as a treatment for colorectal and other cancers that express proGRP-derived peptides.

PPARalpha agonists stimulate progastrin production in human colorectal carcinoma cells.

The three subtypes of peroxisome proliferator activated-receptors (PPARalpha, delta and gamma) control the storage and metabolism of fatty acids. Treatment of rats with the PPARalpha ligand ciprofibrate increases serum gastrin concentrations, and several lines of evidence suggest that non-amidated gastrins act as growth factors for the colonic mucosa. The aim of the present study was to investigate the expression of PPARs and the effect of PPAR ligands on gastrin production and cell proliferation in human colorectal carcinoma (CRC) cell lines. mRNAs for all three PPAR subtypes were detected by PCR in all CRC cell lines tested. The concentrations of progastrin, but not of glycine-extended or amidated gastrin, measured by radioimmunoassay in LIM 1899 conditioned media and cell extracts were significantly increased by treatment with the PPARalpha ligand clofibrate. Similar increases in progastrin were seen following treatment with the PPARalpha ligands ciprofibrate and fenofibrate, but not with bezafibrate, gemfibrozil or Wy 14643. The PPARgamma agonist rosiglitazone had no significant effect on progastrin production. The PPARalpha ligand clofibrate also stimulated proliferation of the LIM 1899 cell line. We conclude that some PPARalpha ligands increase progastrin production by the human CRC cell line LIM 1899, and that clofibrate increases proliferation of LIM 1899 cells. These studies have revealed a relationship between PPARs and gastrin, two regulatory molecules implicated in the pathogenesis of CRC.