gastrins and Colonic-Neoplasms

Gastrin is a peptide hormone that regulates both acid secretion and growth of the gastric oxyntic mucosa. Recent studies suggest that gastrin, in both its amidated, and less processed forms (glycine-extended gastrin and progastrin) may also exert biological activity in other organs in the gastrointestinal tract. This article will review the studies performed to date addressing the physiological role of gastrin outside of the gastric mucosa, with particular emphasis on the information gleaned from gastrin-deficient mice. Most of these studies address the potential role for the less processed forms of gastrin in regulating the proliferation of the colonic mucosa and colon cancers. There is also some data to support a potential role for gastrin in the regulation of the pancreas and the kidney, although the effects of gastrin deficiency on the function of these organs in mice have not yet been rigorously studied.

Topics: Animals; Brain; Colonic Neoplasms; Gastrins; Kidney; Mice; Mice, Knockout; Pancreas; Receptor, Cholecystokinin B; Receptors, Cholecystokinin

Gastrointestinal peptides including mammalian bombesin-like peptides, cholecystokinin (CCK), gastrin, and neurotensin stimulate DNA synthesis and cell proliferation in cultured cells and are implicated as growth factors in a number of fundamental processes including development, inflammation, tissue regeneration, and neoplastic transformation. These agonists bind to G protein-coupled receptors (GPCRs) that promote Galpha q-mediated activation of beta isoforms of phospholipase C to produce two second messengers: Inositol (1,4,5) trisphosphate {Ins (1, 4, 5) P3} that mobilises Ca2+ from internal stores, and diacylglycerol that activates the classic and new isoforms of the protein kinase C (PKC) family. PKCs play a critical part in transducing bombesin/gastrin releasing peptide (GRP) receptor signals into activation of protein kinase cascades. Protein kinase D (PKD), a serine/threonine protein kinase with distinct structural and enzymological properties, is activated by phosphorylation in living cells through a new PKC-dependent signal transduction pathway. GPCR agonists including bombesin/GRP induce a rapid and striking activation of PKD by PKC. These results indicate that PKD functions downstream from PKCs and identify a new phosphorylation cascade that is activated by gastrointestinal peptide agonists. The bombesin/GRP GPCR also promotes rapid Rho-dependent assembly of focal adhesions, formation of actin stress fibres and tyrosine phosphorylation of multiple cellular proteins. We identified p125 focal adhesion kinase (FAK), p130 Crk-associated substrate (CAS) and paxillin as prominent targets of gastrointestinal peptide-stimulated tyrosine phosphorylation and developed a model that envisages a G12/Rho-dependent pathway connecting GPCR activation to the tyrosine phosphorylation of these focal adhesion proteins. Separate pathways mediate gastrointestinal peptide stimulation of additional tyrosine kinase pathways including transactivation of Src and epidermal growth factor receptor (EGFR). Tyrosine phosphorylation has a critical role in gastrointestinal peptide-induced cellular migration and cooperates with Gq-stimulated events to promote mitogenesis. The growth-promoting effects of neuropeptides and the elucidation of the signalling pathways that mediate their effects assume an added importance because these agonists and their receptors are increasingly implicated in sustaining the proliferation of clinically aggressive solid tumours including those from lu

Topics: Animals; Bombesin; Carcinoma, Small Cell; Cholecystokinin; Colonic Neoplasms; ErbB Receptors; Gastrins; Humans; Lung Neoplasms; Mice; Neuropeptides; Neurotensin; Pancreatic Neoplasms; Phosphorylation; Protein Kinase C; Receptors, Leukotriene B4; Receptors, Purinergic P2; rho GTP-Binding Proteins; Signal Transduction; Swiss 3T3 Cells

G17DT or Gastrimmune, as it was formally known, is an antigastrin 17 immunogen producing neutralising high affinity antibodies directed against gastrin-17 (G17). Preclinical studies, initiated to identify biological functionality of G17DT-induced antibodies, confirmed that the antibodies both reduced G17 stimulated gastric acid secretion and inhibited gastrin from interacting with the CCK-2 receptor. Therapeutic efficacy of both passive and active immunisation with G17DT has been established in a number of tumour systems including both primary and metastatic disease. Furthermore, additive effects with 5-fluorouracil (5-FU)/leucovorin have been confirmed in both colon and gastric tumour models. Phase I/II studies in advanced gastrointestinal (GI) malignancies have shown no systemic or autoimmune reactions to active immunisation with G17DT. Use of an optimised dose has yielded a high proportion of responders (> 80%), with minimal side effects and antibody titres measurable within 2-4 weeks. Taken together these results suggest that the G17DT immunogen is a promising agent for the treatment of GI cancer and Phase III trials, currently underway, will definitively evaluate this early promise.

Topics: Adenocarcinoma; Animals; Antibodies; Antigens; Cancer Vaccines; Clinical Trials as Topic; Colonic Neoplasms; Diphtheria Toxoid; Gastrins; Gastrointestinal Neoplasms; Humans; Immunotherapy; Multicenter Studies as Topic; Neoplasm Metastasis; Stomach Neoplasms

Topics: Animals; Cell Division; Colonic Neoplasms; Gastrins; Gastritis; Gastrointestinal Diseases; Humans; Receptors, Cholecystokinin

The safety of proton-pump inhibitors (PPIs) for long-term use is reviewed. PPIs are being used with increasing frequency to inhibit secretion of gastric acid in order to treat acid-related disorders such as gastroesophageal reflux disease and peptic ulcer disease. Some patients may require long-term acid suppressive treatment to control the symptoms of their disease, which raises questions about the long-term safety of PPIs. A thorough literature search was conducted, and the clinical consequences of sustained hypergastrinemia induced by all antisecretory therapy, the consequences of atrophic gastritis in patients infected with Helicobacter pylori, the effects of hypochlorhydria on bacterial overgrowth and nutrient absorption, and possible interactions of PPIs with other drugs were identified as areas of concern with long-term use of PPIs. Short- and long-term studies showed that PPIs have a wide safety margin and a favorable adverse-event profile with few drug interactions. Available data support the short- and long-term safety of PPIs.

Topics: Anti-Ulcer Agents; Colonic Neoplasms; Drug Interactions; Enterochromaffin Cells; Gastric Acid; Gastrins; Gastritis, Atrophic; Humans; Hyperplasia; Proton Pump Inhibitors; Stomach Neoplasms

There is now clear-cut evidence that polypeptide growth factors control the proliferation of the normal gastrointestinal mucosa. Epidermal growth factor (EGF) stimulates normal growth throughout the gastrointestinal tract, and accelerates the healing of ulcerated epithelium. While the effects of gastrin were at first thought to be similarly widespread, the gastrin target now appears to be restricted to the enterochromaffin-like cells in the stomach. Isolated reports suggest that several other hormones, including fibroblast growth factor and the insulin-like growth factors, have similar proliferative effects. In contrast, indirect evidence suggests that somatostatin and transforming growth factor-beta inhibit the growth of the gastrointestinal mucosa. The same growth factors profoundly affect the growth of some gastrointestinal carcinomas. Prolonged hypergastrinaemia increases the risk of development of gastric endocrine tumours, but has no effect on the incidence of gastric adenocarcinoma. Gastrin also stimulates the in vivo growth of 50% of gastric and colorectal carcinoma xenografts, but has no consistent effect on the growth of carcinoma cell lines in vitro. EGF, on the other hand, significantly stimulates proliferation of many gastrointestinal cell lines in culture. Interest has recently focused on autocrine stimulation of gastrointestinal carcinoma growth. Elevated levels of EGF receptor, and of EGF or related mRNAs, have been demonstrated in gastric carcinomas, and the growth of some gastrointestinal cell lines is inhibited by antibodies against EGF, and by antisense oligonucleotides based on EGF mRNA. Similarly gastrin/cholecystokinin antagonists inhibit the growth of several colon carcinoma cell lines, although the spectrum of antagonist potencies suggests that classical gastrin and cholecystokinin receptors are not necessarily involved. Continued research on antagonists may therefore lead to novel therapies for gastrointestinal cancers.

Topics: Adult; Animals; Colonic Neoplasms; Digestive System Physiological Phenomena; Epidermal Growth Factor; ErbB Receptors; Gastrins; Gastrointestinal Hormones; Gastrointestinal Neoplasms; Glucagon-Like Peptides; Humans; Peptide YY; Peptides; Somatostatin; Transforming Growth Factor alpha; Transforming Growth Factor beta; Tumor Cells, Cultured

Controversy exists about the relationship between hypergastrinemia and colon cancer. Conflicting lines of evidence may be interpreted to support a variety of views regarding the link between the two. Although some experimental and clinical data suggest a strong correlation, other studies refute this. It is likely that the actual situation lies between these two viewpoints; the complex nature of the relationship between carcinogenesis and putative gut hormones makes a definitive answer unlikely. Nevertheless, a critical reading of the recent literature suggests that hypergastrinemia does not play a direct role in colorectal carcinogenesis. Certain subgroups of patients with elevated serum gastrin levels and tumors that possess gastrin receptors may have accelerated tumor growth. Further study of this issue is warranted to elucidate the role of the gastrointestinal hormonal milieu in colorectal neoplasia.

Topics: Colonic Neoplasms; Gastrins; Humans

In the high altitude environment the oxygen and air density are decreased, the temperature and humidity are low, there es an increase in radioactivity. These environmental factors influence on the human body; it has been known for many years that people born and living at high altitude have different morphological and physiological characteristic than those at low altitude. The digestive mechanism for adaptation or acclimation to high altitude has interested physiologist and clinicians for many years. The objective of this article is to present a brief overview of the digestive physiology and pathology in the high altitude.

Topics: Altitude; Bolivia; Cholelithiasis; Colonic Neoplasms; Diverticulum, Colon; Dyspepsia; Endopeptidases; Gallbladder Neoplasms; Gastric Mucosa; Gastrins; Gastrointestinal Diseases; Gastrointestinal Hemorrhage; Gastrointestinal Transit; Humans; Intestinal Obstruction; Intestines; Peptic Ulcer; Peru

During the early experience with omeprazole, it was recommended that plasma gastrin levels be monitored to identify patients with 4-5-fold increases above baseline. Such patients were thought to be at an increased risk of developing gastric carcinoid tumours. Studies have established that plasma gastrin levels usually rise 2-4-fold during omeprazole therapy, there being considerable inter- and intra-individual variation. Approximately 3.3% of patients have plasma gastrin levels above 400 pg/ml when treated continuously for 1 year. In clinical practice, it is not cost-effective to screen all patients to detect such a small percentage, particularly given the paucity of realistic treatment options in such patients, and the growing evidence that hypergastrinaemia during omeprazole treatment is of little, if any, clinical significance.

Topics: Colonic Neoplasms; Drug Monitoring; Fasting; Gastric Mucosa; Gastrins; Humans; Omeprazole; Ranitidine; Recurrence; Stomach Neoplasms; Vagotomy, Proximal Gastric

Increased basal serum gastrin level has been described in patients presenting with colorectal cancer. The aim of this work was to study the evolution of serum gastrin levels after cancer treatment. We measured basal serum gastrin levels before and 1 to 2 months after treatment in 15 patients (7 men, 8 women; mean age: 61.6 years). There were 3 malignant polyps, 4 Dukes A, 3 Dukes B, 4 Dukes C and 1 Dukes D colonic cancers. Treatment included 3 endoscopic polypectomies, 2 laser photodestructions, and 10 surgical resections, Mean basal gastrin level after treatment (49.07 +/- 12.65 mIU/l) was significantly lower (P less than 0.002) than before treatment (104.47 +/- 26.98 mIU/l). In the 2 patients treated by laser therapy, recurrences were associated with reincreasing serum gastrin levels. These results suggest an "autocrine" secretion of gastrin.

Topics: Adenocarcinoma; Adult; Aged; Aged, 80 and over; Colonic Neoplasms; Endoscopy, Gastrointestinal; Female; Gastrins; Humans; Laser Therapy; Male; Middle Aged; Neoplasm Recurrence, Local; Postoperative Care; Preoperative Care; Rectal Neoplasms; Time Factors

Achlorhydria has been discussed as a possibly dangerous consequence of therapeutic inhibition of gastric acid secretion since the introduction of H2-receptor antagonists. The risk of long-term hypergastrinaemia has only been considered for about 5 years. The reason for this was the demonstration that gastric carcinoids (ECLomas) observed after life-long treatment of rats with the proton pump inhibitor omeprazole could also be produced in rats by other methods leading to long-lasting profound hypergastrinaemia. Such methods were the 80% resection of the oxyntic mucosa or feeding of ranitidine (2000 mg/day) for 2 years. The endocrine tumours corresponded to the gastric carcinoids found in patients with long-lasting hypergastrinaemia due to pernicious anaemia or with a gastrinoma as part of the MEN I syndrome. Neither in animals nor in man could other endocrine tumours or adenocarcinomas of the gastrointestinal tract be related to hypergastrinaemia. Epidemiologic data do not support gastrin dependence of adenocarcinoma of the stomach or the colon. Experimental findings of gastrin effects on tumour growth in vivo and in vitro have been contradictory and may be explained by the presence of gastrin receptors on tumour cells and the role of gastrin as an autocrine growth factor in some of these tumours. Since acid blockade by proton pump inhibitors or H2-receptor blockers dose-dependently increase serum gastrin levels, patients with ranitidine-resistant peptic ulceration receiving long-term treatment with high-dose omeprazole have been followed up with serial gastric biopsy specimens for up to 5 years. Complete healing, moderate hypergastrinaemia, and a slight hyperplasia but no dysplasia of the ECL cells in the oxyntic mucosa have been observed, which seemed to be correlated to chronic gastritis progressing over the years. Despite these negative findings excessive hypergastrinaemia by overdosage of potent drugs for inhibition of gastric secretion should be avoided and monitoring of plasma gastrin levels is recommended in case of long-term treatment.

Topics: Achlorhydria; Adenocarcinoma; Colonic Neoplasms; Enterochromaffin Cells; Gastric Acid; Gastric Mucosa; Gastrins; Gastritis; Histamine H2 Antagonists; Humans; Stomach Neoplasms

Prolonged intestinal paralysis can be a problem after gastrointestinal surgery. Several systematic reviews and meta-analyses have suggested the efficacy of gum chewing for the prevention of postoperative ileus.. The purpose of this study was to examine the efficacy of gum chewing for the recovery of bowel function after surgery for left-sided colorectal cancer and to determine the physiological mechanism underlying the effect of gum chewing on bowel function.. This was a single-center, placebo-controlled, parallel-group, prospective randomized trial.. The study was conducted at a general hospital in Japan.. Forty-eight patients with left-sided colorectal cancer were included.. The patients were randomly assigned to a gum group (N = 25) and a control group (N = 23). Four patients in the gum group and 1 in the control group were subsequently excluded because of difficulties in continuing the trial, resulting in the analysis of 21 and 22 patients in the respective groups. Patients in the gum group chewed commercial gum 3 times a day for ≥5 minutes each time from postoperative day 1 to the first day of food intake.. The time to first flatus and first bowel movement after the operation were recorded, and the colonic transit time was measured. Gut hormones (gastrin, des-acyl ghrelin, motilin, and serotonin) were measured preoperatively, perioperatively, and on postoperative days 1, 3, 5, 7, and 10.. Gum chewing did not significantly shorten the time to the first flatus (53 ± 2 vs. 49 ± 26 hours; p = 0.481; gum vs. control group), time to first bowel movement (94 ± 44 vs. 109 ± 34 hours; p = 0.234), or the colonic transit time (88 ± 28 vs. 88 ± 21 hours; p = 0.968). However, gum chewing significantly increased the serum levels of des-acyl ghrelin and gastrin.. The main limitation was a greater rate of complications than anticipated, which limited the significance of the findings.. Gum chewing changed the serum levels of des-acyl ghrelin and gastrin, but we were unable to demonstrate an effect on the recovery of bowel function.

Topics: Aged; Aged, 80 and over; Chewing Gum; Colectomy; Colon, Descending; Colonic Neoplasms; Defecation; Female; Flatulence; Gastrins; Gastrointestinal Motility; Ghrelin; Humans; Ileus; Japan; Length of Stay; Male; Middle Aged; Motilin; Postoperative Care; Postoperative Complications; Serotonin; Sigmoid Neoplasms; Treatment Outcome

Overexpression of human progastrin increases colonic mucosal proliferation and colorectal cancer progression in mice. The G-protein coupled receptor 56 (GPR56) is known to regulate cell adhesion, migration, proliferation and stem cell biology, but its expression in the gut has not been studied. We hypothesized that the promotion of colorectal cancer by progastrin may be mediated in part through GPR56. Here, we found that GPR56 expresses in rare colonic crypt cells that lineage trace colonic glands consistent with GPR56 marking long-lived colonic stem-progenitor cells. GPR56 was upregulated in transgenic mice overexpressing human progastrin. While recombinant human progastrin promoted the growth and survival of wild-type colonic organoids in vitro, colonic organoids cultured from GPR56-/- mice were resistant to progastrin. We found that progastrin directly bound to, and increased the proliferation of, GPR56-expressing colon cancer cells in vitro, and proliferation was increased in cells that expressed both GPR56 and the cholecystokinin-2 receptor (CCK2R). In vivo, deletion of GPR56 in the mouse germline abrogated progastrin-dependent colonic mucosal proliferation and increased apoptosis. Loss of GPR56 also inhibited progastrin-dependent colonic crypt fission and colorectal carcinogenesis in the azoxymethane (AOM) mouse model of colorectal cancer. Overall, we found that progastrin binds to GPR56 expressing colonic stem cells, which in turn promotes their expansion, and that this GPR56-dependent pathway is an important driver and potential new target in colorectal carcinogenesis.

Topics: Animals; Apoptosis; Carcinogenesis; Cell Line, Tumor; Cell Proliferation; Colon; Colonic Neoplasms; Gastrins; Humans; Intestinal Mucosa; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Protein Binding; Protein Precursors; Receptor, Cholecystokinin B; Receptors, G-Protein-Coupled; Stem Cells

Subpopulations of cancer stem-like cells (CSC) are thought to drive tumor progression and posttreatment recurrence in multiple solid tumors. However, the mechanisms that maintain stable proportions of self-renewing CSC within heterogeneous tumors under homeostatic conditions remain poorly understood. Progastrin is a secreted peptide that exhibits tumor-forming potential in colorectal cancer, where it regulates pathways known to modulate colon CSC behaviors. In this study, we investigated the role of progastrin in regulating CSC phenotype in advanced colorectal cancer. Progastrin expression and secretion were highly enriched in colon CSC isolated from human colorectal cancer cell lines and colon tumor biopsies. Progastrin expression promoted CSC self-renewal and survival, whereas its depletion by RNA interference-mediated or antibody-mediated strategies altered the homeostatic proportions of CSC cells within heterogeneous colorectal cancer tumors. Progastrin downregulation also decreased the frequency of ALDH(high) cells, impairing their tumor-initiating potential, and inhibited the high glycolytic activity of ALDH(high) CSC to limit their self-renewal capability. Taken together, our results show how colorectal CSC maintain their tumor-initiating and self-renewal capabilities by secreting progastrin, thereby contributing to the tumor microenvironment to support malignancy. Cancer Res; 76(12); 3618-28. ©2016 AACR.

Topics: Aldehyde Dehydrogenase; Animals; Cell Line, Tumor; Cell Survival; Colonic Neoplasms; Gastrins; Humans; Mice; Neoplastic Stem Cells; Protein Precursors; Tumor Microenvironment

The guanine nucleotide exchange factor Rgnef (also known as ArhGEF28 or p190RhoGEF) promotes colon carcinoma cell motility and tumor progression via interaction with focal adhesion kinase (FAK). Mechanisms of Rgnef activation downstream of integrin or G protein-coupled receptors remain undefined. In the absence of a recognized G protein signaling homology domain in Rgnef, no proximal linkage to G proteins was known. Utilizing multiple methods, we have identified Rgnef as a new effector for Gα13 downstream of gastrin and the type 2 cholecystokinin receptor. In DLD-1 colon carcinoma cells depleted of Gα13, gastrin-induced FAK Tyr(P)-397 and paxillin Tyr(P)-31 phosphorylation were reduced. RhoA GTP binding and promoter activity were increased by Rgnef in combination with active Gα13. Rgnef co-immunoprecipitated with activated Gα13Q226L but not Gα12Q229L. The Rgnef C-terminal (CT, 1279-1582) region was sufficient for co-immunoprecipitation, and Rgnef-CT exogenous expression prevented Gα13-stimulated SRE activity. A domain at the C terminus of the protein close to the FAK binding domain is necessary to bind to Gα13. Point mutations of Rgnef-CT residues disrupt association with active Gα13 but not Gαq. These results show that Rgnef functions as an effector of Gα13 signaling and that this linkage may mediate FAK activation in DLD-1 colon carcinoma cells.

Topics: Cell Line, Tumor; Colonic Neoplasms; Focal Adhesion Protein-Tyrosine Kinases; Gastrins; GTP-Binding Protein alpha Subunits, G12-G13; Guanine Nucleotide Exchange Factors; HEK293 Cells; Humans; Paxillin; Phosphorylation; Receptor, Cholecystokinin B; Rho Guanine Nucleotide Exchange Factors; Tyrosine

Over-expression of growth factors can contribute to the development and progression of cancer, and gastrins in particular have been implicated in accelerating the development of gastrointestinal cancers. Previously our group showed that hypoxia, cobalt chloride (a hypoxia mimetic) and zinc chloride could activate the expression of the gastrin gene in vitro. To characterise activation of the gastrin promoter by zinc ions further in vivo, TALEN technology was used to engineer a luciferase reporter construct into the endogenous human gastrin gene promoter in SW480 colon cancer cells. Gastrin promoter activity in the resultant Gast(luc) SW480 colon cancer cells was then measured by bioluminescence in cell culture and in tumour xenografts in SCID mice. Activation of intracellular signalling pathways was assessed by Western blotting. Activation of the gastrin promoter by zinc ions was concentration dependent in vitro and in vivo. Zinc ions significantly stimulated phosphorylation of ERK1/2 (MAPK pathway) but not of Akt (PI3K pathway). We conclude that the endogenous gastrin promoter is responsive to zinc ions, likely via activation of the MAPK pathway.

Topics: Animals; Cell Line, Tumor; Colonic Neoplasms; Gastrins; Humans; Mice; Mice, SCID; Phosphorylation; Promoter Regions, Genetic; Signal Transduction; Xenograft Model Antitumor Assays; Zinc

Recent studies have suggested that Helicobacter pylori (H. pylori) constitutes a risk for the development of colonic neoplasia. Hypergastrinemia can be induced by H. pylori infection, and gastrin can act as putative promoter of colorectal carcinogenesis. Aim of our study was to assess whether H. pylori infection and/or increased serum gastrin levels are associated with the occurrence of colonic neoplasms. For this, we reviewed prospectively collected data of 377 patients with a minimum age of 50 years who underwent colonoscopy. H. pylori and CagA status were determined by serology. Serum gastrin levels were measured in fasting state by commercially available assay. In H. pylori infected patients (n = 138; 36.6%), the overall prevalence of colonic neoplasms was more frequent compared to H. pylori negative patients (n = 239; 63.4%) (OR = 2.73, 95% CI: 1.76-4.24). H. pylori infection occurred more frequently in patients with hyperplastic polyps (OR = 2.66, 95% CI: 1.23-5.74) and adenomas presenting with low grade intraepithelial neoplasia (IEN) (OR = 1.85, 95% CI: 1.14-2.99). Attributable risk for adenomas with high grade IEN and colorectal adenocarcinoma (n = 14) was not assessed due to the low number of cases. The expression of CagA was also associated with an increased risk for colonic neoplasms (OR = 2.25, 95% CI: 1.29-3.94). Hypergastrinemia did not increase the risk for any colonic neoplasms and there was no difference in basal serum gastrin levels between H. pylori positive and negative patients. In conclusion, H. pylori infection, including CagA expression is associated with an increased risk for the development of colonic neoplasm.

Topics: Aged; Antibodies, Bacterial; Antigens, Bacterial; Bacterial Proteins; Colon; Colonic Neoplasms; Colonoscopy; Female; Gastrins; Helicobacter Infections; Helicobacter pylori; Humans; Inflammation; Male; Polyps; Prospective Studies; Risk Factors

Though long-term administration of proton pump inhibitor (PPI) imposed the risk of gastrointestinal track tumorigenesis by accompanied hypergastrinemia, no overt increases of colon cancer risk were witnessed after a long-term cohort study. Our recent investigation revealed that PPI prevented colitis-associated carcinogenesis through anti-inflammatory, anti-oxidative, and anti-mutagenic mechanisms in spite of hypergastrinemia. Therefore, we hypothesized that PPI might either antagonize the trophic action of gastrin on gastrointestinal tumorigenesis or synergize to exert augmented anti-tumorigenic actions. We challenged APCMin/+ mice with gastrin, PPI, PPI and gastrin together for 10 weeks and counted intestinal polyposis accompanied with molecular changes. Gastrin significantly increased intestinal polyposis, but combination of PPI and gastrin markedly attenuated intestinal polyposis compared to gastrin-promoted APCMin/+ mice (P<.001), in which significant β-catenin phosphorylation and inhibition of β-catenin nuclear translocation were observed with PPI alone or combination of PPI and gastrin, whereas gastrin treatment significantly increased β-catenin nuclear translocation. Significant footprints of apoptosis, G0/G1 accumulation, inactivation of p38 and extracellular signal-regulated kinase, decreased expressions of CD31, and inhibition of tumor necrosis factor-α and cyclooxygenase-2 were noted in the combination group. In vitro investigations were similar to in vivo findings as shown that PPI treatment inhibited the binding of gastrin to its receptor, inactivated β-catenin-associated signaling including Tcf/Lef and glycogen synthase kinase β, and paradoxically inhibited β-catenin-associated proliferative activities. Our investigations explain why colon cancer risk has not increased despite long-term use of PPIs and provide a rationale for using PPI to achieve anti-tumorigenesis beyond acid suppression.

Topics: 2-Pyridinylmethylsulfinylbenzimidazoles; Active Transport, Cell Nucleus; Animals; Antineoplastic Agents; Apoptosis; beta Catenin; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Colonic Neoplasms; Cyclooxygenase 2; Gastrins; Gene Expression Regulation, Neoplastic; Inflammation; Intestinal Polyposis; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Neoplasm Transplantation; Neoplasms, Experimental; Pantoprazole; Platelet Endothelial Cell Adhesion Molecule-1; Proton Pump Inhibitors; Signal Transduction; Tumor Necrosis Factor-alpha

Macrophage infiltration is a negative prognostic factor for most cancers but gastrointestinal tumors seem to be an exception. The effect of macrophages on cancer progression depends on their phenotype, which may vary between M1 (pro-inflammatory, defensive) to M2 (tolerogenic, pro-tumoral). Gastrointestinal cancers often become an ectopic source of gastrins and macrophages present receptors for these peptides. The aim of the present study is to analyze whether gastrins can affect the pattern of macrophage infiltration in colorectal tumors. We have evaluated the relationship between gastrin expression and the pattern of macrophage infiltration in samples from colorectal cancer and the influence of these peptides on the phenotype of macrophages differentiated from human peripheral monocytes in vitro. The total number of macrophages (CD68+ cells) was similar in tumoral and normal surrounding tissue, but the number of M2 macrophages (CD206+ cells) was significantly higher in the tumor. However, the number of these tumor-associated M2 macrophages correlated negatively with the immunoreactivity for gastrin peptides in tumor epithelial cells. Macrophages differentiated from human peripheral monocytes in the presence of progastrin showed lower levels of M2-markers (CD206, IL10) with normal amounts of M1-markers (CD86, IL12). Progastrin induced similar effects in mature macrophages treated with IL4 to obtain a M2-phenotype or with LPS plus IFNγ to generate M1-macrophages. Macrophages differentiated in the presence of progastrin presented a reduced expression of Wnt ligands and decreased the number and increased cell death of co-cultured colorectal cancer epithelial cells. Our results suggest that progastrin inhibits the acquisition of a M2-phenotype in human macrophages. This effect exerted on tumor associated macrophages may modulate cancer progression and should be taken into account when analyzing the therapeutic value of gastrin immunoneutralization.

Topics: Aged; Aged, 80 and over; Cell Count; Cell Line, Tumor; Colonic Neoplasms; Female; Gastrins; Humans; Immunohistochemistry; Intestinal Mucosa; Ligands; Macrophage Activation; Macrophages; Male; Middle Aged; Neoplasm Grading; Neoplasm Staging; Phenotype; Protein Precursors; Wnt Proteins

Human anion exchanger 2 (AE2) is a plasma membrane protein that regulates intracellular pH and cell volume. AE2 contributes to transepithelial transport of chloride and bicarbonate in normal colon and other epithelial tissues. We now report that AE2 overexpression in colon cancer cells is correlated with expression of the nuclear proliferation marker, Ki67. Survival analysis of 24 patients with colon cancer in early stage or 33 patients with tubular adenocarcinoma demonstrated that expression of AE2 is correlated with poor prognosis. Cellular and molecular experiments indicated that AE2 expression promoted proliferation of colon cancer cells. In addition, we found that transcription factor EGR1 underlies AE2 upregulation and the AE2 sequester p16INK4a (P16) in the cytoplasm of colon cancer cells. Cytoplasmic P16 enhanced ERK phosphorylation and promoted proliferation of colon cancer cells. Gastrin inhibited proliferation of colon cancer cells by suppressing expression of EGR1 and AE2 and by blocking ERK phosphorylation. Taken together, our data describe a novel EGR1/AE2/P16/P-ERK signaling pathway in colon carcinogenesis, with implications for pathologic prognosis and for novel therapeutic approaches.

Topics: Anion Transport Proteins; Antineoplastic Agents; Antiporters; Cell Line, Tumor; Cell Proliferation; Colonic Neoplasms; Cyclin-Dependent Kinase Inhibitor p16; Early Growth Response Protein 1; Gastrins; Humans; Immunohistochemistry; MAP Kinase Signaling System; Polymerase Chain Reaction; RNA, Messenger; SLC4A Proteins

The most frequently occurring lesions in the colon are the hyperplastic polyps. Hyperplastic polyps have long been considered as lesions with no malignant potential and colonoscopy for these patients is not recommended. However, recent works suggest that hyperplastic polyps may represent precursor lesions of some sporadic colorectal cancers. Until now, no biomarker allows to identify the subset of hyperplastic polyps that may have a malignant potential. Because the hormone precursor progastrin has been involved in colon carcinogenesis, we investigated whether its expression in hyperplastic polyps predicts the occurrence of colonic neoplasm after resection of hyperplastic polyps. We retrospectively analyzed progastrin expression in hyperplastic polyps from 74 patients without history of colorectal pathology. In our study, 41% of patients presenting an initial hyperplastic polyp subsequently developed adenomatous polyps, recognized as precursor lesions for colorectal adenocarcinomas. Progastrin was overexpressed in the hyperplastic polyps in 40% of the patients. We showed a significant association between progastrin overexpression and shortened neoplasm-free survival (P = 0.001). Patients with high overexpression of progastrin had a 5-year neoplasm-free survival rate of 38% as compared with 100% for the patients with low progastrin expression. In addition, we established a predictive test on the basis of progastrin staining and patients' age that predicts occurrence of neoplasm after developing a first hyperplastic polyp with a sensitivity of 100% [95% confidence interval (CI), 79%-100%] and a specificity of 74% (51%-90%). We show that progastrin expression evaluation in hyperplastic polyps is an efficient prognostic tool to determine patients with higher risk of metachronous neoplasms who could benefit from an adapted follow-up.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Cohort Studies; Colonic Neoplasms; Colonic Polyps; Disease-Free Survival; Female; Gastrins; Humans; Immunohistochemistry; Male; Middle Aged; Protein Precursors; Reproducibility of Results; ROC Curve

Transgenic mice overexpressing human progastrin (hGAS) show colonic crypt hyper-proliferation and elevated susceptibility to colon carcinogenesis. We aimed to investigate effects of p53 mutation on colon carcinogenesis in hGAS mice. We show that introducing a p53 gene mutation further increases progastrin dependent BrdU labeling and results in markedly elevated number of aberrant crypt foci (ACF) and colonic tumors. We demonstrate that hGAS/Lgr5-GFP mice have higher number of Lgr5+ colonic stem cells per crypt when compared to Lgr5-GFP mice indicating that progastrin changes crypt biology through increased stem cell numbers and additional p53 mutation leads to more aggressive phenotype in this murine colon cancer model.

Topics: Aberrant Crypt Foci; Animals; Azoxymethane; Carcinogens; Cell Proliferation; Cell Transformation, Neoplastic; Colonic Neoplasms; Disease Models, Animal; Female; Gastrins; Humans; Immunohistochemistry; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Mutation; Protein Precursors; Tumor Suppressor Protein p53

Gastrin is secreted from a subset of neuroendocrine cells residing in the gastric antrum known as G cells, but low levels are also expressed in fetal pancreas and intestine and in many solid malignancies. Although past studies have suggested that antral gastrin is transcriptionally regulated by inflammation, gastric pH, somatostatin, and neoplastic transformation, the transcriptional regulation of gastrin has not previously been demonstrated in vivo. Here, we describe the creation of an enhanced green fluorescent protein reporter (mGAS-EGFP) mouse using a bacterial artificial chromosome that contains the entire mouse gastrin gene. Three founder lines expressed GFP signals in the gastric antrum and the transitional zone to the corpus. In addition, GFP(+) cells could be detected in the fetal pancreatic islets and small intestinal villi, but not in these organs of the adult mice. The administration of acid-suppressive reagents such as proton pump inhibitor omeprazole and gastrin/CCK-2 receptor antagonist YF476 significantly increased GFP signal intensity and GFP(+) cell numbers in the antrum, whereas these parameters were decreased by overnight fasting, octreotide (long-lasting somatostatin ortholog) infusion, and Helicobacter felis infection. GFP(+) cells were also detected in the anterior lobe of the pituitary gland and importantly in the colonic tumor cells induced by administration with azoxymethane and dextran sulfate sodium salt. This transgenic mouse provides a useful tool to study the regulation of mouse gastrin gene in vivo, thus contributing to our understanding of the mechanisms involved in transcriptional control of the gastrin gene.

Topics: Aging; Animals; Azoxymethane; Carcinogens; Chromosomes, Artificial, Bacterial; Colonic Neoplasms; Dextran Sulfate; Down-Regulation; Fasting; Fetus; Gastric Acid; Gastrin-Secreting Cells; Gastrins; Gene Expression Regulation, Developmental; Genes, Reporter; Green Fluorescent Proteins; Helicobacter felis; Helicobacter Infections; Mice; Mice, Transgenic; Pyloric Antrum; Somatostatin; Tissue Distribution; Transcription, Genetic; Transgenes; Up-Regulation

One of the major angiogenic factor released by tumor cells is VEGF. Its high expression is correlated with poor prognosis in colorectal tumors. In colon cancer, gastrin gene expression is also upregulated. In these tumors, gastrin precursors are mainly produced and act as growth factors. Recently, a study has also shown that the gastrin precursor, G-gly induced in vitro tubules formation by vascular endothelial cells suggesting a potential proangiogenic role. Here, we demonstrate that stimulation of human colorectal cancer cell lines with G-gly increases the expression of the proangiogenic factor VEGF at the mRNA and protein levels. In addition, blocking the progastrin autocrine loop leads to a downregulation of VEGF. Although HIF-1 is a major transcriptional activator for VEGF our results suggest an alternative mechanism for VEGF regulation in normoxic conditions, independent of HIF-1 that involves the PI3K/AKT pathway. Indeed we show that G-gly does not lead to HIF-1 accumulation in colon cancer cells. Moreover, we found that G-gly activates the PI3K/AKT pathway and inhibition of this pathway reverses the effects of G-gly observed on VEGF mRNA and protein levels. In correlation with these results, we observed in vivo, on colon tissue sections from transgenic mice overexpressing G-gly, an increase in VEGF expression in absence of HIF-1 accumulation. In conclusion, our study demonstrates that gastrin precursors, known to promote colon epithelial cells proliferation and survival can also contribute to the angiogenesis process by stimulating the expression of the proangiogenic factor VEGF via the PI3K pathway and independently of hypoxia conditions.

Topics: Animals; Blotting, Western; Colon; Colonic Neoplasms; Enzyme-Linked Immunosorbent Assay; Fluorescent Antibody Technique; Gastrins; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Immunoblotting; Immunoenzyme Techniques; Mice; Mice, Transgenic; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Tumor Cells, Cultured; Up-Regulation; Vascular Endothelial Growth Factor A

Hypergastrinemia has been observed in patients suffering from colorectal cancer. Vascular endothelial growth factor (VEGF) is known to play a pivotal role in tumour growth. Therefore, we addressed whether gastrin-17-gly and gastrin-17-amide regulate VEGF-A-gene and protein expression.. Colo-320-cells were stably transfected with a VEGF-Luciferase-reporter gene. Luciferase activity was assessed after stimulation with various gastrin concentrations. Relevant promotor elements were identified by deletion analyses. VEGF protein levels in culture supernatants were quantified by ELISA.. VEGF-A stimulation with gastrin induced a dose- and time-dependent stimulation of luciferase activity. The greatest activities were found 6h after stimulation at concentrations of 10(-)(6)mmol/l. VEGF-promotor expression resulted in significantly (p<0.05) increased VEGF-A protein secretion. These effects were restricted to gastrin-17-amide.. Gastrin-17-amide enhances VEGF-A gene and protein expression in Colo320 cells stably transfected with a wild-type CCK-B/gastrin receptor. The induction of VEGF-A transcription and translation may contribute to the carcinogenic effects of gastrin observed in clinical studies. Therefore, CCK-B receptor antagonists may represent a treatment strategy in patients with colorectal cancer.

Topics: Cell Line, Tumor; Colonic Neoplasms; Gastrins; Hormones; Humans; Promoter Regions, Genetic; Vascular Endothelial Growth Factor A

Mice overexpressing progastrin (PG) in intestinal mucosa (fatty acid-binding protein (Fabp)-PG mice) are at an increased risk of proximal colon carcinogenesis in response to azoxymethane. Here, we report a significant increase in the length of proximal colonic crypts in Fabp-PG mice, associated with potent antiapoptotic effects of PG, which likely contributed to the previously reported increase in colon carcinogenesis in Fabp-PG mice. Phosphorylation of kinase of IkappaBalpha (IKKalpha/beta), inhibitor of kappaB (IkappaB)alpha and p65NF-kappaB was significantly elevated in proximal colonic crypts of Fabp-PG versus wild-type mice, which was associated with degradation of IkappaBalpha and nuclear translocation/activation of p65. Surprisingly, distal colonic crypt cells were not as responsive to elevated levels of PG in Fabp-PG mice. Annexin II, recently described as a high-affinity receptor for PG, strongly co-localized with PG intracellularly and on basolateral membranes of proximal crypt cells, providing evidence that annexin-II binds PG in situ in colonic crypt cells. Proliferative and antiapoptotic effects of PG on proximal crypts of Fabp-PG mice were attenuated to wild-type levels, on treatment with NEMO peptide (an inhibitor of nuclear factor-kappaB (NF-kappaB) activation), demonstrating for the first time a critical role of NF-kappaB in mediating hyperproliferative affects of PG on colonic crypts of Fabp-PG mice, in vivo. Thus, downregulation of NF-kappaB may significantly reduce the increased risk of colon carcinogenesis in response to PG.

Topics: Animals; Annexin A2; Apoptosis; Cell Proliferation; Colon; Colonic Neoplasms; DNA; Extracellular Signal-Regulated MAP Kinases; Fatty Acid-Binding Proteins; Gastrins; I-kappa B Proteins; Mice; NF-kappa B; NF-KappaB Inhibitor alpha; Phosphorylation; Protein Precursors; Transcription Factor RelA

Cyclooxygenase-2 (COX-2) is expressed in colonic neoplasms, where it supports cell proliferation via prostaglandin E(2) (PGE(2)) production. This study investigated the effects of somatostatin-14 on COX-2 expression, PGE(2) production and proliferation in colon cancer cells.. Human colon adenocarcinoma cell lines Caco-2, HT-29 and HCT116 were used. The following techniques were employed: colourimetric assay for cell growth; 5-bromo-2'-deoxyuridine assay for DNA synthesis; enzyme immunoassay for PGE(2); COX-2 mRNA silencing; RT-PCR or Western blot for somatostatin receptor subtypes, cyclooxygenase isoforms, phosphorylated-ERK-1/ERK-2 and phosphorylated-Akt.. HT-29 and Caco-2 cells expressed COX-2 and somatostatin receptors (sst(3/4/5) and sst(3/5), respectively). HCT116 cells did express somatostatin receptors (sst(2/3/5)), but not COX-2. Somatostatin-14 inhibited basal COX-2 expression, PGE(2) production, DNA synthesis and growth in Caco-2 cells and these effects were prevented by BN81658 (sst(3) receptor antagonist). Basal proliferation of HT-29, HCT116 and COX-2-silenced Caco-2 cells was not affected by somatostatin-14. Stimulation of HT-29 cells with gastrin-17 elicited increments of ERK-1/ERK-2 and Akt phosphorylation, COX-2 expression, PGE(2) production, DNA synthesis and cell growth, which were all counteracted by somatostatin-14. Somatostatin-14-induced inhibition of COX-2 expression, PGE(2) production and DNA synthesis were blocked by BIM23056 (sst(5) receptor antagonist).. Somatostatin decreases COX-2 expression and function in colon cancer cells via activation of sst(3) or sst(5) receptors, and these effects contribute to the inhibitory action of somatostatin on cell proliferation. These findings can be relevant to the development of therapeutic strategies based on the modulation of the COX-2 pathway.

Topics: Caco-2 Cells; Cell Proliferation; Colon; Colonic Neoplasms; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Down-Regulation; Gastrins; HT29 Cells; Humans; Mitogen-Activated Protein Kinase 1; Oligopeptides; Protein Kinase C; Protein Tyrosine Phosphatases; Somatostatin

Proton pump inhibitors (PPIs) have become the mainstay of therapy in acid-related upper gastrointestinal disorders including gastroesophageal reflux disease and peptic ulcer disease. Alltough these medications are generally accepted as safe, the long-term clinical consequences of the inducing hypochlorhydria are not completely clear. Gastric acid production is mainly controlled by the hormone gastrin through a negative feedback in which hypochlorhydria induces an increase in serum gastrin. PPIs have been shown to increase serum gastrin levels. Gastric endocrine cell hyperplasia can occur in 10 to 30% of patients without carcinoid tumors. Recent studies indicate no association between PPI use and the risk of colorectal and gastric cancers. Proton pump inhibitor-associated gastric polyps are totally benign tumors that should not be followed. There is an association between PPIs-induced acid suppression and an increased risk of enteric infection. PPIs do not inhibit intestinal absorption of lipids, iron, phosphorus, magnesium or zinc from food but can affect vitamin B12 status in older patients. Despite the undoubted benefits of PPIs, the practitioner always needs to consider risks and benefits before initiating them.

Topics: Bacterial Infections; Colonic Neoplasms; Gastric Acid; Gastrins; Humans; Polyps; Proton Pump Inhibitors; Vitamin B 12 Deficiency

Gastrin and its derivatives are becoming important targets for immunotherapy of pancreatic, gastric and colorectal tumors. This study was conducted to design antibodies able to block gastrin binding to the gastrin/cholecystokinin-2 (CCK-2) receptor in order to delay tumor growth. The authors have used different gastrin molecules, combined with the diphtheria toxoid, to generate and select human single chain variable fragments (scFvs) as well as mouse monoclonal antibodies and scFvs against different regions of gastrin. There was a remarkable conservation in the antibody repertoire against gastrin, independently of the approach and the species. The germlines most frequently used in gastrin antibody formation were identified. Three different epitopes were identified in the gastrin molecule. The resulting mouse monoclonal antibodies and scFvs were analyzed for gastrin neutralization using Colo 320 WT cells, which overexpress the CCK-2 receptor. The gastrin neutralizing activity assay showed that N-terminal specific mouse monoclonal antibodies were more efficient to inhibit proliferation of Colo 320 WT cells than the anti-C terminal antibodies. Moreover, the human antigastrin scFvs obtained in this study inhibited significantly the proliferation of Colo 320 tumoral cells. These findings should contribute to a more rational design of antibody-based antigastrin therapies in cancer, including passive administration of human antibodies with blocking activity.

Topics: Animals; Antibodies, Blocking; Antibodies, Monoclonal; Cell Proliferation; Colonic Neoplasms; Diphtheria Toxoid; Enzyme-Linked Immunosorbent Assay; Gastrins; Humans; Immunization; Immunoglobulin Variable Region; Mice; Peptide Library; Receptor, Cholecystokinin B; Spleen; Surface Plasmon Resonance; Tumor Cells, Cultured

We and others have reported the presence of novel progastrin (PG)/gastrin receptors on normal and cancerous intestinal cells. We had earlier reported the presence of 33-36 kDa gastrin-binding proteins on cellular membranes of colon cancer cells. The goal of the current study was to identify the protein(s) in the 33-36 kDa band, and analyse its functional significance. A carbodiimide crosslinker was used for crosslinking radio-labeled gastrins to membrane proteins from gastrin/PG responsive cell lines. Native membrane proteins, crosslinked to the ligand, were solubulized and enriched by >1000-fold, and analysed by surface-enhanced laser desorption/ionization-time of flight-mass spectrometry. The peptide masses were researched against the NCBInr database using the ProFound search engine. Annexin II (ANX II) was identified, and confirmed by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry. As HCT-116 cells express autocrine PG, the in situ association of PG with ANX II was demonstrated in pulldown assays. Direct binding of PG with ANX II was confirmed in an in vitro binding assay. In order to confirm a functional importance of these observations, sense and anti-sense (AS) ANX II RNA-expressing clones of intestinal epithelial (IEC-18) and human colon cancer (HCT-116) cell lines were generated. AS clones demonstrated a significant loss in the growth response to exogenous (IEC-18) and autocrine (HCT-116) PG. We have thus discovered that membrane-associated ANX II binds PG/gastrins, and partially mediates growth factor effects of the peptides.

Topics: Animals; Annexin A2; Cell Proliferation; Colonic Neoplasms; Cross-Linking Reagents; Epithelial Cells; Fibroblasts; Gastrins; Humans; Hydrogen-Ion Concentration; Intestinal Mucosa; Mice; Peptide Fragments; Protein Precursors; Rats; Receptor, Cholecystokinin B; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Tumor Cells, Cultured; Tumor Stem Cell Assay

Our previous studies have shown that stimulation of proliferation of DLD-1 and HT29 human colonic cancer cells by nanomolar gastrin (G17) and carboxymethyl gastrin (G17Gly) and reversal of growth by micromolar G17 and G17Gly involves binding sites which can neither be CCK1 nor CCK2 receptors; the N terminal fragment, G17(1-12), is sufficient to increase the number of HT-29 cells by binding the higher affinity binding site but is without a suppressing effect through the lower affinity site. In this study with DLD-1 cells, competitive binding using 125I-G17(1-12) showed that G17(1-12) binds both high and low affinity sites, as do G17 and G17Gly. G17(1-6)-NH2, even without the central-to-C-terminal portion of G17, was still able to bind a single site and to promote a dose-dependent increase in cell number at nanomolar concentrations. The results indicate the presence of a non-CCK receptor on human colonic cancer cells which could mediate the tumor-promoting activity of the N-terminal-to-central portion of G17Gly which, unlike G17, is produced by such cells.

Topics: Carcinogens; Cell Line, Tumor; Cell Proliferation; Colonic Neoplasms; Gastrins; Humans; Peptide Fragments; Radioligand Assay; Receptors, Cholecystokinin

To study the effect of gastrin on the mRNA and protein expression of urokinase-type plasminogen activator (u-PA) in human colon cancer cells and detect the role of p38 MAPK in this process.. Lipofectin method was used to transfect pCR3. 1/CCK2R vector expressing gastrin receptor into a colon cancer cell line colo320. Gastrin and gastrin antagonist were used to up-regulate and down-regulate the signaling pathway, respectively. Human colon cancer colo320 cells and colo320/ CCK2,R cells were cultured and then stimulated with gastrin for different time; SB203580 was added into culture medium to prevent p38 kinase pathway before incubating with gastrin; Western blot and RT-PCR were used to examine the u-PA expression. Western blot was employed to detect p38 kinase phosphorylation.. Gastrin increased evidently the mRNA and protein expressions of u-PA and induced p38 kinase phosphorylation in colo320/CCK,R cells time-dependently. However, the extent of enhancement of u-PA and p38 MAPK expression in colo320 cells was much less than that in colo320/CCK2R cells. The gastrin antagonist L-365, 260 showed an effect of competitive inhibition on gastrin-induced u-PA expression and p38 kinase phosphorylation. The inhibitor SB203580 could sufficiently suppress gastrin-induced p38 kinase phosphorylation and significantly attenuate gastrin-induced u-PA mRNA and protein expressions in colo320/ CCK2 R cells in a dose-dependent manner.. Gastrin-gastrin receptor signal transduction pathway can obviously induce u-PA expression in human colon cancer cells via activating the phosphorylation of p38 kinase.

Topics: Benzodiazepinones; Blotting, Western; Cell Line, Tumor; Colonic Neoplasms; Gastrins; Gene Expression Regulation, Neoplastic; Genetic Vectors; Humans; Imidazoles; p38 Mitogen-Activated Protein Kinases; Phenylurea Compounds; Phosphorylation; Pyridines; Receptor, Cholecystokinin B; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Transfection; Urokinase-Type Plasminogen Activator

Cyclooxygenase-2, the inducible enzyme of arachidonic acid metabolism and prostaglandin synthesis, is over expressed in colorectal cancer. Inhibition of COX-1/-2 by non-steroidal anti-inflammatory drugs is associated with a decreased risk for these malignancies, whereas high serum gastrin levels elevate this risk. As gastrin exhibits trophical effects on colonic epithelium we sought to explore whether it is capable to induce COX-2 expression in a human colon cancer cell line. The aim of this study is the description of the gastrin evoked effects on the transcriptional activity of the COX-2 gene in colorectal cancer cells and the identification of regulatory promoter elements. Reporter gene assays were performed with the gastrin-stimulated human colorectal cancer cell-line Colo-320, which was stable transfected with the human cholecystokinin-B/gastrin receptor cDNA and COX-2-promoter-luciferase constructs containing different segments of the 5'-region of the COX-2 gene or with mutated promoter constructs. Transcription factors were characterized with electrophoretic mobility shift assays. Gastrin-dependent induction of COX-2 mRNA was shown using "real-time" PCR. Resulting elevated Prostaglandin E2-levels were measured using ELISA. Gastrin stimulated the PGE2-generation and COX-2-mRNA expression in human Colo-320-B cells potently, obviously by transactivating the COX-2-promoter using a region between - 68 bp and + 70 bp. Further examinations identified a CRE-E-box element between - 56 bp and - 48 bp mediating the gastrin-effects on the COX-2 gene. Transcription factors binding to this promoter element were USF-1 und -2. These results show the necessity to perform succeeding studies, which could describe possible mechanisms in which gastrin and COX-2 contribute to the induction of colorectal carcinomas.

Topics: Binding Sites; Colonic Neoplasms; Cyclic AMP Response Element-Binding Protein; Cyclooxygenase 2; Dinoprostone; Gastrins; Humans; Promoter Regions, Genetic; Response Elements; RNA, Messenger; Transcriptional Activation; Transfection; Tumor Cells, Cultured; Upstream Stimulatory Factors

To explore the molecular mechanism of increasing the invasion of colon cancer cells by gastrin 17.. The plasmid pCR 3.1/GR expressing the gastrin receptor cholecystokinin-2 receptor (CCK-2R) was transfected into colonic carcinoma cells of the line Colo320 by Lipofectamine 2000. The clones expressing stably CCK-2R were screened by G418 and named as Colo320WT cells. The expression levels of gastrin receptor of the Colo320 and Colo320WT cells were assayed by RT-PCR and Western blotting. The Colo320WT cells were treated by gastrin-17, and the expression levels of phosphorylated FAKTyr397 and total focal adhesion kinase (FAK) in the Colo320WT cells at the time points 0, 1, 6, 12, 24, and 48 h were detected by Western blotting. Another Colo320WT cells were treated by L365, 260, gastrin17 receptor blocker, for 30 minutes firstly and then treated by gastrin17 again for 12 hours, and then Western blotting was used to detect the expression levels of phosphorylated FAKTyr397 and total focal adhesion kinase (FAK) at the time points 0, 1, 6, 12, 24, and 48 h. Confocal microscopy was used to observe the phosphorylated FAKTyr397 localizing in the lamellipodia. The information of FAK-Src-p130(Cas)-Dock180 signaling complex was assayed by coimmuniprecipation and immunity blotting. The level of Rac-GTPase was tested by pull down assay.. The level of phosphorylated FAKTyr397 expression in the Colo320WT cells after the gastrin17 intervention increased time-dependently and peaked at the time point of 12 h, and the phosphorylated FAKTyr397 expression in the Colo320WT cells treated by L365, 260 decreased remarkably, but the level of total FAK remained unchanged. The phosphorylated FAKTyr397/FAK levels were 2.82%, 9.28%, 22.62%, 38.59%, 28.41%, and 14.94%, 0, 1, 6, 12, 24, and 48 h after the gastrin17 treatment respectively, and the level was 7.21% after L365, 260 treatment. The amount of phosphorylated FAKTyr397 localizing in the lamellipodia of the Colo320WT cells that were treated by gastrin17 increased time-dependently and peaked at the time-point 12 h. FAK-Src-p130(Cas)-Dock180 signaling complex was formed in the Colo320WT cells stimulated with gastrin17. Gastrin17 activated Rac, but did not affect the total Rac expression.. The mechanism of increasing the colon cancer cells' invasion by gastrin17 is probably that gastrin17 makes FAK-Tyr397 phosphorylated and be localized to lamellipodia, causes the forming of FAK-Src-p130(Cas)-Dock180 signaling complex when it is bound to its receptor CCK-2, and activation of Rac.

Topics: Benzodiazepinones; Blotting, Western; Cell Line, Tumor; Cell Movement; Colonic Neoplasms; Focal Adhesion Protein-Tyrosine Kinases; Gastrins; Humans; Neoplasm Invasiveness; Phenylurea Compounds; Phosphorylation; Receptor, Cholecystokinin B; Reverse Transcriptase Polymerase Chain Reaction; Transfection; Tyrosine

To explore the effects of human FRNK gene on E-cadherin/beta-catenin complex in colon cancer cell line Colo320WT cells stimulated with extrinsic gastrinl7.. AdEasy system was used to construct pAdhFRNK expressing human FRNK gene by recombination in E. coli. BJ5283. pCR3.1/GR plasmid expressing gastrin receptor CCK-2 was transfected into colon cancer cell line Colo320 cells by Lipofectamine 2000 and expressing stably CCK-2R clones were screened by G418 (500 pg/ml). The expression levels of gastrin receptor in Colo320 cells and the transfected Colo320WT cells were assayed by RT-PCR. Colo320WT cells were treated by 10(-8) mol/L gastrinl7 for 12 h; and after Colo320WT cells were infected by pAdhFRNK (MOI: 100) for 2 d the cells were treated by gastrin17 for 12 h again. The expression levels of E-cadherin and beta-catenin in TX-100 soluble fraction and TX-100 insoluble fraction of Colo320WT cells were assayed by co-immunoprecipation and Western blot. E-cadherin and beta-catenin's distribution in Colo320WT cells were detected by immunocytochemistry.. When 10(-8) mol/L gastrin17 stimulated Colo320WT cells for 12 h, the expression levels of E-cadherin and beta-catenin in TX-100-soluble fraction decreased apparently, while the expression levels of E-cadherin and beta-catenin in TX-100-insoluble fraction increased markedly. When pAdhFRNK infected Colo320WT cells for 2 d and 10(-8) mol/L gastrin17 treated the cells for 12 h, the expression levels of E-cadherin and beta-catenin in TX-100-soluble fraction increased apparently again, and the expression levels of E-cadherin and beta-catenin in TX-100-insolutble fraction decreased markedly. Immunocytochemistry showed that the distribution of E-cadherin and beta-catenin was translocated from plasma membrane into cytoplasm and nucleus in the cells stimulated with gastrinl7, and after the cells were infected with pAdhFRNK and stimulated by gastrinl7 again. beta-catenin was mainly observed in cytoplasm and little nuclear immunoreactivity.. An adenovirus vector pAdhFRNK can inhibit abnormal distribution of E-cadherin and beta-catenin in the gastrin17-stimulated cells. The mechanism is probably that hFRNK can disphosphorylate phosphorylated FAK and block FAK pathway.

Topics: Adenoviridae; beta Catenin; Blotting, Western; Cadherins; Cell Line, Tumor; Cell Membrane; Cell Nucleus; Colonic Neoplasms; Cytoplasm; Gastrins; Genetic Vectors; Humans; Immunohistochemistry; Immunoprecipitation; Lipids; Protein Binding; Protein Transport; Protein-Tyrosine Kinases; Receptor, Cholecystokinin B; Transfection

The expression of the human cholecystokinin-2/gastrin receptor (CCK-2R) has been widely reported in human colorectal cancers. Recently, a splice variant of the CCK-2R retaining intron 4 (CCK-2i4svR) has been cloned from human colorectal cancers and postulated to exhibit constitutive activity. But its role in mediating carcinogenic effects of mature-amidated gastrin in colorectal cancers has not been fully explored. The purpose of the present study was to determine whether the activation of CCK-2i4svR by gastrin transactivates the COX-2 promoter in human colon cancer cells and in COS-7 cells. In this study, Colo320 cells and COS-7 cells were transfected with the human CCK-2R wild type (CCK-2wtR) (COS-7WT, Colo320WT) and the human CCK-2i4svR (COS-7SV, Colo320SV) cDNA. After stimulation with gastrin-17 (G-17), transactivation of the COX-2 promoter was determined by luciferase reporter gene assay. 5'deletions of the COX-2 promoter were transfected into Colo320 cells to narrow down the minimally required regulatory element. Induction of COX-2 expression was further explored at the mRNA level using real time RT-PCR. The effects of CCK-2i4svR activation on phosphorylation of ERK1/2, p38(MAPK) and JNK were examined by using immunoblotting. Prostaglandin E(2) (PGE(2)) secretion was measured by ELISA. Our results showed that gastrin transactivates the COX-2 promoter in both Colo320 cells and COS-7 cells expressing the CCK-2i4svR cDNA. Inhibition of p38(MAPK) pathway using specific inhibitor significantly blocked the gastrin-induced COX-2 transactivation. Gastrin time-dependently increased COX-2 mRNA expression, the peak mRNA levels appeared at 10 h after stimulation. PGE(2) secretion from gastrin-treated cells increased significantly 8 h after stimulation. Treatment with gastrin also stimulated PGE(2) secretion in the Colo320 cells expressing CCK-2i4svR. In conclusion, the CCK-2i4svR mediates transactivation of the COX-2 promoter and MAPK pathway is involved in this process.

Topics: Animals; Chlorocebus aethiops; Colonic Neoplasms; COS Cells; Cyclooxygenase 2; Dinoprostone; Enzyme-Linked Immunosorbent Assay; Gastrins; Genes, Reporter; Humans; Introns; Luciferases; Plasmids; Promoter Regions, Genetic; Protein Isoforms; Receptor, Cholecystokinin B; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Transcriptional Activation; Tumor Cells, Cultured

Peroxisome proliferator-activated receptor gamma (PPAR gamma) are members of the largest nuclear hormone receptor family of transcription factors (1). PPAR gamma (PPARgamma) plays an important role in adipogenesis, control of sensitivity to insulin, inflammation and atherosclerosis but recent studies also suggest that PPARgamma is involved in cell cycle withdrawal. PPARgamma can promote cell differentiation, exert an antiproliferative action and inhibit angiogenesis (2, 3). However, there are studies showing that activation of PPARgamma promotes the development of colon cancer (4). These data are in sharp contrast with studies that attribute anticancer effects to PPARgamma in gastrointestinal malignancies. Probably, the action of PPARgamma on cell cycle and proliferation depends on the cell type and presence of other stimuli that predispose cells to cancer development. Amidated and non-amidated gastrins may play an important role in the proliferation and carcinogenesis of GI cancers. It is known that gastrin peptides activate phosphorylation of Protein Kinase B (PKB/Akt) and anti-apoptotic signalling but there is little known about the link between gastrins and PPARgamma receptors in relation to apoptosis.

Topics: Apoptosis; Blotting, Western; Cell Line, Tumor; Cell Survival; Colonic Neoplasms; Gastrins; Humans; Pancreatic Neoplasms; PPAR gamma; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Thiazolidinediones; Transcription, Genetic; Up-Regulation

Glycine-extended gastrin (G-Gly) is an end product of processing of the progastrin precursor peptide that has a different spectrum of activity to amidated gastrin. G-Gly promotes cell proliferation in normal and malignant colonic epithelium but the mechanisms responsible are poorly understood. Prostaglandins produced by the cyclo-oxygenase (COX) enzymes have been implicated as downstream mediators of several growth factors, and COX inhibitors such as non-steroidal anti-inflammatory drugs inhibit the proliferation and invasiveness of colonic cancer and reduce the incidence of colon cancer. We have examined the mechanisms of the actions of G-Gly in HT-29 colon cancer cells. G-Gly induced a dose-dependent increase in cell proliferation that was insensitive to inhibition of either COX-1 or COX-2, but was abolished by inhibition of the p38 MAP kinase, ERK and NF-kappaB pathways. G-Gly did not increase prostaglandin E2 production. Celecoxib induced apoptosis and reduced viable cell numbers in a COX-independent manner. G-Gly significantly reduced serum-starvation and celecoxib-induced apoptosis and this effect was also blocked by inhibition of the p38 MAP kinase, ERK and NF-kappaB pathways. Stimulation of HT-29 cells with G-Gly led to a rapid increase in ERK and p38 MAP kinase phosphorylation and increased nuclear translocation of active NF-kappaB. Activation of NF-kappaB was independent of ERK and p38 MAP kinase. G-Gly stimulates proliferation and inhibits apoptosis in colon cancer cells via COX-independent and ERK-, p38 MAP kinase-, and NF-kappaB-dependant pathways. Locally and systemically produced G-Gly may be important in reducing the beneficial effects of chemopreventative agents in colon cancer.

Topics: Apoptosis; Celecoxib; Cell Count; Cell Proliferation; Colonic Neoplasms; Cyclooxygenase 2; Cyclooxygenase Inhibitors; Dinoprostone; Gastrins; HT29 Cells; Humans; Isoenzymes; Membrane Proteins; Mitogen-Activated Protein Kinases; NF-kappa B; Phosphorylation; Prostaglandin-Endoperoxide Synthases; Pyrazoles; Sulfonamides

Glycine-extended gastrin (G-Gly) is produced by colon cancers and has growth promoting and anti-apoptotic effects in the colonic epithelium. We have examined the anti-apoptotic effects of G-Gly and the signal transduction pathways involved. G-Gly stimulated HT-29 cell proliferation in a concentration dependent manner and inhibited serum-starvation and celecoxib-induced apoptosis. Inhibition of signalling via c-Jun NH2-terminal kinase (JNK) with SP600125 or PI3-kinase/Akt with LY294002 abolished the effects of G-Gly. G-Gly significantly increased phosphorylation of both JNK and Akt. The JAK2 inhibitor AG490 abolished the anti-apoptotic effect of G-Gly and inhibited phosphorylation of Akt but not of JNK. G-Gly stimulated tyrosine phosphorylation of JAK2. G-Gly-increased activation of AP-1 was JNK-dependant and activation of STAT3 was JAK2-dependant. We conclude that G-Gly promotes growth and inhibits apoptosis in colon cancer cells. These effects are mediated via the JAK2, PI3-kinase/Akt and JNK pathways. Activation of JAK2 is upstream of Akt but not of JNK.

Topics: Anthracenes; Apoptosis; Celecoxib; Cell Line, Tumor; Cell Survival; Chromones; Colonic Neoplasms; Gastrins; Humans; Janus Kinase 2; JNK Mitogen-Activated Protein Kinases; Morpholines; Phosphatidylinositol 3-Kinases; Phosphorylation; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Pyrazoles; Signal Transduction; STAT3 Transcription Factor; Sulfonamides; Transcription Factor AP-1; Tyrosine; Tyrphostins

This study examined whether gastrin modulates endothelial cell activity via heparin-binding epidermal growth factor-like growth factor (HB-EGF) expression. Human umbilical vascular endothelial cells (HUVEC) were assessed for tubule formation in the presence of amidated gastrin-17 (G17) and glycine-extended gastrin-17 (GlyG17) peptides. HB-EGF gene and protein expressions were measured by quantitative reverse transcription-PCR, immunocytochemistry, and Western blotting, and HB-EGF shedding by ELISA. Matrix metalloproteinases MMP-2, MMP-3, and MMP-9 were assessed by Western blotting. Chick chorioallantoic membrane studies measured the in vivo angiogenic potential of gastrin and microvessel density (MVD) was assessed in large intestinal premalignant lesions of hypergastrinaemic APC(Min) mice. MVD was also examined in human colorectal tumor and resection margin normals and correlated with serum-amidated gastrin levels (via RIA) and HB-EGF protein expression (via immunohistochemistry). HUVEC cells showed increased tubule and node formation in response to G17 (186%, P < 0.0005) and GlyG17 (194%, P < 0.0005). This was blockaded by the cholecystokinin-2 receptor (CCK-2R) antagonists JB95008 and JMV1155 and by antiserum to gastrin and HB-EGF. Gastrin peptides increased HB-EGF gene expression/protein secretion in HUVEC and microvessel-derived endothelial cells and the levels of MMP-2, MMP-3, and MMP-9. G17 promoted angiogenesis in a chorioallantoic membrane assay, and MVD was significantly elevated in premalignant large intestinal tissue from hypergastrinaemic APC(Min) mice. In terms of the clinical situation, MVD in the normal mucosa surrounding colorectal adenocarcinomas correlated with patient serum gastrin levels and HB-EGF expression. Gastrin peptides, acting through the CCK-2R, enhance endothelial cell activity in models of angiogenesis. This may be mediated through enhanced expression and shedding of HB-EGF, possibly resulting from increased activity of matrix metalloproteinases. This proangiogenic effect translates to the in vivo and human situations and may add to the tumorigenic properties attributable to gastrin peptides in malignancy.

Topics: Animals; Cell Differentiation; Chick Embryo; Colonic Neoplasms; Endothelial Cells; Epidermal Growth Factor; Gastrins; Gene Expression; Heparin-binding EGF-like Growth Factor; Humans; Immune Sera; Intercellular Signaling Peptides and Proteins; Isoenzymes; Metalloendopeptidases; Mice; Neovascularization, Physiologic; Omeprazole; Receptor, Cholecystokinin B

To investigate whether Src, JAK2 and phosphatidylinositol 3-kinase (PI3K) pathways are involved in the proliferation of human colonic tumour cells induced by glycine-extended gastrin (G-gly), the precursor of the mature amidated gastrin and to elucidate the molecular interaction between these three kinases in response to this peptide.. Using the human colonic tumour cell line HCT116 as a model, we first measured the activation of PI3K, p60-Src and JAK2 in response to G-gly by in vitro kinase assays. Then we investigated the involvement of these kinases in G-gly-induced cell proliferation by MTT test.. G-gly stimulation induced p60-Src, JAK2 and PI3K activation in HCT116. The different pathways were involved in proliferation of human colon cancer cells induced by G-gly. Furthermore, we found that both Src and JAK2 were necessary to PI3K regulation by this peptide. However, we did not find any cross-talk between the two tyrosine kinases.. Our results suggest that the p60-Src/PI3K and JAK2/PI3K pathways act independently to mediate G-gly proliferative effect on human colonic tumour cells.

Topics: Cell Proliferation; Colonic Neoplasms; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; Gastrins; HCT116 Cells; Humans; Immunoprecipitation; Janus Kinase 2; Phosphatidylinositol 3-Kinases; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins pp60(c-src); Signal Transduction

Focal adhesion kinase (FAK) is suggested to be intimately involved in the progression of malignancies. Our previous research has demonstrated that activation of cholecystokinin-2 receptor (CCK2R) by gastrin stimulates a rapid activation of FAK pathway in human colon cancer cells. The purpose of this study is to determine the role of CCK2R and FAK in the progression of colon cancer. In this study, matched tissue samples of primary colon cancer and adjacent normal colon mucosa from the same patient were collected from 45 patients with colon cancer undergoing surgical resection. The gastrin expression was detected using reverse transcription polymerase chain reaction (RT-PCR). The CCK2R expression was examined by in situ hybridization and RT-PCR. The expression of FAK and phosphorylated FAK at tyrosine 397 (phospho-FAK) were detected using immunohistochemistry and immunoblotting. Colo320 and SW787, 2 colon cancer cell lines with or without CCK2R expression, were recruited in this study. Antisense oligonucleotide of FAK was used to block the expression of FAK. Invasiveness and motility of colon cancer cells were detected by Boyden chamber. In this series, enhanced expression of gastrin, CCK2R, FAK and phospho-FAK were observed in colon cancer tissues. CCK2R expression correlated with expression of phospho-FAK. Coexpression of CCK2R and phospho-FAK associated with invasion and lymph node metastasis. Increased invasion and motility was induced by gastrin in Colo320 cells. Overexpression of CCK2R by stable transfection of CCK2R plasmid amplified this increase and incubation with 1 microM L-365,260, a specific CCK2R antagonist, completely inhibited the effect of gastrin. FAK antisense largely blocked the increase of invasion and motility in Colo320 cells. Our data represent the evidence for the CCK2R regulating invasion and motility of colon cancer cells, and support a role of CCK2R in the progression of colon cancer. FAK play a critical role in this CCK2R-mediated effect.

Topics: Benzodiazepinones; Cell Line, Tumor; Cell Movement; Colonic Neoplasms; Disease Progression; Enzyme Activation; Female; Focal Adhesion Protein-Tyrosine Kinases; Gastrins; Gene Expression Regulation, Neoplastic; Humans; Immunoblotting; Immunohistochemistry; In Situ Hybridization; Male; Middle Aged; Oligonucleotides, Antisense; Phenylurea Compounds; Phosphorylation; Receptor, Cholecystokinin B; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transfection; Tyrosine

Helicobacter pylori infection is a recognized cause of hypergastrinemia, but the association of blood gastrin levels with colonic adenomas (CAs) is controversial. The aim of this study is to investigate if hypergastrinemia, H. pylori infection and/or cagA protein are risk factors for CAs.. In this prospective case-control study, fasting serum samples from 78 consecutive patients with CAs and 78 demographically matched colonoscopy-negative controls were assayed for anti-H. pylori immunoglobulin G, cagA protein and serum gastrin levels. Multivariate analysis was performed to identify risk factors for colon adenomas.. Though prevalence of H. pylori antibodies was not significantly different, the prevalence of cagA protein was significantly higher in patients with adenomas (42.3%) as compared with controls (25.6%, p < 0.03). Mediangastrin levels were significantly higher in patients with CAs (55, 20-975 pg/ml) than in controls (45.2, 23-529 pg/ml) (p < 0.001). Hypergastrinemia (>110 pg/ml) was commoner in patients with CAs than in controls (29.5 vs. 11.5%, p = 0.006) and was the only independent risk factor for adenomas (odds ratio 3.2, 95% CI 1.4-7.5) by multivariate analysis, but not H. pylori infection or cagA positivity. There was a significant association of hypergastrinemia and distal distribution of adenomas (p < 0.002).. Our study shows that hypergastrinemia is a risk factor for CAs, especially of the distal colon.

Topics: Adenoma; Adult; Aged; Aged, 80 and over; Antigens, Bacterial; Bacterial Proteins; Case-Control Studies; Colonic Neoplasms; Female; Gastrins; Helicobacter Infections; Helicobacter pylori; Humans; Male; Middle Aged; Multivariate Analysis; Prospective Studies; Risk Factors

To explore the effect of gastrin 17 (G17) on beta-catenin/T cell factor-4 (Tcf-4) signaling in colonic cancer cell line Colo320WT.. The pCR3.1/GR plasmid, which expresses gastrin receptor, cholecystokinin-2 receptor (CCK-2R), was transfected into a colonic cancer cell line Colo320 by Lipofectamine (TM)2000 and the stably expressing CCK-2R clones were screened by G418. The expression levels of gastrin receptor in the Colo320 and the transfected Colo320WT cell line were assayed by RT-PCR. Colo320WT cells were treated with G17 in a time-dependent manner (0, 1, 6, 12, 24 and 48 h), then with L365,260 (Gastrin(17) receptor blocker) for 30 min, and with G17 again for 12 h or L365,260 for 12 h. Expression levels of beta-catenin in a TX-100 soluble fraction and TX-100 insoluble fraction of Colo320WT cells treated with G17 were detected by co-immuniprecipation and Western blot. Immunocytochemistry was used to examine the distribution of beta-catenin in CoLoWT320 cells. Expression levels of c-myc and cyclin D1 in Colo320WT cells treated with G17 were assayed by Western blot.. Expression levels of beta-catenin in the TX-100 solution fraction decreased apparently in a time-dependent fashion and reached the highest level after G17 treatment for 12 h, while expression levels of beta-catenin in the TX-100 insoluble fraction were just on the contrary. Immunocytochemistry showed that beta-catenin was translocated from the cell membranes into the cytoplasm and nucleus under G17 treatment. Expression levels of c-myc and cyclin D1 in the G17-treated Colo320WT cells were markedly higher compared to the untreated Colo320WT cells. In addition, the aforementioned G17-stimulated responses were blocked by L365,260.. Gastrin17 activates beta-catenin/Tcf-4 signaling in Colo320WT cells, thereby leading to over-expression of c-myc and cyclin D1.

Topics: Base Sequence; beta Catenin; Cell Line, Tumor; Colonic Neoplasms; Cyclin D; Cyclins; DNA, Complementary; Gastrins; Humans; Proto-Oncogene Proteins c-myc; Receptor, Cholecystokinin B; Recombinant Proteins; TCF Transcription Factors; Transcription Factor 7-Like 2 Protein; Transfection

The present study investigates the effects of gastrin-17 on human colon cancer HT-29 cells to examine whether gastrin receptor (CCK-2), cyclooxygenase (COX-1, COX-2) isoforms and prostaglandin receptor pathways interact to control cell growth. Reverse transcription (RT)-polymerase chain reaction (PCR) analysis demonstrated that HT-29 cells are endowed with the naive expression of CCK-2 receptor (short splice variant), COX-1, COX-2 and prostaglandin EP(4) receptor, but not gastrin. Gastrin-17 significantly promoted cell growth and DNA synthesis. Both these stimulating effects were abolished by L-365,260 or GV150013 (CCK-2 receptor antagonists), but were unaffected by SC-560 (COX-1 inhibitor). L-745,337 (COX-2 inhibitor) or AH-23848B (EP(4) receptor antagonist) partly reversed gastrin-17-induced cell growth, while they fully antagonized the enhancing action on DNA synthesis. HT-29 cells responded to gastrin-17 with a significant increase in prostaglandin E(2) release. This enhancing effect was completely counteracted by L-365,260, GV150013 or L-745,337, while it was insensitive to cell incubation with SC-560. Exposure of HT-29 cells to gastrin-17 was followed by an increased phosphorylation of both extracellular regulated kinases (ERK-1/ERK-2) and Akt. Moreover, gastrin-17 enhanced the transcriptional activity of COX-2 gene promoter and stimulated COX-2 expression. These latter effects were antagonized by L-365,260 or GV150013, and could be blocked also by PD98059 (inhibitor of ERK-1/ERK-2 phosphorylation) or wortmannin (inhibitor of phosphatidylinositol 3-kinase). Analogously, gastrin-17-induced prostaglandin E(2) release was prevented by PD98059 or wortmannin. The present results suggest that (a) in human colon cancer cells endowed with CCK-2 receptors, gastrin-17 is able to enhance the transcriptional activity of COX-2 gene through the activation of ERK-1/ERK-2- and phosphatidylinositol 3-kinase/Akt-dependent pathways; (b) these stimulant actions lead to downstream increments of COX-2 expression, followed by prostaglandin E(2) production and EP(4) receptor activation; (c) the recruitment of COX-2/prostaglandin pathways contributes to the growth-promoting actions exerted by gastrin-17.

Topics: Antimetabolites; Blotting, Western; Bromodeoxyuridine; Cell Proliferation; Cells, Cultured; Colonic Neoplasms; Cyclooxygenase 2; Dinoprostone; DNA; Enzyme Induction; Gastrins; HT29 Cells; Humans; Isoenzymes; Luciferases; Membrane Proteins; Phosphatidylinositol 3-Kinases; Promoter Regions, Genetic; Prostaglandin-Endoperoxide Synthases; Receptor, Cholecystokinin B; Receptors, Prostaglandin; Reverse Transcriptase Polymerase Chain Reaction; RNA; Signal Transduction

To explore the correlation between expression of somatostatin (SS), gastrin (GAS) and cell apoptosis regulation gene bcl-2/bax in large intestine carcinoma.. Sixty-two large intestine cancer tissue samples were randomly and retrospectively selected from patients with large intestine carcinoma. Immunohistochemical staining for bcl-2, bax, GAS, SS was performed according to the standard streptavidin-biotin-peroxidase (S-P) method. According to the semi-quantitative integral evaluation, SS and GAS were divided into three groups as follows. Scores 1-3 were defined as the low expression group, 4-8 as the intermediate expression group, 9-16 as the high expression group. Bax and bcl-2 protein expressions in different GAS and SS expression groups of large intestine carcinoma were assessed.. The positive expression rate of bax had a prominent difference between SS and GAS high, intermediate and low expression groups (P<0.05, chi(2)(SS) = 9.246; P<0.05, chi(2)(GAS) = 6.981). The positive expression rate of bax in SS high (80.0%, 8/10) and intermediate (76.5%, 13/17) expression groups was higher than that in low expression group (40.0%, 14/35) (P<0.05, chi(2)( high vs low ) = 5.242; P<0.05, chi(2)( middle vs low ) = 6.097). The positive expression rate of bax in GAS high expression group (27.3%, 3/8) was lower than that in low expression group (69.4%, 25/36) (P<0.05, chi(2) = 4.594). However, bax expression in GAS intermediate expression group (46.7%, 7/15) was lower than that in low expression group, but not statistically significant. The positive expression rate of bcl-2 had a prominent difference between SS and GAS high, intermediate and low expression groups (P<0.05, chi(2)(SS) = 7.178; P<0.05, chi(2)( GAS ) = 13.831). The positive expression rate of bcl-2 in GAS high (90.9%, 10/11) and intermediate (86.7%, 13/15) expression groups was higher than that in low expression group (44.4%, 16/36) (P<0.05, chi(2)( high vs low ) = 5.600; P<0.05, chi(2)( middle vs low ) = 7.695). However, the positive expression rate of bcl-2 in SS high (40.0%, 4/10) and intermediate (47.1%, 8/9) expression groups was lower than that in low expression group (77.1%, 27/35) (P<0.05, chi(2)( high vs low ) = 4.710; P<0.05, chi(2)( middle vs low ) = 4.706). There was a significant positive correlation between the integral ratio of GAS to SS and the integral of bcl-2 (P<0.01, r = 0.340). However, there was a negative correlation between the integral ratio of GAS to the SS and bax the integral of (P<0.05, r = -0.299).. The regulation and control of gastrin, somatostatin in cell apoptosis of large intestine carcinoma may be directly related to the abnormal expression of bcl-2, bax.

Topics: Adult; Aged; Apoptosis; bcl-2-Associated X Protein; Colon; Colonic Neoplasms; Female; Gastrins; Humans; Immunohistochemistry; Male; Middle Aged; Proto-Oncogene Proteins c-bcl-2; Somatostatin

Gastrin and cholecystokinin (CCK) have trophic action on cells expressing wild type A or B CCK receptors. Potential relevance to pancreatic and colonic cancers was raised by the demonstration of a misspliced type B CCK receptor that, when expressed in Balb3T3 cells, had constitutive activity to stimulate intracellular calcium. We attempted to confirm and extend this observation in CHO cells by establishing lines expressing similar densities of variant or wild type B CCK receptor. While both were capable of normal binding and agonist-induced signaling, neither expressed constitutive signaling and both had similar basal growth. Agonist stimulation of cells expressing misspliced receptor had greater increases in calcium and greater growth rates than control cells despite similar MAP kinase phosphorylation responses. Thus, this variant receptor can potentiate peptide-stimulated signaling and trophic action and may contribute to the proliferation of neoplasms expressing it.

Topics: Alternative Splicing; Animals; Binding, Competitive; Cell Proliferation; CHO Cells; Cholecystokinin; Colonic Neoplasms; Cricetinae; Cricetulus; Gastrins; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mutation; Pancreatic Neoplasms; Phosphorylation; Receptor, Cholecystokinin B; RNA, Messenger; Transfection

The authors recently reported that gastrin gene knockout (GAS-KO) mice had an increased risk for colon carcinogenesis in response to azoxymethane (AOM) compared with their wild type (WT) littermates. In the current report, the authors discuss the predisposition of GAS-KO mice to develop obesity and metabolic hormonal changes that may contribute to their increased risk of colon carcinogenesis.. The weight and deposition of fat was monitored in the mice over a 14 month period, using magnetic resonance imaging and nuclear magnetic resonance techniques. Changes in plasma concentrations of ghrelin, leptin, insulin, and glucose were assessed using radioimmunoassay analysis and enzyme-linked immunosorbent assays. Preneoplastic markers of colon carcinogenesis (aberrant crypt foci [ACFs]), in response to AOM, were measured in a subset of obese versus lean GAS-KO mice and were compared with the markers in WT mice.. Increases in visceral adiposity were evident by age 2 months in GAS-KO mice, resulting in macroscopic obesity by age 7 months. Hyperinsulinemia and insulin:glucose ratios were increased significantly in GAS-KO mice as young as 1 month and preceded alterations in nonfasting leptin and ghrelin levels. The number of ACFs per mouse colon were increased significantly in the following order: obese GAS-KO mice > lean GAS-KO mice > WT mice. Fasting plasma insulin levels were 0.88 +/- 0.1 ng/mL, 1.45 +/- 0.3, and 2.76 +/- 0.9 ng/mL in the WT, GAS-KO lean, and GAS-KO obese mice, respectively.. The current results suggest the novel possibility that loss of amidated gastrins may increase adipogenesis, hyperinsulinemia, and colon carcinogenesis in GAS-KO mice. The increase in colon carcinogenesis may be due in part to hyperinsulinemia, increased obesity, and other associated hormone changes that were measured in GAS-KO mice.

Topics: Animals; Azoxymethane; Body Weight; Carcinogens; Colonic Neoplasms; Gastrins; Gene Expression; Ghrelin; Glucose; Hyperinsulinism; Insulin; Insulin Resistance; Leptin; Magnetic Resonance Imaging; Male; Mice; Mice, Knockout; Obesity; Peptide Hormones; Precancerous Conditions; Radioimmunoassay; Thinness

G17-Gly has been shown to stimulate the growth of DLD-1 human colon cancer cells in a biphasic manner via high and low affinity receptors. In the current study, the existence of heterogeneous receptor populations for G17-Gly on the HT-29 human colon cancer cell line was investigated. The effect of either N- or C-terminal peptide truncation on receptor binding and cell growth stimulation was also explored. [Leu15]G17-Gly bound to both high (nM) and low (microM) affinity sites on HT-29 cells. The peptide stimulated cell growth in a dose-dependent and biphasic manner with maximal stimulation at 10(-9) M peptide concentration, suggesting that, as in the case of DLD-1 cells, it is the high affinity receptor which is responsible for the growth-promoting effects. In contrast, G17(1-12) stimulated the growth of HT-29 cells in a sigmoidal fashion with an EC50 of 4.6x10(-9) M. Sequential N-terminal truncation of [Leu15]G17-Gly results in decreased binding to the high affinity G17-Gly receptor on DLD-1 cells. [Leu15]G17(11-17)Gly bound to the low affinity G17-Gly receptor with an affinity similar to that of the full sequence peptide but was unable to displace the radioligand from high affinity sites. G17(1-6)-NH2 was unable to displace [3H]G17-Gly from either site. These results suggest that the important residues for binding to the low affinity receptor are in the C-terminal region of the peptide while those required for interaction with the high affinity receptor lie further towards the N-terminus.

Topics: Amino Acid Sequence; Binding Sites; Binding, Competitive; Cell Membrane; Colonic Neoplasms; Gastrins; HT29 Cells; Humans; Molecular Sequence Data; Peptides; Protein Binding; Receptor, Cholecystokinin B

Although expression of the gastrin/cholecystokinin-2 receptor (CCK2R) is widely reported in human colorectal cancer, little is known on its role in mediating mature amidated gastrin (gastrin-17 amide, G-17) induced intracellular signal transduction in colon cancer cells. The purpose of this study was to explore the intracellular events of colorectal cancer cells after gastrin binding to CCK2R. Meanwhile, the influence of a natural point mutation 286V-->F in the third intracellular loop of CCK2R on gastrin-envoked intracellular signal transduction was also investigated. Firstly, Colo320 cells were stably transfected with wild type (Colo320 WT) and mutant CCK2R (Colo320 M), respectively. The intracellular signal transduction events in response to gastrin were investigated in both Colo320 WT and Colo320 M cells. In Colo320 WT cells, G-17 induced formation of intracellular cyclic AMP and inositol 1,4,5-trisphosphate, and stimulated intracellular calcium mobilization. G-17 also stimulated tyrosine phosphorylation of ERKl/2, p38, FAK, and paxillin, and up-regulated the mRNA expression of early response gene c-Jun and c-Fos. However, G-17 inhibited proliferation and induced apoptosis in Colo320 WT cells. Mutation 286V-->F in the third intracellular loop of CCK2R blocked G-17 induced biological without affecting binding affinity of CCK2R to G-17. Our results suggest that activation of CCK2R by gastrin stimulates heterotrimeric G-protein Gq and G(12/13) mediated intracellular signal transduction pathway in colon cancer cells. The valine-287 residue in third intracellular loop of CCK2R plays a pivotal role in CCK2R mediated intracellular signal transduction.

Topics: Apoptosis; Cell Line, Tumor; Cell Proliferation; Colonic Neoplasms; Cyclic AMP; Focal Adhesion Kinase 1; Gastrins; Genes, Immediate-Early; Humans; Inositol 1,4,5-Trisphosphate; Paxillin; Phosphorylation; Point Mutation; Protein Structure, Secondary; Receptor, Cholecystokinin B; RNA, Messenger; Signal Transduction; Transfection; Tyrosine; Up-Regulation; Valine

The study investigated the effect of gastrin on tyrosine phosphorylation and protein expression of focal adhesion kinase (FAK) in human colon cancer cells.. The eukaryotic plasmid that expresses cholecystokinin 2 receptor (CCK2R) stably, named pCR3.1/CCK2R, was transfected into Colo320 to construct an up-regulated gastrin-CCK2R signal pathway; the gastrin antagonist was used to down-regulate the signal pathway. Thus, including the normal control, there were three signal pathways with different CCK2R levels. Different doses of gastrin were used to stimulate FAK in the different time. FAK tyrosine phosphorylation and FAK expression in different groups were detected by immunoprecipitation and Western-blotting assays, and analyzed with Labimage software.. RT-PCR result showed that Colo320 transfected with CCK2R had a mRNA level four times higher than that normal Colo320 did, and which suggested that up-regulated signal transduction pathway was constructed. After stimulated by gastrin with 0, 0.1, 1, 10 and 100 nmol/L, tyrosine phosphorylation levels of Colo320 were 24.0%, 39.7%, 46.2%, 50.4% and 44.5%, and those of Colo320 transfected with CCK2R were 24.6%, 70.7%, 90.1%, 100% and 88.6%. When incubated with gastrin at 0, 2.5, 5, 10 and 20 min, the tyrosine phosphorylation levels of Colo320 were 23.9%, 63.6%, 58.6%, 45.5% and 40.9%, and those of Colo320 transfected with CCK2R were 24.5%, 84.6%, 100%, 98.6% and 97.9%. The increases of tyrosine phosphorylation in both Colo320 and Colo320 transfected with CCK2R were dose dependence of gastrin. In Colo320, the time of phosphorylation had a tendency of exhaustion at 2.5 min; but in Colo320 transfected with CCK2R, it was at 10 min. Gastrin had no effects on FAK protein expression in different cell groups. An up-regulated level of CCK2R could enhance the effect of gastrin on FAK tyrosine phosphorylation. The gastrin antagonist showed an effect of competitive inhibition on tyrosine phosphorylation of FAK.. FAK is a signal transducer in downstream of CCK(2)R; FAK exerts its functions by tyrosine phosphorylation, but dose not increase FAK protein. Gastrin-CCK2R-FAK signal pathway is a pivotal one in the cell growth and proliferation caused by gastrin.

Topics: Cell Line, Tumor; Cell Proliferation; Colonic Neoplasms; Dose-Response Relationship, Drug; Down-Regulation; Focal Adhesion Protein-Tyrosine Kinases; Gastrins; Humans; Phosphorylation; Receptor, Cholecystokinin B; RNA, Messenger; Signal Transduction; Tyrosine; Up-Regulation

The role of amidated gastrin17 (G17) and the gastrin/CCKB/CCK2 receptor in colorectal carcinogenesis is still a controversial issue. Here, we investigated the effect of G17 on proliferation and apoptosis of CCK2 receptor-expressing human colon cancer cell lines in vitro and in vivo.. Proliferation was determined by cell counting and cell cycle analysis. Apoptosis was analyzed by annexin V staining, TUNEL staining, caspase-3/7 assay, and JC1 (delta psi) assay. Signal-transduction pathways were analyzed by Western blotting and gel-shift and luciferase assays. An in vivo tumor model with subcutaneously inoculated colon cancer cells in SCID mice was used, and systemic hypergastrinemia was induced by omeprazole.. In Colo320 cells stably transfected with the wild-type CCK2 receptor (Colo320wt) or in Lovo cells endogenously expressing CCK2 receptors, G17 treatment inhibited proliferation along with a G2/M cell cycle arrest. Furthermore, the administration of G17 significantly augmented apoptosis of CCK2 receptor-expressing cells. In contrast, G17 had no effect on proliferation and apoptosis in Colo320 cells stably transfected with a tumor-derived CCK2 receptor mutant (Colo320mut) or in cells lacking CCK2 receptor expression. Systemic hypergastrinemia in severe combined immunodeficiency (SCID) mice suppressed the growth of Colo320wt tumors accompanied by enhanced apoptosis as compared with untreated tumors. In contrast, omeprazole did not affect Colo320mut tumors reflecting a loss-of-function state of the CCK2(mut) receptor. This is supported by the observation that, in Colo320wt cells, but not in Colo320mut cells, G17 treatment induced the MAPK/ERK/AP-1 pathway and inhibited the activity of NF-kappaB.. G17 exerts an antiproliferative and proapoptotic effect on human colon cancer cells expressing the wild-type CCK2 receptor. This supports the view that amidated gastrin prevents rather than promotes colorectal carcinogenesis.

Topics: Animals; Apoptosis; Cell Division; Cell Line, Tumor; Cell Survival; Colonic Neoplasms; Gastrins; Gene Expression Regulation, Neoplastic; Humans; Mice; Mitochondria; Receptor, Cholecystokinin B; Reverse Transcriptase Polymerase Chain Reaction; RNA, Neoplasm; Transfection

Although the expression of CCK(2) receptors is widely reported in human colorectal cancers, little is known on its role in mediating the proliferative effects of mature amidated gastrin (G17 amide) on colorectal cancers. The purpose of the present study was to determine the effects of G17 amide on tyrosine phosphorylation of focal adhesion kinase (FAK), paxillin, and p130 Crk-associated substrate (p130(Cas)) in Colo 320 cells, a human colorectal cancer cell line which expresses CCK(2) receptors. By immunoprecipitation and immunoblotting, an increase in tyrosine phosphorylation of FAK (tyrosine-397), paxillin (tyrosine-31), and p130(Cas) was detected in a time- and dose-dependent manner. Overexpression of CCK(2) receptors in Colo 320 cells (Colo 320 WT) by stable transfection with the human CCK(2) receptor cDNA resulted in an increased tyrosine phosphorylation of FAK, paxillin, and p130(Cas). After incubation with 1 microM L-365,260, a specific CCK(2) receptor antagonist, this increase was completely inhibited. Our results demonstrate that in human colon cancer cells, gastrin caused a rapid tyrosine phosphorylation of FAK, paxillin, and p130(Cas) by activation of CCK(2) receptor. The phosphorylation of these proteins might be important in mediating gastrin effects on proliferation, apoptosis, and metastasis.

Topics: Colonic Neoplasms; Crk-Associated Substrate Protein; Cytoskeletal Proteins; Densitometry; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; Gastrins; Humans; Paxillin; Phosphoproteins; Phosphorylation; Protein-Tyrosine Kinases; Proteins; Receptor, Cholecystokinin B; Retinoblastoma-Like Protein p130; Tumor Cells, Cultured; Tyrosine

The authors recently reported that transgenic mice (hGAS) expressing pharmacologic levels of progastrin (PG) (> 10 nM to 100 nM) exhibited increased susceptibility to colon carcinogenesis in response to azoxymethane (AOM). It is not known whether PG functions as a cocarcinogen at the concentrations observed in patients with hypergastrinemia (approximately 1.0 nM).. The authors generated transgenic mice that overexpressed either wild-type (wtPG) or mutant (mtPG) human PG in the intestinal mucosa using the murine fatty acid binding protein (Fabp) promoter. Fabp-PG mice and their wild-type littermates were treated with AOM, and their colons were examined for preneoplastic (aberrant crypt foci [ACF]) and neoplastic (adenomas [Ads] and adenocarcinomas [AdCas]) lesions after 2 weeks and 6 months of treatment.. ACF and tumors were significantly more common (by a factor of approximately 2) in colon specimens from both Fabp-wtPG mice and Fabp-mtPG mice relative to wild-type mice. It is noteworthy that the multiplicity of ACF and the total number of small and large Ads and AdCas were significantly greater in colon specimens from Fabp-PG mice compared with colon specimens from wild-type mice, irrespective of gender.. The results of the current study suggest that at concentrations (approximately 1.0 nM) far lower than the ones observed in hGAS mice, PG functions as an equally potent cocarcinogen and significantly increases the risk of colon carcinogenesis in response to AOM. Thus, PG may represent a clinically relevant target molecule in patients with hypergastrinemia or colon carcinoma.

Topics: Animals; Azoxymethane; Carcinogens; Colonic Neoplasms; Female; Gastrins; Humans; Male; Mice; Mice, Transgenic; Polymerase Chain Reaction; Precancerous Conditions; Protein Precursors; Radioimmunoassay

The effects of glycine-extended gastrin (G-Gly) on the invasion by colon cancer cells through stromal extracellular matrix and the role of metalloproteinases (MMPs) in this invasion were investigated. We found that 10(-9)-10(-6) M G-Gly significantly increased the invasiveness of 2 human colon cancer cell lines, LoVo and HT-29, both expressing the G-Gly-specific binding site but little gastrin/CCK-B receptor (gastrin receptor). LoVo cells expressed MMP-1, -2, -3 and -9. An amount of 10(-7) M G-Gly enhanced collagenase MMP-1 expression. Overexpression of enhanced green fluorescent protein (EGFP)-fused MMP-1 in LoVo cells, by cDNA transfection, enhanced invasiveness through type I collagen gel. Immunofluorescence study revealed that G-Gly increased the number of cytoplasmic vesicles containing MMP-1, some vesicles being released from the cells. The MMP-1 vesicles contained one of the ubiquitous coat proteins, Golgi-localized, gamma-adaptin ear-containing, ARF-binding proteins-2 (GGA-2). MMP-1 also colocalized with CD147 (EMMPRIN, an extracellular matrix metalloproteinase inducer in adjacent stromal cells). It was suggested that G-Gly increased the number of vesicles containing MMP-1 and that MMP-1 interacted with CD147 to increase invasion. G-Gly significantly enhanced the production of MMP-3, an activator of MMP-1 and -9, as well as gelatinase MMP-9 activity. The G-Gly-mediated MMP-9 increase was inhibited by treatment with anti-MMP-3 IgG and MMP-3 siRNA. Furthermore, G-Gly increased the proMMP-2 level, although no activated MMP-2 was found in conditioned medium in either the presence or the absence of G-Gly. By contrast, gastrin (10(-7) M) had no effect on the levels of these MMPs or the invasiveness of colon cancer cells in type I collagen gel and Matrigel. These effects of G-Gly on the activity and expression of MMPs and the invasiveness of colon cancer cells were inhibited by treating the cells with a broad-spectrum metalloproteinase inhibitor (CGS27023A) and nonselective gastrin/CCK receptor antagonists (proglumide and benzotript). But a gastrin/CCK-B receptor antagonist (YM022) did not inhibit the increased invasion by G-Gly. Together, these results demonstrate that G-Gly renders colon cancer cells more invasive by increasing MMP-1 and MMP-3 expressions via the putative G-Gly receptor and would thus be a good molecular target in a clinical setting.

Topics: Biocompatible Materials; Collagen; Collagen Type I; Colonic Neoplasms; DNA, Complementary; Drug Combinations; Gastrins; Gels; Humans; Laminin; Matrix Metalloproteinase 1; Matrix Metalloproteinase 3; Neoplasm Invasiveness; Proteoglycans; Transfection; Tumor Cells, Cultured

We recently reported that downregulation of gastrin gene expression in colon cancer cells significantly suppresses relative levels of mitochondrial cytochrome c (cyt c) oxidase Vb (Cox Vb) RNA and protein. These unexpected findings suggested the possibility that gastrin gene products [mainly progastrin (PG)] may be directly or indirectly mediating the observed effects in colon cancer cells. Because colon cancer cells do not respond to exogenous PG, we examined the possibility of whether PG regulates Cox Vb expression in gastrin-responsive intestinal epithelial cells (IECs) in vitro. Levels of Cox Vb RNA and protein were significantly increased in a dose-dependent manner in response to PG. Mitochondrial synthesis of ATP was also increased by approximately three- to fivefold in response to optimal concentrations (0.1-1.0 nm) of PG. Possible antiapoptotic effects of PG were additionally examined, because activation of caspases 9 and 3 had been noted in colon cancer cells downregulated for gastrin gene expression. We measured a significant loss in the levels of cyt c in the cytosol of PG-treated vs. control IEC cells, which correlated with a significant loss in the activation of caspases 9 and 3, resulting in a significant loss in DNA fragmentation on PG treatment of the cells. Our results thus suggest the novel possibility that the precursor PG peptide exerts direct antiapoptotic effects on IECs, which may contribute to the observed growth effects of PG on these cells. Additionally, Cox Vb gene appears to be an important intracellular target of PG, resulting in an increase in ATP levels, which may also contribute to the observed increase in the growth of target cells in response to PG.

Topics: Adenosine Triphosphate; Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Camptothecin; Caspase 3; Caspase 9; Caspases; Cell Line; Colonic Neoplasms; Electron Transport Complex IV; Enzyme Activation; Gastrins; Intestinal Mucosa; Mitochondria; Promoter Regions, Genetic; Protein Precursors; Rats; RNA, Messenger; Up-Regulation

Growth hormone (GH), Insulin-like growth factor-I (somatomedine, IGF-I) and gastrin seem to play a significant role in cell proliferation in mammalian and rat cells. The role of these factors in the etiology of gastric and large bowel cancer has not been completely elucidated. The aim of this study was to concurrently estimate the levels of GH, IGF-I and gastrin in a group of patients with gastric and colorectal cancer and to compare the results with those of a group of normal controls.. In 33 consecutive patients with gastric (16 patients) and large bowel (17 patients) cancer, the serum levels of GH, IGF-I and gastrin were measured by radioimmunoassay. Fifty-four normal people were served as controls.. Significantly higher levels of serum GH (3.16 +/- 3.12 ng/ml in gastric cancer patients vs. 3.01 +/- 2.91 ng/ml in colorectal cancer patients vs. 0.69 +/- 1.60 ng/ml in normal controls, adjusted P<0.001) and gastrin (98.2 +/- 87.9 pg/ml in gastric cancer patients vs. 95.3 +/- 85.4 pg/ml in colorectal cancer patients, vs. 47.5 +/- 32.4 pg/ml in normal controls, adjusted P<0.035 and <0.05 respectively) were found in both groups of patients compared with normal controls. The levels of IGF-I in patients with gastric and colorectal cancer although higher compared to normal controls did not reach statistical significance. (98.2 +/- 87.9 pg/ml vs. 95.3 +/- 85.4 vs. 47.5 +/- 32.4 respectively) (adjusted P=0.070).. It is concluded that in patients with gastric and colorectal cancer a significant increase of serum GH and gastrin can be found. This increase is likely to play a role in gastric and colorectal carcinogenesis.

Topics: Aged; Animals; Case-Control Studies; Colonic Neoplasms; Fasting; Female; Gastrins; Human Growth Hormone; Humans; Insulin-Like Growth Factor Binding Protein 1; Male; Middle Aged; Radioimmunoassay; Rats; Stomach Neoplasms

We recently reported that transgenic mice overexpressing progastrin were at a higher risk for developing colon cancers in response to azoxymethane (AOM), whereas mice overexpressing gastrin-17 were at a reduced risk. To examine further the role of gastrins in colon carcinogenesis, we generated gastrin gene knockout mice (GAS-KO).. The height and proliferative index (PI) of colonic crypts were similar in GAS-KO and wild-type (WT) mice, suggesting that the absence of gastrins in GAS-KO mice did not significantly affect the growth of colonic mucosa. GAS-KO and WT mice were treated with AOM for 3-4 weeks; control mice received saline.. Colonic proliferation in response to AOM was significantly increased in GAS-KO vs. WT mice. Aberrant crypt foci (ACFs) were similarly increased significantly by approximately 2-5-fold in GAS-KO vs. WT mice after 2 weeks of AOM treatment. Female GAS-KO mice developed adenomas (Ads) and adenocarcinomas (AdCAs) at earlier times ( approximately 10 months) than the male GAS-KO mice and the male and female WT mice ( approximately 12 months). The total numbers of Ads and AdCAs were significantly higher in GAS-KO than in WT mice.. These results suggest the novel possibility that loss of gastrin expression (and hence amidated gastrins) significantly increases susceptibility to colon carcinogenesis in response to AOM. Previous studies with FVB/N transgenic mice similarly suggested a protective role of amidated gastrins against colon carcinogenesis, which supports the present findings of an increase in colon carcinogenesis in GAS-KO mice lacking normal physiological levels of amidated gastrins.

Topics: Animals; Azoxymethane; Colonic Neoplasms; Female; Gastrins; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Precancerous Conditions

The role of hyper-gastrinaemia in the incidence of colonic cancer remains to be clarified. The aim of this study was to determine whether cholecystokinin-2 (CCK-2) receptor expression predicts the sensitivity of human colonic adenomas to the proliferative effects of serum hyper-gastrinaemia. Gene expression of the classical (74 kDa) CCK-2 receptor in human colonic adenoma specimens and cell lines, was quantified by real-time PCR. Western blotting, using a CCK-2 receptor antiserum, confirmed protein expression. A transformed human colonic adenoma was grown in SCID mice, with hyper-gastrinaemia induced by proton pump inhibitors. CCK-2 receptor blockade was achieved by using neutralising antiserum. Both human colonic adenoma cell lines and biopsies expressed CCK-2 receptor mRNA at levels comparable with CCK-2 receptor transfected fibroblasts and oxyntic mucosa. Western blotting confirmed immunoreactive CCK-2 receptor bands localised to 45, 74 and 82.5 kDa. Omeprazole and lansoprazole-induced hyper-gastrinaemia (resulting in serum gastrin levels of 34.0 and 153.0 pM, respectively) significantly increased the weight of the human adenoma grafts (43% (P=0.016) and 70% (P=0.014), respectively). The effect of hypergastrinaemia on tumour growth was reversed by use of antiserum directed against the CCK-2 receptor. Hyper-gastrinaemia may promote proliferation of human colonic adenomas that express CCK-2 receptor isoforms.

Topics: Adenocarcinoma; Adenoma; Animals; Colonic Neoplasms; Computer Systems; Enzyme Inhibitors; Female; Fibroblasts; Gastric Mucosa; Gastrins; Gene Expression Regulation, Neoplastic; Humans; Introns; Male; Mice; Mice, SCID; Neoplasm Proteins; Neoplasm Transplantation; Omeprazole; Parietal Cells, Gastric; Polymerase Chain Reaction; Protein Isoforms; Protein Processing, Post-Translational; Proton Pump Inhibitors; Receptor, Cholecystokinin B; Receptors, Cholecystokinin; RNA, Messenger; Secretory Rate; Tumor Cells, Cultured

Gastrin is a gastrointestinal peptide that possesses potent trophic properties on both normal and neoplastic cells of gastrointestinal origin. Previous studies have indicated that chronic hypergastrinaemia increases the risk of colorectal cancer and cancer growth and that interruption of the effects of gastrin could be a potential target in the treatment of colorectal cancer. Here we demonstrate that gastrin leads to a dose-dependent increase in colon cancer cell proliferation and tumour growth in vitro and in vivo, and that this increment is progressively reversed by pretreatment with the cyclo-oxygenase-2 inhibitor NS-398. Gastrin was able to induce cyclo-oxygenase-2 protein expression, as well as the synthesis of prostaglandin E2, the major product of cyclo-oxygenase. Moreover, gastrin leads to approximately a two-fold induction of cyclo-oxygenase-2 promoter activity in transiently transfected cells. The results of these studies demonstrate that cyclo-oxygenase-2 appears to represent one of the downstream targets of gastrin and that selective cyclo-oxygenase-2 inhibition is capable of reversing the trophic properties of gastrin and presumably might prevent the growth of colorectal cancer induced by hypergastrinaemia.

Topics: Adenocarcinoma; Animals; Cell Division; Colonic Neoplasms; Cyclin D1; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Cyclooxygenase Inhibitors; Dinoprostone; DNA Replication; Dose-Response Relationship, Drug; Gastrins; Gene Expression Regulation, Neoplastic; Genes, Reporter; Isoenzymes; Male; Mice; Mice, Inbred BALB C; Neoplasm Proteins; Neoplasm Transplantation; Nitrobenzenes; Proliferating Cell Nuclear Antigen; Promoter Regions, Genetic; Prostaglandin-Endoperoxide Synthases; Receptors, Cholecystokinin; Substrate Specificity; Sulfonamides; Transfection; Tumor Cells, Cultured

New compounds, named nuclear aggregates of polyamines, having a molecular mass of 8000, 4800 and < 1000 Da, were found in the nuclear extracts of several replicating cells. Their molecular structure is based on the formation of ionic bonds between polyamine ammonium and phosphate groups. The production of the 4800 Da compound, resulting from the aggregation of five or more < 1000 Da units, was increased in Caco-2 cells treated with the mitogen gastrin. Dissolving single polyamines in phosphate buffer resulted in the in vitro aggregation of polyamines with the formation of compounds with molecular masses identical to those of natural aggregates. After the interaction of the 4800 Da molecular aggregate with the genomic DNA at 37 degrees C, both the absorbance of DNA in phosphate buffer and the DNA mobility in agarose gel increased greatly. Furthermore, these compounds were able to protect the genomic DNA from digestion by DNase I, a phosphodiesterasic endonuclease. Our data indicate that the nuclear aggregate of polyamines interacts with DNA phosphate groups and influence, more efficaciously than single polyamines, both the conformation and the protection of the DNA.

Topics: Adenocarcinoma; Amino Acids; Caco-2 Cells; Cell Division; Cell Nucleus; Chromatography, High Pressure Liquid; Circular Dichroism; Colonic Neoplasms; DNA; DNA Fragmentation; Electrophoresis, Polyacrylamide Gel; Gastrins; Humans; Magnetic Resonance Spectroscopy; Nucleosomes; Osmolar Concentration; Polyamines; Spectrophotometry

Colorectal cancers express significant amounts of immature glycine-extended gastrin (G-Gly) and G-Gly is able to stimulate cell proliferation in colonic cell lines and mucosa. Here we wished to investigate whether G17-Gly promote the invasiveness of LoVo human colonic cancer cells, a process which requires degradation of extracellular matrix by proteases and concomitant induction of cell migration. We confirmed that LoVo cells express gastrin and gastrin/CCK-B receptor mRNAs. We showed that these cells secrete matrix metalloproteinase (MMP)-1, -2, and -9. The function of MMP being to degrade components of extracellular matrix, they may thus favor cell migration. As compared to controls, G17-Gly (10(-7) to 10(-12) M) significantly enhanced about two to three times the LoVo cell migration through Matrigel, an artificial basement matrix barrier. Moreover, G17-Gly increased and gastrin/CCK-B receptor antagonists decreased MMP secretion in conditioned culture media of LoVo cells. Our findings show that physiological doses of incompletely processed form of gastrin induce the invasiveness of tumor cells in vitro and suggest a novel potential role for this peptide in the metastatic process of colonic cancers in vivo.

Topics: Adenocarcinoma; Base Sequence; Colonic Neoplasms; Culture Media, Conditioned; DNA Primers; Gastrins; Humans; Matrix Metalloproteinases; Neoplasm Invasiveness; Receptor, Cholecystokinin B; Receptors, Cholecystokinin; Tumor Cells, Cultured

Evidence is accumulating that gastrin precursors may act as growth factors for the colonic mucosa in vivo and for colorectal carcinoma cell lines in vitro. The effect of short term administration of synthetic gastrins on the colonic mucosa in vivo, however, has not been reported. The aim of our study was to determine whether continuous systemic infusion of glycine-extended gastrin(17) stimulated proliferation and accelerated carcinogenesis in the colorectal mucosa. A significant increase in colonic mucosal proliferation as assessed by metaphase index was seen in the caecum (23%, p < 0.02) and distal colon (27%, p < 0.001), but not the rectum, after treatment of intact rats with glycine-extended gastrin(17) for 1 week using implanted miniosmotic pumps. Defunctioning of the rectum reduced both the proliferative index and crypt height of the rectal mucosa of untreated rats. Treatment of rectally defunctioned animals with glycine-extended gastrin(17) for either 1 or 4 weeks resulted in a significant increase in both the proliferative index (40% and 93%, respectively) and crypt height (11% and 19%, respectively) of the rectal mucosa. The total number of aberrant crypt foci in intact rats treated with the procarcinogen azoxymethane plus glycine-extended gastrin(17) was increased by 48% compared to the value in controls treated with azoxymethane only (p = 0.01). We conclude that short term administration of glycine-extended gastrin(17) to mature rats not only has a proliferative effect upon colonic mucosa, but also increases the number of aberrant crypt foci formed in the colorectal mucosa after treatment with azoxymethane. Glycine-extended gastrin(17) could thus potentially act as a promoter of carcinogenesis.

Topics: Animals; Azoxymethane; Carcinogens; Cell Division; Colon; Colonic Neoplasms; Gastrins; Glycine; Hormones; Male; Mucous Membrane; Precancerous Conditions; Rats; Rats, Sprague-Dawley; Time Factors

Recent studies show that nonamidated gastrins (Gly-gastrin and progastrin) stimulate colonic proliferation. However, the role of nonamidated vs. amidated gastrins in colon carcinogenesis has not been defined. We measured intermediate markers of carcinogenesis in transgenic mice overexpressing either progastrin (hGAS) or amidated gastrin (INS-GAS) in response to azoxymethane (AOM). The hGAS mice showed significantly higher numbers of aberrant crypt foci (140-200% increase) compared with that in wild-type (WT) and INS-GAS mice (P < 0.05) after AOM treatment. The bromodeoxyuridine-labeling index of colonic crypts also was significantly elevated in hGAS mice vs. that in WT and INS-GAS mice. The results therefore provide evidence for a mitogenic and cocarcinogenic role of nonamidated gastrins (progastrin), which is apparently not shared by the amidated gastrins. Although nonamidated gastrins are now believed to mediate mitogenic effects via novel receptors, amidated gastrins mediate biological effects via different receptor subtypes, which may explain the difference in the cocarcinogenic potential of nonamidated vs. amidated gastrins. In conclusion, our results provide strong support for a cocarcinogenic role for nonamidated gastrins in colon carcinogenesis.

Topics: Animals; Azoxymethane; Bromodeoxyuridine; Carcinogens; Colon; Colonic Neoplasms; Disease Susceptibility; Gastrins; Humans; Mice; Mice, Inbred Strains; Mice, Transgenic; Precancerous Conditions; Protein Precursors

Processing intermediates of preprogastrin (gly-gastrin and progastrin), termed nonamidated gastrins, are mitogenic for several cell types including colonic epithelial cells. However, presently it is not known if nonamidated gastrins play a role in colon carcinogenesis and if the effects are similar to those of amidated gastrins.. Colon carcinogenesis in response to azoxymethane (AOM) was examined in transgenic mice overexpressing either progastrin (hGAS) or amidated gastrin (INS-GAS), compared with that in wild-type (WT) mice.. In AOM-treated groups, the total number of tumors per colon was significantly higher in hGAS (4.8+/-0.34) than INS-GAS (3.0+/-0.16) and WT (2.7+/-0.35) mice. Total numbers of adenocarcinomas and adenomas per animal colon were also significantly higher in hGAS than INS-GAS and WT mice. The size of the tumors was greater in hGAS mice, resulting in a significantly higher tumor burden per mouse in the hGAS mice than INS-GAS and WT mice. Although >90% of the tumors were located in the distal half of the colon in INS-GAS and WT mice, a significant number (42%) were present at the proximal end of the colon in hGAS mice.. The results suggest that the risk for developing colon carcinomas and adenomas in response to AOM is significantly increased in mice expressing high levels of progastrin, but not amidated gastrins.

Topics: Adenoma; Amides; Animals; Azoxymethane; Carcinogens; Carcinoma; Colonic Neoplasms; Gastrins; Incidence; Mice; Mice, Transgenic; Neoplasms, Multiple Primary; Protein Precursors; Reference Values; Survival Analysis

Suppression of the gastrin gene in human colon cancer cells by stably expressing antisense (AS) gastrin RNA results in significant growth suppression of AS cells. To understand mechanisms mediating the growth effects of autocrine gastrins, differential expression of transcripts by AS and control (C) clones of a representative cell line (HCT-116) was analyzed to identify target genes of autocrine gastrins. Six differentially expressed transcripts were confirmed and sequenced. Of these, the RNA and protein levels of cytochrome c oxidase (COX) Vb were significantly higher in C versus AS cells. The expression of COX Vb by colon cancer cells was proportional to the expression of gastrin. Higher levels of COX Vb coprecipitated with cytochrome c in the mitochondria of C versus AS cells. Treatment of mitochondria with digitonin resulted in a 2-fold higher release of cytochrome c from AS versus C mitochondria. As a corollary, the cytosolic levels of cytochrome c were significantly higher in AS versus C cells, which correlated with approximately 2- and approximately 3-fold higher activation of caspase-9 and -3, respectively, in AS versus C cells in response to camptothecin. Thus, autocrine gastrins may support growth/survival of cells by up-regulating COX Vb, which may decrease the sensitivity of the cancer cells to apoptotic stimuli by increasing retention of cytochrome c in mitochondria.

Topics: Colonic Neoplasms; Cytochrome c Group; Cytosol; Electron Transport Complex IV; Gastrins; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Mitochondria; RNA, Antisense; Transcription, Genetic; Tumor Cells, Cultured

Topics: Colonic Neoplasms; Gastrin-Secreting Cells; Gastrins; Humans; Receptor, Cholecystokinin B; Receptors, Cholecystokinin

Gastrin and the cholecystokinin type B/gastrin receptor (CCKBR) have been shown to be expressed in colorectal adenocarcinoma. Both exogenous and autocrine gastrin have been demonstrated to stimulate growth of colorectal cancer but it is not known if gastrin affects the growth of colonic polyps. The purpose of this study was to determine if gastrin and CCKBR are expressed in human colonic polyps and to determine at which stage of progression this occurs.. A range of human colonic polyps was assessed for gastrin and CCKBR gene and protein expression.. Normal colonic mucosa did not express gastrin or CCKBR. Gastrin and CCKBR reverse transcription-polymerase chain reaction products were detected and verified by specific hybridisation with an oligo probe on Southern blots. Gastrin and CCKBR were expressed in 78% and 81% of polyps, respectively. Both genes were coexpressed in 97% of cases. Immunohistochemistry identified progastrin in 91%, glycine extended gastrin 17 in 80%, and amidated gastrin 17 in only 47% of polyps. CCKBR was present in 96% of polyps. Expression of gastrin and CCKBR was seen in all histological types and sizes of polyps.. This study is the first to show widespread expression of both gastrin and its receptor in colorectal polyps. Their activation occurs early in the adenoma-carcinoma sequence. Gastrin may promote progression through the adenoma-carcinoma sequence.

Topics: Adenoma; Aged; Carcinoma; Colonic Neoplasms; Colonic Polyps; Disease Progression; Female; Gastrins; Humans; Immunohistochemistry; Male; Middle Aged; Neoplasm Proteins; Precancerous Conditions; Receptor, Cholecystokinin B; Receptors, Cholecystokinin; Reverse Transcriptase Polymerase Chain Reaction

Since human colon cancers often contain significant quantities of progastrin-processing intermediates, we sought to explore the possibility that the biosynthetic precursor of fully processed amidated gastrin, glycine-extended gastrin, may exert trophic effects on human colonic cancer cells.. Binding of radiolabeled glycine-extended and amidated gastrins was assessed on five human cancer cell lines: LoVo, HT 29, HCT 116, Colo 320DM, and T 84. Trophic actions of the peptides were assessed by increases in [3H]thymidine incorporation and cell number. Gastrin expression was determined by northern blot and radioimmunoassay.. Amidated gastrin did not bind to or stimulate the growth of any of the five cell lines. In contrast, saturable binding of radiolabeled glycine-extended gastrin was seen on LoVo and HT 29 cells that was not inhibited by amidated gastrin (10(-6) M) nor by a gastrin/CCKB receptor antagonist (PD 134308). Glycine-extended gastrin induced a dose-dependent increase in [3H]thymidine uptake in LoVo (143 +/- 8% versus control at 10(-10) M) and HT 29 (151 +/- 11% versus control at 10(-10) M) cells that was not inhibited by PD 134308 or by a mitogen-activated protein (MAP) or ERK kinase (MEK) inhibitor (PD 98509). Glycine-extended gastrin did stimulate jun-kinase activity in LoVo and HT 29 cells. The two cell lines expressed the gastrin gene at low levels and secreted small amounts of amidated gastrin and glycine-extended gastrin into the media.. Glycine-extended gastrin receptors are present on human colon cancer cells that mediate glycine-extended gastrin's trophic effects via a MEK-independent mechanism. This suggests that glycine-extended gastrin and its novel receptors may play a role in colon cancer cell growth.

Topics: Animals; Antineoplastic Agents; Binding Sites; Binding, Competitive; Calcium-Calmodulin-Dependent Protein Kinases; Cell Division; Colonic Neoplasms; DNA-Binding Proteins; Dose-Response Relationship, Drug; Enzyme Inhibitors; ets-Domain Protein Elk-1; Flavonoids; Gastrins; Gene Expression Regulation; Humans; Indoles; JNK Mitogen-Activated Protein Kinases; Meglumine; Mitogen-Activated Protein Kinases; Peptide Fragments; Protein Processing, Post-Translational; Proto-Oncogene Proteins; Rats; Receptors, Cholecystokinin; Recombinant Proteins; Response Elements; Signal Transduction; Tetradecanoylphorbol Acetate; Transcription Factors; Tumor Cells, Cultured

Gastrin is a putative promoter of colorectal carcinomas. The aim of this study was to evaluate the temporal relationship between gastrinemia and development of colorectal malignancy.. We conducted a nested case-control study among 128,992 subscribers to a health maintenance program who had participated in a multiphasic health checkup between 1964 and 1969. Serum had been frozen since the checkup and the cohort followed up for cancer. Of 1881 incident colorectal carcinoma cases, 250 were randomly selected; 1 control without cancer was matched to each case by age, sex, education, and date of serum collection. Stored sera were tested for Helicobacter pylori immunoglobulin G and for gastrin and glycine-extended gastrin.. Verified cases included 166 colon cancers, 58 rectal cancers, and 9 with cancer in both locations. A mean of 15.3 years had elapsed between serum collection and diagnosis of cancer. Median gastrin levels were similar in cases and controls (41.7 vs. 40.7 pg/mL). However, a gastrin level above normal was associated with increased risk for colorectal malignancy (odds ratio, 3.9; 95% confidence interval, 1.5-9.8). If this association is causal, 8.6% of colorectal cancers could be attributed to high serum gastrin level.. Hypergastrinemia is associated with an increased risk of colorectal carcinoma.

Topics: Case-Control Studies; Cohort Studies; Colonic Neoplasms; Colorectal Neoplasms; Female; Gastrins; Humans; Incidence; Male; Middle Aged; Multivariate Analysis; Odds Ratio; Prospective Studies; Rectal Neoplasms; Risk Factors

The expression of gastrin, as a tumor growth factor, is significantly increased in some colon cancers compared with the low levels found in normal mucosa. The aim of this study was to elucidate the transcriptional mechanisms of gastrin induction in colon cancer.. Gastrin messenger (mRNA) levels and K-ras genotype were determined in colon cancer cell lines and surgical specimens. Colon cancer cells were transfected with oncogenic ras expression vectors, and transcriptional activity was assayed with gastrin-luciferase reporter genes.. Colon cancer cell lines and tissues with K-ras mutations all had significantly higher gastrin mRNA levels than those that were ras wild type. Treatment of several ras mutant cell lines with PD98059, an inhibitor of mitogen-activated protein kinase kinase, resulted in a decrease in endogenous gastrin mRNA levels. The effects of ras on gastrin expression appeared to be mediated through the gastrin promoter because transfection of oncogenic ras and activated raf expression vectors both induced gastrin-promoter, luciferase-reporter genes. The inductive effects of oncogenic ras could be blocked by the coexpression of dominant negative forms of raf and extracellular regulated kinase.. Oncogenic ras induces gastrin gene expression through activation of the Raf-MEK-ERK signal transduction pathway.

Topics: Calcium-Calmodulin-Dependent Protein Kinases; Colonic Neoplasms; Enzyme Inhibitors; Flavonoids; Gastrins; Gene Expression Regulation, Neoplastic; Genes, ras; Genes, Reporter; Genotype; Humans; Luciferases; Mitogen-Activated Protein Kinase Kinases; Phosphorylation; Promoter Regions, Genetic; Protein Kinase Inhibitors; RNA, Messenger

Gastrin is trophic for the mucosa of the acid-producing part of the rat stomach, notably the histamine-producing ECL cells. Gastrin is said to stimulate growth also of colonic mucosa and colon cancer. The purpose of the present study was to examine whether endogenous hypergastrinaemia had trophic effects on normal colonic mucosa and transplanted colon adenocarcinoma in rats.. Rats were subjected to fundectomy (surgical removal of the acid-producing part of the stomach) or treatments known to induce endogenous hypergastrinaemia. The treatments included refeeding after 48 h of food deprivation or administration of omeprazole (400 micromol/kg/day, orally). Other operations included colostomy and sham operation. A K12-cell line, originally established from a 1,2-dimethylhydrazine-induced colon adenocarcinoma, was used for transplantation. The rates of cell proliferation were determined in the oxyntic and colonic mucosa and in the tumour by measuring the proportion of the cells that accumulated bromodeoxyuridine in their nuclei, i.e. the labelling index (LI). The thickness of the oxyntic mucosa and the activity of histidine decarboxylase (HDC), the histamine-forming enzyme of the ECL cells, were measured. In addition, the thickness of the colonic mucosa and the weight and volume of the tumour were measured.. Refeeding or treatment with a single dose of omeprazole in fasted rats raised the serum gastrin concentration and the LI and HDC activity in the oxyntic mucosa; refeeding but not omeprazole raised the LI in the colonic mucosa. In fed rats, hypergastrinaemia induced by fundectomy or treatment with omeprazole (for 10 days) failed to affect either the LI or the thickness of the mucosa of the proximal colon and the excluded distal colon of the colostomized rats. Fundectomy failed to stimulate the growth of the tumour transplants.. Endogenous hypergastrinaemia did not induce trophic effects on rat colonic mucosa and did not promote growth of a transplanted colon adenocarcinoma in the rat.

Topics: Adenocarcinoma; Animals; Cell Division; Colon; Colonic Neoplasms; Enzyme Inhibitors; Gastrins; Intestinal Mucosa; Male; Neoplasm Transplantation; Omeprazole; Parietal Cells, Gastric; Rats; Rats, Sprague-Dawley

The influence of gastrin on the colonic mucosa is still uncertain. Some authors have suggested a stimulating effect on the growth of normal and malignant colonic epithelium, while others have shown no association between gastrin and neoplastic development.. To evaluate the effect of gastrin on colorectal cell proliferation, patients with chronic endogenous hypergastrinaemia underwent proctoscopy. Biopsy specimens were taken in order to study rectal cell kinetics.. Ten patients with chronic autoimmune gastritis (CAG), six patients with Zollinger-Ellison syndrome (ZES), and 16 hospital controls took part in this study. Patients with CAG and ZES had basal serum gastrin concentrations significantly higher than controls (p < 0.001).. Immunohistochemistry was performed on 3 microns sections of rectal biopsy specimens incubated with 5'-bromodeoxyuridine.. The percentage of proliferating cells in the entire crypts (overall labelling index) was similar in all the groups. However, the labelling frequency in the upper two fifths of the glands (phi h value) was significantly higher in patients with CAG or ZES compared with controls (p < 0.01 in both patient groups versus controls).. Endogenous hypergastrinaemia is associated with rectal cell proliferation defects, similar to those observed in conditions at high risk for colon cancer. The effect of the increased serum concentrations of gastrin on the colorectal mucosa after treatment with drugs inhibiting gastric acid secretion should be investigated.

Topics: Adult; Aged; Autoimmune Diseases; Cell Division; Chronic Disease; Colonic Neoplasms; Female; Gastrins; Gastritis; Humans; Immunohistochemistry; Male; Middle Aged; Rectum; Risk Factors; Zollinger-Ellison Syndrome

Whereas considerable experimental evidence suggests chronic hypergastrinemia can increase the occurrence of colonic neoplasia, the risks in man remain unclear. Zollinger-Ellison syndrome (ZES) is associated with marked plasma elevation of all forms of gastrin and, because of its prolonged course, has been shown to be an excellent model disease to study the effects of chronic hypergastrinemia in man. To determine whether profound chronic hypergastrinemia affects the occurrence of colonic dysplasia and neoplasia, 97 consecutive patients with ZES were studied. All patients underwent colonoscopic examination to the cecum, and the location, size, and type of polyps/tumors were determined. The patients had a mean fasting gastrin level 31 times above normal and a mean disease duration of 10 years; 17/97 (18%) had adenomatous polyps, 67/97 (69%) no adenomatous polyps, and 2/97 (2%) had colonoscopy and/or autopsy studies fo asymptomatic controls. Stratification by age or gender, presence of MEN-I, tumor extent, and duration of degree of hypergastrinemia did not increase prevalence. This study shows that despite prolonged, profound hypergastrinemia, no increased rate of colonic neoplasia (polyps or cancer) was noted. These data suggest that the development of hypergastrinemia secondary to continuous use of H+,K+-ATPase inhibitors for as long as 10 years is unlikely to cause an increased risk of developing colonic neoplasia in man.

Topics: Adenocarcinoma; Adenomatous Polyps; Age Distribution; Chronic Disease; Colonic Neoplasms; Colonic Polyps; Colonoscopy; Fasting; Female; Gastrins; Humans; Incidence; Male; Middle Aged; Risk Factors; Sex Distribution; Zollinger-Ellison Syndrome

The effect of gastrin neutralization was evaluated on the in vivo growth of the rat colon line, DHDK12, which expressed cholecystokinin B/gastrin receptors and secreted glycine-extended gastrin-17 (G17). Gastrin neutralization was achieved by administration of the immunogen, Gastrimmune, which is composed of the amino terminal portion of G17 linked to a diphtheria toxoid. A rat-specific version of Gastrimmune was used to preimmunize rats, with control animals receiving diphtheria toxoid only. The antibodies raised neutralized both carboxy-amidated and glycine-extended G17. The tumor was implanted into the muscle layer of the abdominal wall, and rats immunized with Gastrimmune had significantly reduced median cross-sectional tumor areas (70.2% reduction; P = 0.005) and weights (56.5% reduction; P = 0.0078)) when compared to control rats. Histological analysis revealed that the tumors had an enhanced degree of necrosis, with the area of viable tumor in the Gastrimmune-immunized rat reduced to 40.3% compared to 58.6% in the control rats (P = 0.003). Immunization with Gastrimmune raised antibodies that inhibited the growth of a rat colon tumor. This could have been mediated by neutralization of both serum G17 and cell-associated precursor gastrin molecules.

Topics: Amino Acid Sequence; Animals; Antibody Formation; Antigen-Antibody Reactions; Cancer Vaccines; Cell Division; Colonic Neoplasms; Diphtheria Toxoid; Drug Design; Gastrins; Humans; Immunoglobulin Isotypes; Immunotoxins; Male; Molecular Sequence Data; Rabbits; Rats; Rats, Inbred Strains; Receptor, Cholecystokinin B; Receptors, Cholecystokinin

Topics: Colonic Neoplasms; Gastrins; Humans; Protein Precursors; Receptors, Cholecystokinin

The majority of human colon cancers express the gastrin gene, and a significant percentage bind gastrin-like peptides. However, it is not known if gastrin gene products are physiologically relevant to the growth and proliferation of human colon cancers. To investigate the functional role of gastrin gene expression, we examined the effect of gastrin antisense (AS) RNA expression on the growth and tumorigenicity of colon cancer cells. The full-length human gastrin cDNA was cloned in the AS direction in a retroviral vector under the transcriptional control of human cytomegalovirus promoter. Three representative human colon cancer cell lines that expressed negligible (Colo-205A) to significant (Colo-320 and HCT-116) levels of gastrin mRNA were transfected with either AS or control vectors and subjected to various growth studies in vitro and in vivo. The proliferative and tumorigenic potential of the AS clones from the gastrin-expressing cell lines was significantly suppressed compared to that of the control clones, whereas the growth of Colo-205A-AS cells (the negative control) was similar to that of the Colo-205A-C-cells, indicating the relative specificity of the antitumorigenic effects of AS gastrin RNA expression. We believe that this is the first evidence that supports a possible critical role of gastrin gene expression in the tumorigenicity of human colon cancers that express the gastrin gene. Because > 60-80% of human colon cancers express the gastrin gene, it can be expected that the growth of a significant percentage of these cancers may be critically dependent on the expression of gastrin gene products. Therapeutic measures, such as the AS strategy used in the present study, may therefore prove to be useful in treating human colon cancers in the future.

Topics: Animals; Base Sequence; Cell Division; Cell Line; Colonic Neoplasms; DNA Primers; Gastrins; Humans; Mice; Mice, Nude; Molecular Sequence Data; Polymerase Chain Reaction; Protein Precursors; Protein Processing, Post-Translational; Recombinant Proteins; RNA, Messenger; Transcription, Genetic; Transfection; Transplantation, Heterologous; Tumor Cells, Cultured

This study was designed to evaluate the effects of hypergastrinaemia induced via suppression of gastric acid by omeprazole on carcinogen-induced colon cancer in rats. The carcinogen methylazoxymethanol (MAM), 30 mg/kg, was administered intraperitoneally at 6-weekly intervals to Sprague-Dawley rats. Four weeks after the last MAM injection, the first daily dose of omeprazole, 40 mg/kg, was given by gastric gavage to one group of rats, and the rest were given buffered methylcellulose vehicle. After 10 weeks of daily omeprazole or vehicle, the rats were anaesthetized with ether, blood samples obtained, and animals sacrificed. Gastrin levels in serum from omeprazole-treated rats were elevated nearly six-fold. DNA and RNA levels in gastric mucosa were unchanged by omeprazole, but protein content was somewhat reduced. No biochemical or histological changes related to omeprazole treatment were observed in normal colon. The number of tumours, tumour volumes, and total tumour burden were not significantly different in colons of vehicle- or omeprazole-treated rats. Analysis by flow cytometry revealed that the S phase fraction was lower in tumour cells from omeprazole-treated animals; and that the frequency of DNA aneuploidy was also reduced. The results indicate that while omeprazole-induced suppression of stomach acid in rats elevates levels of gastrin in serum, it does not substantially alter the biochemical or cellular characteristics of carcinogen-induced colon tumours.

Topics: Animals; Anti-Ulcer Agents; Carcinogens; Cell Cycle; Colonic Neoplasms; DNA Replication; Gastric Mucosa; Gastrins; Male; Methylazoxymethanol Acetate; Omeprazole; Rats; Rats, Sprague-Dawley

Topics: Amino Acid Sequence; Colonic Neoplasms; Gastrins; Humans; Molecular Sequence Data; Protein Processing, Post-Translational

The effect of endogenous hypergastrinemia on growth of human colon carcinoma is not known. Our aim was to study the growth of human colon carcinoma in an animal model with endogenous hypergastrinemia.. Human colon carcinoma was transplanted to the colon of 40 athymic rats. Of these, 25 underwent gastric fundectomy to accomplish endogenous hypergastrinemia, and 15 were sham operated to serve as controls. The duration of the study was 8 weeks. During the last week, 12 fundectomized animals received a gastrin (cholecystokinin B) receptor antagonist. Metaphase arrest index, local invasion, and distant spread of the tumor were investigated. Expression of gastrin and cholecystokinin B receptor messenger RNA was examined by reverse-transcription polymerase chain reaction.. Tumor spread by direct extension outside the colon was observed in all animals, and liver metastases were observed in 10 of the 25 fundectomized animals. Sham-operated animals showed none of these features. The metaphase arrest index of the tumor did not differ between fundectomized animals given the cholecystokinin B receptor antagonist and sham-operated animals, whereas it was significantly increased in fundectomized animals not given the antagonist. The tumor expressed both gastrin and cholecystokinin B receptor messenger RNA.. The results indicate that endogenous hypergastrinemia may promote proliferation and spread of human colon carcinoma expressing cholecystokinin B receptor.

Topics: Aged; Animals; Carcinoma; Colonic Neoplasms; Female; Gastric Fundus; Gastrins; Humans; Liver Neoplasms; Male; Neoplasm Transplantation; Rats; Rats, Inbred Lew; Rats, Nude; Receptors, Cholecystokinin

The effects of peptide histidine isoleucine (PHI) on the incidence and histology of colon tumors induced by azoxymethane (AOM), and on the labeling index of colon mucosa were investigated in Wistar rats. Rats received weekly s.c. injections of 7.4 mg/kg body weight of AOM for 10 weeks, and of 1.0 or 4.0 nmol/kg body weight of PHI until the end of the experiment in week 35. Administration of PHI at the higher, but not the lower dosage, significantly increased the incidence of colon tumors. PHI had no influence on the histology of colon tumors or adenocarcinomas. It also caused significant increase in the labeling index of colon epithelial cells. These findings indicate that PHI enhances colon carcinogenesis, and that its effect may be related to increasing proliferation of colon epithelial cells.

Topics: Adenocarcinoma; Animals; Azoxymethane; Body Weight; Colon; Colonic Neoplasms; Drug Synergism; Gastrins; Intestinal Mucosa; Peptide PHI; Rats; Rats, Wistar

The direct effects of omeprazole on colonic cells has not been evaluated. Controversy exists regarding the potential adverse effects of omeprazole on cell proliferation. In order to mimic the in vivo situation in the patient treated with omeprazole, proliferation cell culture experiments were performed, monitoring directly the effects of gastrin and omeprazole both alone and in combination. Three colonic cancer cell lines were used, two with neuroendocrine features (NCI-H716, LCC-18) and one (DLD-1) not known to have these features. In these in vitro proliferation experiments, only the NCI-H716 colorectal cancer cell line responded to omeprazole by decreased proliferation (P < 0.05). The effect was concentration dependent shown for all doses of omeprazole used. Gastrin had a statistically significant effect on increasing proliferation in the NCS-H716 cell line alone but only at the highest concentration (10(-6) M). Omeprazole has a cytostatic effect on one of three colorectal cancer cell lines but the mechanism for this effect of omeprazole and its potential role in treatment awaits elucidation.

Topics: Cell Division; Cell Line; Colonic Neoplasms; Colorectal Neoplasms; Gastrins; Humans; Omeprazole; Tumor Cells, Cultured

Published studies suggest that hypergastrinemia stimulates growth of normal or malignant colon tissue. Other studies dispute these findings. This study was designed to test the hypothesis that hypergastrinemia enhances progression or invasiveness of colon cancer.. Colonic carcinomas were induced in male Sprague-Dawley rats by six weekly intraperitoneal injections of methylazoxymethanol. Four weeks after the last injection of carcinogen, the animals were randomized into four treatment groups, including vehicle control, low- and high-dose omeprazole, and ranitidine. After 10 weeks of treatment, the animals were bled, stomach weights were recorded, and colon tumors were mapped, enumerated, measured, and scored histopathologically by Dukes' classification. Crypt and mucosal heights were determined in colonic mucosa unaffected by tumor.. Drug administration induced a sustained hypergastrinemia that did not enhance tumor burden or invasiveness or crypt height/mucosal height ratios. Ranitidine-treated rats consumed less food, weighed less, and developed fewer tumors. This group also had lower crypt and mucosal heights than rats in the vehicle- or omeprazole-treated rats.. The results suggest that endogenous hypergastrinemia induced by these acid-suppressing drugs has no stimulatory effect on colon mucosal growth or progression or biological behavior of experimental rat colon cancer.

Topics: Analysis of Variance; Animals; Colonic Neoplasms; Gastrins; Intestinal Mucosa; Male; Methylazoxymethanol Acetate; Neoplasm Invasiveness; Omeprazole; Random Allocation; Ranitidine; Rats; Rats, Sprague-Dawley

Gastrin has been suggested to be involved in the promotion and progression of colon cancer. Mice colon cancers and colon-carcinoma cell lines are stimulated to grow by gastrin, and gastrin receptors have been found in the majority of human colon-tumor specimens. High serum gastrin levels have been reported in patients with colon polyps and cancers, together with increased ornithine decarboxylase (ODC) activity. Since gastrin stimulates ornithine decarboxylase in colon cancer cells in vitro it has been suggested that increased synthesis of intracellular polyamines is one of the mechanisms activated by the hormone. In order to confirm the presence of hypergastrinemia in colon cancer and to investigate the relationship between plasma gastrin and tumor growth, we used an animal model of colon carcinogenesis that minimizes the possible bias of human studies, related to varying diet, age and environmental factors. We evaluated blood gastrin levels in 35 rats with colon cancer induced by the carcinogen azoxymethane (AOM), and we correlated gastrinemia with tumor proliferation, assessed by thymidine-labeling index (TLI) and ODC activity; 6 animals constituted the control group. Gastrin levels in rats with AOM-induced tumors were significantly higher than in controls. Significantly higher TLI and ODC activity were found in the tumors of hypergastrinemic rats than in neoplasms of animals with normal gastrin levels. Our data provide additional evidence of a role for gastrin as trophic hormone for colon neoplastic cells.

Topics: Animals; Azoxymethane; Cell Division; Colonic Neoplasms; Gastrins; Male; Ornithine Decarboxylase; Rats; Rats, Wistar; Thymidine

Gastrin is transcriptionally responsive to EGF stimulation (Merchant et al., 1991, Mol. Cell. Biol., 11:2686-2696). Consequently, we hypothesized that previously recognized gastrin autocrine loops (Hoosein et al., 1990, Exp. Cell. Res., 186:15-21), might be controlled by autocrine TGF alpha in human colon carcinoma cells. Therefore, we examined the interaction between these two autocrine growth factors in two colon carcinoma cell lines which utilize TGF alpha. The FET cell line requires exogenous TGF alpha/EGF for optimal growth and has a classical TGF alpha autocrine loop which is disrupted by TGF alpha or epidermal growth factor receptor (EGFr) antibodies. The HCT 116 cell line is not dependent on exogenous TGF alpha/EGF and exhibits a nonclassical TGF alpha autocrine loop which is not disrupted by neutralizing antibodies to either TGF alpha itself or the EGFr. Basal gastrin mRNA production is significantly higher in HCT 116 than FET as measured by RNase protection assay. In the FET cells, exogenous EGF stimulates gastrin mRNA production but not in HCT 116. When the TGF alpha autocrine loop in HCT 116 is disrupted by constitutive expression of antisense TGF alpha mRNA, the gastrin mRNA level is significantly repressed. In xenografts derived from these antisense clones, TGF alpha reverted to high expression, and the gastrin mRNA level was again increased. This interaction between the strong TGF alpha loop in HCT 116 and the gastrin autocrine loop may confer a growth advantage to these colon cells. Such interactions between growth factors may promote enhanced tumorigenicity to transformed cells with these strong, nonclassical autocrine loops.

Topics: Animals; Colonic Neoplasms; DNA, Antisense; Epidermal Growth Factor; Gastrins; Gene Expression Regulation; Humans; Mice; Mice, Nude; Neoplasm Transplantation; Polymerase Chain Reaction; RNA, Messenger; Transfection; Transforming Growth Factor alpha; Transplantation, Heterologous; Tumor Cells, Cultured

Gastrin is mitogenic for several colon cancers and is postulated as an autocrine growth factor for colon cancer cells. In the present study we report the development of a simple competitive polymerase chain reaction (PCR) method for measuring relative abundance of gastrin gene expression in colon cancer cells. Primers flanking exons 2 and 3 of the gastrin gene were utilized for co-amplification of cDNA and genomic DNA. The amplification of genomic DNA was distinguished from that of cDNA by the presence of the 130 bp intron sequence which was resolved by electrophoresis on agarose gels. A standard reaction of competitive PCR, using known concentrations of genomic DNA and cDNA, was first established. The steady state levels of gastrin mRNA were next quantitated in three human colon cancer cell lines (HCT-116, Colo-205 and DLD-1) by competitive PCR. Gastrin mRNA levels in these cell lines ranged from approximately 0.1 to 1.0 fmoles/mg total RNA (approximately 2-25 copies of gastrin mRNA per cell). Thus low to moderate levels of gastrin were expressed by human colon cancer cell lines which may function as autocrine growth factors for colon cancers.

Topics: Base Sequence; Colonic Neoplasms; DNA Primers; DNA, Complementary; DNA, Neoplasm; Gastrins; Humans; Molecular Sequence Data; Neoplasm Proteins; Polymerase Chain Reaction; RNA-Directed DNA Polymerase; RNA, Messenger; RNA, Neoplasm; Tumor Cells, Cultured

Cell growth is regulated by various peptide growth factors through receptor-linked multiple intracellular signal-transduction pathways, such as the cyclic adenosine monophosphate (cAMP) pathway. cAMP activates cAMP-dependent protein kinase A (PKA) either to stimulate or inhibit cell growth. The effect on growth is determined by the presence of two isoforms of the regulatory (R) subunit of PKA; activation of RI alpha-type PKA leads to stimulation of growth, activation of RII beta-type inhibits cell growth. We determined whether the effect of gastrin on the growth of human colon cancer cells is determined by cell-specific content of PKA. We utilized two human colon cancer cell lines: LoVo, growth of which is stimulated by gastrin, and HCT116, growth of which is inhibited by gastrin. Activation of both types of PKA with 8-Br-cAMP mimicked the regulation of growth by gastrin; preferential activation of RII beta-type PKA with 8-Cl-cAMP inhibited growth of both cell lines. LoVo cells possess the predominantly RI alpha isoform of PKA at the mRNA and protein level; HCT116 cells possess predominantly the RII beta-type PKA. The cAMP-mediated regulation of growth (either stimulatory or inhibitory) by gastrin on these human colon cancer cells was determined by the predominant isoform of PKA.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Cell Division; Colonic Neoplasms; Cyclic AMP-Dependent Protein Kinase RIalpha Subunit; Cyclic AMP-Dependent Protein Kinase RIIbeta Subunit; Cyclic AMP-Dependent Protein Kinases; Gastrins; Humans; Isoenzymes; Signal Transduction; Tumor Cells, Cultured

Nude mice bearing xenografts of HT-29 human colon cancer cell line were treated for 4 weeks with somatostatin analog (RC-160), bombesin/gastrin releasing peptide (GRP) antagonists (RC-3095 and RC-3440). In three separate experiments somatostatin analog RC-160 (50 micrograms/day) released from microgranules significantly reduced tumor growth. Bombesin/GRP antagonists, RC-3095 and RC-3440 injected subcutaneously (s.c.) twice daily at a dose of 10 micrograms had the greatest and consistently significant inhibitory effect on tumor growth. RC-3095 given once daily s.c. at a dose of 20 micrograms was less effective. RC-3095 also inhibited metastatic tumor growth after intrasplenic injection of HT-29 cells in nude mice. Specific binding sites of somatostatin, bombesin and epidermal growth factor (EGF) were detected on intact HT-29 cells or on the membranes from HT-29 tumor xenografts. The inhibitory effects of bombesin antagonists on tumor growth were consistently linked with a significant down-regulation of EGF receptors. Bombesin/GRP antagonists and somatostatin analogs could be considered for the development of new hormonal therapies for colon cancer.

Topics: Adenocarcinoma; Animals; Antineoplastic Agents; Bombesin; Colonic Neoplasms; Drug Screening Assays, Antitumor; ErbB Receptors; Gastrin-Releasing Peptide; Gastrins; Growth Hormone; Humans; Insulin-Like Growth Factor I; Liver Neoplasms; Male; Mice; Mice, Nude; Neoplasm Transplantation; Peptide Fragments; Peptides; Radioligand Assay; Somatostatin

Inhibition of colon carcinoma cell growth by the nonselective gastrin/cholecystokinin (CCK) receptor antagonists proglumide and benzotript provided evidence that gastrin functions as an autocrine growth factor. However, the molecular properties of the receptor mediating the antagonist effects have not been identified. A 78-kDa gastrin-binding protein (GBP), the sequence of which is related to the family of enzymes possessing enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase activities, has been previously purified from porcine gastric mucosal membranes. I now report that covalent cross-linking of 125I-labeled [Nle15]gastrin2,17 to the 78-kDa GBP is inhibited by crotonyl-CoA and by acetoacetyl-CoA. Gastrin, CCK, and their analogues also inhibit cross-linking, and the spectrum of analogue affinities correlates better with the values previously reported for binding to the gastrin/CCK-C receptor than with the values reported for binding to either the CCK-A or the gastrin/CCK-B receptor. Cross-linking is also inhibited by proglumide and benzotript, but no inhibition is seen with either the CCK-A receptor-selective antagonist L364,718 or the gastrin/CCK-B receptor-selective antagonist L365,260. The affinities of antagonists for the GBP correlate well with their affinities for the gastrin/CCK-C receptor and with their potencies for inhibition of colon carcinoma cell growth. I conclude that the 78-kDa gastrin-binding protein is (i) a member of the hydratase/dehydrogenase family of fatty acid oxidation enzymes, (ii) the gastrin/CCK-C receptor, and (iii) the target for the antiproliferative action of two gastrin/CCK receptor antagonists.

Topics: 3-Hydroxyacyl CoA Dehydrogenases; Acyl Coenzyme A; Animals; Carcinoma; Carrier Proteins; Colonic Neoplasms; Enoyl-CoA Hydratase; Gastrins; Humans; Mitochondrial Trifunctional Protein; Multienzyme Complexes; Protein Binding; Receptors, Cholecystokinin; Swine

Two receptors for cholecystokinin (CCK) have been isolated which also bind gastrin: CCK-A type and CCK-B type, both are coupled to phospholipase C (PLC) activation. However, identification of the "true" gastrin receptor remains controversial. We determined which CCK receptor mediated the trophic effect of gastrin on human colon cancer cells (LoVo). LoVo cells lack mRNA for either CCK receptor by Northern hybridization. Gastrin stimulated cyclic AMP production, not PLC activity, in LoVo cells. The trophic effect was not blocked by receptor antagonists for CCK-A (L364,718) or CCK-B (L365,260). The gastrin receptor pharmacology on LoVo cells and the lack of appropriate transcripts suggest that gastrin stimulated growth of these cells by a receptor other than CCK-A or CCK-B type and there likely exists another receptor for gastrin.

Topics: Cell Division; Cholecystokinin; Colonic Neoplasms; Cyclic AMP; Gastrins; Humans; Receptors, Cholecystokinin; Tumor Cells, Cultured

Gastrin is mitogenic for several colon cancers. To assess a possible autocrine role of gastrin in colon cancers, we examined human colon cancer cell lines for expression of gastrin mRNA and various forms of gastrin. Gastrin mRNA was not detected in the majority of colon cancer cell lines by Northern hybridization but was detected in all human colon cancer lines by the sensitive method of reverse transcriptase-polymerase chain reaction (PCR). Gastrin mRNA was quantitated by the competitive PCR method. The majority of cell lines expressed very low levels of gastrin mRNA (< 1-5 copies/cell); only one cell line expressed > 20 copies/cell. The mature carboxyamidated form of gastrin was not detected in any of the cell lines by radioimmunoassay or immunocytochemistry. Results suggested that either gastrin mRNA expressed by colon cancer cells was altered (mutated) or posttranslational processing of progastrin was incomplete. Gastrin cDNA from all the colon cancer cell lines had an identical sequence to the published sequence of human gastrin cDNA. Specific antibodies against precursor forms of gastrin were used, and significant concentrations of nonamidated (glycine-extended) and prepro forms of gastrin were measured in tumor extracts of representative colon cancer cell lines. The presence of precursor forms of gastrin suggested a lack of one or more of the processing enzymes and/or cofactors. Significant concentrations of the processing enzyme (peptidylglycine alpha-amidating monooxygenase) were detected in colon cancer cells by immunocytochemistry. Therefore, lack of other cofactors or enzymes may be contributing to incomplete processing of precursor forms of gastrin, which merits further investigation. Since low levels of gastrin mRNA were expressed by the majority of human colon cancer cell lines and progastrin was incompletely processed, it seems unlikely that gastrin can function as a viable autocrine growth factor for colon cancer cells. High concentrations of glycine-extended gastrin-17 (GG) (> 10(-6) M) were mitogenic for a gastrin-responsive human colon cancer (DLD-1) cell line in vitro. It remains to be seen if GG or other precursor forms of gastrin are similarly mitogenic in vivo, which may then lend credibility to a possible autocrine role of gastrinlike peptides in colon cancers.

Topics: Base Sequence; Colonic Neoplasms; Gastrins; Gene Expression; Humans; Immunohistochemistry; Mixed Function Oxygenases; Molecular Sequence Data; Multienzyme Complexes; Oligonucleotide Probes; Polymerase Chain Reaction; Protein Precursors; Protein Processing, Post-Translational; RNA, Messenger; Tissue Distribution; Transcription, Genetic; Tumor Cells, Cultured

Gastrin is a trophic factor for some human colon cancer cells. However, the signal-transduction pathways by which gastrin regulates growth are still unknown. We examined the effect of synthetic human gastrin-17 (G-17) on signal-transduction pathways and cell growth using 4 different human colon cancer cell lines (LoVo, COLO 320, HT-29, and HCT116). G-17 stimulated the production of cyclic AMP in LoVo, COLO 320, and HCT116 cells, while G-17 stimulated phosphatidylinositol hydrolysis and mobilization of intracellular calcium in HT-29 cells. The growth-regulatory effect of G-17 on these colon cancer cells (stimulatory on LoVo, COLO 320, and HT-29 cells; inhibitory on HCT116 cells) was well correlated with the effect of G-17 on the signal-transduction pathway in each cell line. We further examined the effect of a selective cholecystokinin-B type receptor antagonist, JMV 320, on G-17-induced signal-transduction pathways and G-17-regulated growth. In each cell line, the effect of JMV 320 on G-17-induced signal-transduction pathways was well correlated with that on G-17-regulated growth. G-17 appears to regulate, at least to some extent, growth of human colon cancer cells through gastrin receptor-linked signal-transduction pathways that are cell-specific.

Topics: Calcium; Cell Division; Cell Line; Colonic Neoplasms; Cyclic AMP; Dose-Response Relationship, Drug; Gastrins; Hormones; Humans; Hydrolysis; Kinetics; Phosphatidylinositols; Signal Transduction; Time Factors; Tumor Cells, Cultured

To investigate the hypothesis that gastrin might be synthesized by tumour tissues in cancer of the colon, samples from six human colon tumours, one hepatic metastasis, four normal colonic mucosal samples and two antral and one fundic gastric mucosal samples from nine patients were analysed to determine whether gastrin mRNA was present. RNA was extracted from surgical specimens by ultracentrifugation on a CsCl cushion, purified using the guanidinium thiocyanate method, reverse-transcribed and amplified by polymerase chain reaction. Gastrin mRNA was detected in each colonic carcinoma sample (including the hepatic metastasis), while no such signal was observed in normal colon biopsies. Positive and negative controls (gastric antrum and fundus respectively) gave the expected results. In each of the positive samples, the chemiluminescent revelation of amplified products after Southern blotting corresponded to gastrin mRNA without the intron. These findings demonstrate the ability of primary and metastatic human colonic tumours to produce gastrin mRNA. Since malignant cell lines have been reported to produce gastrin peptide, and since gastrin receptors were present in some cases, our results support the idea that gastrin may be involved in an autocrine mechanism.

Topics: Base Sequence; Colon; Colonic Neoplasms; Gastric Fundus; Gastric Mucosa; Gastrins; Humans; Intestinal Mucosa; Liver Neoplasms; Luminescent Measurements; Molecular Sequence Data; Neoplasm Proteins; Organ Specificity; Polymerase Chain Reaction; Pyloric Antrum; RNA-Directed DNA Polymerase; RNA, Messenger

Numerous clinical and experimental studies suggest that gastrin plays an important part in the development of colorectal cancer in humans. This study was done to assess the influence of omeprazole induced hypergastrinaemia on the development of colorectal tumours in an experimental animal model. Forty female Sprague-Dawley rats received either omeprazole (40 mumol/kg) or vehicle (0.25% methylcellulose) by once daily oral gavage throughout the experiment. All animals received 12 consecutive weekly subcutaneous injections of azoxymethane (10 mg/kg/week) beginning at week 6. Serum gastrin concentrations were measured during weeks 1 and 5 and at death (week 27). Chronic omeprazole treatment resulted in appreciable hypergastrinaemia during the study, mean gastrin concentrations in omeprazole treated rats being raised by up to nine to 10 fold, compared with vehicle treated control rats (p < 0.001). Despite this, tumour incidence in the omeprazole group was significantly lower at 63%, compared with 95% in the vehicle only group (p < 0.02). The median number of tumours in the omeprazole group (1) compared with the vehicle group (3) was also significantly lower (p = 0.02). Average tumour size, site distribution, and the comparative frequencies of adenomas and adenocarcinomas were similar in the two groups. This study shows that omeprazole protects against colorectal carcinogenesis in this model despite causing appreciable hypergastrinaemia. The mechanism by which this occurs is unclear and merits further investigation. Because of the compounding protective effects of omeprazole, this model is not a suitable one for studying the longterm trophic effects of gastrin on the colon.

Topics: Animals; Azoxymethane; Body Weight; Colonic Neoplasms; Eating; Female; Gastrins; Omeprazole; Rats; Rats, Sprague-Dawley; Rectal Neoplasms

We have previously reported mitogenic effects of gastrin on a mouse colon cancer (MC-26) cell line in vivo. The present studies were undertaken to determine if gonadal hormones can influence the mitogenic response of MC-26 cells to gastrin. The female gonadal hormone, estradiol (E2), was determined to be as mitogenic as pentagastrin (PG) for the growth of MC-26 tumors in mice; the mitogenic effects of E2 and PG were not additive. Female gonadal hormones were furthermore as effective as PG in maximally up-regulating gastrin receptor (GR) concentrations on MC-26 tumor membranes, which was confirmed in autoradiographic studies. Since PG and E2 had similar and non-additive trophic effects it was hypothesized that gastrin may be mediating the trophic effects of E2. Serum gastrin concentrations were significantly increased in E2 treated ovariectomized mice that correlated with an increase in tumor weights; E2 however was ineffective in stimulating the release of gastrin from perfused rat stomachs indicating that the increase in serum gastrin concentration on long-term treatment with E2 was mediated by some other mechanism. Saturable high-affinity E2 binding sites were not measured in MC-26 cells and tumors, supporting the possibility that mitogenic effects of E2 were probably mediated via indirect mechanisms. In summary our results indicate that both E2 and PG are equally mitogenic for colon cancer cells in vivo which may explain the sex- and age-related discrepancy in the incidence of human colon cancers.

Topics: Animals; Cell Division; Colonic Neoplasms; Estradiol; Female; Gastrins; Male; Mice; Mice, Inbred BALB C; Ovariectomy; Pentagastrin; Rats; Receptors, Cholecystokinin; Sex Characteristics; Tumor Cells, Cultured

To evaluate the hypothesis that gastrin is a local growth factor in colonic carcinomas, the expression of gastrin messenger RNA (mRNA) and peptides were examined in five human colon carcinoma cell lines, 12 solid colon carcinomas, and normal colonic tissue.. Northern analysis, reverse-transcription PCR, and a library of sequence-specific radioimmunoassays were the principal methods.. Cell lines, tumors, and normal tissue all expressed a gastrin mRNA of 0.7 kilobases, and all cell lines contained incompletely processed progastrin (range, 17-54 fmol/10(6) cells). Two cell lines secreted progastrin into the media (LoVo, 25 +/- 3 pmol/L; HCT116; 12 +/- 2 pmol/L). Normal colonic tissue and all the solid tumors also contained progastrin, the concentration being higher in tumors (range, 0.4-2 pmol/g) than in normal tissue (range, 0.1-0.2 pmol/g). Only one tumor contained carboxyamidated gastrins.. Normal and neoplastic colonic mucosa both express the gastrin gene, but the posttranslational phase of expression is attenuated. The incomplete processing and low level of expression suggest that autocrine gastrin secretion has only minor significance for normal adult and most neoplastic colonic tissue.

Topics: Actins; Adenocarcinoma; Blotting, Northern; Colon; Colonic Neoplasms; Colorectal Neoplasms; Exons; Gastric Mucosa; Gastrins; Humans; Oligodeoxyribonucleotides; Polymerase Chain Reaction; Protein Precursors; Rectal Neoplasms; RNA Probes; RNA, Messenger; Tumor Cells, Cultured

Information concerning the influence that gastrin may exert on the colon is fragmentary and somewhat controversial. This study analyzed the effect of chronic endogenous hypergastrinemia on cell proliferation and tumor occurrence in the human colonic mucosa.. Twenty-three consecutive hypergastrinemic patients presenting with the Zollinger-Ellison syndrome and 18 normogastrinemic subjects were studied. All had fasting serum gastrin determination, colonoscopy, and cell kinetic measurement in two colonic sites using in vitro 5'-bromodeoxyuridine incorporation.. Macroscopic tumors, one endocrine and five adenomas, were found in 5 of 23 hypergastrinemic patients, without apparent relationship with the level of gastrin. The labeling indices were significantly higher in hypergastrinemic than in normogastrinemic individuals in the right and left colon, (P < 0.002 to P < 0.001) without resulting in colonic cell hyperplasia. There was no correlation between labeling indices and serum gastrin concentrations or duration of hypergastrinemia. The DNA labeling distribution along the crypt was normal in the two groups, without expansion of the proliferative zone towards the surface.. These results showed for the first time that long-lasting endogenous hypergastrinemia is accompanied by increased in vivo cell proliferation in the human colonic mucosa. However, the prevalence of adenomas (17.4%) in patients, all more than 50 years old, may not be different from that in the general population.

Topics: Adenoma; Adult; Aged; Cell Division; Chronic Disease; Colon; Colonic Neoplasms; Colonoscopy; DNA; Female; Gastrins; Humans; Intestinal Mucosa; Male; Middle Aged; Zollinger-Ellison Syndrome

Topics: Colonic Neoplasms; Gastrins; Humans

Two previous studies have shown higher circulating gastrin levels in subjects with colonic neoplasia than in colonoscopy-negative controls. In this much larger study, sera were collected from fasting subjects undergoing colonoscopy. Colonoscopy with biopsy classified participants as having colonic adenomas (N = 139), colon carcinoma (N = 29), or controls without colonic neoplasia (N = 150). Frozen, stored sera were later analyzed for gastrin by radioimmunoassay. Serum gastrin values were no higher in subjects with colonic adenomas or carcinoma than in colonoscopy-negative controls. We conclude that elevated serum gastrin levels play little, if any, role in the initiation of colonic neoplasia.

Topics: Adenocarcinoma; Adenoma; Case-Control Studies; Colonic Neoplasms; Colonoscopy; Female; Gastrins; Humans; Male; Middle Aged; Radioimmunoassay

The hormone gastrin is mainly produced by the G cells of the antral mucosa and plays a major role in the regulation of digestive mucosal growth. Since it permits identification of cell types containing mRNA, in situ hybridization (ISH) appears to be an interesting method for studying gastrin-producing tissues. In this study, in situ detection of gastrin mRNA has been carried out on frozen sections of four human normal antral mucosa samples and of six colonic carcinomas removed from patients with high levels of plasma gastrin, using a gastrin oligonucleotidic DNA probe. We have compared the results provided respectively by the [35s] labelling and the digoxigenin labelling of the synthetic probe. Positive cells were found in each normal sample analysed with radioactive- as well as digoxigenin-labelled antisense probes. The total number of cells expressing gastrin mRNA appeared slightly higher with the [35s]-labelled probe, while the digoxigenin-labelled probe gave a better definition of positive signals. In contrast, neither radioactive nor cold probes gave positive signals in the six colonic carcinoma samples, although gastrin expression had been demonstrated in these tumours using a reverse transcriptase-PCR method. These results show that, although ISH does not seem sensitive enough to allow the detection of very low levels of gastrin expression, it would appear to be a reliable method for visualizing gastrin mRNA in human antral mucosa.

Topics: Base Sequence; Colonic Neoplasms; Digoxigenin; DNA Probes; Gastric Mucosa; Gastrins; Humans; In Situ Hybridization; Molecular Sequence Data; RNA, Messenger; Sulfur Radioisotopes

Gastrin has been postulated to stimulate proliferation in colorectal neoplasms. Although gastrin mRNA has been demonstrated to be present in colon cancer cell lines, the intact peptide had not been recovered from human colorectal neoplasms. We demonstrate that gastrin and its precursors are present in both colorectal neoplasia and adjacent normal-appearing colonic mucosa. In colonic tissue, the glycine-extended precursor form of the peptide is over 10-fold more abundant than the amidated gastrin, and progastrin is more than 700-fold more abundant. In contrast, amidated gastrin in the human antrum is the predominant form of gastrin by a factor of 10. Furthermore, the ratio of gastrin precursors to gastrin is significantly increased in neoplastic colonic mucosa when compared with normal colonic tissue. These data suggest that the processing of gastrin is unique in the human colon and that further differences in processing occur in neoplastic colonic tissue.

Topics: Amino Acid Sequence; Colon; Colonic Neoplasms; Gastrins; Humans; Intestinal Mucosa; Molecular Sequence Data; Protein Precursors; Protein Processing, Post-Translational; Rectal Neoplasms; Tumor Cells, Cultured

Topics: Adult; Aged; Aged, 80 and over; Carcinoma; Colonic Neoplasms; Female; Gastrins; Humans; Male; Middle Aged; Postoperative Care; Preoperative Care; Prospective Studies; Rectal Neoplasms; Reference Values; Time Factors

The effects of bombesin on the incidence, number and histology of colon tumors induced by azoxymethane (AOM), and on their metastases to the peritoneum and/or lymph nodes, were investigated in Wistar rats. Rats received weekly s.c. injections of AOM for 10 weeks, and s.c. injections of bombesin in depot form every other day until the end of the experiment in week 30. Administration of bombesin significantly increased the incidence of colon tumors in week 30. It had no influence on the histological features or depths of involvement of colon adenocarcinomas, but significantly increased the incidence of cancer metastasis to the peritoneum and/or lymph nodes. It also caused a significant increase in the labeling index of the colon epithelial cells. Our findings indicate that bombesin enhances the development and metastasis of colon tumors, and that this effect may be related to its effect in increasing proliferation of epithelial cells of the colon.

Topics: Adenocarcinoma; Animals; Azoxymethane; Bombesin; Colon; Colonic Neoplasms; Gastrins; Male; Neoplasm Metastasis; Norepinephrine; Rats; Rats, Inbred Strains

Colonic mucosa and adenocarcinoma are known to possess gastrin receptors. Recent studies have suggested that some patients with large intestinal cancers and polyps have elevated serum gastrin levels and that gastrin may stimulate growth of colonic neoplasms. The aim of the present investigation was to determine whether endogenous hypergastrinemia--induced by the proton pump inhibitor omeprazole--would influence growth in a subcutaneously implanted murine colonic cancer. The results show that despite a fivefold increase in serum gastrin levels (193 pg/ml median value, range 186-252, in the omeprazole-treated group vs 36 pg/ml median value, range 28-37 in controls), there were no differences in tumor size or survival of tumor-bearing animals. Additionally, there were no differences in serum gastrin values between tumor- (29 pg/ml, range 25-38) and non-tumor- (34 pg/ml, range 25-30) bearing, untreated animals. Endogenous elevation of the serum gastrin hormone to five times the normal level does not demonstrate trophic effects on the murine colon tumor MC-26.

Topics: Adenocarcinoma; Animals; Cell Division; Colonic Neoplasms; Gastrins; Liver Neoplasms; Mice; Mice, Inbred BALB C; Neoplasm Transplantation; Omeprazole

A synthetic DNA template has been constructed that is suitable for the quantitation of mRNAs encoding gastrin, transforming growth factor alpha (TGF alpha), cholecystokinin, and the 78-kDa gastrin-binding protein. The template was used to measure levels of gastrin and TGF alpha mRNA in 7 colonic and 2 gastric carcinoma cell lines by the polymerase chain reaction. All lines produced detectable gastrin and TGF alpha mRNA with amounts varying between 2.1 and 540 molecules of gastrin mRNA/10(3) cells and 1.1 and 28 molecules of TGF alpha mRNA/10(3) cells. These results are consistent with the hypothesis that both gastrin and TGF alpha act as autocrine growth factors in colon carcinoma cell lines.

Topics: 3T3 Cells; Animals; Base Sequence; CHO Cells; Colonic Neoplasms; Cricetinae; Gastrins; Humans; Mice; Molecular Sequence Data; Polymerase Chain Reaction; RNA, Messenger; RNA, Neoplasm; Stomach Neoplasms; Transforming Growth Factor alpha; Tumor Cells, Cultured

The effects of ad libitum feeding of synthetic, low-protein diets on the incidence, number and histology of colon tumors induced by azoxymethane (AOM), on the norepinephrine concentration in the colon wall tissue and on the labelling index of colon mucosa were investigated in Wistar rats. Rats received 10 weekly injections of 7.4 mg/kg body weight of AOM and were given synthetic diets of equal calorie content containing 25% casein (normal-protein diet), 10% casein (low-protein diet) or 5% casein (very-low-protein diet). Administration of the low- and very-low-protein diets resulted in significant increases in the incidence and number of colon tumors at week 30. However, it did not affect the histology of the colon tumors. The low- and very-low-protein diets also resulted in significant increases in norepinephrine concentration in the proximal and distal portions of the colon wall and in the labelling indices of both parts of the colon mucosa. Our findings indicate that low- and very-low-protein diets enhance colon carcinogenesis and that this may be related to their effects in increasing the norepinephrine level in the colon wall and in stimulating proliferation of colon epithelial cells.

Topics: Adenocarcinoma; Adenoma; Animals; Azoxymethane; Cell Division; Colon; Colonic Neoplasms; Dietary Proteins; Gastrins; Intestinal Mucosa; Male; Norepinephrine; Rats; Rats, Inbred Strains

The effect of a long-acting somatostatin analogue SMS 201.995 (SMS; Sandoz) on basal and gastrin-stimulated growth of 4 human colon cancer lines was studied in vitro and in vivo. Proliferation assay was done with overnight [75Se]selenomethionine uptake after 5 days of incubation. Gastrin concentrations used were 5e-10 M and 1e-7 M. SMS concentrations were from 2e-12 M to 2e-7 M. Cell lines LIM 1215, LIM 2405, and LIM 2412 were inhibited dose-dependently in both basal and gastrin-stimulated groups. LIM 1863 was slightly stimulated. Based on in vivo growth characteristics, LIM 2412 and LIM 2405 were selected for xenograft study. The dose of 50 micrograms/kg/day was arrived at after a preliminary experiment showed it to be safe and effective. The LIM 2412 xenografts in the SMS-treated animals were 473.3 +/- 99.9 (SD) versus 838.1 +/- 111.3 mm3 in control (P less than 0.05) after 20 days. The LIM 2405 tumors were also significantly inhibited (81.2 +/- 30.0 versus 245.7 +/- 48.3 mm3, P less than 0.01). The effect of SMS appeared to be reversible. Oral SMS at 200 micrograms/kg/day was not absorbed. This study suggests that SMS may have direct antitumor effects in human colon cancer.

Topics: Animals; Antineoplastic Agents; Cell Division; Cell Line; Colonic Neoplasms; Gastrins; Humans; Kinetics; Male; Mice; Mice, Nude; Neoplasm Transplantation; Octreotide; Selenomethionine; Transplantation, Heterologous

Nude mice bearing xenografts of HT-29 human colon cancer cell line were treated for 4 weeks with a [D-Trp6] agonist of luteinizing hormone releasing hormone (LH-RH), somatostatin analogue (RC-160), and bombesin/gastrin releasing peptide antagonist (RC-3095). Some inhibitory effect of [D-Trp6] LH-RH microcapsules releasing 25 micrograms/day on tumor growth was observed that could be due to sex steroid deprivation, but the inhibition was not statistically significant. Microcapsules of RC-160, releasing 50 micrograms/day, significantly reduced tumor volume after 21 and 24 days of treatment, but at the end of the experiment the inhibition in tumor volume and weight was not significant. Bombesin/gastrin releasing peptide antagonist RC-3095, at a dose of 20 micrograms/day administered by daily s.c. injections or by continuous infusion using Alzet osmotic minipumps, had the greatest inhibitory effect on tumor growth. Tumor volume, percentage change in tumor volume, and tumor weights were significantly decreased in both groups treated with RC-3095. This is the first report on inhibition of human colon cancer growth in vivo by bombesin antagonists.

Topics: Animals; Antineoplastic Agents; Bombesin; Cell Division; Cell Line; Colonic Neoplasms; Gastrins; Growth Hormone; Humans; Insulin-Like Growth Factor I; Kinetics; Mice; Mice, Nude; Neoplasm Transplantation; Peptide Fragments; Somatostatin; Transplantation, Heterologous

Topics: Adenocarcinoma; Colonic Neoplasms; Colonic Polyps; Gastrins; Humans

Gastrin stimulates the growth of some human colon adenocarcinomas grown in vitro or as xenografts in nude mice. To evaluate the possibility of elevated plasma gastrin levels in patients with adenomatous polyps or colorectal cancer, we carried out a radioimmunoassay in subjects fasting overnight and undergoing colonoscopy. The study included 190 patients who were divided into three groups: controls (n = 65), those with benign adenomas (n = 63), and those with adenocarcinomas (n = 62). The mean values of plasma gastrin in the cancer group (112.71 +/- 16.65 pg/ml) were significantly higher than those of the control group (40.41 +/- 1.88 pg/ml) as well as those of the polyp group. Mean plasma gastrin values in the polyp group (54.27 +/- 5.29 pg/ml) were also significantly higher than those of the control group. In the cancer group, 32 of 62 patients (51.6%) had gastrin levels greater than the control mean +2 SD, as opposed to only 10 of 63 (15.9%) in the polyp group. The number, size, histologic type, and presence of dysplasia in the polyp group and the location or Dukes' stage in the cancer group had no significant influence on gastrin levels in this study. Preliminary results in cancer patients with elevated preoperative gastrin levels show a postoperative reduction in six of seven patients. The exact cause and role of hypergastrinemia in tumor growth in such patients remains to be determined. Measurements taken both before and after colectomy coupled with a systematic search for specific gastrin receptors would be useful.

Topics: Adenocarcinoma; Aged; Colonic Neoplasms; Colonic Polyps; Female; Gastrins; Humans; Male; Middle Aged; Radioimmunoassay; Rectal Neoplasms

Epidermal growth factor receptors and insulin like growth factor-I receptors were co-expressed on two gastric and three colorectal tumour cell lines. Previous studies have shown that gastrin receptors were also expressed at a low level or two of these cell lines. Both TGF alpha and IGF-I promoted cell growth in all of the cell lines tested. The cell doubling time of a colorectal cell line was reduced from 48 to 30-34 h. Furthermore the effects of the growth factors were additive. Each growth factor also increased the response of the cells to gastrin, but a combination of both growth factors and gastrin did not further increase growth.

Topics: Cell Division; Colonic Neoplasms; Drug Synergism; Drug Therapy, Combination; ErbB Receptors; Gastrins; Humans; Insulin-Like Growth Factor I; Mitogens; Receptors, Cell Surface; Receptors, Somatomedin; Stomach Neoplasms; Transforming Growth Factor alpha; Tumor Cells, Cultured

We studied the effects of dietary calcium, fat and fiber on serum gastrin in Fischer-344 rats in a full factorial experiment as part of a larger study of diet and colon cancer risk factors. Nine- to 10-wk-old male rats were fed standard or experimental diets for 4 wk. Wheat bran was the sole source of fiber. Wheat bran levels were 0, 2.5, 10 and 20%; fat levels were 1, 5 and 10%; calcium levels were 0.18, 0.52 and 1.04% of diet weight. On d 29 serum was collected and stored at -80 degrees C until analyzed. There was a significant (P less than 0.0001) dose-dependent increase in serum gastrin from 102 to 173 ng/L, with increasing calcium. No other significant changes in serum gastrin were noted with the dietary changes. A long-term change in the level of serum gastrin, caused by dietary modification, will influence the trophic effect that gastrin has on colonic mucosa as well as on colon carcinomas. We speculate that calcium supplementation, although slowing colonic proliferation, might have an undesirable effect on the growth of early undetected colonic tumors.

Topics: Adenocarcinoma; Animals; Calcium, Dietary; Colonic Neoplasms; Dietary Fats; Dietary Fiber; Gastrins; Male; Rats; Rats, Inbred F344; Risk Factors; Triticum; Weight Gain

The effects of gastrin (G-17), proglumide (a gastrin receptor antagonist), and enprostil (a synthetic analog of prostaglandin E2) used alone or in association were studied in colonic cancer Prob and Regb cell growth. The Prob (progressive in BD IX rats) and Regb (regressive) cell lines were cloned from a single chemically-induced rat colonic cancer. After a serum-free period corresponding to one doubling cell time, cells were incubated with 100 to 1,200 pM G-17, 40 or 80 mM proglumide, and 2.5 to 5 micrograms/ml enprostil for 8 h. Cell growth was measured 48 h later by colorimetric MTT assay. Two and four hundred pM G-17 gave a growth stimulation of 17.4 percent and 31 percent for Prob cells respectively or 35.5 percent and 49 percent for Regb cells. Growth stimulation was found to be statistically different (P less than 0.01) for Prob and Regb cells. Proglumide partially inhibited this growth stimulation whereas enprostil inhibited in totally. These results suggest that growth of some colonic cancer cell lines may be G-17 dependent. However the intensity of cell-growth stimulation depends on the level of cell malignancy or differentiation in a single tumor.

Topics: Adenocarcinoma; Animals; Colonic Neoplasms; Dimethylhydrazines; Drug Combinations; Enprostil; Gastrins; Proglumide; Rats; Stimulation, Chemical; 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

The effects of neurotensin on the incidence, number, size, and histology of colon tumours induced by azoxymethane (AOM) were investigated in Wistar rats. Rats were given 10 weekly injections of AOM (7.4 mg kg-1 body weight) and were also given 200 micrograms kg-1 of neurotensin in depot form every other day until the end of the experiment. In week 40, prolonged alternate-day administration of neurotensin resulted in significant increases in the number and size of colon tumours and the incidence of adenocarcinomas penetrating muscle layer and deeper. However, neurotensin did not influence the incidence of tumour-bearing rats and the histological appearance of colon tumours. Administration of neurotensin caused a significant increase in the labelling index of the colon cancers but not that of colon mucosa. These findings indicate that neurotensin enhanced the growth of colon tumours, possibly related to its effect in increasing proliferation of colon cancer cells.

Topics: Adenocarcinoma; Adenoma; Animals; Azoxymethane; Body Weight; Colonic Neoplasms; Drug Synergism; Gastrins; Male; Neurotensin; Rats; Rats, Inbred Strains

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 peptide gastrin has been shown to have growth stimulatory effects on normal as well as malignant gastrointestinal tissue. In this study, we have examined the possibility of autocrine growth-stimulation of cultured colon tumor cells by a gastrin-like peptide. The gastrin/CCK receptor antagonist dibutyryl cGMP inhibited the proliferation of two human colon carcinoma cell lines HCT 116 (EC50 = 1.3 mM) and CBS (EC50 = 2.5 mM) in a dose-dependent manner. Marked morphological changes were observed in HCT 116 cells after treatment with 1 mM dibutyryl cGMP. In receptor binding assays, dibutyryl cGMP competed with 125I-labeled gastrin for binding to HCT 116 cells (IC50 = 1.8 mM). Another derivative of cyclic GMP, 8-Bromo cGMP used as control due to its considerably weaker affinity for the gastrin/CCK receptor, did not compete with radiolabeled gastrin for binding to HCT 116 cells and had no effect on the morphology or proliferation in monolayer cultures of HCT 116 or CBS cells at concentrations up to 10 mM. Antigastrin/CCK antisera was also found to have dose-dependent cytostatic effects on HCT 116 and CBS cells adapted to growth in serum-free medium. The antiproliferative effect of the gastrin/CCK receptor antagonist and antigastrin/CCK antibodies suggested that a gastrin-like peptide secreted by these cells may promote growth. Radioimmunoassay of the conditioned medium of these two cell lines indicated the presence of a gastrin-like peptide (10-50 pg/10(6) cells/72 h). Northern analysis using an oligonucleotide DNA probe complementary to the nucleotide sequence coding the dodecapeptide carboxyl terminal of human gastrin showed three transcripts (0.7, 3.3, and 3.7 kb) that hybridized with the probe. These data provide, for the first time, evidence for an autocrine growth stimulatory role of a gastrin/CCK-like peptide in cultured colon tumor cells.

Topics: Blotting, Northern; Cell Line; Cell Transformation, Neoplastic; Cholecystokinin; Colonic Neoplasms; Dibutyryl Cyclic GMP; Gastrins; Humans; Immune Sera; Radioimmunoassay; Receptors, Cholecystokinin; Tumor Cells, Cultured

The hypothesis that a gastrin-like peptide is acting as an autocrine growth factor in gastric and colonic carcinoma cell lines requires that the cells should synthesize a gastrin-like mRNA. Although no gastrin mRNA was observed in the gastric line Okajima or the colonic lines HCT 116 or LIM 1215 by Northern blotting, gastrin mRNA was detected by application of the polymerase chain reaction. Two products were observed corresponding to mRNA with and without a 130 bp intron. The sequences of both products were identical to the sequences predicted from the normal human gastrin gene.

Topics: Base Sequence; Blotting, Northern; Cloning, Molecular; Colonic Neoplasms; DNA; DNA Replication; Gastrins; Humans; Molecular Sequence Data; Oligonucleotide Probes; Polymerase Chain Reaction; RNA, Messenger; Stomach Neoplasms; Tumor Cells, Cultured

Topics: Adenocarcinoma; Animals; Cell Division; Cell Line; Colonic Neoplasms; DNA Replication; Gastrins; Glutamine; Humans; Mice; Mice, Nude; Neoplasm Transplantation; Proglumide; Somatostatin; Tumor Cells, Cultured

The effects of cysteamine (2-aminoethanethiol hydrochloride) on the incidence, number and histology of colon tumors induced by azoxymethane (AOM), and on the norepinephrine concentration in the colon wall tissue and the labelling indices of colon mucosa and colon cancers were investigated in Wistar rats. Rats received 10 weekly injections of 7.4 mg/kg body weight of AOM and alternate-day subcutaneous injections of 25 mg/kg of cysteamine in 0.9% NaCl solution until the end of the experiment. At week 40, prolonged administration of cysteamine significantly reduced the incidence and number of colon tumors. Histologically, the adenocarcinomas that did develop in rats treated with cysteamine exhibited high mucin-producing activity. Administration of cysteamine caused significant decreases in the norepinephrine concentration in colon tissue and in the labelling indices of colon mucosa and cancers. Our findings indicate that cysteamine inhibits the development of colon tumors. This action may be related to its effect in decreasing norepinephrine concentration in the colon wall tissues and subsequently in decreasing proliferation of colon cancer cells.

Topics: Adenocarcinoma; Adenoma; Animals; Azoxymethane; Carcinoma; Cell Division; Colon; Colonic Neoplasms; Cysteamine; Gastrins; Male; Norepinephrine; Rats; Rats, Inbred Strains

Human and mouse colon cancers have specific binding sites for gastrin and demonstrate a trophic response to gastrin. In the present study we used radiolabeled gastrin (2-17), to determine the molecular weight of gastrin binding proteins (receptors) on mouse and human colon cancers, by cross-linking methods. Crude membrane aliquots prepared from the tumors were radiolabeled with [125I]gastrin (2-17) +/- 1000 fold excess of unlabeled gastrin and cross-linked with 1 mM disuccinimidyl suberate. The cross-linked radiolabeled binding protein complexes were solubilized and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis. The autoradiographs of the gels demonstrated the presence of a predominant band of approximately 33-40 KDa gastrin binding protein, that was specific for gastrin analogs. Our present findings thus indicate that specific gastrin binding proteins/gastrin receptors on colon cancers are primarily present as one band with a molecular mass of approximately 33-40 KDa and are specific for gastrin-like peptides.

Topics: Animals; Autoradiography; Carrier Proteins; Colonic Neoplasms; Cross-Linking Reagents; Gastrins; Humans; Male; Mice; Mice, Inbred BALB C; Molecular Weight

Gastrin has been shown to stimulate the growth of carcinogenic-induced colon cancer in animals, and some human colon cancers grown in vitro or as xenografts in nude mice. We determined fasting plasma gastrin levels in control subjects and patients with adenomatous polyps or adenocarcinoma of the colon to determine whether abnormal levels occurred in either patient group. Blood samples were obtained from 73 patients undergoing colonoscopy, primarily for evaluation of Hemoccult-positive stools. Fasting plasma gastrin was significantly greater in patients with adenomatous polyps (24.2 +/- 5.7 pM, N = 25) or colon cancer (84.5 +/- 28.5 pM, N = 20) than in controls (9.9 +/- 0.9 pM, N = 28). Elevations were due to gastrin values greater than control mean + 2 SD in nine patients with polyps (19.5-150.2 pM) and eight with cancer (20.7-403.2 pM). None of the patients had identifiable causes (drugs, prior surgery) for elevated gastrin levels. Our results indicate that elevated plasma gastrin occurs in subgroups of patients with adenomatous polyps or adenocarcinoma of the colon. The cause and potential role of elevated gastrin for polyp and tumor growth in these patients is not known.

Topics: Adenocarcinoma; Aged; Colonic Neoplasms; Colonic Polyps; Fasting; Female; Gastrins; Humans; Male; Middle Aged

The effect of dietary wheat bran consumption on the anticarcinogenic action of tetragastrin upon colon carcinogenesis induced by azoxymethane was investigated in 122 inbred Wistar rats. Rats were given a control fiber-free diet or the same basal diet plus 20% wheat bran. From week 5, they were given 250 micrograms per kg body weight of tetragastrin in depot form every other day until the end of the experiment at week 45. Prolonged administration of tetragastrin resulted in a significant reduction of the incidence and number of colonic tumors per rat in the group given the fiber-free diet. The adenocarcinomas that did develop in this group had high mucin-producing activity, unlike the cancers produced in controls without tetragastrin. However, administration of tetragastrin had little or no influence on the incidence, number or histology of colonic tumors in the group given basal diet plus wheat bran. Dietary supplementation with wheat bran alone had little or no effect on the development or histology of colonic tumors. Before and during the administration of carcinogens, addition of fiber to the diet resulted in a significant fall in the colonic pH and a significant increase in the crypt column length, but administration of tetragastrin did not have an additive effect on the crypt column length in rats fed diet supplemented with fiber.

Topics: Adenocarcinoma; Animals; Colon; Colonic Neoplasms; Dietary Fiber; Gastrins; Hydrogen-Ion Concentration; Male; Rats; Tetragastrin

The effects of gastrin, proglumide (a gastrin receptor antagonist), and somatostatin on growth of human colon adenocarcinoma cell lines CX1, X56, and HT29 were examined in two experimental models. Nude mice bearing xenografts of colon cancer CX1 or X56 were treated for 14-25 days subcutaneously with saline, pentagastrin (0.5 or 1.0 mg/kg), proglumide (250 or 500 mg/kg), or somatostatin 14 (33, 100, or 300 micrograms/kg) twice daily. Tumor volume, weight, protein, and deoxyribonucleic acid were measured. HT29 cells were grown in vitro and the effects of gastrin 17, proglumide, and somatostatin on growth were evaluated by cell counts or [3H]thymidine incorporation. The larger dose of pentagastrin significantly increased tumor growth in the nude mouse (p less than 0.005) and gastrin induced a biphasic effect on deoxyribonucleic acid synthesis in tissue culture with significant increases of up to 39% (p less than 0.025). Somatostatin alone significantly inhibited tumor growth in two of the cell lines and also inhibited the gastrin-induced growth. Proglumide had no effect by itself but significantly inhibited gastrin-stimulated growth. These findings suggest that growth of some human colon cancers may be hormone-dependent.

Topics: Adenocarcinoma; Animals; Cell Line; Colonic Neoplasms; DNA, Neoplasm; Gastrins; Glutamine; Humans; Mice; Mice, Nude; Neoplasm Transplantation; Neoplasms, Hormone-Dependent; Proglumide; Somatostatin; 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

We have recently demonstrated that gastrin stimulates growth of mouse colon cancer (MC-26) in vivo by regulation of gastrin receptors (GR). In the present study, we have tested the effect of proglumide (PGL), a GR antagonist, on the trophic and GR-regulatory effects of gastrin on MC-26 tumors. Four groups of 12 mice each were inoculated with 5 X 10(4) MC-26 cells and given injections of either normal saline (control), pentagastrin (PG), PGL, or both PG + PGL for 21 days. At the end of the treatment period, body, tumor, fundic, and colon weights were noted and GR measured. Two types of specific gastrin-binding sites were found on tumor cell membranes of control mice, one with high binding affinity (Kd = less than 1.0 nM) and low capacity (GR), and the other with a very high capacity and a low affinity (Kd = greater than 0.1 microM) (type 2 gastrin-binding sites). Only the type 1 GR were observed on the fundic mucosal and colon membranes. PG treatment resulted in a significant weight increase of the tumors with an up-regulation of only type 1 GR. On the other hand, PG had no significant effect on fundic mucosal and colonic GR levels, but caused a significant increase in fundic mucosal weights. PGL completely inhibited both the trophic and GR up-regulatory effects of PG on tumors, but incompletely reduced the PG-stimulated fundic mucosal weight gain, indicating differential sensitivity of tumor and normal tissues to PGL. PGL, in the absence of PG, was slightly trophic for normal fundic mucosa, but had no effect on MC-26 tumors and normal colon. The one striking effect of PGL, in the presence of PG, was the significant lowering of the binding affinity of type 1 GR for gastrin on both the tumor and normal gastrointestinal tissues. This effect may be another mechanism by which PGL interferes with the actions of PG on MC-26 tumors and fundic mucosa of mice.

Topics: Animals; Colon; Colonic Neoplasms; Gastrins; Glutamine; Male; Mice; Pentagastrin; Proglumide; Receptors, Cholecystokinin

We recently reported trophic response of transplantable mouse colon cancer cells (MC-26) to pentagastrin, in vivo, and demonstrated gastrin receptors on MC-26 cells, in vitro. In the present study, growth of MC-26 cells in mice, in response to pentagastrin, was studied in relation to binding kinetics and capacity of gastrin receptor. Gastrin receptor levels on mouse fundic and colonic membranes and on MC-26 cellular membranes were determined before MC-26 cell inoculation and designated as Day 0 levels. Four groups of mice were next inoculated with MC-26 cells and given injections of either pentagastrin (treated) or normal saline (control) for 10 or 15 days. At the end of the treatment periods, body, tumor, fundic, and colon weights were noted and gastrin receptor measured. tumor and fundic weights increased significantly within 15 days of pentagastrin treatment, compared to control values. In control (non-pentagastrin treated) mice, the binding affinity of gastrin receptor on tumor membranes was significantly decreased and associated with the complete loss of high-affinity gastrin receptor (Kd = less than 0.5 nM) by Day 15 of tumor growth. On the other hand, both the binding affinity and gastrin receptor levels of tumor membranes were maintained at Day 0 values by pentagastrin treatment. Endogenous gastrin was therefore ineffective in maintaining high-affinity gastrin receptor on control tumors. A significant number of low-affinity gastrin-binding sites (Kd = less than 2 nM) appeared in control tumors by Day 15, which could reflect rapid dedifferentiation or conformational changes of gastrin receptor in the absence of high levels of normal regulatory hormones. These studies demonstrate that the trophic effects of gastrin on MC-26 cells are probably mediated by its regulation and maintenance of the binding affinity and capacity of gastrin receptor on the cancer cells, in vivo.

Topics: Animals; Body Weight; Colon; Colonic Neoplasms; Gastric Fundus; Gastrins; Mice; Mice, Inbred BALB C; Neoplasm Transplantation; Pentagastrin; Receptors, Cell Surface; Receptors, Cholecystokinin; Time Factors

The effects of the C-terminal tetrapeptide of gastrin, tetragastrin, on the colonic mucosa on Days 15 and 25 during intrarectal administration of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and its effects on the incidences of colonic tumors in experimental Wk 20 and 35 were investigated in Wistar rats. Administration of tetragastrin in depot form during instillation of MNNG resulted in significant decreases in the incidences of mucosal erosions, ulcerations, and atypical regenerative glandular hyperplasias in the colonic mucosa, most of these lesions being greater in the distal half of the colon. Administration of tetragastrin also significantly decreased the incidences and/or numbers of colonic tumors in Wk 20 and 35. The distribution of colonic tumors induced in Wk 20 and 35 corresponded well to those of erosions, ulcerations, and atypical regenerative glandular hyperplasias induced during the administration of MNNG. These findings suggest that the effect of tetragastrin in decreasing the incidences of erosions, ulcerations, and atypical regenerative glandular hyperplasias in the colonic mucosa during instillation of MNNG is related to its effect in reducing the development of colonic tumors.

Topics: Animals; Colonic Neoplasms; Drug Administration Schedule; Gastrins; Intestinal Diseases; Intestinal Mucosa; Methylnitronitrosoguanidine; Rats; Tetragastrin; Time Factors

The trophic effects of the hormone gastrin-17 were examined on a human colon cancer cell line. LoVo cells were obtained from the American Type Culture Collection and grown in minimal essential medium in the presence of 10% bovine fetal serum. To demonstrate the trophic effect of gastrin, synchronization was necessary. The effect of gastrin was optimal after 26-h exposure to 0.6 mM thymidine. In the presence of serum the optimal dose of gastrin for stimulation of DNA synthesis was 7.2 X 10(-10) M. Under these conditions gastrin caused a 220% increase in [3H]thymidine incorporation. In the absence of serum the optimal dose of gastrin (3.6 X 10(-9) M) increased DNA synthesis approximately 200%. Twenty-four hours after gastrin treatment (1.8 X 10(-10) M gastrin 17) cell numbers increased 50.8% compared with control. At 48 h this increase was maintained at 44%. Maximum stimulation by gastrin occurred 7-8 h after release from synchronization and exposure to gastrin. This corresponded to the S phase of the cell cycle. Significant stimulation occurred a second time at 22-24 h, presumably during the second S phase in a still synchronous or partially synchronous cell population. These data demonstrate that physiological concentrations of gastrin-17 can stimulate the growth of a human cancer cell line and that some degree of synchronization may be necessary to demonstrate similar effects in other cell lines. Such cell lines may provide a source of rapidly growing cells in which the mechanisms of the trophic effect of gastrin can be examined.

Topics: Adenocarcinoma; Blood; Cell Division; Cell Line; Colonic Neoplasms; Culture Media; DNA; Gastrins; Humans

Gastrin is trophic for normal gastric and colonic mucosa. We examined the potential trophic effects of chronic gastrin administration on the growth of mouse colon adenocarcinoma (MC-26). Thirty-three mice bearing transplantable MC-26 colon cancers were treated with varying doses (125, 250, or 500 micrograms/kg/day) of pentagastrin. Significant increases in tumor weight and DNA content were observed. Fundic mucosal weight and DNA content in these mice showed a dose-related trophic response. The weight of control fundic mucosa was 10 mg and rose to 20, 45, and 65 mg with increasing doses of gastrin. The DNA content of control fundic mucosa was 155 micrograms and rose to 220, 340, and 480 micrograms as the dose of gastrin was increased. Pentagastrin stimulated growth of the MC-26 colon cancer, but the threshold for gastrin-stimulated tumor growth was different from that of normal mucosal growth. The hyperplastic response of the fundic mucosa was increased by increasing gastrin doses; whereas, colon cancer hyperplasia was maximal at the lowest dose tested (125 micrograms/kg/day) and did not increase further with increasing doses of hormone. Mice bearing gastrin-stimulated tumors died at a significantly greater rate than did mice with untreated tumors (80% of control mice and none of the treated mice were alive at day 55). The effects of gastrin treatment on the growth of MC-26 colon cancer persist after treatment is discontinued; mice with tumors that were treated with gastrin for either 7 or 14 days and in which the treatment was stopped were all dead by 35 or 28 days, respectively, after the end of treatment.

Topics: Adenocarcinoma; Animals; Colon; Colonic Neoplasms; DNA, Neoplasm; Gastrins; Intestinal Mucosa; Male; Mice; Mice, Inbred BALB C; Neoplasm Transplantation; Pentagastrin; Stimulation, Chemical

Some tumors are responsive to hormone manipulation. Some gastric and colonic adenocarcinomas from both humans and animals have specific gastrin receptors. A transplantable mouse colon adenocarcinoma cell line (MC-26) contains gastrin receptors; growth of MC-26 colon cancer in vivo is stimulated by pentagastrin (PG). The purpose of this study was to determine whether a gastrin-receptor antagonist, proglumide (PGL), would inhibit growth of MC-26 colon cancer and prolong survival in tumor-bearing mice. Subcutaneous tumors were induced by injecting single-cell suspensions of MC-26 cells into 50 mice divided into 10/group. In Experiment 1, all mice received 1 X 10(5) tumor cells and treatment groups were divided as follows: Group A received intraperitoneal (IP) saline (0.2 ml tid beginning on day 1); B, IP, PGL (250 mg/kg tid) from day of tumor cell inoculation; and C, IP PGL (250 mg/kg tid) from day 7 after tumor implantation. In Experiment 2, mice were inoculated with half the number of tumor cells. Group I mice received saline and Group II received PGL in the same manner starting on day 1. Tumors were measured and all mice were sacrificed on day 23. In Experiment 1, mean tumor area in Group B (PGL-treated) was significantly smaller than Group A on days 11, 14, 17, and 21. Tumors of Group C were significantly smaller than controls on day 21. Survival of PGL-treated mice was significantly prolonged. In Experiment 2, mean tumor area, mean tumor weight, and tumor DNA and RNA content were significantly less in the PGL-treated group than control. It was concluded that growth of a gastrin-responsive colon cancer was inhibited and host survival was enhanced by treatment with a gastrin-receptor antagonist. Hormone manipulation may be a useful treatment for gastrointestinal cancers.

Topics: Adenocarcinoma; Animals; Body Weight; Cell Division; Cell Line; Colon; Colonic Neoplasms; Gastric Fundus; Gastrins; Glutamine; Mice; Organ Size; Pancreas; Proglumide; Receptors, Cell Surface

The effects of ad libitum feeding of a chemically defined diet in liquid form on the incidence and histology of colon cancer induced by 10 weekly sc injections of 7.4 mg/kg of azoxymethane [(AOM) CAS: 25843-45-2] were investigated in W-rats. The chemically defined diet was adjusted once every 24 hours from 4 weeks before injection of the carcinogen to the end of the experiment at week 40. Oral administration of the defined diet resulted in significant increase in the incidence of colon cancer at week 40. Histologic examination showed that unlike adenocarcinomas with high mucin-producing activity, which were common in rats on pellet diet, most of the adenocarcinomas that developed in rats fed on defined diet were highly or well differentiated, with a typical glandular pattern. Administration of the chemically defined diet also resulted in marked colon mucosal hypoplasia and reduced gastrin levels in the serum at weeks 4 and 40.

Topics: Adenocarcinoma; Animals; Colonic Neoplasms; Dietary Fiber; Food, Formulated; Gastric Mucosa; Gastrins; Male; Rats; Rats, Inbred Strains

Synthetic human gastrin 17-I (MG) and an analogue, [Leu15]gastrin-17-I (LG), were radiolabeled with Na125I by Iodo-Gen, EnzymoBead, and chloramine-T methods, and the characteristics of the radiolabeled peptides were determined. When 125I-MG was iodinated by chloramine-T, its biological activity and its binding activity were almost abolished, whereas the biological activity of 125I-LG, iodinated by either of the methods, and of 125I-MG, iodinated by Iodo-Gen and EnzymoBeads, was not significantly affected. The kinetics, affinity, and specificity of binding of 125I-MG, iodinated by Iodo-Gen, to crude and purified membranes from rat fundic mucosa were examined and found to be similar to that for 125I-LG. Age-associated changes in the number and affinity of gastrin receptors (GR) on the crude membranes of gastrointestinal mucosa of rats was also examined. Significantly fewer GR were observed on the crude membranes of fundic mucosa of aged (24 mo old) compared with young (3- and 6-mo-old) rats. In addition, specific gastrin-binding sites (4.7 +/- 0.9 fmol/mg prot) with low affinity (Kd = 3.7 +/- 1.2 nM) were observed in the antrum of aged rats, the significance of which is not understood. There were, however, no differences in the number and characteristics of GR in other regions of the intestine of old and young rats. The presence of GR was additionally assessed in cell lines of gastrointestinal cancers from humans, mice, and hamsters.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Age Factors; Animals; Cell Line; Cell Membrane; Colonic Neoplasms; Cricetinae; Dogs; Dose-Response Relationship, Drug; Gastric Fundus; Gastric Mucosa; Gastrins; Gastrointestinal Neoplasms; Humans; Intestinal Mucosa; Kinetics; Male; Mice; Rats; Rats, Inbred Strains; Receptors, Cell Surface; Receptors, Cholecystokinin; Stomach Neoplasms

A study was done to determine if continuous administration of exogenous secretin would inhibit the trophic effect of elevated endogenous gastrin on colon neoplasms in rats. Colon tumors were induced in 37 Fisher rats by subcutaneous injection of 1,2-symdimethylhydrazine, 20 mg/kg, weekly for 18 weeks. Elevation of endogenous gastrin was achieved by antral exclusion surgery. Control rats received a sham operation. Subcutaneous osmotic minipumps delivered 35.5 U/kg/day of secretin for 7 days before the rats were killed. Rats receiving antral exclusion had significantly elevated serum gastrin levels and increased 3H-thymidine incorporation into tumor DNA compared with sham rats (P less than 0.05). There was a significant decrease in tumor DNA uptake in antral exclusion rats that received secretin as compared with antral exclusion rats without secretin (P less than 0.05). Therefore it appears that gastrin does have a trophic effect on rat colon neoplasms and that secretin does inhibit this effect, in a dose-related manner. The results of this study imply that hormonal manipulation of colon cancer may eventually become a viable therapeutic modality.

Topics: Animals; Colonic Neoplasms; Dimethylhydrazines; DNA; Gastric Mucosa; Gastrins; Injections, Subcutaneous; Intestinal Mucosa; Male; Pyloric Antrum; Rats; Secretin

The effects of prolonged administration of tetragastrin from the beginning of intrarectal instillation of 1 ml of 0.25% N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and after MNNG-treatment on the incidence and histology of colonic tumors were compared in inbred Wistar rats. In week 35 prolonged administration of testragastrin in depot form from the beginning of MNNG-treatment resulted in a significant reduction in the incidence of colonic tumors and a significant increase in the incidence of mucinous adenocarcinoma, unlike the well-differentiated adenocarcinoma produced in controls without gastrin. In contrast, prolonged administration of tetragastrin after MNNG-treatment had little or no influence on the incidence, size or histology of colonic tumors. Thus tetragastrin had no promoting effect on colonic tumors.

Topics: Adenocarcinoma; Animals; Cocarcinogenesis; Colonic Neoplasms; Drug Administration Schedule; Gastrins; Male; Methylnitronitrosoguanidine; Neoplasm Metastasis; Rats; Rats, Inbred Strains; Tetragastrin

Topics: Adenocarcinoma; Aged; Animals; Colonic Neoplasms; Female; Gastrins; Humans; Leucine; Male; Mice; Mice, Nude; Middle Aged; Neoplasm Proteins; Secretin; Stomach Neoplasms

Our objective was to determine whether chronic hypergastrinemia enhances chemical induction of rat colorectal cancers. Forty-five rats were randomized to sham operation or antral exclusion. Following a 2-week postoperative recovery period all rats were treated with 1,2-dimethylhydrazine (10 mg/kg) intraperitoneally for 20 weekly doses. Seven weeks later, the rats were killed. Blood was assayed for gastrin by radioimmunoassay. Tumor number, location, size, weight and histology were determined. The 23 rats receiving antral exclusion were hypergastrinemic compared with the 22 sham operated rats. All hypergastrinemic rats developed tumors while only 72.7% of normogastrinemic rats developed tumors. Hypergastrinemia increased the number of tumors/rat, total tumor weight/rat and total tumor volume/rat.

Topics: Animals; Colonic Neoplasms; Dimethylhydrazines; DNA, Neoplasm; Gastrins; Male; Neoplasm Proteins; Radioimmunoassay; Rats; Rats, Inbred Strains; Rectal Neoplasms; RNA, Neoplasm; Time Factors

This study deals with the growth effect of gastrin on two xenotransplantable human gastric carcinomas (SC-6-JCK, poorly differentiated adenocarcinoma; and St-15, mucinous adenocarcinoma) and on one colonic carcinoma (Co-3, well-differentiated adenocarcinoma). In SC-6-JCK, the treatment with s.c. injection of pentagastrin at a dose of 10 micrograms/mouse once daily for 25 days promoted the growth of the tumor transplanted in nude mice, but gastrin had no effect at all on St-15 and Co-3. In SC-6-JCK, the weight, size, and labeling index of [3H]thymidine of the tumor were significantly increased in comparison with those of the control (p less than 0.05). In SC-6-JCK, cyclic adenosine 3':5'-monophosphate (cAMP) in the tumor was increased by a single i.p. injection of pentagastrin at a dose of 20 micrograms/mouse in nude mice, but such an increase was not observed in St-15 and Co-3. Cyclic guanosine 3':5'-monophosphate in SC-6-JCK was slightly increased by gastrin treatment but was not affected in the other tumors. In SC-6-JCK, at 30 min after gastrin treatment when cAMP showed a maximum increase, the activity ratio of cAMP-dependent protein kinase in the tumor was also elevated. In vitro also, gastrin stimulated cAMP production and cAMP-dependent protein kinase activation. The data suggest that some human gastric carcinomas may have receptor for gastrin.

Topics: Adenocarcinoma; Adult; Animals; Cell Division; Cell Line; Colonic Neoplasms; Cyclic AMP; Cyclic GMP; Female; Gastrins; Humans; Kinetics; Male; Mice; Mice, Nude; Middle Aged; Neoplasm Transplantation; Protein Kinases; Stomach Neoplasms; Transplantation, Heterologous

The effect of tetragastrin on the incidence and histology of colonic tumors induced by intrarectal instillation of N-methyl-N'-nitro-N-nitrosoguanidine was investigated in Wistar rats. Prolonged administration of tetragastrin in depot form during and after treatment with N-methyl-N'-nitro-N-nitrosoguanidine resulted in a significant reduction in the incidence of colonic tumors in Experimental Week 35. Histological examinations showed that, unlike the well-differentiated adenocarcinomas with a typical glandular pattern in control groups, the adenocarcinomas that developed in rats treated with tetragastrin had high mucin-producing activity.

Topics: Adenocarcinoma; Adenoma; Animals; Colonic Neoplasms; Gastrins; Injections, Subcutaneous; Male; Methylnitronitrosoguanidine; Neoplasms, Experimental; Rats; Rats, Inbred Strains; Sarcoma; Tetragastrin

The hepatic uptake of unlabeled synthetic human gastrin I (1-17) was determined in four unanesthetized patients who did not have hepatic or gastroduodenal disease. The hormone was given by brief infusion and at rates producing concentrations of immunoreactive gastrin within the physiologic range. We estimated the first-pass fractional hepatic uptake by comparing the results after a portal and a peripheral infusion in each patient. It was -0.01 +/- 0.14 (mean +/- SD), demonstrating lack of hepatic uptake of gastrin I (1-17) in humans.

Topics: Colonic Neoplasms; Gastrins; Half-Life; Humans; Kinetics; Liver; Middle Aged

This study was designed to determine the effect of gastrin on colorectal neoplasms in the rat. Multiple colon and rectal cancers were induced in each of 57 rats with methylazoxymethanol. Animals were randomly assigned to groups: (1) antral exclusion, (2) antrectomy, (3) sham, or (4) sham with subsequent pentagastrin injections. Resulting tumors were analyzed for concentration and synthesis of DNA, RNA, and protein. The number of tumors per rat and distribution of tumors within the colons did not vary among groups. Antrectomy did not alter the gastrin level, and tumors developing in these animals did not differ from those of sham controls. Antral exclusion markedly raised serum gastrin levels. Both chronic endogenous hypergastrinemia and administration of exogenous pentagastrin significantly increased tumor synthesis and concentration of DNA, RNA, and protein. We conclude that gastrin exerts a trophic effect on colorectal neoplasms in rats. This biological phenomenon suggests a tumor regulatory role for the hormone.

Topics: Animals; Colonic Neoplasms; DNA; Gastrectomy; Gastrins; Male; Neoplasms, Experimental; Pentagastrin; Proteins; Rats; Rectal Neoplasms; RNA

The potential tropic effect of gastrin in promoting colonic carcinogenesis was tested in rats (n = 133) with 6- to 16-fold variations in endogenous gastrin resulting from antrectomy or fundectomy, followed by 12 injections of 1,2-dimethylhydrazine (10 mg/kg). The incidence and grade of differentiation of resulting tumors, as well as the thickness, content of nucleic acids, and specific activity of DNA in colonic mucosa, were similar in both high-gastrin and low-gastrin animals and were unchanged from data in unoperated control animals receiving only the carcinogen. Long-term changes in endogenous gastrin concentration are not followed by colonic mucosal tropic effects, and gastrin does not act as a cofactor in dimethylhydrazine-induced carcinogenesis at this dosage.

Topics: 1,2-Dimethylhydrazine; Animals; Colonic Neoplasms; Dimethylhydrazines; Gastric Fundus; Gastrins; Male; Neoplasms, Experimental; Pyloric Antrum; Rats

Topics: Colon; Colonic Neoplasms; Gastrins; Humans; Male; Middle Aged; Zollinger-Ellison Syndrome

The distributions of acid alpha1-glycoprotein, alpha1-fetoprotein, beta-galactosidase and gastrin in gastric carcinoma and gastric ulcer as well as in the neighbourhood of these lesions were studied by means of immunohistochemical methods on imprint preparation. We could not find significant differences between gastric carcinoma and the nonneoplastic lesions, except for the acid alpha1-glycoprotein. The results of this first study indicate that the immunochemical and immunohistological assay of acid alpha1-glycoprotein might be of practical value in diagnosing malignant changes of gastric mucosa.

Topics: ABO Blood-Group System; alpha-Fetoproteins; Animals; Colonic Neoplasms; Duodenal Ulcer; Fluorescent Antibody Technique; Galactosidases; Gastric Mucosa; Gastrins; Goats; Guinea Pigs; Histocytochemistry; Humans; Peptic Ulcer; Rabbits; Stomach Neoplasms