big-gastrin and Pancreatic-Neoplasms

Accumulating evidence in literature suggests that amidated and non-amidated gastrins (gastrin precursors) may play an important role in the proliferation and carcinogenesis of gastrointestinal and pancreatic cancers, especially in the presence of DNA damaging agents and/or infectious agents. Amidated gastrins appear to have a protective role, while progastrins exert growth promoting effects in cancers. Several receptor subtypes and signal transduction pathways mediate the biological effects of the gastrin peptides. Progastrin and gastrins also exert anti-apoptotic effects, which may additionally contribute to the growth and co-carcinogenic effects of these peptides on GI mucosal cells in vivo. Amidated gastrins additionally play an important role in the migration of GI epithelial cells, and in glandular morphogenesis, while progastrins may play an important role in invasion and metastasis. Therefore, targeting progastrins, gastrins, and their cognate receptors may provide a therapeutic tool for treating GI and pancreatic cancers. Targeting CCK2-receptors has, so far, not provided optimal beneficial effects. However, targeting gastrins via a vaccine approach has provided some encouraging results for treating GI and pancreatic cancers. It is expected that targeting precursor gastrins (progastrins and gly-gastrins), exclusively rather than amidated gastrins, may be more effective for treating GI cancers. Since GI cancers at advanced stages are largely responsive to autocrine and intracrine progastrins, down-regulation of intracellular progastrins will likely be more effective at this stage.

Topics: Animals; Antineoplastic Agents; Apoptosis; Gastrins; Gastrointestinal Neoplasms; Humans; Immunotherapy; Neoplasm Metastasis; Pancreatic Neoplasms; Protein Precursors; Receptor, Cholecystokinin B; Signal Transduction

The interaction of gastrin with the cholecystokinin 2 (CCK2)/gastrin receptor has been studied extensively in relation to gastric acid secretion. However, not much is known about the contribution of individual amino acids of gastrin interacting with the CCK2 receptor, when gastrin is acting as a tumor growth factor. The purpose of the present study was to determine the significance of each individual amino acid residue of human gastrin-17 with respect to CCK2 receptor-mediated cell proliferation. Activation of this receptor was assessed using an in vitro bioassay based on gastrin-induced expression of a c-fos-luciferase reporter, transfected in AR42JB13 and Colo 320 cells, a rat pancreatic and human colorectal cell line respectively. Gastrin-17 dose dependently increased c-fos induction in both cancer cell lines. L365,260, a known CCK2 receptor antagonist, completely blocked the gastrin signal, demonstrating the specificity of this assay. We demonstrated for the first time that four carboxy-terminal amino acids of gastrin-17 are essential for activation of the CCK2 receptor with respect to c-fos induction. Also other residues of gastrin-17, notably glycine-2 for the rat CCK2 receptor and glutamic acid 8-10 and tyrosine-12 for the human receptor, were found to be important, although to a lesser extent. Alanine-substitution variants of each of the four carboxy-terminal amino acids of gastrin-17 showed strongly reduced receptor activation but did not act as competitive inhibitors of gastrin-17. Identification of the essential role of the carboxy-terminal tetrapeptide of gastrin-17 in CCK2 receptor-mediated c-fos induction indicates that gastrin inhibitory therapeutic strategies should mainly be targeted toward this region of gastrin.

Topics: Alanine; Amino Acid Substitution; Animals; Cell Proliferation; Colorectal Neoplasms; DNA Primers; Gastrins; Genes, fos; Humans; Luciferases; Pancreatic Neoplasms; Pentagastrin; Promoter Regions, Genetic; Protein Precursors; Rats; Receptor, Cholecystokinin B; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tumor Cells, Cultured

Progastrin (PG) exerts proliferative and antiapoptotic effects on intestinal epithelial and colon cancer cells via Annexin II (ANX-II). In here, we show that ANX-II similarly mediates proliferative and antiapoptotic effects of PG on a pancreatic cancer cell line, AR42J. The role of several signaling molecules was examined in delineating the biological activity of PG. PG (0.1-1.0 nmol/L) caused a significant increase (2- to 5-fold) in the phosphorylation of phosphatidylinositol 3-kinase (PI3K), Akt (Thr(308)), p38 mitogen-activated protein kinase (MAPK; Thr(180)/Tyr(182)), extracellular signal-regulated kinases (ERK; Thr(202)/Tyr(204)), IkappaB kinase alpha/beta (IKKalpha/beta; Ser(176)/(180)), IkappaBalpha (Ser(32)), and p65 nuclear factor-kappaB (NF-kappaB; Ser(536)). Inhibition of p44/42 ERKs (PD98059), p38 MAPK (SB203580), Akt, and PI3K (LY294002), individually or combined, partially reversed antiapoptotic effects of PG. The kinetics of phosphorylation of IKKalpha/beta in response to PG matched the kinetics of phosphorylation and degradation of IkappaBalpha and correlated with phosphorylation, nuclear translocation, and activation of p65 NF-kappaB. NF-kappaB essential modulator-binding domain peptide (an inhibitor of IKKalpha/beta) effectively blocked the activity of p65 NF-kappaB in response to PG. Activation of p65 NF-kappaB, in response to PG, was 70% to 80% dependent on phosphorylation of MAPK/ERK and PI3K/Akt molecules. Down-regulation of p65 NF-kappaB by specific small interfering RNA resulted in the loss of antiapoptotic effects of PG on AR42J cells. These studies show for the first time that the canonical pathway of activation of p65 NF-kappaB mediates antiapoptotic effects of PG. Therefore, targeting PG and/or p65 NF-kappaB may be useful for treating cancers, which are dependent on autocrine or circulating PGs for their growth.

Topics: Annexin A2; Apoptosis; Blotting, Western; Cell Line, Tumor; Cell Proliferation; Gastrins; Gene Expression Regulation, Neoplastic; Humans; I-kappa B Kinase; Immunoprecipitation; NF-kappa B; Pancreatic Neoplasms; Phosphorylation; Promoter Regions, Genetic; Protein Precursors; RNA, Small Interfering; Transcription Factors

The gastrin gene is expressed widely in pancreatic adenocarcinomas and the study aimed to assess its role in both the resistance of cancer cells to apoptosis and the sensitivity of cells to chemotherapeutic agents. Two human pancreatic cell lines, PAN1 and BXPC3, expressed gastrin at both the RNA and protein levels and are shown to be representative of human pancreatic adenocarcinomas in terms of gastrin expression. Inhibition of endogenous gastrin production by tumor cells was achieved with neutralizing gastrin antiserum and transfection with a gastrin antisense plasmid. Gastrin antiserum synergized with both taxotere and gemcitabine in inhibiting the in vitro growth of the PAN1 cell line with the inhibitory effect of the antiserum increasing from 12.7% to 70.2% with taxotere (P < 0.05) and 28.6% with gemcitabine (P < 0.01) after controlling for the effects of the cytotoxics. Synergy was only achieved with taxotere in BXPC3 cells with the inhibitory effect of gastrin antiserum increasing from 22.9% to 50.0% (P < 0.005). Cells transfected with gastrin antisense had reduced in vitro growth in low serum conditions and were poorly tumorigenic in nude mice at an orthotopic site. Gastrin antisense-transfected PAN1 cells had increased sensitivity to the antiproliferative effects of both gemcitabine (IC50 of > 100 microg/ml reduced to 0.1 microg/ml) and taxotere (IC50 of 20 microg/ml reduced to < 0.01 microg/ml) when compared with vector controls. The increased sensitivity of PAN1 antisense coincided with increased caspase-3 activity and reduced protein kinase B/Akt phosphorylation in response to both gemcitabine and taxotere. Gastrin gene circumvention may be an optimal adjunct to chemotherapeutic agents, such as taxotere and gemcitabine, in pancreatic adenocarcinoma.

Topics: Adenocarcinoma; Animals; Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Deoxycytidine; DNA, Antisense; Docetaxel; Gastrins; Gemcitabine; Gene Expression; Genetic Therapy; Humans; Male; Mice; Mice, Nude; Pancreatic Neoplasms; Phosphorylation; Protein Precursors; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Taxoids; Transfection

This study evaluates whether a new analytic principle, processing-independent analysis (PIA), offers better specificity and sensitivity than the conventional gastrin radioimmunoassay in the diagnosis of gastrinomas.. Plasma concentrations of alpha-amidated gastrins and the total progastrin product were measured with radioimmunoassay and with PIA, respectively, in 512 samples taken for gastrin measurement and in a selected group of gastrinoma patients (n=10).. Among the 512 patients were 9 with gastrinomas. In plasma from these patients the median degree of amidation (ratio of alpha-amidated gastrins to total progastrin product) was 75% (range, 25-98%), whereas in the other groups the medians varied from 41% to 86%. In the second group of gastrinoma patients all had a degree of amidation of less than 50%.. In screening for gastrinomas PIA offered no diagnostic advantages in comparison with conventional gastrin radioimmunoassay. However, in selected patients who in spite of normal or slightly increased concentrations of amidated gastrins were still suspected of having gastrinoma, additional measurement of the total progastrin product showed incomplete processing of progastrin and thus proved helpful in establishing the diagnosis.

Topics: Anti-Ulcer Agents; Case-Control Studies; Female; Gastrinoma; Gastrins; Humans; Male; Middle Aged; Pancreatic Neoplasms; Peptic Ulcer; Protein Precursors; Radioimmunoassay; Sensitivity and Specificity; Zollinger-Ellison Syndrome

Functional gastrin-containing tumor cells were maintained for up to 8 wk without fibroblastoid cell overgrowth. Short-term cultures consisted mainly of colonies composed of small polygonal cells, 70%-90% of which stained positive for immunoreactive gastrin. Cultures exhibited limited growth but viability remained high for 2-3 wk. Culture medium contained component I, and gastrin 34, 17, and 14. With time the major C-terminal gastrin species in medium changed from gastrin 17 at 3 days to gastrin 34 at 5 wk. Extracts of cultured cells contained gastrin 34, 17, and 14; gastrin 17 was the major form detected at all times. Ultrastructurally, cultured tumor cells retained morphological integrity for several weeks; however, with time changes in the appearance of the secretory granules accompanied by evidence of cellular retrodifferentiation were gradually observed. Secretin, gastrin-releasing peptide, 8-bromoadenosine 3':5'-cyclic monophosphate, and phorbol, 12-myristate, 13-acetate stimulated the release of gastrin from cultured cells in a time-dependent fashion. Secretin, bombesin, gastrin-releasing peptide, L-tryptophan, and ethylamine stimulated gastrin release in a dose-dependent fashion. Somatostatin 14 inhibited secretin, bombesin, and gastrin-releasing peptide stimulated gastrin release but did not alter basal release. Cultured cells demonstrated de novo gastrin synthesis, evidenced by their ability to incorporate radiolabeled amino acids into immunoadsorbable gastrinlike material. Primary cultures of gastrin-containing tumor cells free from stromal contamination offer unique advantages for studies of factors that regulate the synthesis and secretion of gastrin and may prove of potential value for studies on cell differentiation and growth.

Topics: Culture Media; Gastrinoma; Gastrins; Humans; Immunoenzyme Techniques; Pancreatic Neoplasms; Protein Precursors; Time Factors; Tumor Cells, Cultured

Processing-independent radioimmunoanalysis for progastrin showed that extracts of normal pancreatic tissue from normal subjects (n = 5) and from patients with adenocarcinoma of the papilla of Vater (n = 4) contain progastrin and its products. The concentrations varied from 0.1 to 5.8 pmol/g tissue, of which carboxyamidated bioactive gastrins constituted 0.03-1.9 pmol/g. In histologically normal and nonneoplastic pancreatic tissue from patients with duodenal (n = 3) and pancreatic (n = 2) gastrinomas the expression of gastrin was significantly higher-14.5 pmol/g (median), of which 28% was bioactive amidated gastrins. Gastrin-17 was the main bioactive product, but its immediate precursor, glycine-extended gastrin-17, constituted the predominant part of the preprogastrin product in pancreatic tissue. Proper gastrinoma tissue contained several precursor forms, including intact unprocessed progastrin. Progastrins were also found in high concentrations in plasma from the gastrinoma patients. The results raise the possibility that increased expression of progastrin and its products in non-neoplastic pancreatic tissue is a primary defect predisposing to neoplasia.

Topics: Adenocarcinoma; Ampulla of Vater; Common Bile Duct Neoplasms; Gastrinoma; Gastrins; Gene Expression; Humans; Pancreas; Pancreatic Neoplasms; Protein Precursors; Protein Processing, Post-Translational; Zollinger-Ellison Syndrome

The characterization of the tumors and their metastasis in patients with the Zollinger-Ellison syndrome is currently based on the immunohistochemical identification of gastrin cells. However, sometimes tumoral cells fail to react with common C-terminal gastrin antibodies. In order to clarify this failure, we carried out morphologic, morphometric and immunocytochemical analyses performed on light and electron microscope levels of 6 pancreatic and 1 metastatic gastrinomas, using antibodies raised against various sequences of human progastrin. On the basis, in light microscopy, of qualitative analysis of immunostaining within cells and of immunostained cell numbers, gastrin 34 residue seemed to be the prominent form in 2 of the tumor tissues, G-17 in 1 tumor which was not responsive with C terminus progastrin and N terminus G-34 antisera, and progastrin in the metastatic tissue that did not contain typical gastrin (G-like) cells. Two tumors failed to react with all antisera used. At the electron microscope level, immunogold staining revealed that progastrin was present only in the progranules and gastrin 34 in both progranules and intermediate granules. Quantitative studies performed on 3 tumors showed that, within a given tumoral cell, about 25 percent of progranules contained progastrin while 75 percent contained gastrin 34. We concluded that different forms of gastrin can be immunodetected in a gastrinoma tissue, depending on the regions, and that the distribution of progastrin fragments is variable from tumor to tumor. So, specific antibodies to different fragments of progastrin may help to the characterization of gastrinomas.

Topics: Antibodies; Epitopes; Gastrinoma; Gastrins; Humans; Immune Sera; Immunohistochemistry; Microscopy, Electron; Pancreatic Neoplasms; Protein Precursors

There is a general agreement on the cell specificity of gastrin processing. In order to investigate this processing in Zollinger-Ellison (ZE) patients, we have studied in two primary gastrinoma cultures (one from a pancreatic tumor, the other from a liver metastasis) the proportion of progastrin fragments using immunochemical and immunohistological methods. In tumor extracts as well as in sera, the predominant gastrin form differed between the two patients (i.e. being G17 and G34, respectively). In the two gastrinoma cultures, RIA determinations and electron microscopic observations indicated that the proportion of progastrin increased with time while that of G17 and G34 decreased. On the other hand, as the culture time extended, an increasing proportion of nonimmunostained secretory granules was observed suggesting the presence of other gastrin precursors (e.g. Gly-extended progastrin). From these findings, we suggest that gastrinoma culture cells could be a valuable tool in the biochemical approach to gastrin processing in ZE tumors.

Topics: Cells, Cultured; Gastrinoma; Gastrins; Humans; Pancreatic Neoplasms; Protein Precursors; Staining and Labeling

Topics: Amino Acid Sequence; Animals; Base Sequence; DNA; Exons; Gastrinoma; Gastrins; Gene Expression Regulation; Humans; Introns; Pancreatic Neoplasms; Protein Precursors; Species Specificity

Tissue pieces of a metastatic human gastrinoma (ultrastructural Type II) were successfully transplanted to the anterior eye-chamber of rats immunosuppressed with Cyclosporin A. Immunocytochemical investigation of the transplants showed evidence for preserved endocrine activity of tumour cells with immunoreactivity towards the C-terminal of the gastrin/cholecystokinin molecule. Studies of gastric acid secretion in tumour-bearing rats and sham-operated controls with chronic gastric fistulas showed that the basal acid output did not differ between the groups during 3 weeks of study. However, the stimulated gastric acid secretion decreased after 5 days in both groups to remain significantly depressed throughout the study, an effect probably due to Cyclosporin A treatment of the groups. The concentration of immunoreactive gastrin in plasma from rats with tumours in oculo was 5 times higher than in sham-operated rats. Gastrin-34 was the major immunoreactive component in both patient serum and rat plasma. An immunoreactive fraction corresponding to component I was found in the patient serum, but not in the rat plasma, although present in the chamber fluid. Components corresponding to gastrin-17 were found both in the patient serum and in the rat plasma. The chromatographic pattern of the tumour was similar to that in rat chamber fluid. The dominating component corresponded to gastrin-17, while gastrin-34 represented the quantitatively smaller component. Gastrin-34 was, however, relatively more abundant in the tumour extract than in the chamber fluid. The study also indicates that a gastrin-producing tumour transplanted in oculo in immunosuppressed rats may increase the rat plasma concentration of the same molecular forms of gastrin as seen in the clinical situation.

Topics: Adolescent; Animals; Anterior Chamber; Chromatography, Gel; Female; Gastric Acid; Gastrinoma; Gastrins; Humans; Immunosuppression Therapy; Male; Microscopy, Electron; Neoplasm Transplantation; Pancreatic Neoplasms; Protein Precursors; Radioimmunoassay; Rats; Rats, Inbred Strains

Topics: Duodenal Neoplasms; Gastric Mucosa; Gastrinoma; Gastrins; Glycine; Humans; Pancreatic Neoplasms; Protein Precursors