cholecystokinin and Colonic-Neoplasms

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

Cholecystokinin (CCK) receptors are overexpressed in numerous human cancers, such as pancreatic, colon and gastric cancers. Previous studies have shown that the specific receptor-binding property of CCK for CCK receptors (CCKRs) can be exploited to produce immunotoxins (ITs) that target cancer cells overexpressing CCK receptors.. Construct a new IT-targeting CCKR-overexpressing colon cancers.. To construct the CCKR-targeted IT, a reverse CCK8 peptide was fused with a modified 38-kDa truncated form of the Pseudomonas exotoxin (PE38KDEL). An efficient immunoaffinity purification procedure was used to produce a PE38-based IT. Several analyses, including CCK8 competition and indirect immunofluorescence assays, were performed to confirm the interaction between rCCK8 and CCKR. After cytotoxic assays on several cell lines, the anti-tumor activity of the new IT was detected in nude mice.. The rCCK8PE38 IT showed specific cytotoxicity for two colon cancer cell lines and one gastric cancer cell line. After purification, 18-26 mg of pure rCCK8PE38 per 1 L of culture was obtained. Purified rCCK8PE38 showed high cytotoxicity in colon cancer cell lines with IC50 values of 0.8-3.5 ng/mL. The results of the CCK8 competition and indirect immunofluorescence assays showed that rCCK8 had a specific interaction with CCKR. Nude mice inoculated with HCT-8 tumor xenografts were treated with rCCK8PE38, which efficiently decreased the tumor size in those mice.. All of these data suggest that rCCK8PE38 has potential as a new immunotherapy agent. Furthermore, the results of this study further support the high value of the immunoaffinity method for IT purification procedures.

Topics: Animals; Cell Line, Tumor; Cholecystokinin; Colonic Neoplasms; Exotoxins; Female; Fluorescent Antibody Technique, Indirect; Humans; Immunotherapy; Immunotoxins; Mice; Mice, Nude; Peptide Fragments; Pseudomonas; Receptors, Cholecystokinin; Stomach Neoplasms; Xenograft Model Antitumor Assays

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

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

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

Topics: Adult; Age Factors; Aged; Aging; Cholecystokinin; Colonic Neoplasms; Female; Gallbladder Diseases; Gastrectomy; Hernia, Diaphragmatic; Histocytochemistry; Humans; Male; Microscopy, Electron; Middle Aged; Pancreas; Secretin

Topics: Amyloidosis; Biochemical Phenomena; Biochemistry; Cholangitis; Cholecystitis; Cholecystokinin; Colonic Neoplasms; Drug Therapy; Duodenum; Electrophoresis; Enteritis; Gastritis; Hemosiderosis; Humans; Intestinal Secretions; Leucyl Aminopeptidase; Liver Cirrhosis; Liver Diseases; Melanoma; Mucous Membrane; Nephrosis; Pancreatitis