Discovery of once-weekly, peptide-based selective GLP-1 and cholecystokinin 2 receptors co-agonizts.

A group of long-acting, peptide-based, and selective GLP-1R/CCK-2R dual agonizts were identified by rational design. Guided by sequence analysis, structural elements of the CCK-2R agonist moiety were engineered into the GLP-1R agonist Xenopus GLP-1, resulting in hybrid peptides with potent GLP-1R/CCK-2R dual activity. Further modifications with fatty acids resulted in novel metabolically stable peptides, among which 3d and 3 h showed potent GLP-1R and CCK-2R activation potencies and comparable stability to semaglutide. In food intake tests, 3d and 3 h also showed a potent reduction in food intake, superior to that of semaglutide. Moreover, the acute hypoglycemic and insulinotropic activities of 3d and 3 h were better than that of semaglutide and ZP3022. Importantly, the limited pica response following 3d and 3 h administration in SD rats preliminarily indicated that the food intake reduction effects of 3d and 3 h are independent of nausea/vomiting. In a 35-day study in db/db mice, every two days administration of 3d and 3 h increased islet areas and numbers, insulin contents, ß-cell area, ß-cell proliferation, as well as improved glucose tolerance, and decreased HbA1c, to a greater extent than ZP3022 and semaglutide. In a 21-day study in DIO mice, once-weekly administration of 3d and 3 h significantly induced body weight loss, improved glucose tolerance, and normalized lipid metabolism, to a greater extent than semaglutide. The current study showed the antidiabetic and antiobesity potentials of GLP-1R/CCK-2R dual agonizts that warrant further investigation.

Cholecystokinin release triggered by NMDA receptors produces LTP and sound-sound associative memory.

Memory is stored in neural networks via changes in synaptic strength mediated in part by NMDA receptor (NMDAR)-dependent long-term potentiation (LTP). Here we show that a cholecystokinin (CCK)-B receptor (CCKBR) antagonist blocks high-frequency stimulation-induced neocortical LTP, whereas local infusion of CCK induces LTP. CCK-/- mice lacked neocortical LTP and showed deficits in a cue-cue associative learning paradigm; and administration of CCK rescued associative learning deficits. High-frequency stimulation-induced neocortical LTP was completely blocked by either the NMDAR antagonist or the CCKBR antagonist, while application of either NMDA or CCK induced LTP after low-frequency stimulation. In the presence of CCK, LTP was still induced even after blockade of NMDARs. Local application of NMDA induced the release of CCK in the neocortex. These findings suggest that NMDARs control the release of CCK, which enables neocortical LTP and the formation of cue-cue associative memory.

Cholecystokinin is involved in triglyceride fatty acid uptake by rat adipose tissue.

The incorporation of plasma triglyceride (TG) fatty acids to white adipose tissue (WAT) depends on lipoprotein lipase (LPL), which is regulated by angiopoietin-like protein-4 (ANGPTL-4), an unfolding molecular chaperone that converts active LPL dimers into inactive monomers. The production of ANGPTL-4 is promoted by fasting and repressed by feeding. We hypothesized that the postprandial hormone cholecystokinin (CCK) facilitates the storage of dietary TG fatty acids in WAT by regulating the activity of the LPL/ANGPTL-4 axis and that it does so by acting directly on CCK receptors in adipocytes. We report that administration of CCK-8 (a bioactive fragment of CCK) to rats: (i) reduces plasma ANGTPL-4 levels; (ii) represses Angptl-4 expression in WAT and (iii) simultaneously enhances LPL activity in this tissue without inducing Lpl expression. In vivo CCK-8 effects are specifically antagonized by the CCK-2 receptor (CCK-2R) antagonist, L-365,260. Moreover, CCK-8 downregulates Angptl-4 expression in wild-type pre-adipocytes, an effect that is not observed in engineered pre-adipocytes lacking CCK-2R. These effects have functional consequences as CCK-8 was found to promote the uptake of dietary fatty acids by WAT, as demonstrated by means of proton nuclear magnetic resonance (1H-NMR). The efficacy of acute CCK-8 administration was not reduced after chronic CCK-8 treatment. Moreover, the effects of CCK-8 on WAT were not associated to the increase of circulating insulin. Our results show that cholecystokinin promotes lipid storage in WAT by acting on adipocyte CCK-2R, suggesting a pivotal role for CCK in TG homeostasis.

Cholecystokinin receptors regulate sperm protein tyrosine phosphorylation via uptake of HCO3-.

Cholecystokinin (CCK), a peptide hormone and a neurotransmitter, was detected in mature sperm two decades ago. However, the exact role of CCK and the types of CCK receptors (now termed CCK1 and CCK2) in sperm have not been identified. Here, we find that CCK1 and CCK2 receptors are immunolocalized to the acrosomal region of mature sperm. The antagonist of CCK1 or CCK2 receptor strongly activated the soluble adenylyl cyclase/cAMP/protein kinase A signaling pathway that drives sperm capacitation-associated protein tyrosine phosphorylation in dose- and time-dependent manners. But these actions of stimulation were abolished when sperm were incubated in the medium in the absence of HCO3-. Further investigation demonstrated that the inhibitor of CCK1 or CCK2 receptor could accelerate the uptake of HCO3- and significantly elevate the intracellular pH of sperm. Interestingly, the synthetic octapeptide of CCK (CCK8) showed the same action and mechanism as antagonists of CCK receptors. Moreover, CCK8 and the antagonist of CCK1 or CCK2 receptor were also able to accelerate human sperm capacitation-associated protein tyrosine phosphorylation by stimulating the influx of HCO3-. Thus, the present results suggest that CCK and its receptors may regulate sperm capacitation-associated protein tyrosine phosphorylation by modulating the uptake of HCO3-.

Differential sensitivity of types 1 and 2 cholecystokinin receptors to membrane cholesterol.

Recent studies indicate that membrane cholesterol can associate with G protein-coupled receptors (GPCRs) and affect their function. Previously, we reported that manipulation of membrane cholesterol affects ligand binding and signal transduction of the type 1 cholecystokinin receptor (CCK1R), a Class A GPCR. We now demonstrate that the closely related type 2 cholecystokinin receptor (CCK2R) does not share this cholesterol sensitivity. The sequences of both receptors reveal almost identical cholesterol interaction motifs in analogous locations in transmembrane segments two, three, four, and five. The disparity in cholesterol sensitivity between these receptors, despite their close structural relationship, provides a unique opportunity to define the possible structural basis of cholesterol sensitivity of CCK1R. To evaluate the relative contributions of different regions of CCK1R to cholesterol sensitivity, we performed ligand binding studies and biological activity assays of wild-type and CCK2R/CCK1R chimeric receptor-bearing Chinese hamster ovary cells after manipulation of membrane cholesterol. We also extended these studies to site-directed mutations within the cholesterol interaction motifs. The results contribute to a better understanding of the structural requirements for cholesterol sensitivity in CCK1R and provides insight into the function of other cholesterol-sensitive Class A GPCRs.

Highly improved metabolic stability and pharmacokinetics of indium-111-DOTA-gastrin conjugates for targeting of the gastrin receptor.

The development of metabolically stable radiolabeled gastrin analogues with suitable pharmacokinetics is a topic of recent research activity. These imaging vectors are of interest because the gastrin/CCK2 receptor is highly overexpressed in different tumors such as medullary thyroid cancer, neuroendocrine tumors, and SCLC. The drawback of current targeting agents is either their metabolic instability or their high kidney uptake. We present the synthesis and in vitro and in vivo evaluation of 11 (111)In-labeled DOTA-conjugated peptides that differ by their spacer between the peptide and the chelate. We introduced uncharged but hydrophilic spacers such as oligoethyleneglycol, serine, and glutamine. The affinity of all radiopeptides was high with IC(50) values between 0.5 and 4.8 nM. The improvement of human serum stability is 500-fold within this series of compounds. In addition the kidney uptake could be lowered distinctly and the tumor-to-kidney ratio improved almost 60-fold if compared with radiotracers having charged spacers such as glutamic acid.

Cholecystokinin action on layer 6b neurons in somatosensory cortex.

Layer 6b in neocortex is a distinct sublamina at the ventral portion of layer 6. Corticothalamic projections arise from 6b neurons, but few studies have examined the functional properties of these cells. In the present study we examined the actions of cholecystokinin (CCK) on layer 6b neocortical neurons using whole-cell patch clamp recording techniques. We found that the general CCK receptor agonist CCK8S (sulfated CCK octapeptide) strongly depolarized the neurons, and this action persisted in the presence of tetrodotoxin, suggesting a postsynaptic site of action. The excitatory actions of CCK8S were mimicked by the selective CCK(B) receptor agonist CCK4, and attenuated by the selective CCK(B) receptor antagonist L365260, indicating a role for CCK(B) receptors. Voltage-clamp recordings revealed that CCK8S produced a slow inward current associated with a decreased conductance with a reversal potential near the K(+) equilibrium potential. In addition, intracellular cesium also blocked the inward current, suggesting the involvement of a K(+) conductance, likely K(leak). Our data indicate that CCK, acting via CCK(B) receptors, produces a long-lasting excitation of layer 6b neocortical neurons, and this action may play a critical role in modulation of corticothalamic circuit activity.

Stress-induced analgesia in mice: evidence for interaction between endocannabinoids and cholecystokinin.

Stress-induced analgesia (SIA) is an important endogenous mechanism in response to stressful stimuli. Opioid peptides, as well as endocannabinoids, are known mediators of SIA. We were interested in whether the endocannabinoid tone and the resulting SIA could be influenced by changing the activity of cholecystokinin (CCK) in mice. This hypothesis is related to recent evidence showing a cellular colocalization of neuropeptide CCK and cannabinoid type 1 (CB(1)) receptors in many central nervous system structures. However, the physiological relevance of this colocalization is unknown. Our aim was to test whether abolishing the endogenous CCK tone might influence the cannabinoid-mediated form of SIA. As expected, the CB(1) antagonist rimonabant prevented the development of analgesia in response to footshock-induced stress in wild-type mice. In contrast, CCK type 2 (CCK(2)) receptor-deficient mice developed SIA regardless of rimonabant treatment. Naloxone, an opioid antagonist, antagonized SIA in both wild-type and CCK(2) receptor-deficient homozygous mice. This finding suggests that CCK, through CCK(2) receptors, modulates the action of endocannabinoids. Gene expression analysis revealed an up-regulation of CCK(2) receptor gene in the lumbar spinal cord and mesolimbic area in wild-type mice in response to stress. In addition, wild-type mice displayed up-regulation of genes implicated in endocannabinoid-mediated neurotransmission (elevation of CB(1) receptor, 2-arachidonoylglycerol and the anandamide-synthesizing enzymes sn-1-diacylglycerol lipase alpha and N-acyl-phosphatidylethanolamine-hydrolysing phospholipase D) in response to stress in the lumbar spinal cord and mesolimbic area. We did not find any of these changes in CCK(2) receptor-deficient homozygous mice. Altogether, behavioural and gene expression studies reflect an involvement of CCK(2) receptors in the development of endocannabinoid-mediated SIA.

Synthesis of a novel pentagastrin-drug conjugate for a targeted tumor therapy.

The synthesis of the novel pentagastrin seco-CBI conjugate 3, which is based on the highly cytotoxic antitumor antibiotic (+)-duocarmycin SA (1), is reported. A key step in the synthesis is the palladium-catalyzed carbonylation of aryl bromide 7 to give the benzyl ester 16, which is transformed into the new seco-CBI derivative 21 bearing a carboxylic acid ester moiety. Subsequent transformation of 21 into an activated ester followed by the introduction of beta-alanine and tetragastrin led to the new pentagastrin drug 3 that contains a peptide moiety for targeting cancer cells expressing CCK-B/gastrin receptors.

Regulation of mammalian gastrin/CCK receptor (CCK2R) expression in vitro and in vivo.

The gastrin/CCK receptor (CCK2R) mediates the physiological functions of gastrin in the stomach, including stimulation of acid secretion and cellular proliferation and migration, but little is known about the factors that regulate its expression. We identified endogenous CCK2R expression in several cell lines and used luciferase promoter-reporter constructs to define the minimal promoter required for transcription in human gastric adenocarcinoma, AGS, and rat gastric mucosa, RGM1, cells. Consensus binding sites for SP1, C/EBP and GATA were essential for activity. Following serum withdrawal from RGM1 and AR42J cells, endogenous CCK2R mRNA abundance and the activity of a CCK2R promoter-reporter construct were significantly elevated. Transcription of CCK2R was also increased in AGS-G(R) and RGM1 cells by gastrin through mechanisms partly dependent upon protein kinase C (PKC) and mitogen/extracellular signal-regulated kinase (MEK). Gastrin significantly increased endogenous CCK2R expression in RGM1 cells, and CCK2R protein expression was elevated in the stomach of hypergastrinaemic animals. In mice with cryoulcers in the acid-secreting mucosa, CCK2R expression increased progressively in the regenerating mucosa adjacent to the ulcer repair margin, evident at 6 days postinjury and maximal at 13 days. De novo expression of CCK2R was observed in the submucosa beneath the repairing ulcer crater 6-9 days postinjury. Many of the cells in mucosa and submucosa that expressed CCK2R in response to cryoinjury were identified as myofibroblasts, since they coexpressed vimentin and smooth muscle alpha-actin but not desmin. The data suggest that increased CCK2R expression might influence the outcome of epithelial inflammation or injury and that the response may be mediated in part by myofibroblasts.

Progress in developing cholecystokinin (CCK)/gastrin receptor ligands that have therapeutic potential.

Gastrin and cholecystokinin (CCK) are two of the oldest hormones and within the past 15 years there has been an exponential increase in knowledge of their pharmacology, cell biology, receptors (CCK1R and CCK2R), and roles in physiology and pathological conditions. Despite these advances there is no approved disease indication for CCK receptor antagonists and only a minor use of agonists. In this review, the important factors determining this slow therapeutic development are reviewed. To assess this it is necessary to briefly review what is known about the roles of CCK receptors (CCK1R and CCK2R) in normal human physiology, their role in pathologic conditions, the selectivity of available potent CCKR agonists/antagonists as well as to review their use in human conditions to date and the results. Despite extensive studies in animals and in humans, recent studies suggest that monotherapy with CCK1R agonists will not be effective in obesity, nor CCK2R antagonists in panic disorders or CCK2R antagonists to inhibit growth of pancreatic cancer. Areas that require more study include the use of CCK2R agonists for imaging tumors and radiotherapy, CCK2R antagonists in hypergastrinemic states especially with long-term PPI use and for potentiation of analgesia as well as use of CCK1R antagonists for a number of gastrointestinal disorders [motility disorders (irritable bowel syndrome, dyspepsia, and constipation) and pancreatitis (acute and chronic)].

Cholecystokinin CCK2 receptors mediate the peptide's inhibitory actions on the contractile activity of human distal colon via the nitric oxide pathway.

BACKGROUND AND PURPOSE: Cholecystokinin is known to exert stimulant actions on intestinal motility via activation of type 1 cholecystokinin receptors (CCK(1)). However, the role played by cholecystokinin 2 (CCK(2)) receptors in the regulation of gut motility remains undetermined. This study was designed to examine the influence of CCK(2) receptors on the contractile activity of human distal colon. EXPERIMENTAL APPROACH: The effects of compounds acting on CCK(2) receptors were assessed in vitro on motor activity of longitudinal smooth muscle, under basal conditions as well as in the presence of KCl-induced contractions or transmural electrical stimulation. KEY RESULTS: Cholecystokinin octapeptide sulphate induced concentration-dependent contractions which were enhanced by GV150013 (CCK(2) receptor antagonist; +57% at 0.01 microM). These effects were unaffected by tetrodotoxin. The enhancing actions of GV150013 on contractions evoked by cholecystokinin octapeptide sulphate were unaffected by N(omega)-propyl-L-arginine (NPA, neuronal nitric oxide synthase inhibitor), while they were prevented by N(omega)-nitro-L-arginine methylester (L-NAME, non-selective nitric oxide synthase inhibitor). In the presence of KCl-induced contractions, cholecystokinin octapeptide sulphate elicited concentration-dependent relaxations (-36%), which were unaffected by NPA, but were counteracted by GV150013 or L-NAME. The application of electrical stimuli evoked phasic contractions which were enhanced by GV150013 (+41 % at 0.01 microM). CONCLUSIONS AND IMPLICATIONS: CCK(2) receptors mediate inhibitory actions of cholecystokinin on motor activity of human distal colon. It is suggested that CCK(2) receptors exert their modulating actions through a nitric oxide pathway, independent of the activity of the neuronal nitric oxide synthase isoform.

Cholecystokinin B-type receptors mediate a G-protein-dependent depolarizing action of sulphated cholecystokinin ocatapeptide (CCK-8s) on rodent neonatal spinal ventral horn neurons.

Reports of cholecystokinin (CCK) binding and expression of CCK receptors in neonatal rodent spinal cord suggest that CCK may influence neuronal excitability. In patch-clamp recordings from 19/21 ventral horn motoneurons in neonatal (PN 5-12 days) rat spinal cord slices, we noted a slowly rising and prolonged membrane depolarization induced by bath-applied sulfated CCK octapeptide (CCK-8s; 1 microM), blockable by the CCK B receptor antagonist L-365,260 (1 microM). Responses to nonsulfated CCK-8 or CCK-4 were significantly weaker. Under voltage clamp (V H -65 mV), 22/24 motoneurons displayed a CCK-8s-induced tetrodotoxin-resistant inward current [peak: -136 +/- 28 pA] with a similar time course, mediated via reduction in a potassium conductance. In 29/31 unidentified neurons, CCK-8s induced a significantly smaller inward current (peak: -42.8 +/- 5.6 pA), and I-V plots revealed either membrane conductance decrease with net inward current reversal at 101.3 +/- 4.4 mV (n = 16), membrane conductance increase with net current reversing at 36.1 +/- 3.8 mV (n = 4), or parallel shift (n = 9). Intracellular GTP-gamma-S significantly prolonged the effect of CCK-8s (n = 6), whereas GDP-beta-S significantly reduced the CCK-8s response (n = 6). Peak inward currents were significantly reduced after 5-min perfusion with N-ethylmaleimide. In isolated neonatal mouse spinal cord preparations, CCK-8s (30-300 nM) increased the amplitude and discharge of spontaneous depolarizations recorded from lumbosacral ventral roots. These observations imply functional postsynaptic G-protein-coupled CCK B receptors are prevalent in neonatal rodent spinal cord.

Gastrin regulates the TFF2 promoter through gastrin-responsive cis-acting elements and multiple signaling pathways.

Trefoil family factor 2 (TFF2) is expressed in gastrointestinal epithelial cells where it serves to maintain mucosal integrity and promote epithelial repair. The peptide hormone, gastrin, stimulates acid secretion but also induces proliferation of the acid-secreting mucosa. Because the relationship between these peptides of overlapping function is not understood, we chose to investigate the regulatory effect of gastrin on TFF2 expression. The expression of mRNA and protein of TFF2 was determined by RT-PCR and immunohistochemical staining, respectively. A series of truncated and mutant murine TFF2 promoter constructs was generated. Promoter activity was assessed using dual luciferase reporter assays. Gastrin-responsive DNA-binding sites in the TFF2 promoter were evaluated by electrophoretic mobility shift assay. Gastrin significantly increased the level of endogenous mRNA of TFF2 in the gastrin receptor-expressing AGS-E gastric cancer cell line in a time- and dose-dependent manner. TFF2 protein expression in the gastric fundus was elevated in hypergastrinemic (INS-GAS) transgenic mice and reduced in gastrin-deficient mice. Gastrin treatment increased TFF2 promoter activity through cis-acting regions, containing CCAATA- and GC-rich enhancers. Pretreatment with Y-F476, a gastrin/CCK(B) receptor antagonist, abolished gastrin-dependent promoter activity. Inhibitors of protein kinase C (PKC), mitogen/extracellular signal-regulated kinase (MEK1), and phosphatidylinositol 3-kinase (PI 3-kinase) reduced gastrin-dependent TFF2 promoter activity, whereas an epithelial growth factor receptor (EGFR) inhibitor had no effect. We found that gastrin regulates TFF2 transcription through a GC-rich DNA-binding site and a PKC-, MEK1- and PI 3-kinase-dependent but EGFR-independent pathway. Regulation of TFF2 by gastrin may play a role in the maintenance and repair of the gastrointestinal mucosa.

Differences in behavioural effects of amphetamine and dopamine-related gene expression in wild-type and homozygous CCK2 receptor deficient mice.

Neuropeptide cholecystokinin (CCK) interacts with dopamine in the regulation of motor activity and motivations. Therefore, in CCK(2) receptor deficient mice the behavioural effects of repeated amphetamine administration and changes in dopamine-related gene expression were studied. Four-day amphetamine (1 mg/kg) treatment induced a significantly stronger motor sensitization in homozygous mice compared to their wild-type littermates. However, in the conditioned place preference test the action of amphetamine was more pronounced in wild-type animals. As opposed to wild-type mice, amphetamine (1-3 mg/kg) did not cause a significant conditioned place preference in homozygous mice. The expression of Tyhy gene was elevated in the mesolimbic structures and Drd2 gene was down-regulated in the mesencephalon of saline-treated homozygous mice in comparison with respective wild-type group. Four-day treatment with amphetamine induced a significant increase in the expression of Tyhy in the mesencephalon, striatum and mesolimbic structures of wild-type mice, whereas in homozygous mice a similar change was evident only in the mesencephalon. Also, the expression of Drd1 gene in the striatum and Drd2 gene in the mesolimbic structures of wild-type mice were up-regulated under the influence of amphetamine. In conclusion, the present study established differences in the behavioural effects of amphetamine in wild-type and homozygous mice. The increased tone of dopaminergic projections from the mesencephalon to mesolimbic structures is probably related to increased amphetamine-induced motor sensitization in homozygous mice. The lack of development of up-regulation of Drd1 and Drd2 genes after repeated treatment with amphetamine probably explains the reduced place conditioning in CCK(2) receptor deficient mice.

Gastrin suppresses growth of CCK2 receptor expressing colon cancer cells by inducing apoptosis in vitro and in vivo.

BACKGROUND & AIMS: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Synergistic inhibitory effects of gastrin and histamine receptor antagonists on Helicobacter-induced gastric cancer.

BACKGROUND & AIMS: Apart from its importance as an acid secretogogue, the role of histamine as a downstream target of gastrin has not been fully explored. Previous studies have shown that the combination of hypergastrinemia and Helicobacter infection resulted in accelerated gastric cancer in mice. We used this model to examine the role of cholecystokinin 2 (CCK2)/gastrin receptor and histamine H2-receptor signaling in the development of gastric atrophy and cancer. METHODS: Male hypergastrinemic mice (INS-GAS mice) were infected with Helicobacter felis and given the CCK2/gastrin receptor antagonist YF476 and/or the histamine H2-receptor antagonist loxtidine for 3 or 6 months. In addition, mice were treated with omeprazole alone or in combination with either YF476 or loxtidine for 3 months. RESULTS: Mice treated with YF476 or loxtidine alone showed partial suppression of both gastric acid secretion and progression to neoplasia. The combination of YF476 plus loxtidine treatment resulted in nearly complete inhibition of both parameters. YF476 and/or loxtidine treatment did not alter the overall level of H. felis colonization but did result in significant down-regulation of the growth factors regenerating gene I and amphiregulin. Loxtidine treatment, with or without YF476, induced a mild shift in T-helper cell polarization. In contrast, omeprazole treatment resulted in mild progression of gastric hyperplasia/dysplasia, which was ameliorated by the addition of YF476 or loxtidine. CONCLUSIONS: The combination of CCK2/gastrin- and histamine H2-receptor antagonists has synergistic inhibitory effects on development of gastric atrophy and cancer in H. felis/INS-GAS mice, while the proton pump inhibitor showed no such effects. These results support an important role for the gastrin-histamine axis in Helicobacter-induced gastric carcinogenesis.

Gastrin promotes human colon cancer cell growth via CCK-2 receptor-mediated cyclooxygenase-2 induction and prostaglandin E2 production.

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.

Role of progastrins and gastrins and their receptors in GI and pancreatic cancers: targets for treatment.

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.

A phase II study of G17DT in gastric carcinoma.

PURPOSE: G17DT is a gastrin immunogen, raising antibodies that blockade gastrin-stimulated growth. The aim of the study was to characterise antibody response and assess safety and tolerability of G17DT given to patients with gastric cancer. EXPERIMENTAL DESIGN: G17DT was administered to 52 patients with gastric adenocarcinoma at weeks 0, 2 and 6 by intramuscular injection at doses of 10, 100 and 250 microg. Antibody levels were measured by an ELISA assay. A radioligand displacement assay determined the ability of G17DT-immunised patients' sera to inhibit binding of 125IG17 to cholecystokinin (CCK)-2 receptors. RESULTS: By week 12 of the study, 6/12 evaluable stage I-III patients achieved an antibody response in the 10 microg group, 7/11 in the 100 microg group, and 11/12 in the 250 microg group. Stage IV patients dosed at 250 microg achieved a similar response rate to stage I-III patients dosed at 10 or 100 microg. G17DT was well tolerated in 47/52 patients. Two patients suffered significant adverse reactions including injection site pain and abscess. G17DT antibodies displaced iodinated gastrin from CCK-2 receptors, with the level of displacement correlating with antibody titre. CONCLUSIONS: G17DT immunisation is a well-tolerated method of raising functional antibodies to 17 amino acid gastrin forms in patients with gastric carcinomas.