Gastrin mRNA Research Articles

Abstract 3250: LNA gapmer to gastrin inhibits growth and metastases of human pancreatic cancer in mice

Abstract Background: Pancreatic cancer has a poor prognosis and novel strategies are needed to improve the survival. Gastrin stimulates growth of pancreatic cancer by an autocrine mechanism and RNAi techniques that decrease gastrin expression have been shown to inhibit growth of this malignancy in vitro. Locked nucleic acid (LNA) Gapmers are antisense oligonucleotides (ASO) that have long half-life and stability in vivo, hence represent a potential method to deliver RNAi for cancer therapeutics. We developed an LNA Gapmer that selectively downregulates gastrin expression in pancreatic cancer cells in culture. The goal of this project was to test this anti-gastrin Gapmer in vivo as a novel therapy for pancreatic cancer. Methods: The anti-gastrin LNA Gapmer was modified using thiol-click chemistry to conjugate a peptide to the 5’ end and render the Gapmer target-specific with the ability to bind selectively to the cholecystokinin-B receptor that is over-expressed in pancreatic cancer. Forty athymic nude mice with established AsPC-1 human pancreatic orthotopic tumors were divided into four groups of equal tumor size. Mice (N=10 per group) were treated with PBS (controls), an untargeted LNA-Gapmer, the CCK-BR targeted Gapmer (60nM), and the CCK-BR targeted Gapmer (120nM) intraperitoneally twice a week for 4 weeks. Growth was monitored by luciferase IVIS imaging. At necropsy, tumors were excised, and metastases counted. Results: Mean tumor mass was 43% smaller in mice treated with the targeted Gapmer-120nM (P=0.0007). Untargeted and targeted Gapmer 60nM slightly decreased tumor mass but this difference was not significant. Compared to PBS control-treated mice, metastases were significantly less in mice treated with the untargeted Gapmer by -39% (p=0.01); in the targeted Gapmer-60nM by -44% (p=0.003), and with the targeted Gapmer-120nM by -72% (p<0.0001). Gastrin mRNA analysis of the tumors by qRT-PCR confirmed downregulation of gastrin. Conclusion: Novel LNA Gapmers that downregulate gastrin mRNA expression decrease growth and metastases of pancreatic cancer in a dose-related fashion. Citation Format: Godhanjali Chekuri, Wenqiang Chen, Narayan Shivapurkar, Hong Cao, Ruvanthi N. Kularatne, Stephan Stern, Jill P. Smith. LNA gapmer to gastrin inhibits growth and metastases of human pancreatic cancer in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3250.

Gastrin/CCK-B Receptor Signaling Promotes Cell Invasion and Metastasis by Upregulating MMP-2 and VEGF Expression in Gastric Cancer.

Accumulated evidence suggests that a functional loop composed of gastrin and cholecystokinin B receptor (CCK-BR) may exist in gastric carcinogenesis. However, this suggestion is not completely supported due to a lack of direct evidence, and the underlying mechanism is not completely understood. Here, we evaluated the effects of gastrin/CCK-BR signaling on the cell growth, invasion, and expression of MMP-2 and VEGF, as well as xenograft growth in vivo. Furthermore, we detected gastrin mRNA content in human gastric cancer tissues, metastatic lymph nodes, and adjacent nontumor tissues. We found that the forced gastrin could promote the proliferation, migration, and invasion of gastric cancer cells by upregulating the expression of MMP-2 and VEGF. Blocking gastrin/CCK-BR signal using either Proglumide, a CCK-BR antagonist, or shRNA against GASTRIN significantly inhibited the gastrin-promoting effects. In vivo study revealed that the tumor growth in nude mice inoculated with gastrin-overexpressed cells was significantly faster than control cells. The gastrin mRNA content in metastatic lymph nodes was higher in patients with gastric cancer than in primary gastric cancer and adjacent nontumor tissues. In conclusion, we provided direct evidence and possible mechanism of gastrin/CCK-BR signaling in the initiation and progression of gastric cancer.

Maillard Reaction Products of Stir Fried Hordei Fructus Germinatus Are Important for Its Efficacy in Treating Functional Dyspepsia.

Hordei Fructus Germinatus (HFG) has been used as a traditional medicine to treat functional dyspepsia (FD) in China. Stir fried HFG (F-HFG) containing Maillard reaction products (MRPs) is used more widely than the raw HFG (R-HFG). However, the exact mechanisms in its functionality remain unclear. This article investigated the effect of R-HFG, F-HFG, and MRPs on brain-gut peptides, gut microbiota, and digestive enzymes using an FD animal mode. After administration of R-HFG, F-HFG, and MRPs, higher mRNA expression level of gastrin (GAS) and lower mRNA expression level of vasoactive intestinal peptide (VIP) were exhibited in F-HFG and MRPs rats than R-HFG rats (P < .05). Furthermore, compared with the R-HFG group, the contents of motilin (MTL) and GAS showed an upward tendency, whereas the contents of VIP and chokcystokinin (CCK) showed a downward tendency in the F-HFG group. In addition, bacterial communities in the control, F-HFG, and MRPs groups clustered closely to one another, and bacterial communities in the model and recovery groups clustered together, whereas the bacterial communities in the R-HFG group were clustered into a category. Moreover, there were no apparent differences in brain-gut peptides and gut microbiota between the F-HFG and MRPs groups. However, after the oral administration of R-HFG, F-HFG, and MRPs, the level of digestive enzyme did not show a significant change as compared with the recovery group. These results indicated that the stronger effect of F-HFG could be attributed to the MRPs produced during stir frying, and MRPs possessed the effect of regulating brain-gut peptides and gut microbiota.

Stomach gastrin is regulated by sodium via PPAR-α and dopamine D1 receptor

Gastrin, secreted by stomach G cells in response to ingested sodium, stimulates the renal cholecystokinin B receptor (CCKBR) to increase renal sodium excretion. It is not known how dietary sodium, independent of food, can increase gastrin secretion in human G cells. However, fenofibrate (FFB), a peroxisome proliferator-activated receptor-α (PPAR-α) agonist, increases gastrin secretion in rodents and several human gastrin-secreting cells, via a gastrin transcriptional promoter. We tested the following hypotheses: (1.) the sodium sensor in G cells plays a critical role in the sodium-mediated increase in gastrin expression/secretion, and (2.) dopamine, via the D1R and PPAR-α, is involved. Intact human stomach antrum and G cells were compared with human gastrin-secreting gastric and ovarian adenocarcinoma cells. When extra- or intracellular sodium was increased in human antrum, human G cells, and adenocarcinoma cells, gastrin mRNA and protein expression/secretion were increased. In human G cells, the PPAR-α agonist FFB increased gastrin protein expression that was blocked by GW6471, a PPAR-α antagonist, and LE300, a D1-like receptor antagonist. LE300 prevented the ability of FFB to increase gastrin protein expression in human G cells via the D1R, because the D5R, the other D1-like receptor, is not expressed in human G cells. Human G cells also express tyrosine hydroxylase and DOPA decarboxylase, enzymes needed to synthesize dopamine. G cells in the stomach may be the sodium sensor that stimulates gastrin secretion, which enables the kidney to eliminate acutely an oral sodium load. Dopamine, via the D1R, by interacting with PPAR-α, is involved in this process.

Abstract P146: Dopaminergic Stimulation Increases Gastrin Secretion via a PKA-PPAR-Alpha Pathway

Dietary sodium stimulates renal excretion of sodium long before the sodium is enterally absorbed. Moreover, intravenously administered sodium does not stimulate as large an excretory response as sodium taken by mouth. We previously showed that sodium stimulates an increase in gastrin mRNA and protein in stomach G-cells via PPAR-α and dopamine D1 receptor (D 1 R). Studies have shown that PKA pathway is important in the regulation of PPAR-α activity, and PKA is also involved in the D 1 R pathway. Thus, we hypothesized that D 1 R may regulate PPAR-α through PKA in human G-cells in response to sodium. Human G-cells were treated with 8-Br-cAMP, a cell-permeable second messenger analog, and Rp-cAMP (RPCA), a PKA inhibitor. Image analysis showed there was more PPAR-α in the nucleus with 8-Br-cAMP treatment, and less PPAR-α in the nucleus with RPCA treatment. Therefore, PKA is involved in regulating PPAR-α (VEH, 1194±74; 8-Br-cAMP, 1490±44; RPCA, 740±75; 8-Br-cAMP+ RPCA, 1037±54; VEH vs 8-Br-cAMP, P&lt;0.01; 8-Br-cAMP vs 8-Br-cAMP+ RPCA, P&lt;0.001; one-way ANOVA, n=13). Moreover, gastrin mRNA was increased by 8-Br-cAMP stimulation while the increase was blocked by RPCA (VEH, 1.01±0; 8-Br-cAMP, 1.32±0.07; RPCA, 0.82±0.08; 8-Br-cAMP+ RPCA, 0.9±0.05; VEH vs 8-Br-cAMP, P&lt;0.05; 8-Br-cAMP vs 8-Br-cAMP+ RPCA, p&lt;0.01; one-way ANOVA, n=3). Gastrin protein level was also increased by 8-Br-cAMP treatment with image analysis (VEH, 1.14±0.01; 8-Br-cAMP, 1.28±0.01; RPCA, 1.17±0.01; 8-Br-cAMP+ RPCA, 1.38±0.03; VEH vs 8-Br-cAMP, P&lt;0.01; one-way ANOVA, n=3). These data indicate that PKA is involved in the D 1 R and PPAR-α pathways to regulate G-cell gastrin expression stimulated by sodium.

Evidence for the Direct Effect of the NPFF Peptide on the Expression of Feeding-Related Factors in Spotted Sea Bass (Lateolabrax maculatus).

Neuropeptide FF (NPFF) is a family member of RF-amide peptides, which are suggested to be involved in the control of vertebrate feeding behavior. However, little is known about the effect of the NPFF peptide on feeding-related processes in basal vertebrates. In this study, four full-length cDNAs, npff, npffr1, npffr2-1, and npffr2-2, were cloned from spotted sea bass and characterized. The conserved NPFF peptide is biologically active because it functionally interacts with different receptors expressed in cultured eukaryotic cells to enhance CRE promoter activity. Tissue distribution analysis showed that the highest npff mRNA expression occurred in the telencephalon, hypothalamus, medulla, gonad and muscle, but the npffrs mRNAs were mainly distributed within the central nervous system (CNS). In situ hybridization (ISH) detected npff-expressing cells in several specific regions ranging across the telencephalon and midbrain to the hypothalamus. Incubation of the spotted sea bass conserved NPFF peptide significantly increased the expression of orexin (orx) and neuropeptide Y (npy) mRNA and decreased the expression of leptin (lep), somatostatin (ss), and cholecystokinin (cck) mRNA in brain cells. Similarly, the conserved NPFF peptide also heightened the expression of gastrin (gas), ghrelin (ghrl), and motilin (mtl) mRNA and significantly reduced the expression of cck mRNA in the intestine and stomach. Taken together, these data suggest that the NPFF peptide may play a stimulating role in regulating feeding-related processes in spotted sea bass.

Endogenous Gastrin Collaborates With Mutant KRAS in Pancreatic Carcinogenesis.

The KRAS gene is the most frequently mutated gene in pancreatic cancer, and no successful anti-Ras therapy has been developed. Gastrin has been shown to stimulate pancreatic cancer in an autocrine fashion. We hypothesized that reactivation of the peptide gastrin collaborates with KRAS during pancreatic carcinogenesis. LSL-Kras; P48-Cre (KC) mutant KRAS transgenic mice were crossed with gastrin-KO (GKO) mice to develop GKO/KC mice. Pancreata were examined for 8 months for stage of pancreatic intraepithelial neoplasia lesions, inflammation, fibrosis, gastrin peptide, and microRNA expression. Pancreatic intraepithelial neoplasias from mice were collected by laser capture microdissection and subjected to reverse-phase protein microarray, for gastrin and protein kinases associated with signal transduction. Gastrin mRNA was measured by RNAseq in human pancreatic cancer tissues and compared to that in normal pancreas. In the absence of gastrin, PanIN progression, inflammation, and fibrosis were significantly decreased and signal transduction was reversed to the canonical pathway with decreased KRAS. Gastrin re-expression in the PanINs was mediated by miR-27a. Gastrin mRNA expression was significantly increased in human pancreatic cancer samples compared to normal human pancreas controls. This study supports the mitogenic role of gastrin in activation of KRAS during pancreatic carcinogenesis.

Abstract P290: Human Stomach Gastrin is Regulated by Sodium Human Stomach Gastrin Secretion is Regulated by Ingested Sodium and the Dopamine 1 Receptor

Dietary sodium stimulates the renal excretion of sodium long before there is expansion of the extracellular fluid volume. We have previously shown that an increase in sodium concentration in the incubation buffer in human SW626 colon adenocarcinoma cells that express gastrin-secreting G- cells increased gastrin mRNA and protein. Thus, we hypothesized that increasing extracellular sodium would also increase gastrin expression in normal human stomach G-cells. The highest sodium concentration (170 mM) increased gastrin protein expression significantly (all cell lines were normalized to their gastrin expression at 90 mM, sodium). In normal human G-cells cultured in non-polarized conditions, the normalized gastrin expression in cells incubated in 143mM sodium was 0.98 ± 0.06; 170mM sodium increased gastrin expression 1.515 ± 0.106-fold (n=4 different cell lines, P&lt;0.01 one-way ANOVA). Cultured human renal tubule cells demonstrate more physiologically relevant phenotype when grown in a 3D cell culture (3DCC) system. Therefore, we also studied human G-cells grown in 3DCC. Normal human G-cells grown in 3D also had a greater gastrin expression when incubated in 170 than143 mM sodium: 1.08 ± 0.12 vs. 0.62 ± 0.04, n=4, P&lt;0.05, t-test). We have also shown that the dopamine type 1 receptor (D 1 R) is expressed in G-cells and may play a role in regulation of gastrin expression. We now report the presence of the complete dopamine biosynthetic pathway in human G-cells: tyrosine hydroxylase is expressed ubiquitously in the stomach while DOPA decarboxylase is expressed selectively in G-cells where D 1 R is also expressed. We have reported that in the kidney, gastrin, via CCKBR and dopamine, via D 1 R synergistically increase renal sodium excretion. Thus, the pathway that is involved in the increase in sodium excretion after the ingestion sodium involves sodium- and D 1 R-mediated increase in gastrin expression in G-cells, followed by secretion of gastrin into the circulation to interact with kidney mediated gastrin pathways.

Abstract 2162: Novel anti-gastrin nanoparticle inhibits growth of pancreatic cancer

Abstract Background: Receptor-targeted therapies or inhibition of growth factors have improved cancer survival. Pancreatic ductal adenocarcinoma (PDAC) markedly over-expresses the cholecystokinin-B (CCK-B) receptor and re-expression gastrin stimulates growth of PDAC by an autocrine mechanism through the CCK-B receptor. When gastrin mRNA is down regulated by RNAi techniques, PDAC growth and metastases are inhibited in animal models. However, anti-gastrin gene therapy cannot be readily used in humans unless nontoxic gene delivery strategies are implemented. The purpose of this project was to develop a unique drug delivery nanoparticle (NP) that selectively binds to the CCK-B receptor and safely delivers siRNA to down-regulate gastrin expression and inhibit growth of PDAC. Methods: In order to develop the targeted NP, a thiol functionalized polyethylene glycol-block-poly(L-lysine) (SH-PEG-PLL) polymer was synthesized. To render the NP target-specific for the CCK-B receptor we used a maleimide link to conjugate Gastrin-10 to the PEG via Michael addition reaction. The resulting Ga-PEG-PLL was extensively purified using a PD-10 column and by dialysis. The polyplex micelle was prepared by mixing 1mg/mL of the Ga-PEG-b-PLL with a gastrin siRNA (si286 GUGCUGAGGAUGAGAACUA), which decreases gastrin mRNA 90%. The PEG protects the siRNA from degradation in solution or blood and the lysine polymer forms a micelle shielding the positive charge and eliminating toxicity. The NP was analyzed by dynamic light scattering (DLS) and zeta potential. Efficacy of the NPs to inhibit growth was tested on PANC-1 human PDAC cells that have a high number of CCK-B receptors. Cells were plated into 6-well plates overnight and then treated for 72h with PBS, 100x NP (10nM siRNA) or 50x NPs (5nM siRNA) in serum-free DMEM media. Viable cell counts were performed by trypan blue exclusion. Results: Characterization of the functionalized polyplex NP confirmed a molecular weight of 9700 Da. Trityl deprotection and conjugation of Ga-10 to the SH-PEG-PLL polymer were confirmed by NMR which demonstrated complete removal of the trityl group and greater than 70% conjugation of the peptide to target the receptor. The polyplex NP complex was confirmed by DLS measurement, which demonstrated size distributions of 44.3 ± 0.3 and 48.2 ± 0.3 nm for receptor-targeted and untargeted polyplex respectively. Treatment of PANC-1 cancer cells with the anti-gastrin NPs significantly inhibited growth by 98% compared to untreated controls (p = 0.005). Conclusion: A novel NP that targets the over-expressed CCK-B receptor on PDAC was successfully synthesized and characterized. The NP can deliver anti-gastrin gene therapy in the form of siRNA into human pancreatic cancer cells to significantly inhibit cell growth by downregulation of gastrin expression. This technique may provide a safe and novel gene therapy delivery method to treat those with advanced PDAC. Supported by: Otto J Ruesch Center for the Cure of GI Cancers Citation Format: Julian Burks, Sandeep Nadella, Abdullah Mahmud Mahmud, Scott McNeil, Jong-IN Hahm, Stephan Stern, Jill Smith. Novel anti-gastrin nanoparticle inhibits growth of pancreatic cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2162.

Abstract 087: Human Stomach Cell Gastrin Inhibits Renal NHE3 and NaKATPase in Concert With the Renal D1R

The digestive track secretes gastrin in response to sodium ingestion stimulating increased renal sodium excretion. We previously showed that gastrin secreted by human colon cancer cells (SW626) can bind to cell surface cholecystokinin receptors on renal proximal tubule cells (RPTC), modulating the natriuretic dopaminergic system. We tested the hypothesis that a similar gastro/renal axis exists in humans. Novel human stomach antrum G-cell lines from 6 separate individuals were isolated and each shown to express gastrin mRNA and protein, and bind Phaseolus vulgaris Leucoagglutinin (PHA-l) by fluorescence lectin affinity (marker for gastrin secreting cells). It was determined that the D1 receptor (D1R) is also found on G-Cells. In human stomach tissue, gastrin expression was increased by Fenofibrate treatment, (PPARα agonist, p= 0.07, in 3 live ex-vivo cultured human stomachs). Moreover, we tested the effect of gastrin on CCKB2 receptors in human RPTC. Gastrin (100 nM 15 min.) increased RPTC phospholipace-C (PLC) activity by 1.07± 0.01 fold, N=35, p&lt;0.0001, using a PLC-FRET biosensor, CYPHR, but did not increase cAMP levels using the specific cAMP-FRET biosensor, ICUE-YR. Both gastrin and SKF83822 (100 nM, a cAMP specific D1R agonist, 15 min) alone reduced sodium influx into RPTC via NHE3 (from VEH 100± 4.7% to 73.5± 6.2% or 74.1± 4.3% respectively N=6, p&lt;0.05), but gastrin along with SKF83822 decreased sodium influx more than either alone (57.2 ± 5.8% N=6, p&lt;0.05). Sodium efflux via NaKATPase was reduced by SKF83822 (from VEH 100± 3.9% to 84.2± 3.3%, N=6, p&lt;0.05), but not gastrin alone, however SKF83822 along with gastrin reduced sodium efflux more than SKF83822 alone (72.3±5.1% vs N=6, p&lt;0.05). Additionally we found that Angiotensin II (AngII, 10 nM, 15 min.) increased NHE3 activity (12.3± 3.6% N=6, p&lt;0.01) and this increase was completely blocked by gastrin (N=6, p&lt;0.01). The PLC inhibitor U73122 reversed the inhibitory effect of gastrin on NHE3 and NaKATPase. Gastrin was also found to decrease the amount of fluorescent AngII binding to RPTCs (27.4± 6.1% N=6, p&lt;0.01) and this decrease was completely blocked by U73122. Thus, both stomach gastrin and the renal D1R inhibit NHE3 and NaKATPase, to increase sodium elimination from the body after salt is ingested.

Remodeling of the residual gastric mucosa after roux-en-y gastric bypass or vertical sleeve gastrectomy in diet-induced obese rats.

Whereas the remodeling of intestinal mucosa after bariatric surgeries has been the matter of numerous studies to our knowledge, very few reported on the remodeling of the residual gastric mucosa. In this study, we analyzed remodeling of gastric mucosa after Roux-en-Y gastric bypass (RYGB) and vertical sleeve gastrectomy (VSG) in rats. Diet-induced obese rats were subjected to RYGB, VSG or sham surgical procedures. All animals were assessed for food intake, body-weight, fasting blood, metabolites and hormones profiling, as well as insulin and glucose tolerance tests before and up to 5 weeks post-surgery. Remodeling of gastric tissues was analyzed by routine histology and immunohistochemistry studies, and qRT-PCR analyses of ghrelin and gastrin mRNA levels. In obese rats with impaired glucose tolerance, VSG and RYGB caused substantial weight loss and rats greatly improved their oral glucose tolerance. The remaining gastric mucosa after VSG and gastric pouch (GP) after RYGB revealed a hyperplasia of the mucous neck cells that displayed a strong immunoreactivity for parietal cell H+/K+-ATPase. Ghrelin mRNA levels were reduced by 2-fold in remaining fundic mucosa after VSG and 10-fold in GP after RYGB. In the antrum, gastrin mRNA levels were reduced after VSG in line with the reduced number of gastrin positive cells. This study reports novel and important observations dealing with the remaining gastric mucosa after RYGB and VSG. The data demonstrate, for the first time, a hyperplasia of the mucous neck cells, a transit cell population of the stomach bearing differentiating capacities into zymogenic and peptic cells.

Gastrokine 1 inhibits gastrin-induced cell proliferation.

Gastrokine 1 (GKN1) acts as a gastric tumor suppressor. Here, we investigated whether GKN1 contributes to the maintenance of gastric mucosal homeostasis by regulating gastrin-induced gastric epithelial cell growth. We assessed the effects of gastrin and GKN1 on cell proliferation in stable AGS(GKN1) and MKN1(GKN1) gastric cancer cell lines and HFE-145 nonneoplastic epithelial cells. Cell viability and proliferation were analyzed by MTT and BrdU incorporation assays, respectively. Cell cycle and expression of growth factor receptors were examined by flow cytometry and Western blot analyses. Gastrin treatment stimulated a significant time-dependent increase in cell viability and proliferation in AGS(mock) and MKN1(mock), but not in HFE-145, AGS(GKN1), and MKN1(GKN1), cells, which stably expressed GKN1. Additionally, gastrin markedly increased the S-phase cell population, whereas GKN1 significantly inhibited the effect of gastrin by regulating the expression of G1/S cell-cycle regulators. Furthermore, gastrin induced activation of the NF-kB and β-catenin signaling pathways and increased the expression of CCKBR, EGFR, and c-Met in AGS and MKN1 cells. However, GKN1 completely suppressed these effects of gastrin via downregulation of gastrin/CCKBR/growth factor receptor expression. Moreover, GKN1 reduced gastrin and CCKBR mRNA expression in AGS and MKN1 cells, and there was an inverse correlation between GKN1 and gastrin, as well as between GKN1 and CCKBR mRNA expression in noncancerous gastric mucosae. These data suggest that GKN1 may contribute to the maintenance of gastric epithelial homeostasis and inhibit gastric carcinogenesis by downregulating the gastrin-CCKBR signaling pathway.

Inhibitory Effects of Deoxynivalenol on Gastric Secretion in Rats

Deoxynivalenol (DON) is a common mycotoxin produced by Fusarium sp. in cereals and foods. Ingestion of contaminated foodstuffs can cause digestive disorders in various animals. Many researchers focus on its toxicity and the pathological damage and absorptive function in the intestines. However, the effect of DON on gastric function is still unclear. The objective of the current study was to evaluate the impact of DON on gastric secretion. Rats were gavaged with DON at the dose of 0, 1, 5, and 25 mg/kg of body weight (bw). Gastric fluids were gathered by pylorus ligation 0.5 h after DON exposure. The results indicate that the volume of gastric fluid decreased by 25, 51, and 61% compared with the control, respectively. The pH increased to 3.2, 3.81, and 6.65 in the 1, 5, and 25 mg/kg bw DON group, compared with the control (1.9). To examine the mucosal injuries, the stomach tissues were made into hematoxylin and eosin slides. Histopathology observations suggest that no mucosal lesions were observed until DON exposure at 25 mg/kg bw. Additionally, the gastrin secretion in the fluids and mRNA expression in tissues were determined by the radioimmunoassay and real-time PCR assay, respectively. The results indicated that both significantly decreased in DON-exposed rats compared with the control. Taken together, DON exposure reduced gastric secretion in rats. Low gastrin secretion and mRNA expression play a major role, unless mucosal lesions by high DON exposure are present.

Demonstration and biological significance of a gastrin-P21-activated kinase 1 feedback loop in colorectal cancer cells.

Gastrins, including amidated gastrin17 and glycine‐extended gastrin17, are important growth factors in colorectal cancer (CRC). The p21‐activated kinase 1 (PAK1) plays key roles in cellular processes including proliferation, survival, and motility, and in cell transformation and tumor progression. PAK1 expression increases with the progression of CRC, and knockdown of PAK1 blocks CRC cell growth and metastasis both in vitro and in vivo. The aim of this study was to determine the interaction between PAK1 and gastrins in CRC cells. PAK1 expression and activation were assayed by Western blots, and concentrations of gastrin mRNA and peptides by real‐time PCR and radioimmunoassay, respectively. Proliferation of CRC cells was measured by 3H‐thymidine incorporation, and vascular endothelial growth factor (VEGF) secretion was measured by ELISA. Gastrins activated PAK1 via PI3K‐dependent pathways. Activated PAK1 in turn mediated gastrin‐stimulated activation of β‐catenin and VEGF secretion in CRC cells, as knockdown of PAK1 blocked stimulation of these cellular processes by gastrins. Downregulation of gastrin reduced the expression and activity of PAK1, but in contrast there was a compensatory increase in gastrins either when PAK1 was downregulated, or after treatment with a PAK inhibitor. Our results indicate that PAK1 is required for the stimulation of CRC cells by gastrins, and suggest the existence of an inhibitory feedback loop by which PAK1 downregulates gastrin production in CRC cells.

Zinc ions upregulate the hormone gastrin via an E-box motif in the proximal gastrin promoter

Gastrin and its precursors act as growth factors for the normal and neoplastic gastrointestinal mucosa. As the hypoxia mimetic cobalt chloride upregulates the gastrin gene, the effect of other metal ions on gastrin promoter activity was investigated. Gastrin mRNA was measured by real-time PCR, gastrin peptides by RIA, and gastrin promoter activity by dual-luciferase reporter assay. Exposure to Zn(2)(+) ions increased gastrin mRNA concentrations in the human gastric adenocarcinoma cell line AGS in a dose-dependent manner, with a maximum stimulation of 55 ± 14-fold at 100 μM (P<0.05). Significant stimulation was also observed with Cd(2)(+) and Cu(2)(+), but not with Ca(2)(+), Mg(2)(+), Ni(2)(+), or Fe(3)(+) ions. Activation of MAPK and phosphatidylinositol 3-kinase pathways is necessary but not sufficient for gastrin induction by Zn(2)(+). Deletional mutation of the gastrin promoter identified an 11 bp DNA sequence, which contained an E-box motif, as necessary for Zn(2)(+)-dependent gastrin induction. The fact that E-box binding transcription factors play a crucial role in the epithelial-mesenchymal transition (EMT), together with our observation that Zn(2)(+) ions upregulate the gastrin gene in AGS cells by an E-box-dependent mechanism, suggests that Zn(2)(+) ions may induce an EMT, and that gastrin may be involved in the transition.

Cholecystokinin receptors in Atlantic salmon: molecular cloning, gene expression, and structural basis

The peptide hormone cholecystokinin (CCK) exerts a wide range of digestive and CNS-related physiological signaling via CCK receptors in brain and gut. There is very limited information available on these receptors in Atlantic salmon. The aim of this study was to characterize CCK receptors in gut and brain of salmon. We have identified and cloned one CCK-1 receptor and duplicates of CCK-2 receptor in salmon. The phylogenetic analysis indicates the existence of one common ancestor gene for all CCK receptors. CCK-1R mRNA is highly expressed in pancreas followed by midgut, hindgut, gallbladder, and stomach indicating an involvement in pancreatic regulation and gallbladder contractions. CCK-2R1/gastrin mRNA is expressed at high levels in midgut and at relatively low levels in stomach, gallbladder, and pancreas. We postulate CCK-2R1/gastrin receptor to have gastrin-related functions because of its distribution and abundance in gastro-intestinal (GI) tissues. CCK-2R2 is relatively abundant in brain but has low expression levels in gut tissues supporting the hypothesis for involvement in the gut-brain signaling. Major functional motifs and ligand interaction sites in salmon are conserved with that of mammals. This information will be instrumental for comparative studies and further targeting receptor activation and selectivity of biological responses of CCK in salmon.

Full-length cDNAs cloning and expression characteristics of gastrin and cholecystokinin in the mandarin fish (Siniperca chuatsi)

We evaluated the factors regulating the secretion of digestive juices in fish.We cloned two intestinal hormone genes,gastrin(GAS) and cholecystokinin(CCK),from mandarin fish(Siniperca chuatsi).The GAS cDNA consists of 581 bp nucleotides,including a 100 bp 5′ untranslated region(UTR),a 336 bp open reading frame(ORF) encoding 111 amino-acid residues,and a 145 bp 3′ UTR.The CCK gene has two isoforms,CCK1 and CCK2,consisting of 843 bp and 846 bp nucleotides,respectively.The CCK1 gene consists of a 60 bp 5′ untrans-lated region(UTR),a 414 bp ORF encoding a polypeptide of 137 amino-acid residues and a 369 bp 3′ UTR,while the CCK2 gene consists of a 112 bp 5′ UTR,a 403 bp ORF encoding a polypeptide of 134 amino-acid residues and a 332 bp 3′ UTR.The C-terminal of the GAS polypeptide has a similar octapeptide structure(DYQGWVDF/ DYLGWMDF) to that of CCKs'.The only differences occur in the third and the sixth amino acid in the C-terminal.Val3(GAS) and Glu6(GAS) are replaced by Met3(CCK) and Leu6(CCK),respectively.Real-time PCR analysis revealed that GAS mRNA was primarily expressed in the intestine and pyloric caecum.The expression of CCK1 and CCK2 mRNA was highest in the brain,but expression was also detected in intestine and pyloric caecum.Our results suggest that GAS and CCKs may be involved in regulating digestion,while CCKs appear to be associated with digestive and neurosecretory processes.The GAS and CCK genes are constitutively expressed during larval development(0?22 days post hatching,DPH).Expression was higher during the early stage(4–8 DPH for GAS and 2–4 DPH for CCK),possibly linked to development and growth of the digestive tract,then gradually de-creased to a stable level during the later stages of development.

Characterization of gastrins and their receptor in solid human gastric adenocarcinomas

Objective. The gastrin and the gastrin/CCK-B receptor genes are co-expressed in several carcinomas. The primary translational product, progastrin, however, is processed to several peptides of which only those that are α-amidated at their C-terminus are receptor ligands. So far, characterization of the progastrin-derived peptides in gastric cancer has not been reported. The authors therefore examined the molecular nature of gastrin and its receptor in human gastric carcinomas. Materials and methods. Twenty patients with adenocarcinoma underwent partial or total gastrectomy. In samples from each carcinoma, gastrin peptides were characterized, using a library of sequence-specific immunoassays. Expression was also demonstrated by immunohistochemistry. In addition, the gastrin and gastrin/CCK-B receptor gene expression was quantitated using real-time PCR, and the receptor protein demonstrated by western blotting. Results. α-Amidated gastrins were detectable in 16 of 20 carcinomas (median concentration 2.1 pmol/g tissue; range 0–386 pmol/g tissue). The tissue concentrations correlated closely to the gastrin mRNA contents (r = 0.75, p < 0.0001). Moreover, progastrin and non-amidated processing intermediates, including glycine-extended gastrins, were detected in 19 carcinomas. Immunohistochemistry corroborated gastrin expression in carcinoma cells. Chromatography revealed extensive progastrin processing with α-amidated gastrin-34 and -17 (tyrosyl-sulfated as well as non-sulfated) as major products. Finally, gastrin/CCK-B receptor mRNA and protein were detected in all tumors. Conclusions. The results show that the elements for a local loop of α-amidated gastrins and their receptor are detectable in 80% of human gastric adenocarcinomas. Therefore, the results support the contention that locally expressed gastrin may be involved in the tumorigenesis of gastric adenocarcinomas.

P-21-Activated kinase 1 mediates gastrin-stimulated proliferation in the colorectal mucosa via multiple signaling pathways

Gastrins, including amidated (Gamide) and glycine-extended (Ggly) forms, function as growth factors for the gastrointestinal mucosa. The p-21-activated kinase 1 (PAK1) plays important roles in growth factor signaling networks that control cell motility, proliferation, differentiation, and transformation. PAK1, activated by both Gamide and Ggly, mediates gastrin-stimulated proliferation and migration, and activation of β-catenin, in gastric epithelial cells. The aim of this study was to investigate the role of PAK1 in the regulation by gastrin of proliferation in the normal colorectal mucosa in vivo. Mucosal proliferation was measured in PAK1 knockout (PAK1 KO) mice by immunohistochemistry. The expression of phosphorylated and unphosphorylated forms of the signaling molecules PAK1, extracellular signal-regulated kinase (ERK), and protein kinase B (AKT), and the expression of β-catenin and its downstream targets c-Myc and cyclin D1, were measured in gastrin knockout (Gas KO) and PAK1 KO mice by Western blotting. The expression and activation of PAK1 are decreased in Gas KO mice, and these decreases are associated with reduced activation of ERK, AKT, and β-catenin. Proliferation in the colorectal mucosa of PAK1 KO mice is reduced, and the reduction is associated with reduced activation of ERK, AKT, and β-catenin. In compensation, antral gastrin mRNA and serum gastrin concentrations are increased in PAK1 KO mice. These results indicate that PAK1 mediates the stimulation of colorectal proliferation by gastrins via multiple signaling pathways involving activation of ERK, AKT, and β-catenin.

Induction of Gastrin Expression in Gastrointestinal Cells by Hypoxia or Cobalt Is Independent of Hypoxia-Inducible Factor (HIF)

Gastrin and its precursors have been shown to promote mitogenesis and angiogenesis in gastrointestinal tumors. Hypoxia stimulates tumor growth, but its effect on gastrin gene regulation has not been examined in detail. Here we have investigated the effect of hypoxia on the transcription of the gastrin gene in human gastric cancer (AGS) cells. Gastrin mRNA was measured by real-time PCR, gastrin peptides were measured by RIA, and gastrin promoter activity was measured by dual-luciferase reporter assay. Exposure to a low oxygen concentration (1%) increased gastrin mRNA concentrations in wild-type AGS cells (AGS) and in AGS cells overexpressing the gastrin receptor (AGS-cholecystokinin receptor 2) by 2.1 ± 0.4- and 4.1 ± 0.3-fold (P < 0.05), respectively. The hypoxia mimetic, cobalt chloride (300 μM), increased gastrin promoter activity in AGS cells by 2.4 ± 0.3-fold (P < 0.05), and in AGS-cholecystokinin receptor 2 cells by 4.0 ± 0.3-fold (P < 0.05), respectively. The observations that either deletion from the gastrin promoter of the putative binding sites for the transcription factor hypoxia-inducible factor 1 (HIF-1) or knockdown of either the HIF-1α or HIF-1β subunit did not affect gastrin promoter inducibility under hypoxia indicated that the hypoxic activation of the gastrin gene is likely HIF independent. Mutational analysis of previously identified Sp1 regulatory elements in the gastrin promoter also failed to abrogate the induction of promoter activity by hypoxia. The observations that hypoxia up-regulates the gastrin gene in AGS cells by HIF-independent mechanisms, and that this effect is enhanced by the presence of gastrin receptors, provide potential targets for gastrointestinal cancer therapy.