Gene Summary

Gene:CCKBR; cholecystokinin B receptor
Aliases: GASR, CCK-B, CCK2R
Summary:This gene encodes a G-protein coupled receptor for gastrin and cholecystokinin (CCK), regulatory peptides of the brain and gastrointestinal tract. This protein is a type B gastrin receptor, which has a high affinity for both sulfated and nonsulfated CCK analogs and is found principally in the central nervous system and the gastrointestinal tract. A misspliced transcript variant including an intron has been observed in cells from colorectal and pancreatic tumors. [provided by RefSeq, Jul 2008]
Databases:OMIM, VEGA, HGNC, Ensembl, GeneCard, Gene
Protein:gastrin/cholecystokinin type B receptor
Source:NCBIAccessed: 27 February, 2015


What does this gene/protein do?
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Pathways:What pathways are this gene/protein implicaed in?
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Cancer Overview

Research Indicators

Publications Per Year (1990-2015)
Graph generated 27 February 2015 using data from PubMed using criteria.

Literature Analysis

Mouse over the terms for more detail; many indicate links which you can click for dedicated pages about the topic.

Tag cloud generated 27 February, 2015 using data from PubMed, MeSH and CancerIndex

Specific Cancers (4)

Data table showing topics related to specific cancers and associated disorders. Scope includes mutations and abnormal protein expression.

Note: list is not exhaustive. Number of papers are based on searches of PubMed (click on topic title for arbitrary criteria used).

Latest Publications: CCKBR (cancer-related)

Theissen J, Oberthuer A, Hombach A, et al.
Chromosome 17/17q gain and unaltered profiles in high resolution array-CGH are prognostically informative in neuroblastoma.
Genes Chromosomes Cancer. 2014; 53(8):639-49 [PubMed] Related Publications
The prognostic relevance of chromosome 17 gain in neuroblastoma is still discussed. This investigation specifies the frequency, type, size, and transcriptional relevance in a large patient cohort. Primary tumor material of 202 patients was analyzed using high-resolution oligonucleotide array-based comparative genomic hybridization (aCGH) and correlated with clinical and survival data. A subset (n = 145) was correlated for differentially expressed genes (DEG) by microarray analysis. Chromosome 17 aCGH analysis showed numerical gain in 94/202 patients (47%), partial gain in 93/202 patients (46%), and no gain in 15/202 patients (7%). The frequency of partial gain was higher in stage 4 neuroblastoma (stage 1 15%; stage 2 12%; stage 3 16%; stage 4S 7%; and stage 4 50%). Overall survival (OS) was superior in patients with numerical gain compared with patients with partial gain or no gain (5-y-OS: 0.95 ± 0.02 vs. 0.63 ± 0.05 vs. 0.60 ± 0.13; P < 0.001). Gene expression analysis demonstrated 95/130 DEGs between tumors with numerical or partial chromosome/no gain. Only one DEG (CCKBR) was detected comparing tumors with partial gain and those with no gain. In patients with partial gain, the distribution of breakpoints did not correlate with stage and 11q status, but with MYCN amplification and 1p status. The "best" breakpoints in cases with partial 17q gain were at 42.5 Mb for event-free and 26.6 Mb for OS. Numerical gain of chromosome 17 is associated with a better prognosis than partial and no gain. The group of tumors with partial gain was similar to the group without gain with respect to stage distribution, outcome, and gene expression profile.

He Y, Meng XM, Huang C, et al.
Long noncoding RNAs: Novel insights into hepatocelluar carcinoma.
Cancer Lett. 2014; 344(1):20-7 [PubMed] Related Publications
Recent advances in non-protein coding part of human genome analysis have discovered extensive transcription of large RNA transcripts that lack of coding protein function, termed long noncoding RNAs (lncRNAs). It is becoming evident that lncRNAs may be an important class of pervasive genes involved in carcinogenesis and metastasis. However, the biological and molecular mechanisms of lncRNAs in diverse diseases are not yet fully understood. Thus, it is anticipated that more efforts should be made to clarify the lncRNAs world. Moreover, accumulating studies have demonstrated that a class of lncRNAs are dysregulated in hepatocellular carcinoma(HCC) and closely related with tumorigenesis, metastasis, prognosis or diagnosis. In this review, we will briefly discuss the regulation and functional role of lncRNAs in HCC, therefore evaluating the potential of lncRNAs as prospective novel therapeutic targets in HCC.

Zhang R, Li M, Zang W, et al.
MiR-148a regulates the growth and apoptosis in pancreatic cancer by targeting CCKBR and Bcl-2.
Tumour Biol. 2014; 35(1):837-44 [PubMed] Related Publications
Our previous studies have revealed that miR-148a is downregulated in pancreatic cancer. Bioinformatics analysis has shown cholecystokinin-B receptor (CCKBR) and B cell lymphoma (Bcl-2) to be potential targets of miR-148a. But the pathophysiologic role of miR-148a and its relevance to the growth and development of pancreatic cancer are yet to be investigated. The purpose of this study is to elucidate the molecular mechanisms where miR-148a acts as a tumor suppressor in pancreatic cancer. Our results showed significant downregulation of miR-148a in 28 pancreatic cancer tissue samples and five pancreatic cancer cell lines, compared with their non-tumor counterparts by qRT-PCR. MiR-148a was found to not only inhibit the proliferation of pancreatic cancer cells (PANC-1 and AsPC-1) in vitro by MTT assay and colony formation assay, but also to promote cells apoptosis in vitro by Annexin V-FITC apoptosis detection and caspase activity assay. Using western blot and luciferase activity assay, CCKBR and Bcl-2 were identified as targets of miR-148a. Moreover, we also found that the expression of Bcl-2 lacking in 3'UTR could abrogate the pro-apoptosis function of miR-148a. These findings suggest the importance of miR-148a's targeting of CCKBR and Bcl-2 in the regulation of pancreatic cancer growth and apoptosis.

Goetze JP, Eiland S, Svendsen LB, et al.
Characterization of gastrins and their receptor in solid human gastric adenocarcinomas.
Scand J Gastroenterol. 2013; 48(6):688-95 [PubMed] Related Publications
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.

Song J, Ren H, Li Y, et al.
rG17PE38, a novel immunotoxin target to gastric cancer with overexpressed CCK-2R.
J Drug Target. 2013; 21(4):375-82 [PubMed] Related Publications
BACKGROUND: Gastrin/cholecystokinin subtype 2 receptor (CCK2R) is overexpressed in several types of tumors. Gastrin-17 (G17) peptide has a high affinity with CCK2R. These characters suggest that G17 may be useful for target cancer therapy.
PURPOSE: Construct a new immunotoxin (IT) targeting of CCK2R overexpressed gastric cancer.
METHODS: Two ITs were generated using forward and reverse G17 peptides fused with PE38. To get a high yield, codon optimized gene and optimized fermentation parameters were used in large-scale protein expression. An immunoaffinity technique was introduced into pseudomonas exotoxin (PE)-derived IT purification procedure. G17 competition, GST pull-down and indirect immunoflourescence assays were carried out to confirm the interaction between rG17 and CCK2R. Then, several cytotoxic assays were carried out on 18 cell lines, and an in vivo antitumor activity experiment was tested in nude mice.
RESULTS: The rG17PE38 showed specific cytotoxicity on three gastric cancer cells, while G17PE38 did not. After optimization, the expression level reached about 40% in medium deprived of NaCl. Next, 15-27.5 mg of pure rG17PE38 per 1 L of cultures was obtained. Results of G17 competition, GST pull-down and indirect immunoflourescence assays demonstrated that rG17 have a specific interact with CCK2R. Purified rG17PE38 showed high cytotoxicity on gastric cancer cell lines with the IC50 value of 0.6-4 ng·mL(-1). Treatment of nude mice inoculated with BGC-823 tumor xenografts with rG17PE38 efficiently inhibited tumor size.
CONCLUSIONS AND DISCUSSION: The present study demonstrates that reversed G17 could be used as target moiety of PE-derived IT and the rG17PE38 could be developed as a new immunotherapy agent. Codon optimized gene could increase the rG17PE38 expression level in E. coli and furthermore NaCl inhibits the rG17PE38 expression in large scale. Meanwhile, our present study inducts an immunoaffinity method in the IT purification procedure, which could purify the PE-derived ITs in native form.

Wang T, Zhao L, Yang Y, et al.
EGR1 is critical for gastrin-dependent upregulation of anion exchanger 2 in gastric cancer cells.
FEBS J. 2013; 280(1):174-83 [PubMed] Related Publications
The essential anion exchanger (AE) involved in bicarbonate secretion is AE2/SLC4A2, a membrane protein recognized to be relevant for the regulation of the intracellular pH in several cell types. Here we report that gastrin, a major gastrointestinal hormone, upregulates the expression of AE2 mRNA and protein in a cholecystokinin B receptor dependent manner in gastric cancer cells. The upregulated species of AE2 mRNA originates from the classical upstream promoter of the AE2 gene (here referred to as AE2a1) which provides the binding site for transcription factors early growth response 1 (EGR1) and SP1. EGR1 upregulated the AE2 expression that can be competitively inhibited by SP1 in co-transfection experiments. This competitive inhibition was avoided in cells because the SP1 expression was time-staggered to EGR1 in response to gastrin. Overexpression or knockdown of EGR1 consistently increased or decreased the expression of AE2. Our data linked a novel signal pathway involved in gastrin-stimulated AE2 expression.

Takamura A, Ito M, Boda T, et al.
High expression of gastrin receptor protein in injured mucosa of Helicobacter pylori-positive gastritis.
Dig Dis Sci. 2013; 58(3):634-40 [PubMed] Related Publications
BACKGROUND AND AIM: Gastrin is a growth factor for the gastric epithelial cells. However, it is unknown how gastric receptor (GR) expression is regulated in the gastric mucosa. We studied GR expression using a newly raised antibody and investigated the relationship between GR expression and gastritis.
METHODS: Gastric receptor expression in 63 human gastric mucosa was studied. Helicobacter pylori infection and histological gastritis status were evaluated in gastric biopsy samples. In gastric ulcer cases, additional biopsy specimens were taken from injured mucosa. Fasting sera were collected and serum gastrin level evaluated. MKN-28 cells were cultured at various pH conditions, and the change in GR expression was determined.
RESULTS: Gastric receptor expression was detected in the foveolar epithelium of the gastric mucosa, and its expression was stronger in patients infected with H. pylori. In particular, higher expression was detected in regenerating injured mucosa. There was no association between gastritis score/serum gastrin level and GR expression in H. pylori-positive cases. In MKN-28 cells, GR protein expression was lower in neutral conditions than in acidic or alkaline conditions.
CONCLUSION: Gastric mucosal injury with H. pylori infection destroys the pH barrier on the foveolar epithelium and may induce GR expression through pH changes.

Dockray GJ, Moore A, Varro A, Pritchard DM
Gastrin receptor pharmacology.
Curr Gastroenterol Rep. 2012; 14(6):453-9 [PubMed] Related Publications
C-terminally amidated gastrins act at cholecystokinin-2 receptors (CCK2R), which are normally expressed by gastric parietal and enterochromaffin-like (ECL) cells and smooth muscle; there is also extensive expression in the CNS where the main endogenous ligand is cholecystokinin. A variety of neoplasms express CCK2R, or splice variants, including neuroendocrine, pancreatic, medullary thyroid and lung cancers. Other products of the gastrin gene (progastrin, the Gly-gastrins) may stimulate cell proliferation but are not CCK2R ligands. Depending on the cell type, stimulation of CCK2R evokes secretion, increases proliferation and cell migration, inhibits apoptosis, and controls the expression of various genes. These effects are mediated by increased intracellular calcium and activation of protein kinase C, MAPkinase and other protein kinase cascades. There has been recent progress in developing CCK2R ligands that can be used for imaging tumours expressing the receptor. New antagonists have also been developed, and there is scope for using these for suppression of gastric acid and for treatment of neuroendocrine and other CCK2R-expressing tumours.

Quattrone A, Dewaele B, Wozniak A, et al.
Promoting role of cholecystokinin 2 receptor (CCK2R) in gastrointestinal stromal tumour pathogenesis.
J Pathol. 2012; 228(4):565-74 [PubMed] Related Publications
The cholecystokinin 2 receptor (CCK2R/CCKBR) is expressed in gastrointestinal stromal tumours (GISTs). We sought to investigate the role of CCK2R in GIST pathogenesis. Molecular characterization of CCK2R was performed on a heterogeneous cohort of 50 GISTs. In addition, CCK2R expression was evaluated by immunohistochemistry (IHC), using tissue microarray (TMA) containing 292 GISTs, two cases of hyperplasia of interstitial Cajal's cells (ICC) and six gastric microscopic GISTs. Mono-allelic loss of the CCK2R/11p15 allele was identified in 13.7% of GISTs, having no impact on the level of CCK2R transcript expression. No CCK2R mutations were found. The CCK2Ri4sv, CCK2R splice variant with retention of intron 4 was detected in six of 20 tumours analysed. Wild-type CCK2R transcripts were commonly expressed (57.1% of cases) and this expression was highly correlated with gastric primary site of GISTs (p < 0.001). At the protein level, expression of CCK2R in incidental ICC hyperplasia and early stages of gastric GIST development was documented, and its gastric association was confirmed on GIST-TMA by IHC. To explore the in vivo effect of CCK2R activation on tumour growth, gastrin versus placebo was administered intraperitoneally in nude mice carrying human GIST xenografts. The tumour volume was followed for 10 weeks. The effect of this stimulation on tumour cell proliferation/apoptosis was assessed by IHC and KIT/PKC-θ signalling was evaluated by western blotting (WB). In vivo experiments showed a two-fold increase in the volume of tumours which were exposed to gastrin in comparison with non-exposed controls (p = 0.03), with a significant increase in mitotic activity (p = 0.04) and Ki-67 proliferation index (p = 0.008). By WB, gastrin stimulation resulted in hyper-activation of KIT and PKC-θ kinases, and in evident PI3K-AKT pathway over-activation. Our results indicate a promoting role of CCK2R on GIST tumourigenesis, particularly in tumours of gastric origin.

Fino KK, Matters GL, McGovern CO, et al.
Downregulation of the CCK-B receptor in pancreatic cancer cells blocks proliferation and promotes apoptosis.
Am J Physiol Gastrointest Liver Physiol. 2012; 302(11):G1244-52 [PubMed] Free Access to Full Article Related Publications
Gastrin stimulates the growth of pancreatic cancer cells through the activation of the cholecystokinin-B receptor (CCK-BR), which has been found to be overexpressed in pancreatic cancer. In this study, we proposed that the CCK-BR drives growth of pancreatic cancer; hence, interruption of CCK-BR activity could potentially be an ideal target for cancer therapeutics. The effect of CCK-BR downregulation in the human pancreatic adenocarcinoma cells was examined by utilizing specific CCK-BR-targeted RNA interference reagents. The CCK-BR receptor expression was both transiently and stably downregulated by transfection with selective CCK-BR small-interfering RNA or short-hairpin RNA, respectively, and the effects on cell growth and apoptosis were assessed. CCK-BR downregulation resulted in reduced cancer cell proliferation, decreased DNA synthesis, and cell cycle arrest as demonstrated by an inhibition of G(1) to S phase progression. Furthermore, CCK-BR downregulation increased caspase-3 activity, TUNEL-positive cells, and decreased X-linked inhibitor of apoptosis protein expression, suggesting apoptotic activity. Pancreatic cancer cell mobility was decreased when the CCK-BR was downregulated, as assessed by a migration assay. These results show the importance of the CCK-BR in regulation of growth and apoptosis in pancreatic cancer. Strategies to decrease the CCK-BR expression and activity may be beneficial for the development of new methods to improve the treatment for patients with pancreatic cancer.

Smith JP, Harms JF, Matters GL, et al.
A single nucleotide polymorphism of the cholecystokinin-B receptor predicts risk for pancreatic cancer.
Cancer Biol Ther. 2012; 13(3):164-74 [PubMed] Free Access to Full Article Related Publications
There currently are no tests available for early diagnosis or for the identification of patients at risk for development of pancreatic cancer. We report the discovery of single nucleotide polymorphism (SNP) in the cholecystokinin B receptor (CCKBR) gene predicts survival and risk of pancreatic cancer. Growth of human pancreatic cancer is stimulated by gastrin through the CCKBR and an alternatively spliced isoform of the CCKBR gene called CCKCR. One hundred and ten surgically resected benign and malignant pancreatic tissues as well as normal pancreas were prospectively evaluated for CCKBR genotype and protein expression. Analysis demonstrated the expression of the spliced isoform, CCKCR, was associated with a (SNP) (C > A) at position 32 of the intron 4 (IVS 4) of the CCKBR gene. Since the SNP is within an intron, it has not previously been identified in the GWAS studies. Only patients with the A/A or A/C genotypes, exhibited immunoreactivity to a selective CCKCR antibody. Survival among pancreatic cancer patients with the A-SNP was significantly shorter (p = 0.0001, hazard ratio = 3.63) compared with individuals with C/C genotype. Other variables such as surgical margins, lymph node status, histologic grade or adjuvant chemotherapy were not associated with survival. Furthermore, having one or two of the A-alleles was found to increase the risk of pancreatic adenocarcinoma by 174% (p = 0.0192) compared with the C/C wild type. Cancer cells transfected to overexpress the CCKCR demonstrated increased proliferation over controls. Genetic screening for this SNP may aid in early detection of pancreatic cancer in high risk subjects.

Cayrol C, Bertrand C, Kowalski-Chauvel A, et al.
αv integrin: a new gastrin target in human pancreatic cancer cells.
World J Gastroenterol. 2011; 17(40):4488-95 [PubMed] Free Access to Full Article Related Publications
AIM: To analyse αv integrin expression induced by gastrin in pancreatic cancer models.
METHODS: αv integrin mRNA expression in human pancreatic cancer cells was analysed using a "cancer genes" array and confirmed by real-time reverse transcription-polymerase chain reaction (PCR). Western blotting and semi-quantitative immunohistochemistry were used to examine protein levels in human pancreatic cancer cell lines and pancreatic tissues, respectively. The role of αv integrin on gastrin-induced cell adhesion was examined using blocking anti-αv integrin monoclonal antibodies. Adherent cells were quantified by staining with crystal violet.
RESULTS: Using a "cancer genes" array we identified αv integrin as a new gastrin target gene in human pancreatic cancer cells. A quantitative real-time PCR approach was used to confirm αv integrin gene expression. We also demonstrate that Src family kinases and the PI 3-kinase, two signalling pathways specifically activated by the CCK-2 receptor (CCK2R), are involved in gastrin-mediated αv integrin expression. In contrast, inhibition of the ERK pathway was without any effect on αv integrin expression induced by gastrin. Our results also show that gastrin modulates cell adhesion via αv integrins. Indeed, in vitro adhesion assays performed on fibronectin show that gastrin significantly increases adhesion of pancreatic cancer cells. The use of blocking anti-αv integrin monoclonal antibodies completely reversed the increase in cell-substrate adhesion induced by gastrin. In addition, we showed in vivo that the targeted CCK2R expression in the pancreas of Elas-CCK2 mice, leads to the overexpression of αv integrin. This process may contribute to pancreatic tumour development observed in these transgenic animals.
CONCLUSION: αv integrin is a new gastrin target in pancreatic cancer models and contributes to gastrin effects on cell adhesion.

Song Y, Xu Y, Wang Z, et al.
MicroRNA-148b suppresses cell growth by targeting cholecystokinin-2 receptor in colorectal cancer.
Int J Cancer. 2012; 131(5):1042-51 [PubMed] Related Publications
MicroRNAs (miRNAs) play an important role in the regulation of a variety of cellular processes, including cell growth, differentiation, apoptosis and carcinogenesis. The purpose of this study was to elucidate the molecular mechanisms by which miR-148b acts as a tumor suppressor in colorectal cancer. The expression of miR-148b was significantly downregulated in 96 pairs of human colorectal cancer tissues (p<0.0001) and three cell lines (p<0.01) compared with non-tumor adjacent tissues by quantitative real-time PCR. The results of in situ hybridization highlighted that miR-148b was important in the cancer transformation process. Using statistical analysis, we found that the expression level of miR-148b was associated with tumor size (p=0.033) in colorectal cancer patients. Moreover, overexpression of miR-148b in HCT-116 and HT-29 cells could inhibit cell proliferation in vitro and suppress tumorigenicity in vivo. Importantly, the result of luciferase activity assay and western blot showed that the cholecystokinin-2 receptor gene (CCK2R) was a target of miR-148b and was downregulated by miR-148b at the translational level. Then, we used siRNA, radioimmunoassay and ELISA to demonstrate that miR-148b might have an effect on cell proliferation by regulating the expression of CCK2R which functioned depending on the gastrin in colorectal cancer. Taken together, our data provides the first evidences that miR-148b acts as a tumor suppressor in colorectal cancer and should be further evaluated as a biomarker and therapeutic tool against colorectal cancer.

Kato H, Seto K, Kobayashi N, et al.
CCK-2/gastrin receptor signaling pathway is significant for gemcitabine-induced gene expression of VEGF in pancreatic carcinoma cells.
Life Sci. 2011; 89(17-18):603-8 [PubMed] Related Publications
AIMS: As activation and overexpression of the cholecystokinin-2 (CCK-2)/gastrin receptor can lead to carcinogenesis, it has been explored as a therapeutic target in pancreatic cancer. We demonstrated that Z-360, a CCK-2/gastrin receptor antagonist, combined with gemcitabine prolonged survival and reduced gemcitabine-induced vascular endothelial growth factor (VEGF) expression in a pancreatic carcinoma orthotopic xenograft mouse. In this study, we investigated the role of the CCK-2/gastrin signaling pathway on gemcitabine-induced VEGF expression in PANC-1 human pancreatic carcinoma cells.
MAIN METHODS: In PANC-1 cells treated with Z-360, anti-gastrin IgG or kinase inhibitors, the gene expression levels were analyzed by quantitative real-time RT-PCR, and the protein levels of Akt and phosphorylated Akt (p-Akt) in cellular extracts were measured by ELISA.
KEY FINDINGS: Gemcitabine-induced expression of VEGF and hypoxia-inducible factor-1 alpha (HIF-1 alpha) were suppressed by the treatment with an anti-gastrin antibody. In addition, VEGF and HIF-1 alpha gene expression was inhibited by treatment with an inhibitor of phosphatidylinositol 3-kinase (PI3K), which is involved in the downstream signaling pathway of the CCK-2/gastrin receptor, and was also suppressed by treatment with Z-360. Moreover, although Akt phosphorylation was increased by treatment with gemcitabine, this elevation was partially, but significantly, inhibited by an exposure of Z-360.
SIGNIFICANCE: Gemcitabine might induce gene expression of VEGF via the PI3K/Akt signaling pathway in the downstream of the CCK-2/gastrin receptor. The suppression of the CCK-2/gastrin signaling pathway by treatment with Z-360 could be a useful approach for potentiating prolonged survival of pancreatic cancer patients receiving gemcitabine therapy.

Song YX, Yue ZY, Wang ZN, et al.
MicroRNA-148b is frequently down-regulated in gastric cancer and acts as a tumor suppressor by inhibiting cell proliferation.
Mol Cancer. 2011; 10:1 [PubMed] Free Access to Full Article Related Publications
BACKGROUND: MicroRNAs (miRNAs) are involved in cancer development and progression, acting as tumor suppressors or oncogenes. Our previous studies have revealed that miR-148a and miR-152 are significantly down-regulated in gastrointestinal cancers. Interestingly, miR-148b has the same "seed sequences" as miR-148a and miR-152. Although aberrant expression of miR-148b has been observed in several types of cancer, its pathophysiologic role and relevance to tumorigenesis are still largely unknown. The purpose of this study was to elucidate the molecular mechanisms by which miR-148b acts as a tumor suppressor in gastric cancer.
RESULTS: We showed significant down-regulation of miR-148b in 106 gastric cancer tissues and four gastric cancer cell lines, compared with their non-tumor counterparts by real-time RT-PCR. In situ hybridization of ten cases confirmed an overt decrease in the level of miR-148b in gastric cancer tissues. Moreover, the expression of miR-148b was demonstrated to be associated with tumor size (P = 0.027) by a Mann-Whitney U test. We also found that miR-148b could inhibit cell proliferation in vitro by MTT assay, growth curves and an anchorage-independent growth assay in MGC-803, SGC-7901, BGC-823 and AGS cells. An experiment in nude mice revealed that miR-148b could suppress tumorigenicity in vivo. Using a luciferase activity assay and western blot, CCKBR was identified as a target of miR-148b in cells. Moreover, an obvious inverse correlation was observed between the expression of CCKBR protein and miR-148b in 49 pairs of tissues (P = 0.002, Spearman's correlation).
CONCLUSIONS: These findings provide important evidence that miR-148b targets CCKBR and is significant in suppressing gastric cancer cell growth. Maybe miR-148b would become a potential biomarker and therapeutic target against gastric cancer.

Muiños-Gimeno M, Espinosa-Parrilla Y, Guidi M, et al.
Human microRNAs miR-22, miR-138-2, miR-148a, and miR-488 are associated with panic disorder and regulate several anxiety candidate genes and related pathways.
Biol Psychiatry. 2011; 69(6):526-33 [PubMed] Related Publications
BACKGROUND: The involvement of microRNAs (miRNAs) in neuronal differentiation and synaptic plasticity suggests a role for miRNAs in psychiatric disorders; association analyses and functional approaches were used to evaluate the implication of miRNAs in the susceptibility for panic disorder.
METHODS: Case-control studies for 712 single-nucleotide polymorphisms (SNPs) tagging 325 human miRNA regions were performed in 203 Spanish patients with panic disorder and 341 control subjects. A sample of 321 anxiety patients and 642 control subjects from Finland and 102 panic disorder patients and 829 control subjects from Estonia was used as a replica. Reporter-gene assays and miRNA overexpression experiments in neuroblastoma cells were used to functionally evaluate the spectrum of genes regulated by the associated miRNAs.
RESULTS: Two SNPs associated with panic disorder: rs6502892 tagging miR-22 (p < .0002), and rs11763020 tagging miR-339 (p < .00008). Other SNPs tagging miR-138-2, miR-488, miR-491, and miR-148a regions associated with different panic disorder phenotypes. Replication in the north-European sample supported several of these associations, although they did not pass correction for multiple testing. Functional studies revealed that miR-138-2, miR-148a, and miR-488 repress (30%-60%) several candidate genes for panic disorder--GABRA6, CCKBR and POMC, respectively--and that miR-22 regulates four other candidate genes: BDNF, HTR2C, MAOA, and RGS2. Transcriptome analysis of neuroblastoma cells transfected with miR-22 and miR-488 showed altered expression of a subset of predicted target genes for these miRNAs and of genes that might be affecting physiological pathways related to anxiety.
CONCLUSIONS: This work represents the first report of a possible implication of miRNAs in the etiology of panic disorder.

Kovac S, Xiao L, Shulkes A, et al.
Gastrin increases its own synthesis in gastrointestinal cancer cells via the CCK2 receptor.
FEBS Lett. 2010; 584(21):4413-8 [PubMed] Related Publications
The involvement of the gastrointestinal hormone gastrin in the development of gastrointestinal cancer is highly controversial. Here we demonstrate a positive-feedback loop whereby gastrin, acting via the CCK2 receptor, increases its own expression. Such an autocrine loop has not previously been reported for any other gastrointestinal hormone. Gastrin promoter activation was dependent on the MAP kinase pathway and did not involve Sp1 binding sites or epidermal growth factor receptor transactivation. As the treatment of gastrointestinal cancer cells with amidated gastrin led to increased expression of non-amidated gastrins, the positive-feedback loop may contribute to the sustained increase in circulating gastrins observed in colorectal cancer patients.

Ellrichmann M, Ritter PR, Schrader H, et al.
Gastrin stimulates the VEGF-A promotor in a human colon cancer cell line.
Regul Pept. 2010; 165(2-3):146-50 [PubMed] Related Publications
INTRODUCTION: Hypergastrinemia has been observed in patients suffering from colorectal cancer. Vascular endothelial growth factor (VEGF) is known to play a pivotal role in tumour growth. Therefore, we addressed whether gastrin-17-gly and gastrin-17-amide regulate VEGF-A-gene and protein expression.
MATERIALS AND METHODS: Colo-320-cells were stably transfected with a VEGF-Luciferase-reporter gene. Luciferase activity was assessed after stimulation with various gastrin concentrations. Relevant promotor elements were identified by deletion analyses. VEGF protein levels in culture supernatants were quantified by ELISA.
RESULTS: VEGF-A stimulation with gastrin induced a dose- and time-dependent stimulation of luciferase activity. The greatest activities were found 6h after stimulation at concentrations of 10(-)(6)mmol/l. VEGF-promotor expression resulted in significantly (p<0.05) increased VEGF-A protein secretion. These effects were restricted to gastrin-17-amide.
CONCLUSION: Gastrin-17-amide enhances VEGF-A gene and protein expression in Colo320 cells stably transfected with a wild-type CCK-B/gastrin receptor. The induction of VEGF-A transcription and translation may contribute to the carcinogenic effects of gastrin observed in clinical studies. Therefore, CCK-B receptor antagonists may represent a treatment strategy in patients with colorectal cancer.

Chen Y, Song Y, Wang Z, et al.
Altered expression of MiR-148a and MiR-152 in gastrointestinal cancers and its clinical significance.
J Gastrointest Surg. 2010; 14(7):1170-9 [PubMed] Related Publications
BACKGROUND: MicroRNAs are endogenous small noncoding RNAs that aberrantly expressed in various carcinomas. MiR-148a and miR-152, which have the same "seed region", have not been comprehensively investigated in gastrointestinal cancers.
METHODS: Total RNA was extracted from the tissues of 101 patients with gastric cancer and 101 patients with colorectal cancer as well as their matched nontumor adjacent tissues. After polyadenylation and reverse transcription, the expression of miR-148a and miR-152 was determined using quantitative real-time polymerase chain reaction. The protein level of cholecystokinin B receptor, which might be the target gene of miR-148a and miR-152, was analyzed by Western blot in 40 patients with gastric cancer.
RESULTS: Expression levels of miR-148a and miR-152 in human gastric (p < 0.001 and p = 0.038, respectively, t-test) and colorectal (all p < 0.001) cancers were significantly lower than that in their matched nontumor adjacent tissues. Moreover, their low expression was also found in several gastrointestinal cancer cell lines compared with normal gastric epithelial cell line and normal colorectal tissue, respectively. A strong correlation was found between the expression of miR-148a and miR-152 (all p < 0.001, Pearson's correlation). Furthermore, low expression of miR-152 was correlated with increased tumor size (p = 0.023 and 0.004, respectively, Mann-Whitney U test) and advanced pT stage (p = 0.018 and 0.002, respectively) in gastrointestinal cancers. Low expression of miR-148a was also correlated with increased tumor size (p = 0.045 and 0.018, respectively) in gastrointestinal cancers, but only correlated with advanced pT stage (p = 0.023) in colorectal cancer. We also found the expression of miR-148a (p < 0.001, chi-square test) and miR-152 (p = 0.002) inversely correlated with cholecystokinin B receptor protein in gastric cancer.
CONCLUSION: MiR-148a and miR-152 may be involved in the carcinogenesis of gastrointestinal cancers and might be potential biomarkers in these cancers.

Kobayashi N, Seto K, Orikawa Y, et al.
Z-360, a novel cholecystokinin-2/gastrin receptor antagonist, inhibits gemcitabine-induced expression of the vascular endothelial growth factor gene in human pancreatic cancer cells.
Biol Pharm Bull. 2010; 33(2):216-22 [PubMed] Related Publications
Z-360 is a novel cholecystokinin (CCK)-2/gastrin receptor antagonist that is being developed for the treatment of pancreatic adenocarcinoma in combination with gemcitabine. A previous study shows that the co-administration of Z-360 with gemcitabine significantly prolonged the survival of mice with orthotopically implanted human pancreatic adenocarcinoma cell lines. To clarify the therapeutic effects of Z-360 in combined with gemcitabine, we analyzed gene expression. When gemcitabine was administered, CCK-2/gastrin receptor expression was induced in an orthotropic xenograft model; the result indicating that Z-360 could act on gemcitabine-sensitive cells. Both in vitro and in vivo studies showed that gemcitabine increased the expression of vascular endothelial growth factor A (VEGFA), a prognostic factor for survival in pancreatic cancer, while Z-360 suppressed this induction of VEGFA gene expression. These results help to explain how Z-360 prolongs survival when used in combination with gemcitabine.

Jin G, Ramanathan V, Quante M, et al.
Inactivating cholecystokinin-2 receptor inhibits progastrin-dependent colonic crypt fission, proliferation, and colorectal cancer in mice.
J Clin Invest. 2009; 119(9):2691-701 [PubMed] Free Access to Full Article Related Publications
Hyperproliferation of the colonic epithelium, leading to expansion of colonic crypt progenitors, is a recognized risk factor for colorectal cancer. Overexpression of progastrin, a nonamidated and incompletely processed product of the gastrin gene, has been shown to induce colonic hyperproliferation and promote colorectal cancer in mice, but the mechanism of pathogenesis has not been defined. Cholecystokinin-2 receptor (CCK2R) is the primary receptor for cholecystokinin (CCK) and amidated gastrin. Here, we show that Cck2r was expressed in murine colonic crypts and upregulated in the transgenic mice that overexpress human progastrin. Murine deletion of Cck2r abrogated progastrin-dependent increases in colonic proliferation, mucosal thickness, and beta-catenin and CD44 expression in the colon tumor. In addition, either deletion or antagonism of Cck2r resulted in the inhibition of progastrin-dependent increases in progenitors expressing doublecortin and CaM kinase-like-1 (DCAMKL1), stem cells expressing leucine rich repeat-containing G protein-coupled receptor 5 (LgR5), and colonic crypt fission. Furthermore, in the azoxymethane mouse model of colorectal carcinogenesis, Cck2r deletion in human progastrin-overexpressing mice resulted in markedly decreased aberrant crypt foci formation and substantially reduced tumor size and multiplicity. Taken together, these observations indicate that progastrin induces proliferative effects, primarily in colonic progenitor cells, through a CCK2R-dependent pathway. Moreover, our data suggest that CCK2R may be a potential target in the treatment or prevention of colorectal cancer.

Obszynska JA, Atherfold PA, Nanji M, et al.
Long-term proton pump induced hypergastrinaemia does induce lineage-specific restitution but not clonal expansion in benign Barrett's oesophagus in vivo.
Gut. 2010; 59(2):156-63 [PubMed] Related Publications
BACKGROUND: Barrett's oesophagus is a common premalignant lesion caused partly by acid reflux. Although the requisite therapy, proton pump inhibitors (PPIs), have been implicated in the progression of Barrett's oesophagus in animal models, harmful effects of prolonged PPI therapy in Barrett's oesophagus is both inconclusive and controversial. We therefore aimed to test the role of PPI-induced hypergastrinaemia in vitro and see whether any biological parameters were useful surrogates of long-term therapy in man.
METHODS: We undertook detailed serological and tissue assessment of gastrin and CCK(2) receptors in 90 patients randomised to different doses of PPI therapy during a detailed 2-year follow-up. We also undertook a comprehensive study of cell models to study the consequential biological effects of gastrin on the mucosa.
RESULTS: Gastrin and its cognate receptor CCK(2)R were expressed highest in the stomach, then less in Barrett's oesophagus and least in squamous oesophagus (SqE) (n=20 paired t-test, p<0.01). Analysis of the change in Barrett's oesophagus segment length change in 70 patients who were randomised to high or low PPI dose showed no difference over 2 years (n=70 t-test, p=0.8). Prolonged PPI use did, however, increase the serum gastrin, (36 pg/ml+/-57 pg/ml to 103 pg/ml+/-94 pg/ml (paired t test, p<0.05)). In vitro gastrin also induced changes in OE33(E)(cckr) Barrett's oesophagus cells, but not OE21(E)(cckr) squamous cells, transfected with CCK(2)R; migration was induced by 1 ng/ml of gastrin but proliferation only increased with 100 ng/ml (paired t-test, p<0.01) and both were abolished by antagonists.
CONCLUSION: While the short-term effects of gastrin enhance epithelial restitution in Barrett's oesophagus (but not squamous mucosa) there is no clinical evidence that Barrett's oesophagus length expands over time. This study, which is the largest and longest term randomised controlled trial of gastrin biology in Barrett's oesophagus, is further proof of the clinical safety of PPI therapy.

Körner M, Waser B, Reubi JC, Miller LJ
CCK(2) receptor splice variant with intron 4 retention in human gastrointestinal and lung tumours.
J Cell Mol Med. 2010; 14(4):933-43 [PubMed] Free Access to Full Article Related Publications
The wild-type cholecystokinin type 2 (CCK(2)) receptor is expressed in many gastrointestinal and lung tumours. A splice variant of the CCK(2) receptor with retention of intron 4 (CCK(2)Ri4sv) showing constitutive activity associated with increased tumour growth was described in few colorectal, pancreatic and gastric cancers. Given the potential functional and clinical importance of this spliceoform, its occurrence was quantitatively characterized in a broad collection of 81 gastrointestinal and lung tumours, including insulinomas, ileal carcinoids, gastrointestinal stromal tumours (GIST), gastric, colorectal and pancreatic ductal adenocarcinomas, cholangiocellular and hepatocellular carcinomas, small cell lung cancers (SCLC), non-SCLC (nSCLC) and bronchopulmonary carcinoids, as well as 21 samples of corresponding normal tissues. These samples were assessed for transcript expression of total CCK(2) receptor, wild-type CCK(2) receptor and CCK(2)Ri4sv with end-point and real-time RT-PCR, and for total CCK(2) receptor protein expression on the basis of receptor binding with in vitro receptor autoradiography. Wild-type CCK(2) receptor transcripts were found in the vast majority of tumours and normal tissues. CCK(2)Ri4sv mRNA expression was present predominantly in insulinomas (incidence 100%), GIST (100%) and SCLC (67%), but rarely in pancreatic, colorectal and gastric carcinomas and nSCLC. It was not found in wild-type CCK(2) receptor negative tumours or any normal tissues tested. CCK(2)Ri4sv transcript levels in individual tumours were low, ranging from 0.02% to 0.14% of total CCK(2) receptor transcripts. In conclusion, the CCK(2)Ri4sv is a marker of specific gastrointestinal and lung tumours. With its high selectivity for and high incidence in SCLC and GIST, it may represent an attractive clinical target.

Oikonomou E, Buchfelder M, Adams EF
Cholecystokinin (CCK) and CCK receptor expression by human gliomas: Evidence for an autocrine/paracrine stimulatory loop.
Neuropeptides. 2008; 42(3):255-65 [PubMed] Related Publications
Cholecystokinin (CCK) is a gut-brain peptide has been described to be able to induce mitosis according to recent studies. Additionally, conflicting data has been published on whether tumours of the central and peripheral nervous system in general, and gliomas in particular, express CCK receptors. In the present in vitro study we employed reverse transcription followed by the polymerase chain reaction (RT-PCR) to investigate whether mRNA for CCK-A and CCK-B receptors as well as CCK peptide itself is present in primary human gliomas and the U-87 MG GBM cell line. The data show that 14/14 (100%) of the primary gliomas exhibited mRNA expression for the CCK peptide gene and the B receptor including the U-87 MG cells, whereas, only 2/14 (14%) showed presence of the CCK-A receptor. The presence of CCK receptors together with CCK peptide expression itself suggests presence of an autocrine loop controlling glioma cell growth. In support of this conclusion, a neutralizing antibody against the CCK peptide exhibited a dose dependent inhibition of cell growth whereas, antagonists to CCK caused a dose depend inhibition of exogenous stimulated glioma cell growth in vitro, via the CCK-B receptor which is PKC activated. Assessment of apoptosis and proteasome activity were undertaken and we report that treatment with CCK antagonists decreased proteasome and increased caspase-3 activity. These data indicate that CCK peptide and CCK-B are abundant in human gliomas and they act to stimulate cell growth in an autocrine manner, primarily via the high affinity CCK-B receptor, which was blocked by antagonists to CCK, perhaps via apoptosis.

Hollestelle MJ, Timmerman P, Meloen RH, Höppener JW
Characterization of gastrin-cholecystokinin 2 receptor interaction in relation to c-fos induction.
Endocr Relat Cancer. 2008; 15(1):301-9 [PubMed] Related Publications
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.

Oikonomou E, Charlton A, Buchfelder M, Adams EF
Cholecystokinin (CCK) receptor and CCK gene expression in human pituitary adenomas and in vitro effects of CCK peptides on GH and gonadotrophin secretion.
Exp Clin Endocrinol Diabetes. 2007; 115(10):683-9 [PubMed] Related Publications
There is growing evidence that cholecystokinin (CCK) affects growth and differentiation of anterior pituitary cells, via the CCK-B receptor. The possibility of an autocrine / paracrine role for CCK to modulate hormone secretion in human pituitary tumour cells is demonstrated here by RT-PCR and direct sequencing. In support of this conclusion, a neutralising antibody against the CCK peptide exhibited a dose dependent inhibition of hormone secretion by functionless pituitary adenomas. Total RNA was extracted from human pituitary adenomas, reverse transcribed into cDNA and subjected to PCR using primers specific for the gene for CCK, CCK-A and CCK-B receptors. PCR bands of the predicted length were observed in all tumours using human CCK gene and CCK-B receptor primers. Restriction digestion and direct sequence analysis provided further evidence that they represented both the human CCK peptide along with the CCK-A and/B receptor mRNA. CCK-33 and CCK octapeptide sulphate (CCK-8s) both powerfully stimulated phosphatidylinositol hydrolysis, providing evidence for functional activity of the CCK-A and/B receptors. A direct stimulatory effect of CCK peptides on both LH and FSH secretion is reported for the first time, whereas stimulatory effects on GH were blocked by antagonists to CCK. These results may indicate an autocrine role for CCK in the functioning and perhaps development of human pituitary tumours.

Huang H, Ansorge N, Schrader H, et al.
The CCK-2/gastrin splice variant receptor retaining intron 4 transactivates the COX-2 promoter in vitro.
Regul Pept. 2007; 144(1-3):34-42 [PubMed] Related Publications
The expression of the human cholecystokinin-2/gastrin receptor (CCK-2R) has been widely reported in human colorectal cancers. Recently, a splice variant of the CCK-2R retaining intron 4 (CCK-2i4svR) has been cloned from human colorectal cancers and postulated to exhibit constitutive activity. But its role in mediating carcinogenic effects of mature-amidated gastrin in colorectal cancers has not been fully explored. The purpose of the present study was to determine whether the activation of CCK-2i4svR by gastrin transactivates the COX-2 promoter in human colon cancer cells and in COS-7 cells. In this study, Colo320 cells and COS-7 cells were transfected with the human CCK-2R wild type (CCK-2wtR) (COS-7WT, Colo320WT) and the human CCK-2i4svR (COS-7SV, Colo320SV) cDNA. After stimulation with gastrin-17 (G-17), transactivation of the COX-2 promoter was determined by luciferase reporter gene assay. 5'deletions of the COX-2 promoter were transfected into Colo320 cells to narrow down the minimally required regulatory element. Induction of COX-2 expression was further explored at the mRNA level using real time RT-PCR. The effects of CCK-2i4svR activation on phosphorylation of ERK1/2, p38(MAPK) and JNK were examined by using immunoblotting. Prostaglandin E(2) (PGE(2)) secretion was measured by ELISA. Our results showed that gastrin transactivates the COX-2 promoter in both Colo320 cells and COS-7 cells expressing the CCK-2i4svR cDNA. Inhibition of p38(MAPK) pathway using specific inhibitor significantly blocked the gastrin-induced COX-2 transactivation. Gastrin time-dependently increased COX-2 mRNA expression, the peak mRNA levels appeared at 10 h after stimulation. PGE(2) secretion from gastrin-treated cells increased significantly 8 h after stimulation. Treatment with gastrin also stimulated PGE(2) secretion in the Colo320 cells expressing CCK-2i4svR. In conclusion, the CCK-2i4svR mediates transactivation of the COX-2 promoter and MAPK pathway is involved in this process.

Cramer T, Jüttner S, Plath T, et al.
Gastrin transactivates the chromogranin A gene through MEK-1/ERK- and PKC-dependent phosphorylation of Sp1 and CREB.
Cell Signal. 2008; 20(1):60-72 [PubMed] Related Publications
Our previous work revealed that gastrin regulates chromogranin A (CgA) transcription through enhanced binding of Sp1, CREB and Egr-1 to a proximal gastrin-responsive promoter element (Gas-RE). Here, we provide a detailed characterization of the signalling pathways transmitting the effect of gastrin on the CgA promoter. Gastrin treatment of gastric AGS-B cells potently stimulated MEK-1 as well as MAP kinases ERK-1/-2, JNK and p38 in a time-dependent manner. Interruption of ERK-1/-2/MEK-1 pathways abolished the transactivating effect of gastrin, whereas blockade of JNK or p38 activity was without effect. Functional promoter analysis revealed that the minimal element CgA-85/-64 was sufficient and necessary to confer MEK-1/ERK responsiveness. Analysis of proximal signalling pathways showed that activation of the MEK-1/ERK-1/2 module by gastrin does not require Ras, PI3-kinase or intracellular calcium signals, but depends on activation of kinases of the PKC family. This report demonstrates that a pathway comprising PKCs>Raf-1>MEK-1>ERK-1/-2 mediates the effect of gastrin on the CgA promoter, and strongly suggests that enhanced phosphorylation of Sp1 and CREB is crucial for CgA transactivation through the G protein-coupled CCK-B/gastrin receptor.

Sebens Müerköster S, Rausch AV, Isberner A, et al.
The apoptosis-inducing effect of gastrin on colorectal cancer cells relates to an increased IEX-1 expression mediating NF-kappa B inhibition.
Oncogene. 2008; 27(8):1122-34 [PubMed] Related Publications
Addressing the puzzling role of amidated gastrin(17) (G17) and the gastrin/CCKB/CCK2 receptor in colorectal carcinogenesis, we analysed potential candidate genes involved in G17-dependent NF-kappaB inhibition and apoptosis. The colorectal carcinoma cell line Colo320 overexpressing the wild-type CCK2 receptor (Colo320wt) underwent G17-induced apoptosis along with suppressed NF-kappaB activation and decreased expression of the antiapoptotic NF-kappaB target genes cIAP1 and cIAP2, whereas G17 was without effect on Colo320 cells expressing a CCK2 receptor bearing a loss of function mutation (Colo320mut). Gene microarray analysis revealed an elevated expression of the stress response gene IEX-1 in G17-treated Colo320wt but not Colo320mut cells. Quantitative real-time PCR and conventional RT-PCR confirmed this G17-dependent increase of IEX-1 expression in Colo320wt cells. If these cells were subjected to IEX-1 knockdown by small interfering RNA transfection, the apoptosis-inducing effect of G17 was abolished. Moreover, tumor necrosis factor alpha (TNFalpha)- or 5-FU-induced apoptosis that is greatly enhanced by G17 treatment in Colo320wt cells was prevented if IEX-1 expression was repressed. Under these conditions of blocked IEX-1 expression, the NF-kappaB activity remained unaffected by G17, in particular in Colo320wt cells co-treated with TNFalpha and also the suppressive effect of G17 on cIAP1 and cIAP2 expression was not observed anymore if IEX-1 expression was blocked. Conversely, IEX-1 overexpression in Colo320mut cells caused an increase of basal and TNFalpha- or 5-FU-induced apoptosis, an effect not further triggered by G17 treatment. Using a xenograft tumor model in severe combined immune deficiency mice, we could show that experimental systemic hypergastrinemia induced by the administration of omeprazole led to enhanced apoptosis as well as to a marked increase of IEX-1 expression in Colo320wt tumors, but not in Colo320mut tumors. These observations indicate that the proapoptotic effect of G17 on human colon cancer cells expressing the wild-type CCK2 receptor is mediated by IEX-1, which modulates NF-kappaB-dependent antiapoptotic protection and thereby exerts tumor-suppressive potential.

von Guggenberg E, Dietrich H, Skvortsova I, et al.
99mTc-labelled HYNIC-minigastrin with reduced kidney uptake for targeting of CCK-2 receptor-positive tumours.
Eur J Nucl Med Mol Imaging. 2007; 34(8):1209-18 [PubMed] Related Publications
PURPOSE: Different attempts have been made to develop a suitable radioligand for targeting CCK-2 receptors in vivo, for staging of medullary thyroid carcinoma (MTC) and other receptor-expressing tumours. After initial successful clinical studies with [DTPA(0),D: Glu(1)]minigastrin (DTPA-MG0) radiolabelled with (111)In and (90)Y, our group developed a (99m)Tc-labelled radioligand, based on HYNIC-MG0. A major drawback observed with these derivatives is their high uptake by the kidneys. In this study we describe the preclinical evaluation of the optimised shortened peptide analogue, [HYNIC(0),D: Glu(1),desGlu(2-6)]minigastrin (HYNIC-MG11).
METHODS: (99m)Tc labelling of HYNIC-MG11 was performed using tricine and EDDA as coligands. Stability experiments were carried out by reversed phase HPLC analysis in PBS, PBS/cysteine and plasma as well as rat liver and kidney homogenates. Receptor binding and cell uptake experiments were performed using AR4-2J rat pancreatic tumour cells. Animal biodistribution was studied in AR4-2J tumour-bearing nude mice.
RESULTS: Radiolabelling was performed at high specific activities and radiochemical purity was >90%. (99m)Tc-EDDA-HYNIC-MG11 showed high affinity for the CCK-2 receptor and cell internalisation comparable to that of (99m)Tc-EDDA-HYNIC-MG0. Despite high stability in solution, a low metabolic stability in rat tissue homogenates was found. In a nude mouse tumour model, very low unspecific retention in most organs, rapid renal excretion with reduced renal retention and high tumour uptake were observed.
CONCLUSION: (99m)Tc-EDDA-HYNIC-MG11 shows advantages over (99m)Tc-EDDA-HYNIC-MG0 in terms of lower kidney retention with unchanged uptake in tumours and CCK-2 receptor-positive tissue. However, the lower metabolic stability and impurities formed in the labelling process still leave room for further improvement.

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