The Role Of Gastrin And Its Receptor In The Proliferation Of Human Esophageal Adenocarcinoma Cells

Barrett's esophagus (BE) is defined as the migration of squamous columnar epithelium of distal esophagus to gastroesophageal junction, with intestinal metaplasia accompanied. BE is an important precancerous change, which is closely correlated with esophageal adenocarcinoma (EAC). Esophageal adenocarcinoma progressed from Barrett's metaplasia has an obviously increased occurrence rate in Western countries, and it is the malignant tumor with the highest growth rate. Though BE and adenocarcinorma are not so common in Asia, their incidences tend to increase as reported in recent years.Gastrin is a polypeptide hormone secreted from gastrointestinal G-cells, which can stimulate gastric secretion and promote epithelial cell growth. Gastrin receptor is presented as cholecystokinin type-2 (CCK-2) in gaster, a member of G-protein-coupled receptor superfamily. It can regulate gastric acid secretion, release of histamine from enterochromaffin like cell and contraction of contractile fiber cells, so it is named as CCK-2/ gastrin receptor along with CCK2R. After gastrin binds to its receptor, multiple signal transduction pathways would be activated, and mitoschisis signal will be transmitted into cell nucleus to proliferate cells. Recently, more and more evidences indicate that gastrin also participates in the occurrrence and development of some malignant tumors. Previous studies reported the gastrin's trophic effect on pancreas and gaster, and many tumor tissues expressed cholecystokinin type-2 (CCK-2). Studies abroad also demonstrated the presence of CCK-2 (gastrin) receptor in Barrett's metaplasia, which is thought to be a precursor of esophageal adenocarcinoma.At present, it is not clear about the mechanism of CCK2R signal transduction. It has been found that, in addition to the classic inositide lipositol and cAMP signal pathways, mitogen-activated protein kinase (MAPK) pathway may also be involved. MAPK is a member of serine streth/threonine protien kinase family, which has been found in the research on growth factor phosphorylation production in the end of 1980s. It is an important intracellular signal transduction pathway for regulating inflammation proliferation, differentiation and apoptosis. Its member ERK1/2 (i.e. p42/44 MAPK) is a key kinase of cell signal to transmit mitogen signal. By activating it, many transcription factors can be stimulated, thus to influence the expression of target gene. It also has a close relationship with cell growth, differentiation and proliferation.Therefore, we set about our research from gastrin. By blocking its specific receptor, the roles of gastrin and its receptor in proliferation of human esophageal adenocarcinoma cells were studied,to explore its mechanism of action, and providding theoretical evidence in clinical prevention and cure.Objective: To explore the role of gastrin and its receptor in the growth of esophageal adenocarcinoma cells, and providding theoretical evidence in clinical prevention and cure.Methods:1 By utilizing in vitro cell culture technique, proliferation of SEG-1 was assayed by MTT.2 Changes of CCK2R mRNA in different interventions were examined by reverse transcription-polymerase chain reaction (RT-PCR).3 Cells were stimulated by gastrin (10-7 mol/L) based on time gradient, expression of total p42/44MAPK and p42/44MAPK phosphorylation were determined by Western blot to identify the time point of maximal phosphorylation of p42/44MAPK.4 Expression of total p42/44MAPK and p42/44MAPK phosphorylation were determined by Western blot.Results:①Effect of gastrin on the proliferation of SEG-1: compared with control group, G-17 at concentration of 10-9 mol/L - 10-5 mol/L promoted the proliferation of SEG-1, and the growth rates were 108.79%, 117.23%, 125.38%, 138.53% and 150.33%, respectively (P<0.05).②Effect of proglumide on proliferation of SEG-1: compared with control group, PGL at concentration of 10-8 mol/L ~ 10-5 mol/L inhibited the proliferation of SEG-1, and the inhibition rates were 14.23%, 18.37%, 34.24% and 55.97%, respectively, (P<0.05).③Effect of gastrin and proglumide on proliferation of SEG-1: compared with control group, PGL at concentration of 10-8 mol/L-10-5 mol/L inhibited the proliferation of SEG-1 incubated in G-17 (10-7 mol/L) for 24h, and the inhibition rates were 0.57%, 3.50%, 5.00% and 12.22%, respectively, (P<0.05).④RT-PCR results: CCK2R mRNA was expressed in normal SEG-1; CCK2R/GAPDH was decreased to 0.78 after G-17 was added, and increased to 1.59 after PGL was added. Under the combined effects of G-17 and PGL, the gray scale ratio was 1.37, still higher than 1.03, the basal expression gray scale ratio.⑤Western blot results: phosphorylation of p42/44 MAPK protein in G-17 groups was increased than that in control group. The phosphorylation level of p42/44 MAPK protein in SEG-1 changed as G-17 action time prolonged. The phosphorylation rates at 0, 5, 10, 20 and 40 min were 3.92%, 19.62%, 39.58%, 22.41% and 14.93%, respectively. Therefore, 10 min is the time point of maximal phosphorylation. Phosphorylation of p42/44 MAPK protein in PGL groups and G-17 + PGL groups were reduced by 1.74% and 21.68% respectivley than that in control group.Conclusions:1 Gastrin could promote the proliferation of SEG-1 in a dose-dependent manner, while proglumide could obviously inhibit the effect.2 CCK2R mRNA expressed in SEG-1, and was downregulated under the effects of G-17, and up-regulated under the effects of PGL.3 The phosphorylation level is in a time-dependent manner. Gastrin-CCK2R-MAPK signal pathway may play an important role in growth of SEG-1.4 Gastrin could induce phosphorylation of p42/44 MAPK in SEG-1, which suggested activation of MAPK pathway.