[A study of gastrin-focal adhesion kinase signal pathway in human colon cancer cells].

Abstract

OBJECTIVE:

The study investigated the effect of gastrin on tyrosine phosphorylation and protein expression of focal adhesion kinase (FAK) in human colon cancer cells.

METHODS:

The eukaryotic plasmid that expresses cholecystokinin 2 receptor (CCK2R) stably, named pCR3.1/CCK2R, was transfected into Colo320 to construct an up-regulated gastrin-CCK2R signal pathway; the gastrin antagonist was used to down-regulate the signal pathway. Thus, including the normal control, there were three signal pathways with different CCK2R levels. Different doses of gastrin were used to stimulate FAK in the different time. FAK tyrosine phosphorylation and FAK expression in different groups were detected by immunoprecipitation and Western-blotting assays, and analyzed with Labimage software.

RESULTS:

RT-PCR result showed that Colo320 transfected with CCK2R had a mRNA level four times higher than that normal Colo320 did, and which suggested that up-regulated signal transduction pathway was constructed. After stimulated by gastrin with 0, 0.1, 1, 10 and 100 nmol/L, tyrosine phosphorylation levels of Colo320 were 24.0%, 39.7%, 46.2%, 50.4% and 44.5%, and those of Colo320 transfected with CCK2R were 24.6%, 70.7%, 90.1%, 100% and 88.6%. When incubated with gastrin at 0, 2.5, 5, 10 and 20 min, the tyrosine phosphorylation levels of Colo320 were 23.9%, 63.6%, 58.6%, 45.5% and 40.9%, and those of Colo320 transfected with CCK2R were 24.5%, 84.6%, 100%, 98.6% and 97.9%. The increases of tyrosine phosphorylation in both Colo320 and Colo320 transfected with CCK2R were dose dependence of gastrin. In Colo320, the time of phosphorylation had a tendency of exhaustion at 2.5 min; but in Colo320 transfected with CCK2R, it was at 10 min. Gastrin had no effects on FAK protein expression in different cell groups. An up-regulated level of CCK2R could enhance the effect of gastrin on FAK tyrosine phosphorylation. The gastrin antagonist showed an effect of competitive inhibition on tyrosine phosphorylation of FAK.

CONCLUSIONS:

FAK is a signal transducer in downstream of CCK(2)R; FAK exerts its functions by tyrosine phosphorylation, but dose not increase FAK protein. Gastrin-CCK2R-FAK signal pathway is a pivotal one in the cell growth and proliferation caused by gastrin.

• Authors: Jian Ding, Jie-ping Yu, Dan Li, He-sheng Luo, Hong-gang Yu
• Journal: Zhonghua nei ke za zhi (Zhonghua Nei Ke Za Zhi) Vol. 44 Issue 6 Pg. 434-7 (Jun 2005) ISSN: 0578-1426 [Print] China
• PMID: 16008856 (Publication Type: English Abstract, Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Gastrins
• RNA, Messenger
• Receptor, Cholecystokinin B
• Tyrosine
• Focal Adhesion Protein-Tyrosine Kinases

Topics

• Cell Line, Tumor
• Cell Proliferation
• Colonic Neoplasms (metabolism, pathology)
• Dose-Response Relationship, Drug
• Down-Regulation
• Focal Adhesion Protein-Tyrosine Kinases (metabolism)
• Gastrins (antagonists & inhibitors, pharmacology)
• Humans
• Phosphorylation
• RNA, Messenger (genetics)
• Receptor, Cholecystokinin B (genetics, metabolism)
• Signal Transduction (physiology)
• Tyrosine (metabolism)
• Up-Regulation