Role of cyclooxygenase-2 in the proliferative activity of gastrin on human colon cancer cells

Introduction. Gastrin exerts trophic actions on mucosal epithelial cells in the gastrointestinal tract. A number of experimental observations supports also a role of this peptide hormone in the stimulation of colon cancer cell growth, but the underlying mechanisms have not been conclusively demonstrated. Cyclooxygenase-2 (COX-2), the inducible isoform of prostaglandin synthase, has been frequently detected in colorectal carcinoma and seems also to be implicated in the maintenance and progression of tumor growth. The present study investigates whether gastrin/CCK receptors (CCK-2) and the COX-2 pathway are involved in the proliferative actions of gastrin on colon cancer cells. Methods. Experiments were performed in vitro on the human colon cancer cell line HT29. The presence of COX-2 and CCK-2 receptor mRNA transcripts in these cells was assayed by RT-PCR. The expression of COX-2 protein was also determined by western blot analysis. Cells were exposed for 3, 5 and 8 days to human gastrin (0.1/~M), either alone or in the presence of CCK-2 receptor and COX-2 blockers. They were then harvested with trypsin-EDTA in order to assess proliferation by cell counting. In experiments on COX-2 transcription regulation, cells were transfected with a COX-2 promoter-luciferese reporter gene construct, and were then treated with gastrin (0.1/~M) for 24 hours. Results. RT-PCR assay showed a moderate level of CCK-2 receptor expression in HT29 cells. A marked expression of COX-2 was also detected by both RT-PCR and western blot analysis. In proliferation experiments, exposure of cancer ceils to gastdn for 3-B days caused an increment of cell growth (25-30%). This stimulant effect was completely blocked by the CCK- 2 receptor antagonists GV150013 (0.1/~M) and L-365,260 (1/~M), and partly prevented by L-745,337 (1 p.M), a selective inhibitor of COX-2. By RT-PCR and western blot analysis it was observed that gastrin enhanced COX-2 expression. In addition, the treatment of transfected HT29 cells with gastrin for 24 hours was associated with a 4-5 fold increase in the activity of COX-2 promoter. These latter effects were antagonized by GV150013 or L-365,260. Conclusions. The present results indicate that gastrin promotes the induction of COX-2 expression in human colon cancer cells, and that this effect may account, at least in part, for the proliferative action of gastdn on tumor growth. It is also suggested that the effects of gastdn on both COX-2 induction and proliferation of HT29 cells are mediated by activation of CCK- 2 receptors