Characterization of mechanisms accounting for COX-2-mediated proliferative actions of gastrin on human colon cancer cells

Introduction. Cyclooxygenase-2 (COX-2) plays a pivotal role in tumorigenesis and neoplastic proliferation. Previous evidence suggests that COX-2 may contribute to trophic actions of gastrin on colon carcinoma. The present study examines the mechanisms accounting for COX-2-mediated proliferative effects of gastrin on human colon cancer cells. Methods, Experiments were performed in vitro on the human colon adenocarcinoma cell line HT-29. The following techniques were used: colorimetric assay for measurement of cell viability; enayme immunoassay to determine PGE2 levels in culture medium; reverse transcriptionpolymerase chain reaction (RT-PCR) to examine mRNA expression of CCK-2 receptor, COX- 1, COX-2, prostagfandin EP receptor subtypes (from 1 to 4), cyclin D1 and bd-2; western blot to assess ERK-1/ERK-2 and Akt phosphorylatiom Results. RT-PCR demonstrated the expression of CCK-2 receptor, both COX isoforms and EP4 receptor. Human gastrin-17 0.1 ~M enhanced cell viability (+57.6% vs control, following 72-hour incubation). This effect was abolished by L-365,260 1 ~M or GV150013 0.1 ~M (CCK-2 receptor antagonists), unaffected by SC-560 0.1 ~M (COX-1 inhibitor), and partly prevented by L-745,337 0.1 p~M (COX-2 inhibitor; + 27.7%) or AH-23848B 1 ~M (EP4 receptor antagonist; + 31.2%). PGE2 release was also increased by gastrin-17 (from 115.2 + 13.7 to 240.3-+ 21.6 pg/10 s cells; + 108.7%), this action being suppressed by L-365,260, GV150013 or L-745,337, but unaffected by SC-560. Gastrin-17 caused an increment of COX-2 expression which was counteracted by L-365,260 or GV150013. Western blot analysis showed that gastrin-17 enhanced the phosphorylation of ERK- 1/ERK-2 and Akt. Moreover, gastrin- 17-induced COX- 2 expression was blocked by PD98059 50 ~M (MEK inhibitor) or wortmaninn 0.1 ~M (Pl3-kinase inhibitor). GV150013-sensitive increments of cyclin D1 and bcl-2 expression were elicited by gastrin-17. Cyclin D1 induction was antagonized by L-745,337 or AH- 23848B, whereas bcl-2 expression was not modified by these drugs. Conclusions. 1) Gastrin can upregnlate COX-2 expression through CCK-2 receptors and downstream activation of ERK-1/ERK-2 and PI3-kinase/Akt signalling pathways; such effects result in increments of PGE2 production leading to subsequent enhancement of cell viability; 2) gastrin elicits both mitogenic and anti-apoptotic signals, and COX-2-dependent PGE2 biosynthesis is implicated in the former action via recruitment of EP4 receptors