Expression of Cyclooxygenase-2 Is Regulated by Glycogen Synthase Kinase-3β in Gastric Cancer Cells

Alexandra Thiel,Mira Heinonen,Johanna Rintahaka,Tuija Hallikainen,Annabrita Hemmes,Dan A Dixon,Caj Haglund,Ari Ristimäki

doi:10.1074/jbc.m512722200

Abstract

Cyclooxygenase-2 (COX-2) expression is a marker of poor prognosis in gastric cancer patients, and its inhibition suppresses gastric tumorigenesis in experimental animal models. The mechanism that leads to COX-2 overexpression in this tumor type is unknown. We have now shown that inhibition of phosphatidylinositol 3-kinase by LY294002 suppresses both basal and phorbol myristate acetate-induced COX-2 expression in TMK-1 and MKN-28 gastric cancer cells. Furthermore, inhibition of glycogen synthase kinase-3beta (GSK-3beta) by SB415286 induced expression of COX-2 mRNA and protein as well as the enzyme activity in the gastric cancer cells. The effect of SB415286 was confirmed by the use of two additional GSK-3beta inhibitors, lithium chloride and SB216763. SB415286 had a modest 1.6-fold stimulatory effect on a 2-kb COX-2 promoter reporter construct, but more importantly, it was shown to block the decay of COX-2 mRNA. In contrast to modulation of phosphatidylinositol 3-kinase/Akt/GSK-3beta pathway, inhibitors of mitogen-activated protein kinases (MEK 1/2, p38, JNK) or the mammalian target of rapamycin did not alter COX-2 expression in gastric cancer cells. Our data show that inhibition of GSK-3beta stimulates COX-2 expression in gastric cancer cells, which seems to be primarily facilitated via an increase in mRNA stability and to a lesser extent through enhanced transcription.

Highlights

Gastric cancer is the second most lethal cancer worldwide [1], and risk factors for this disease include diet, Helicobacter pylori infection, and genetic background [2, 3]
The effects of PMA appeared to be independent of phospholipase A2 activation, because it induced a 12.9fold increase in prostaglandin E2 (PGE2) production during a 24-h incubation period, which was comparable with a 10.8-fold induction when these cells were exposed to exogenous arachidonic acid (AA) for 20 min after the initial 24-h incubation period
Because the selective COX-2 inhibitor NS-398 (5 ␮M) inhibited ϳ90% of AA-induced PGE2 release (Fig. 1B), our results show that the principal COX isoenzyme in TMK-1 cells is COX-2

Summary

Introduction

Gastric cancer is the second most lethal cancer worldwide [1], and risk factors for this disease include diet, Helicobacter pylori infection, and genetic background [2, 3]. The PI3K inhibitor LY294002 suppressed both basal and PMA-induced expression of COX-2 protein in TMK-1 and MKN-28 cells (Fig. 2, A and B). Expression is dependent on modulation of COX-2 transcript levels (Fig. 1C), we found that PMA-induced COX-2 mRNA expression was inhibited by LY294002 in TMK-1 (2.7-fold) and MKN-28 (4.6-fold) cells as detected by real-time RT-PCR (Fig. 2, D and E).

Results

Conclusion