The up-regulation of cyclooxygenase-2 expression in human buccal mucosal fibroblasts by arecoline: a possible role in the pathogenesis of oral submucous fibrosis

Background: Aberrant and persistent tissue inflammation are believed to play an important role on the occurrence of tissue fibrosis. Cyclooxygenase (COX)-2 is an inducible enzyme responsible for prostaglandin synthesis in certain inflammatory diseases. The purpose of this study was to compare COX-2 expression in normal human buccal mucosa and oral submucous fibrosis (OSF) specimens and further explore the potential mechanism that may lead to induce COX-2 expression. Methods: Fifteen OSF specimens and six normal buccal mucosa were examined by immunohistochemistry. Primary human buccal mucosa fibroblasts (BMFs) were established and challenged with arecoline analyzed by reverse transcriptase polymerase chain reaction. Furthermore, to elucidate whether induction of COX-2 is associated with cytotoxicity, aspirin (a non-selective inhibitor of COX enzyme) and NS-398 (a selective COX-2 inhibitor), were added to test their protective effects. Results: COX-2 expression was significantly higher in OSF specimens and expressed mainly by epithelial cells, endothelial cells, and cells with fibroblast morphology. In vitro studies indicated that BMFs did not express COX-2 constitutively. However, when the cells were treated with 80 mug/ml arecoline, COX-2 expression was up-regulated as early as half an hour. This indicates that COX-2 expression is an early cellular response and regulated by arecoline at transcriptional level. In addition, pre-treatment with glutathione (GSH) precursor, 2-oxothiazolidine-4-carboxylic acid (OTZ), led to a decrease in induction of COX-2 mRNA by arecoline. GSH synthesis inhibitor, buthionine sulfoximine (BSO), was found to increase arecoline-induced COX-2 mRNA levels. Moreover, both of aspirin and NS-398 at non-cytotoxic doses are not able to prevent arecoline-induced cytotoxicity. This indicates that arecoline cytotoxicity is not directly via the induction of COX-2 expression. Conclusions: Taken together, these results suggest that COX-2 expression is significantly up-regulated in OSF tissues from areca quid chewers and arecoline may among other constituents be responsible for the enhanced COX-2 expression in vivo . The regulation of COX-2 expression induced by arecoline is critically dependent on the cellular GSH concentration.