P138. Activation of Chk1/Chk2, and the induction of cell cycle arrest, IL-6 and metalloproteinase-9 production of SAS epithelial cells by areca nut ingredients

Abstract

Introduction: There are about 600 million betel quid (BQ) chewers around the world and 3 million BQ chewers in Taiwan. A number of epidemiological studies indicate that BQ chewing is one of the major risk factors of oral leukoplakia, oral submucous fibrosis (OSF) and oral cancer. Areca nut (AN) is the main component of BQ. AN components exhibit cytotoxicity and genotoxicity, that may potentially lead activation of ATM/ATR-Chk1/Chk2 signaling pathways to regulate cell cycle progression. The objective of this study was to clarify the mechanisms of AN-induced oral mucosal inflammatory response and the roles of Chk1 and Chk2. Methods: Cytotoxicity of AN extract (ANE) and arecoline to SAS epithelial cells was determined by trypan blue dye exclusion technique or MTT assay. Cell cycle distribution and apoptosis were analyzed by propidium iodide (PI) staining flow cytometry. Chk1 and Chk2 activation was analyzed by Pathscan p-chk1 and p-Chk2 ELISA or western blotting. Cellular interleukin-6 and metalloproteinase-9 (MMP-9) production was measured by enzyme-linked immunosorbant assay (ELISA). Results: ANE (200–800 g/ml) and arecoline (0.1–0.4 mM), the major ingredient of AN, induced the morphological changes of SAS tongue cancer epithelial cells. Cells became round with intracellular vacuoles formation. ANE (800 g/ml) and arecoline ( 0.4 mM) caused cytotoxicity of SAS cells, and ANE further induced cellular apoptosis and G2/M cell cycle arrest. Interestingly, exposure to ANE (800 and 1200 g/ml) and arecoline (0.8 mM) for 24 h markedly stimulated the Chk1 and Chk2 phosphorylation in SAS cells. ANE also stimulated IL-6 and MMP-9 production in SAS cells and primary gingival keratinocytes (GK). However, arecoline lacked of this stimulatory effect. Pretreatment and then co-incubation by PD407824 (a Chk1 inhibitor) or 2-(4-(4-chlorophenoxy) phenyl-1H-benzimidazole-5carboxamide (a Chk2 inhibitor) showed little preventive effect on the ANEinduced IL-6 and MMP-9 production. Discussion: These results indicate that AN components may contribute to the pathogenesis of oral cancer via induction of tissue inflammation and injury. This can be associated with the activation of Chk1/Chk2, stimulation of IL-6 and MMP-9 production, cell cycle deregulation and apoptosis.