Areca Nut Components Affect COX-2, Cyclin B1/cdc25C and Keratin Expression, PGE2 Production in Keratinocyte Is Related to Reactive Oxygen Species, CYP1A1, Src, EGFR and Ras Signaling

Aims: Chewing of betel quid (BQ) increases the risk of oral cancer and oral submucous fibrosis (OSF), possibly by BQ-induced toxicity and induction of inflammatory response in oral mucosa. Methods: Primary gingival keratinocytes (GK cells) were exposed to areca nut (AN) components with/without inhibitors. Cytotoxicity was measured by 3-(4,5-dimethyl- thiazol- 2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. mRNA and protein expression was evaluated by reverse transcriptase-polymerase chain reaction (RT-PCR) and western blotting. PGE(2)/PGF(2 alpha) production was measured by enzyme-linked immunosorbent assays. Results: Areca nut extract (ANE) stimulated PGE(2)/PGF(2 alpha) production, and upregulated the expression of cyclooxygenase-2 (COX-2), cytochrome P450 1A1 (CYP1A1) and hemeoxygenase-1 (HO-1), but inhibited expression of keratin 5/14, cyclinB1 and cdc25C in GK cells. ANE also activated epidermal growth factor receptor (EGFR), Src and Ras signaling pathways. ANE-induced COX-2, keratin 5, keratin 14 and cdc25C expression as well as PGE(2) production were differentially regulated by alpha-naphthoflavone (a CYP 1A1/1A2 inhibitor), PD153035 (EGFR inhibitor), pp2 (Src inhibitor), and manumycin A (a Ras inhibitor). ANE-induced PGE(2) production was suppressed by piper betle leaf (PBL) extract and hydroxychavicol (two major BQ components), dicoumarol (a NAD(P)H:Quinone Oxidoreductase - NQO1 inhibitor) and curcumin. ANE-induced cytotoxicity was inhibited by catalase and enhanced by dicoumarol, suggesting that AN components may contribute to the pathogenesis of OSF and oral cancer via induction of aberrant differentiation, cytotoxicity, COX-2 expression, and PGE(2)/PGF(2 alpha)production. Conclusions: CYP4501A1, reactive oxygen species (ROS), EGFR, Src and Ras signaling pathways could all play a role in ANE-induced pathogenesis of oral cancer. Addition of PBL into BQ and curcumin consumption could inhibit the ANE-induced inflammatory response.