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production by 1.1-1.7-fold of gingival keratinocytes (GK), changes such as inflammation or dermal papilla atrophy are respectively, following 24 h of exposure. Exposure of GK considered to be secondary effects due to cytokines or other to AN extract (400 µg/ml) led to cell retraction and mediators produced by activated keratinocytes (10). Among intracellular vacuoles formation. At concentrations of 800 numerous inflammatory mediators, prostaglandins (PGs) are and 1200 µg/ml, AN extract induced cell death at 21-24 considered to be important for tumor initiation, promotion and and 32-52% as detected by MTT assay and cellular lactate metastasis (11). PGs can suppress the humoral, cellular immune dehydrogenase release, respectively. Interestingly, AN- responses and the killing of malignant cells (11,12). These induced morphological changes of GK are reversible. GK well-established findings prompted us to elucidate whether BQ can still proliferate following exposure to AN extract. ingredients could take part in the oral carcinogenic and fibrotic Cytotoxicity of AN extract cannot be inhibited by indo- processes by inducing PG production of gingival keratinocytes methacin (1 µM) and aspirin (50 µM), indicating that (GK). In the present study, we therefore tested the effects of prostaglandin (PG) production is not the major factor AN extract on the PG production, cyclooxygenase-2 (COX-2) responsible for AN cytotoxicity. PGE2 exhibited little effect mRNA and protein production of cultured human GK. on the growth of GK at concentrations ranging from 100- 1000 pg/ml. Stimulating GK production of PGs by AN Materials and methods extract could be due to induction of cyclooxygenase-2 Materials (COX-2) mRNA expression and protein production. These Lactate dehydrogenase (LDH) assay kits, 3-(4,5-dimethyl-thiazol-2-yl)-2,5- results suggest that AN ingredients are critical in the diphenyl-tetrazolium bromide (MTT), calf skin type I collagen and bovine pathogenesis of oral submucous fibrosis and oral cancer plasma fibronectin were obtained from Sigma (Sigma Chemical Co., St Louis, via their stimulatory effects on the PGs, COX-2 production MO). PGE2, 6-keto-PGF1α and thromboxane B2 assay kits and PGE2 were purchased from Cayman Chemical Co. (Ann Arbor, MI). Keratinocyte growth and associated tissue inflammatory responses. AN cyto- medium (KGM-SFM), pituitary gland extract and epidermal growth factor toxicity to GK is not directly mediated by COX-2 were obtained from Gibco BRL, Life Technologies (Grand Island, NY).

Areca nut extract up-regulates prostaglandin production, cyclooxygenase-2 mRNA and protein expression of human oral keratinocytes