Betel quid (BQ) chewing is an etiologic factor of oral cancer and submucus fibrosis (OSF). Keratinocyte inflammation is crucial for the pathogenesis of cancer and tissue fibrosis. We found that areca nut (AN) extract (100–400 µg/ml) induced PGE ₂ production by KB cells by 2.34- to 23.1-fold and also TNF-α production by gingival keratinocytes (GK). Arecoline (0.2–1.2 mM) elevated PGE ₂ production by KB cells by 2.5- to 6.1-fold. AN extract (200–400 µg/ml) also induced IL-6 production by GK (7.5- to 8.4-fold) and KB cells. In contrast, arecoline (0.1–1.2 mM) suppressed IL-6 production by GK and KB cells, with 42–81 and 41–63% inhibition, respectively. A 48 h exposure of GK to 800–1200 µg/ml AN extract led to 37–69% cell death. Arecoline cytotoxicity to GK was noted at concentrations of 0.8–1.2 mM, which led to 28–38% cell death. AN extract (400–800 µg/ml) induced Cox-2 and IL-6 mRNA expression and also COX-2 protein production by KB cells. IL-6 (5–100 ng/ml) suppressed GK growth by 20–33%, but enhanced oral fibroblast (OMF) and KB cell growth. PGE ₂ (0.05–5 µg/ml) and anti-IL-6 antibody (ab) (50–1000 ng/ml) showed little effect on GK and KB cell growth. Incubation of GK and KB cells with aspirin, anti-IL-6 ab and anti-TNF-α ab showed little effect on arecoline- and AN-induced cytotoxicity, cell cycle arrest and apoptosis. Exposure to anti-TNF-α ab slightly affected arecoline- and AN-modulated PGE ₂ and IL-6 production by GK and KB cells. Arecoline- and AN-conditioned medium decreased phytohemagglutinin-mediated CD4 ⁺ and CD8 ⁺ T cell activation. These results indicate that BQ chewing contributes to the pathogenesis of cancer and OSF by impairing T cell activation and by induction of PGE ₂ , TNF-α and IL-6 production, which affect oral mucosal inflammation and growth of OMF and oral epithelial cells.

There are 600 million betel quid (BQ) chewers in the world. BQ chewing is a major etiologic factor of oral cancer. Areca nut (AN) and arecoline may inhibit the growth of oral mucosal fibroblasts (OMF) and keratinocytes. In this study, AN extract (100–800 μg/ml) and arecoline (20–120 μM) inhibited the growth of oral KB cells by 36–90 and 15–75%, respectively. Exposure to arecoline (>0.2 mM) for 24 h induced G ₂ /M cell cycle arrest of OMF and KB cells. Areca nut extract (>400 μg/ml) also induced G ₂ /M arrest of KB cells, being preceded by S-phase arrest at 7-h of exposure. No evident sub-G ₀ /G ₁ peak was noted. Marked retraction and intracellular vacuoles formation of OMF and KB cells were observed. Glutathione (GSH) level, mitochondrial membrane potential (Δβm) and H ₂ O ₂ production of KB cells were measured by flow cytometry. GSH level [indicated by 5-chloromethyl-fluorescein (CMF) fluorescence] was depleted by 24-h exposure of KB cells to arecoline (0.4–1.2 mM) and AN extract (800–1200 μg/ml), with increasing the percentage of cells in low CMF fluorescence. By contrast, arecoline (0.1–1.2 mM) and AN extract (800–1200 μg/ml) induced decreasing and increasing H ₂ O ₂ production (by 2′,7'-dichloro- fluorescein fluorescence), respectively. Hyperpolarization of Δβm (increasing of rhodamine uptake) was noted by 24-h exposure of KB cells to arecoline (0.4–1.2 mM) and AN extract (800–1200 μg/ml). AN extract (100– 1200 μg/ml) and arecoline (0.1–1.2 mM) induced little DNA fragmentation on KB cells within 24 h. These results indicate that AN ingredients are crucial in the pathogenesis of oral submucous fibrosis (OSF) and oral cancer by differentially inducing the dysregulation of cell cycle control, Δβm, GSH level and intracellular H ₂ O ₂ production, these events being not coupled with cellular apoptosis.

There are about 200–600 million betel quid (BQ) chewers in the world. BQ chewing is one of the major risk factor of hepatocarcinoma, oropharyngeal, and esophagus cancers in Taiwan, India, and Southeast Asian countries. Thus, the precise molecular mechanisms deserve investigation. We used cultured primary keratinocytes and KB cells, RT-PCR, flow cytometry, Western blotting, and ELISA to evaluate whether alterations in early gene expression is crucial in the carcinogenic processes of BQ. We observed the induction of c-Fos mRNA expression in human gingival keratinocyte (GK) and KB carcinoma cells by areca nut (AN) extract and arecoline. A maximal increment in c-fos gene expression was shown at about 30 min after challenge. AN extract (100–800 μg/ml) and arecoline (0.1–0.8 mm) also stimulated ERK1/ERK2 phosphorylation with a maximal stimulation at 5–10 min of exposure. Pretreatment by U0126 (30 μm), a MEK inhibitor, markedly inhibited the c-Fos, cyclooxygenase-2 (COX-2), and IL-6 mRNA expression of the KB epithelial cells. In addition, U0126 and PD98059 (50 μm) also decreased AN extract- and arecoline-associated PGE₂ and IL-6 production in GK and KB cells. However, U0126 by itself arrested the cells in G₀/G₁ phase, but was not able to prevent AN- and arecoline-induced cell death or apoptosis. In contrast, U0126 enhanced the AN-induced apoptosis of KB cells. AN ingredients thus play a significant role in the pathogenesis of oropharyngeal cancer by activation of MEK1/ERK/c-Fos pathway, which promotes keratinocyte inflammation, cell survival, and affects cell cycle progression.