Areca nut extract and arecoline induced the cell cycle arrest but not apoptosis of cultured oral KB epithelial cells: association of glutathione, reactive oxygen species and mitochondrial membrane potential

Abstract

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 microg/ml) and arecoline (20-120 microM) 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 microg/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 (Deltabetam) 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 microg/ml), with increasing the percentage of cells in low CMF fluorescence. By contrast, arecoline (0.1-1.2 mM) and AN extract (800-1200 microg/ml) induced decreasing and increasing H(2)O(2) production (by 2',7'-dichloro- fluorescein fluorescence), respectively. Hyperpolarization of Deltabetam (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 microg/ml). AN extract (100- 1200 microg/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, Deltabetam, GSH level and intracellular H(2)O(2) production, these events being not coupled with cellular apoptosis.