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.

Effects of arecoline on hepatic cytochrome P450 activity and oxidative stress

“…It is known that thiol depletion as well as ROS attack should be mainly responsible for arecoline-induced hepatoxicity (Dasgupta et al, 2006;Chang et al, 2001), and CYP enzymes play critical roles in mutagenic activation of the areca-specific N-nitrosamines (Miyazaki et al, 2005). The present work found that AH (4 and 20 mg/kg/d) significantly increased the hepatic CYP2E1 activity.…”

The pharmacology, toxicology and potential applications of arecoline: a review

“…Oxidative stress by arecoline could cause acute cell trauma, and is an important factor in epithelial cell death ). Arecoline was also proved to produce ROS in oral cancer and submucosal fibrosis (Chang et al 2001). Besides, previous researches indicated that inflammatory reactions and epithelial-mesenchymal transition (EMT) are also the important reasons for the development of OSF induced by arecoline (Arakeri & Brennan 2013;Zheng et al 2015).…”

Synergistic Effect of Arecoline in Combination with Doxorubicin on HeLa Cervical Cancer Cells

“…(Drummond, 2007). Arecoline also reported to induce the DNA strand break in mamalia cells (Chang, et al, 2001). The synergistic effect of these two compounds are probably by the DNA strand break mechanism.…”

“…Arecoline is a promising compound to be used as co-chemotherapeutic agent of doxorubicin. Previous study showed that arecoline is able to induce cell cycle arrest in KB cell line (Chang, et al, 2001). It is recently reported by Cheng, et al in 2010 that arecoline may induce apotosis in human hepatoma HA22T/VGH cell line through the by supressing the production of Bcl-XL and Bcl-2 protein, increasing p53 production, and inducing the release of cytochrom c from mitochondrial membrane.…”

“…Some in vitro study showed that arecolin have cytotoxicity towards oral and liver cancer cells, and induce apoptosis in basal cancer cells (Chang, et al, 2001;Cheng, et al, 2010). Arecoline is a promising compound to be used as co-chemotherapeutic agent of doxorubicin.…”