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
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
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DOI：
10.1093/carcin/22.9.1527
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发表时间：
2001-09-01
期刊：
影响因子：
4.7
通讯作者：
Jeng, JH
中科院分区：
文献类型：
--
作者：
Chang, MC;Ho, YS;Jeng, JH
世界上有6亿槟榔咀嚼者。BQ咀嚼是口腔癌的主要病因之一。槟榔果（AN）和槟榔碱可能抑制口腔黏膜成纤维细胞（OMF）和角化细胞的生长。在本研究中，AN提取物（100-800马克/毫升）和槟榔碱（20-120马克/毫升）对口腔KB细胞生长的抑制作用分别为36- 90%和15-75%。暴露于槟油碱（>0.2 mM） 24小时可诱导OMF和KB细胞的G(2)/M细胞周期阻滞。槟榔提取物（>400杯/ml）也能诱导KB细胞G2/M阻滞，在暴露7小时后出现s期阻滞。未见明显的亚G(0)/G(1)峰。观察到OMF和KB细胞明显的收缩和细胞内液泡形成。流式细胞术检测KB细胞谷胱甘肽（GSH）水平、线粒体膜电位（Delta betam）和H2O2产量。将KB细胞暴露于茴香碱（0.4-1.2 mM）和AN提取物（800-1200杯/ml） 24小时后，GSH水平[用5-氯甲基荧光素（CMF）荧光表示]降低，低CMF荧光的细胞比例增加。槟榔碱（0.1 ~ 1.2 mM）和AN提取物（800 ~ 1200杯/ml）分别诱导H2O2生成减少和增加（2′，7′-二氯荧光素荧光）。将KB细胞暴露于槟榔碱（0.4-1.2 m-M）和AN提取物（800-1200杯/ml）中24小时，观察到Apm的超极化（罗丹明摄取增加）。AN提取物（100-1200杯/毫升）和槟油碱（0.1-1.2毫米）在24小时内对KB细胞的DNA片段化作用很小。这些结果表明AN成分在口腔粘膜下纤维化（OSF）和口腔癌的发病机制中起着至关重要的作用，通过不同程度地诱导细胞周期控制、Delta β、GSH水平和细胞内H2O2产生的失调，这些事件与细胞凋亡无关。
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 mug/ml) and arecoline (20-120 muM) 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 mug/ml) also induced G2/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 (Delta betam) and H2O2 production of KB cells were measured by flow cytometry. GSH level [indicated by 5-choromethyl-fluorescein (CMF) fluorescence] was depleted by 24-h exposure of KB cells to arecoline (0.4-1.2 mM) and AN extract (800-1200 mug/ml), with increasing the percentage of cells in low CMF fluorescence. By contrast, arecoline (0.1-1.2 mM) and AN extract (800-1200 mug/ml) induced decreasing and increasing H2O2 production (by 2',7'-dichlorofluorescein fluorescence), respectively. Hyperpolarization of Apm (increasing of rhodamine uptake) was noted by 24-h exposure of KB cells to arecoline (0.4-1.2 m-M) and AN extract (800-1200 mug/ml). AN extract (100-1200 mug/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, Delta betam, GSH level and intracellular H2O2 production, these events being not coupled with cellular apoptosis.