蓝莓花青素通过下调p53基因DNA甲基化抑制口腔癌KB细胞增殖及诱导细胞凋亡

Blueberry anthocyanins induce G_2/M cell cycle arrest and apoptosis of oral cancer KB cells through down-regulation methylation of p53

文章从内蒙古野生蓝莓(Vaccinium uliginosum Lim)中提取花青素,观察其对口腔癌细胞株KB的增殖及凋亡的作用,探讨其作用机制与p53基因甲基化的相关性。利用含0.1%盐酸的甲醇提取花青素,用高效液相色谱-质谱(High performance liquid chromatography-mass spectrometry,HPLC-MS)鉴定花青素的成分。利用四甲基偶氮唑蓝(Methylthiazolyl-tetrazolium,MTT)比色法、流式细胞术、免疫荧光法、免疫细胞化学法和Western blot法分析蓝莓花青素对KB细胞增殖、细胞周期、细胞凋亡和p53蛋白表达的影响;利用甲基化特异性PCR法(Methylation-specific PCR,MSP)分析蓝莓花青素诱导细胞凋亡与p53基因甲基化的关系。结果显示,内蒙古自治区的野生蓝莓中至少存在14种花青素成分;蓝莓花青素呈剂量依赖的方式抑制KB细胞增殖,诱导细胞周期阻滞在G2/M期,而且能诱导细胞凋亡;蓝莓花青素处理后Caspase-9蛋白和细胞色素C的表达明显增加;Western blot结果表明蓝莓花青素可以诱导KB细胞中p53蛋白表达上调;MSP结果表明随蓝莓花青素浓度增加,未甲基化的p53的比例增加,说明p53的甲基化状态有所下调。

Blueberries are an excellent source of dietary polyphenols such as anthocyanins and phenolic acids. In this study, we investigated the ability of anthocyanins from the wild blueberries of Inner Mongolia to suppress the growth of the oral cancer cell line KB. The blueberry anthocyanins were extracted with methanol-containing 0.1%（v/v） hydrochloric acid. Fourteen unique anthocyanins were identified using high-performance liquid chromatography-mass spectrometry （HPLC-MS）. The anticancer bioactivity of the extracts on KB cells was analyzed using methylthiazolyl-tetrazolium （MTT）, flow cytometry （FCM） and immunocytochemistry. It was shown that the blueberry anthocyanins suppressed the prolifera-tion of KB cells in a dose-dependent manner, as well as induced G2/M cell cycle arrest and apoptosis of oral cancer KB cells. Immunocytochemistry analysis showed that the expression of caspase-9 and cytochrome c were obviously increased after the anthocyanins treatment. Western blot analysis also indicated that the expression of p53 was increased. Methyla-tion-specific PCR （MSP） showed that the amount of unmethylated p53 increased, indicating that the anthocyanins can down-regulate the methylation of p53.