槐定碱对人肝癌 HepG2 细胞的促凋亡作用。
Pro-apoptotic effects of tectorigenin on human hepatocellular carcinoma HepG2 cells.
机构信息
The First Clinical Medical College, Nanjing University of Chinese Medicine, Nanjing 210046, Jiangsu Province, China.
出版信息
World J Gastroenterol. 2012 Apr 21;18(15):1753-64. doi: 10.3748/wjg.v18.i15.1753.
AIM
To investigate the effects of tectorigenin on human hepatocellular carcinoma (HCC) HepG2 cells.
METHODS
Tectorigenin, one of the main components of rhizome of Iris tectorum, was prepared by simple methods, such as extraction, filtration, concentration, precipitation and recrystallization. HepG2 cells were incubated with tectorigenin at different concentrations, and their viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Apoptosis was detected by morphological observation of nuclear change, agarose gel electrophoresis of DNA ladder, and flow cytometry with Hoechst 33342, Annexin V-EGFP and propidium iodide staining. Generation of reactive oxygen species was quantified using DCFH-DA. Intracellular Ca(2+) was monitored by Fura 2-AM. Mitochondrial membrane potential was monitored using Rhodamine 123. Release of cytochrome c from mitochondria to cytosol was detected by Western blotting. Activities of caspase-3, -8 and -9 were investigated by Caspase Activity Assay Kit.
RESULTS
The viability of HepG2 cells treated by tectorigenin decreased in a concentration- and time-dependent manner. The concentration that reduced the number of viable HepG2 cells by 50% (IC(50)) after 12, 24 and 48 h of incubation was 35.72 mg/L, 21.19 mg/L and 11.06 mg/L, respectively. However, treatment with tectorigenin at 20 mg/L resulted in a very slight cytotoxicity to L02 cells after incubation for 12, 24 or 48 h. Tectorigenin at a concentration of 20 mg/L greatly inhibited the viability of HepG2 cells and induced the condensation of chromatin and fragmentation of nuclei. Tectorigenin induced apoptosis of HepG2 cells in a time- and dose-dependent manner. Compared with the viability rate, induction of apoptosis was the main mechanism of the anti-proliferation effect of tectorigenin in HepG2 cells. Furthermore, tectorigenin-induced apoptosis of HepG2 cells was associated with the generation of reactive oxygen species, increased intracellular Ca(2+), loss of mitochondrial membrane potential, translocation of cytochrome c, and activation of caspase-9 and -3.
CONCLUSION
Tectorigenin induces apoptosis of HepG2 cells mainly via mitochondrial-mediated pathway, and produces a slight cytotoxicity to L02 cells.
目的
研究鸢尾黄素对人肝癌 HepG2 细胞的作用。
方法
采用提取、过滤、浓缩、沉淀、重结晶等简单方法制备鸢尾黄素，一种鸢尾根茎的主要成分。将 HepG2 细胞与不同浓度的鸢尾黄素孵育，通过 3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四唑溴盐法评估细胞活力。通过细胞核形态变化的形态学观察、琼脂糖凝胶电泳 DNA 梯、Hoechst 33342、Annexin V-EGFP 和碘化丙啶染色的流式细胞术检测细胞凋亡。使用 DCFH-DA 定量测定活性氧的产生。通过 Fura 2-AM 监测细胞内 Ca(2+)。用 Rhodamine 123 监测线粒体膜电位。通过 Western blot 检测细胞色素 c 从线粒体到细胞质的释放。通过 Caspase 活性测定试剂盒研究 caspase-3、-8 和 -9 的活性。
结果
鸢尾黄素处理的 HepG2 细胞活力呈浓度和时间依赖性下降。孵育 12、24 和 48 h 后，将存活的 HepG2 细胞数量减少 50%的浓度（IC(50)）分别为 35.72、21.19 和 11.06 mg/L。然而，在孵育 12、24 或 48 h 后，用 20 mg/L 的鸢尾黄素处理对 L02 细胞仅有轻微的细胞毒性。浓度为 20 mg/L 的鸢尾黄素可显著抑制 HepG2 细胞活力，并诱导染色质浓缩和核碎裂。鸢尾黄素呈时间和剂量依赖性诱导 HepG2 细胞凋亡。与活力率相比，诱导凋亡是鸢尾黄素抑制 HepG2 细胞增殖的主要机制。此外，鸢尾黄素诱导 HepG2 细胞凋亡与活性氧的产生、细胞内[Ca(2+)]i 增加、线粒体膜电位丧失、细胞色素 c 易位和 caspase-9 和 -3 的激活有关。
结论
鸢尾黄素主要通过线粒体介导的途径诱导 HepG2 细胞凋亡，并对 L02 细胞产生轻微的细胞毒性。
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