荭草素诱导肝癌细胞凋亡涉及死亡受体 5 的上调。
Casticin-induced apoptosis involves death receptor 5 upregulation in hepatocellular carcinoma cells.
机构信息
Department of Pathology, The Third Xiangya Hospital of Central South University, Changsha 410013, Hunan Province, China.
出版信息
World J Gastroenterol. 2011 Oct 14;17(38):4298-307. doi: 10.3748/wjg.v17.i38.4298.
AIM
To investigate the apoptotic activities of casticin in hepatocellular carcinoma (HCC) cells and its molecular mechanisms.
METHODS
PLC/PRF/5 and Hep G2 cell lines were cultured in vitro and the inhibitory effect of casticin on the growth of cells was detected by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolim bromide (MTT) assay. The apoptotic cell death was examined using the cell apoptosis enzyme linked immunosorbent assay (ELISA) detection kit, flow cytometry (FCM) after propidium iodide (PI) staining and DNA agarose gel electrophoresis. The caspase activities were measured using ELISA. Reactive oxygen species (ROS) production was evaluated by FCM after dichlorodihydrofluorescein diacetate (DCFH-DA) probe labeling. Intracellular glutathione (GSH) content was measured using a glutathione assay kit. The expression of death receptor (DR)4 and DR5 proteins was analyzed by Western blotting and FCM.
RESULTS
Casticin significantly inhibited the growth of human HCC (PLC/PRF/5 and Hep G2) cells in a dose-dependent manner (P < 0.05). Casticin increased the percentage of the sub-G1 population in HCC cells in a concentration-dependent manner. The potency of casticin to PLC/PRF/5 cells was higher than that of 5-flurouracil (26.8% ± 4.8% vs 17.4% ± 5.1%) at 10 μmol/L for 24 h. Casticin increased the levels of Histone/DNA fragmentation and the levels of active caspase-3, -8 and -9 in a concentration-dependent manner (P < 0.05). Treatment with 30 μmol/L casticin for 24 h resulted in the formation of a DNA ladder. Casticin reduced the GSH content (P < 0.05), but did not affect the level of intracellular ROS in PLC/PRF/5 and Hep G2 cells. The thiol antioxidants, acetylcysteine (NAC) and GSH restored GSH content and attenuated casticin-induced apoptosis. In contrast, the nonthiol antioxidants, butylated hydroxyanisole and mannitol failed to do so. In the HCC cells treated with casticin for 24 h, DR5 protein level was increased. The expression of DR5 protein induced by casticin was inhibited by NAC. Pretreatment with DR5/Fc chimera protein, a blocking antibody, effectively attenuated the induction of apoptosis by casticin.
CONCLUSION
Casticin-induced apoptosis of HCC cells is involved in GSH depletion and DR5 upregulation.
目的
研究金雀异黄素对肝癌细胞凋亡的作用及其分子机制。
方法
体外培养 PLC/PRF/5 和 Hep G2 细胞，用 3-[4,5-二甲基噻唑-2-基]-2,5-二苯基四氮唑溴盐（MTT）比色法检测金雀异黄素对细胞生长的抑制作用。用细胞凋亡酶联免疫吸附试验（ELISA）检测试剂盒、碘化丙啶（PI）染色后流式细胞术（FCM）和 DNA 琼脂糖凝胶电泳检测细胞凋亡。用 ELISA 法检测半胱氨酸天冬氨酸蛋白酶（caspase）的活性。用二氯二氢荧光素二乙酸酯（DCFH-DA）探针标记后用 FCM 法评估活性氧（ROS）的产生。用谷胱甘肽测定试剂盒测定细胞内谷胱甘肽（GSH）含量。用 Western blot 和 FCM 分析死亡受体（DR）4 和 DR5 蛋白的表达。
结果
金雀异黄素呈剂量依赖性地显著抑制人肝癌（PLC/PRF/5 和 Hep G2）细胞的生长（P < 0.05）。金雀异黄素以浓度依赖性方式增加 HCC 细胞亚 G1 期细胞的比例。在 10 μmol/L 时，金雀异黄素对 PLC/PRF/5 细胞的作用强度高于 5-氟尿嘧啶（26.8% ± 4.8%对 17.4% ± 5.1%）24 h。金雀异黄素呈浓度依赖性地增加组蛋白/DNA 片段化水平和活性 caspase-3、-8 和 -9 的水平（P < 0.05）。用 30 μmol/L 金雀异黄素处理 24 h 后形成 DNA 梯。金雀异黄素降低 GSH 含量（P < 0.05），但不影响 PLC/PRF/5 和 Hep G2 细胞内 ROS 的水平。硫醇抗氧化剂乙酰半胱氨酸（NAC）和 GSH 恢复 GSH 含量并减轻金雀异黄素诱导的细胞凋亡。相反，非硫醇抗氧化剂丁基羟基茴香醚和甘露醇则不能。在金雀异黄素处理 HCC 细胞 24 h 后，DR5 蛋白水平增加。金雀异黄素诱导的 DR5 蛋白表达被 NAC 抑制。用 DR5/Fc 嵌合蛋白，即阻断抗体预处理，可有效减轻金雀异黄素诱导的细胞凋亡。
结论
金雀异黄素诱导肝癌细胞凋亡涉及 GSH 耗竭和 DR5 上调。
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