Objective: To investigate the inhibitory effect of giganteaside D (GD) on hepatocellular carcinoma and its molecular mechanisms.
Methods: The inhibitory effects of GD on Hep 3b cells were determined using MTT assay and colony formation assay. The morphological changes of Hep 3b cells after GD treatment were observed by electron microscopy, and the cell cycle changes was analyzed using flow cytometry. The cell apoptosis and mitochondrial potential collapse in the treated cells were tested with Hoechst staining assay and flow cytometry. The expression levels of Bcl-2, PARP and key proteins in MAPK pathway were detected using Western blotting.
Results: GD showed a significant inhibitory effect on Hep 3b cells with an IC50 value of 16.08 µmol/L at 72 h. Flow cytometric analysis demonstrated that the phases of cell cycle remained unchanged and a sub-G1 peak (from 3.3% to 33.6%) appeared as GD concentration increased. GD-induced apoptosis was further conformed by Hoechst staining assay, and flow cytometry showed increased mitochondrial potential collapse in the cells. Western blotting demonstrated the cleavage of PARP, decrease of Bcl-2 and p-Erk1/2 (Thr202/Tyr204), and activation of p-p38 (Thr180/Tyr182) and p-JNK (Thr183/Tyr185) in GD-treated cells.
Conclusions: GD has significant inhibitory effect against hepatocellular carcinoma cells in vitro by inducing apoptosis possibly in association with the MAPK signaling pathway.
目的: 探讨巨头刺草皂甙D（GD）对人原发性肝癌Hep 3b细胞的抑制作用及其作用机制。
方法: 采用MTT法及细胞克隆法检测GD对肝癌细胞Hep 3b的抑制作用；运用光学显微镜和Hoechst荧光染色观察细胞形态的变化和凋亡现象；应用流式细胞术检测细胞周期的变化以及线粒体膜电位的变化；运用Western blotting检测细胞内Bcl-2、PARP蛋白及MAPK通路关键蛋白的变化。
结果: GD具有较强的抗肝癌细胞Hep 3b作用，并且其抑制作用具有时间和剂量依赖性，在72 h处的半数抑制浓度IC50=16.08 μmol·L-1。细胞周期分析表明，随着GD浓度增加，Hep 3b细胞出现sub-G1凋亡峰，凋亡率从3.3%增加到33.6%。但细胞周期无明显改变。Hoechst荧光染色及流式细胞术分析发现GD作用Hep 3b细胞后，细胞核能发出明显的蓝色荧光点，并降低该细胞线粒体膜电位。通过Western Blotting发现，GD作用Hep 3b细胞后，Bcl-2蛋白表达减少，凋亡标志分子PARP活化。MAPK通路蛋白Erk磷酸化形式表达下降，p38和JNK磷酸化形式表达增加。
结论: GD可通过诱导肝癌细胞凋亡发挥抗肝癌活性，该作用可能与MAPK通路有关。