Liu Ming-Jie, Wang Zhao, Ju Yong, Wong Ricky Ngok-Shun, Wu Qing-Yu
Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing, People's Republic of China.
Cancer Chemother Pharmacol. 2005 Jan;55(1):79-90. doi: 10.1007/s00280-004-0849-3. Epub 2004 Sep 14.
Diosgenin is a steroidal sapogenin with estrogenic and antitumor properties. In order to elucidate the mechanism of its antiproliferative activity, we investigated its effects on the cell cycle and apoptosis in human chronic myelogenous leukemia K562 cells.
Cell viability was assessed via an MTT assay. Apoptosis was investigated in terms of nuclear morphology, DNA fragmentation, and phosphatidylserine externalization. Cell cycle analysis was performed via PI staining and flow cytometry (FCM). Western blotting and immunofluorescence methods were used to determine the levels of p53, cell cycle-related proteins and Bcl-2 family members. FCM was also used to estimate the changes in mitochondrial membrane potential (MMP), intracellular Ca2+ concentration and reactive oxygen species (ROS) generation.
Cell cycle analysis showed that diosgenin caused G2/M arrest independently of p53. The levels of cyclin B1 and p21Cip1/Waf1 were decreased, whereas cdc2 levels were increased. Subsequent apoptosis was demonstrated with the dramatic activation of caspase-3. A dramatic decline in intracellular Ca2+ concentration was observed as an initiating event in the process of cell cycle arrest and apoptosis, which was followed by the hyperpolarization and depolarization of MMP. Generation of ROS was observed in the progression of apoptosis. The antiapoptotic Bcl-2 and Bcl-xL proteins were downregulated, whereas the proapoptotic Bax was upregulated.
Diosgenin inhibits K562 cell proliferation via cell cycle G2/M arrest and apoptosis, with disruption of Ca2+ homeostasis and mitochondrial dysfunction playing vital roles.
薯蓣皂苷元是一种具有雌激素样和抗肿瘤特性的甾体皂苷元。为阐明其抗增殖活性机制，我们研究了其对人慢性髓性白血病K562细胞的细胞周期和凋亡的影响。
通过MTT法评估细胞活力。从核形态、DNA片段化和磷脂酰丝氨酸外翻方面研究凋亡情况。通过PI染色和流式细胞术（FCM）进行细胞周期分析。采用蛋白质免疫印迹法和免疫荧光法测定p53、细胞周期相关蛋白和Bcl-2家族成员的水平。FCM还用于评估线粒体膜电位（MMP）、细胞内Ca2+浓度和活性氧（ROS）生成的变化。
细胞周期分析表明，薯蓣皂苷元导致G2/M期阻滞，且与p53无关。细胞周期蛋白B1和p21Cip1/Waf1水平降低，而cdc2水平升高。随后通过caspase-3的显著激活证明了凋亡的发生。观察到细胞内Ca2+浓度急剧下降是细胞周期阻滞和凋亡过程中的起始事件，随后是MMP的超极化和去极化。在凋亡进程中观察到ROS的生成。抗凋亡蛋白Bcl-2和Bcl-xL下调，而促凋亡蛋白Bax上调。
薯蓣皂苷元通过细胞周期G2/M期阻滞和凋亡抑制K562细胞增殖，其中Ca2+稳态破坏和线粒体功能障碍起关键作用。
