Yun Young-Gab, Jeon Byung-Hun, Lee Jong-Hyun, Lee Sun-Kyung, Lee Hwa-Jeong, Jung Kyung-Hee, Jun Chang-Duk, Lee Suk-Keun, Kim Eun-Cheol
Department of Prescription, College of Oriental Medicine, Wonkwang University, Iksan, South Korea.
Phytother Res. 2008 Mar;22(3):416-23. doi: 10.1002/ptr.2345.
Although verticinone, a major alkaloid isolated from the bulbus of Fritillaria ussuriensis, has been shown to induce differentiation in human leukemia cells, the exact mechanism of this action is not completely understood in cancer cells. Verticinone was used to conduct growth and apoptosis-related experiments for two stages of oral cancer on immortalized human oral keratinocytes (IHOKs) and primary oral cancer cells (HN4). The procedures included MTT assay, three-dimensional (3-D) raft cultures, Western blotting, cell cycle analysis, nuclear staining and cytochrome c expression related to the apoptosis signaling pathway. Verticinone inhibited the proliferation of immortalized and malignant oral keratinocytes in a dose- and time-dependent manner. In 3-D organotypic culture, verticinone-treated cells were less mature than the control cells, displaying low surface keratinization and decreased epithelial thickness. The major mechanism by which verticinone inhibits growth appears to be induced apoptosis and G(0)G(1) cell cycle arrest. This finding is supported by the results of the cell cycle analysis, FITC-Annexin V staining, DNA fragmentation assay and Hoechst 33258 staining. Furthermore, the cytosolic level of cytochrome c was increased, while the expression of Bcl-2 protein was gradually down-regulated and Bax was up-regulated, accompanied by caspase-3 activation. The data suggests that verticinone may induce apoptosis through a caspase pathway mediated by mitochondrial damage in immortalized keratinocytes and oral cancer cells.
尽管从川贝母鳞茎中分离出的主要生物碱贝母辛已被证明可诱导人白血病细胞分化，但在癌细胞中这种作用的确切机制尚未完全明确。贝母辛用于对永生化人口腔角质形成细胞（IHOKs）和原发性口腔癌细胞（HN4）进行口腔癌两个阶段的生长和凋亡相关实验。实验步骤包括MTT法、三维（3-D）筏式培养、蛋白质印迹法、细胞周期分析、细胞核染色以及与凋亡信号通路相关的细胞色素c表达检测。贝母辛以剂量和时间依赖性方式抑制永生化和恶性口腔角质形成细胞的增殖。在3-D器官型培养中，经贝母辛处理的细胞比对照细胞成熟度低，表现为表面角质化程度低且上皮厚度减小。贝母辛抑制生长的主要机制似乎是诱导凋亡和G(0)G(1)期细胞周期阻滞。细胞周期分析、FITC-Annexin V染色、DNA片段化检测和Hoechst 33258染色结果支持了这一发现。此外，细胞色素c的胞质水平升高，而Bcl-2蛋白表达逐渐下调，Bax上调，同时伴有caspase-3激活。数据表明，贝母辛可能通过线粒体损伤介导的caspase途径在永生化角质形成细胞和口腔癌细胞中诱导凋亡。
