Department of Radiological Sciences, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Sugitani 2630, Toyama 930-0194, Japan.
Chem Biol Interact. 2012 Sep 30;199(3):154-60. doi: 10.1016/j.cbi.2012.07.005. Epub 2012 Aug 8.
Cinobufotalin (CB), one of the bufadienolides prepared from toad venom, was investigated for its cytotoxicity, and the underneath mechanism involved. We primarily utilized DNA fragmentation assay and microscopic observation to assess the effect of various doses of CB in human lymphoma U937 cells. Following that, we investigated other parameters involved in cell death mechanism such as reactive oxygen species (ROS), mitochondrial membrane potential (MMP), and apoptotic proteins activation. HeLa cells were concomitantly used to generalize the data observed. Our results show that CB caused significant DNA fragmentation, decrease of MMP, and an increase in the intracellular Ca(2+) ion and ROS production. In addition, CB induced upregulation of Fas protein, proteolytic activation of cytochrome c, caspase-2, -3, -8 and -9 together with the activation of Bid and Bax. Our findings were further validated using either Fas/FasL antagonist or pan-caspase inhibitor to significantly inhibit CB-induced DNA fragmentation. In our study, we suggest that CB induces caspase dependent cell death in U937 cells, and that Fas plays a role in CB-induced apoptosis. Altogether, our data provides novel insights of the mechanism of action of CB and its potential as a future chemotherapeutic agent.
华蟾酥毒基（CB）是从蟾酥中提取的一种蟾毒配基，本研究旨在探讨其细胞毒性及其作用机制。我们主要利用 DNA 片段化检测和显微镜观察评估不同浓度 CB 对人淋巴瘤 U937 细胞的影响。然后，我们研究了细胞死亡机制中涉及的其他参数，如活性氧（ROS）、线粒体膜电位（MMP）和凋亡蛋白的激活。我们同时使用 HeLa 细胞对观察到的数据进行概括。结果表明，CB 可引起明显的 DNA 片段化、MMP 降低以及细胞内钙离子和 ROS 生成增加。此外，CB 诱导 Fas 蛋白上调、细胞色素 c 水解激活、caspase-2、-3、-8 和 -9 的激活以及 Bid 和 Bax 的激活。我们使用 Fas/FasL 拮抗剂或泛半胱天冬酶抑制剂进一步验证，结果表明 Fas 拮抗剂和泛半胱天冬酶抑制剂可显著抑制 CB 诱导的 DNA 片段化。本研究表明，CB 通过 Fas 依赖途径诱导 U937 细胞发生细胞凋亡，提示 Fas 在 CB 诱导的凋亡中发挥作用。总之，本研究为 CB 的作用机制及其作为未来化疗药物的潜力提供了新的见解。
