Department of Public Health, China Medical University, Taichung 404, Taiwan, R.O.C.
Department of Pathology, Show Chwan Memorial Hospital, Changhua 500, Taiwan, R.O.C.
Oncol Rep. 2018 Feb;39(2):703-710. doi: 10.3892/or.2017.6123. Epub 2017 Nov 29.
Glycyrrhizic acid (GA), the main component of licorice root extracts, has been shown to suppress cell proliferation and induce apoptosis in various types of cancers. However, the molecular mechanism of its anticancer activity remains poorly understood and warrants further investigation. MDA-MB‑231 cells were treated with various concentrations of GA and the cytotoxic effects of GA were determined using the CCK-8 assay. Apoptosis and cell cycle status were detected by flow cytometry. Reactive oxygen species (ROS) levels and mitochondrial membrane potential (∆Ψm) were assessed using DCFDA, MitoSOX and JC-1 staining. Western blot analysis was used to quantify the expression of autophagy-related proteins. In the present study, induction of autophagic cell death was observed in GA-treated MDA-MB‑231 cells. Downregulation of p62- and beclin 1-associated proteins occurred after GA treatment, and, the conversion of LC3 and increased ROS without significant changes in caspase‑associated proteins and intracellular responses were detected. Furthermore, loss of mitochondria and its membrane potential in cells demonstrated that mitochondria were involved in the GA-regulated MDA-MB-231 cell death. The addition of a pan-caspase inhibitor (z-VAD-fmk) did not suppress the GA-induced apoptotic effect, and GA-induced apoptosis was not accompanied by processing of procaspase-8, -9 and -3. However, GA triggered the translocation of the apoptosis-inducing factor (AIF) from the mitochondria into the nucleus. In contrast, GA-induced LC3 conversion was significantly inhibited by 3 methlyadenine (3MA), an inhibitor of PI3K‑regulated autophagy. Therefore, these results suggest that enhancement of both AIF- and LC3-dependent GA-derived ROS generation plays an important role in the inhibition of the growth of MDA-MB-231 human breast cancer cells.
甘草酸（GA）是甘草根提取物的主要成分，已被证明可抑制多种类型癌症的细胞增殖并诱导细胞凋亡。然而，其抗癌活性的分子机制仍知之甚少，需要进一步研究。用不同浓度的 GA 处理 MDA-MB-231 细胞，并通过 CCK-8 测定法测定 GA 的细胞毒性作用。通过流式细胞术检测细胞凋亡和细胞周期状态。使用 DCFDA、MitoSOX 和 JC-1 染色评估活性氧（ROS）水平和线粒体膜电位（∆Ψm）。使用 Western blot 分析来定量测定自噬相关蛋白的表达。在本研究中，观察到 GA 处理的 MDA-MB-231 细胞中诱导自噬性细胞死亡。GA 处理后，p62-和 beclin 1 相关蛋白下调，LC3 转化和 ROS 增加而 caspase 相关蛋白和细胞内反应无明显变化。此外，细胞中线粒体及其膜电位的丧失表明线粒体参与了 GA 调节的 MDA-MB-231 细胞死亡。加入泛半胱天冬酶抑制剂（z-VAD-fmk）不能抑制 GA 诱导的凋亡作用，并且 GA 诱导的凋亡不伴有 procaspase-8、-9 和-3 的加工。然而，GA 触发凋亡诱导因子（AIF）从线粒体转移到细胞核。相反，GA 诱导的 LC3 转化被 PI3K 调节的自噬抑制剂 3 甲基腺嘌呤（3MA）显著抑制。因此，这些结果表明，增强 AIF-和 LC3 依赖性 GA 衍生的 ROS 生成在抑制 MDA-MB-231 人乳腺癌细胞的生长中发挥重要作用。
