Department of Biological Science and Technology, China Medical University, Taichung, Taiwan.
Department of Medical Research, China Medical University Hospital, China Medical University, Taichung, Taiwan.
Environ Toxicol. 2019 Apr;34(4):443-456. doi: 10.1002/tox.22698. Epub 2019 Jan 7.
Genistein, a major isoflavone compound in soybeans, has been shown to have biological activities including anti-cancer activates. In the present, we investigated the anti-leukemia activity of genistein on HL-60 cells in vitro. The percentage of viable cell, cell cycle distribution, apoptotic cell death, reactive oxygen species (ROS), and Ca production and the level of ΔΨ were measured by flow cytometric assay. Cell apoptosis and endoplasmic reticulum (ER) stress associated protein expressions were examined by Western blotting assay. Calpain 1, GRP78, and GADD153 expression were measured by confocal laser microscopy. Results indicated that genistein-induced cell morphological changes, decreased the total viable cells, induced G /M phase arrest and DNA damage and fragmentation (cell apoptosis) in HL-60 cells. Genistein promoted ROS and Ca productions and decreased the level of ΔΨ in HL-60 cells. Western blotting assay demonstrated that genistein increased ER stress-associated protein expression such as IRE-1α, Calpain 1, GRP78, GADD153, caspase-7, caspase-4, and ATF-6α at 20-50 μM treatment and increased apoptosis associated protein expression such as pro-apoptotic protein Bax, PARP-cleavage, caspase-9, and -3, but decreased anti-apoptotic protein such as Bcl-2 and Bid in HL-60 cells. Calpain 1, GRP78, and GADD153 were increased in HL-60 cells after exposure to 40 μM of genistein. In animal xenografted model, mice were intraperitoneally injected with genistein (0, 0.2, and 0.4 mg/kg) for 28 days and the body weight and tumor volume were recorded. Results showed that genistein did not affect the body weights but significantly reduced the tumor weight in 0.4 mg/kg genistein-treated group. Genistein also increased the expressions of ATF-6α, GRP78, Bax, Bad, and Bak in tumor. In conclusion, genistein decreased cell number through G /M phase arrest and the induction of cell apoptosis through ER stress- and mitochondria-dependent pathways in HL-60 cells and suppressed tumor properties in vivo.
染料木黄酮是大豆中的一种主要异黄酮化合物，具有多种生物学活性，包括抗癌活性。本研究旨在探讨染料木黄酮在体外对 HL-60 细胞的抗白血病作用。采用流式细胞术检测细胞活力、细胞周期分布、细胞凋亡、活性氧（ROS）和 Ca 生成及ΔΨ水平；Western blot 法检测细胞凋亡及内质网（ER）应激相关蛋白表达；激光共聚焦显微镜检测钙蛋白酶 1（Calpain 1）、葡萄糖调节蛋白 78（GRP78）和生长停滞及 DNA 损伤基因 153（GADD153）的表达。结果表明，染料木黄酮诱导 HL-60 细胞形态改变，降低总活细胞数，诱导 G/M 期阻滞和 DNA 损伤及片段化（细胞凋亡）。染料木黄酮促进 ROS 和 Ca 生成，降低 HL-60 细胞的ΔΨ水平。Western blot 法检测结果显示，20-50μM 染料木黄酮处理可增加 ER 应激相关蛋白如 IRE-1α、Calpain 1、GRP78、GADD153、caspase-7、caspase-4 和 ATF-6α的表达，并增加促凋亡蛋白如 Bax、PARP 裂解、caspase-9 和 caspase-3 的表达，同时降低抗凋亡蛋白如 Bcl-2 和 Bid 的表达。HL-60 细胞暴露于 40μM 染料木黄酮后，Calpain 1、GRP78 和 GADD153 表达增加。在动物异种移植模型中，小鼠腹腔注射染料木黄酮（0、0.2 和 0.4mg/kg）28 天，记录体重和肿瘤体积。结果表明，染料木黄酮不影响体重，但可显著降低 0.4mg/kg 染料木黄酮处理组的肿瘤重量。染料木黄酮还增加了肿瘤中 ATF-6α、GRP78、Bax、Bad 和 Bak 的表达。综上所述，染料木黄酮通过 ER 应激和线粒体依赖性途径抑制 HL-60 细胞的 G/M 期阻滞和细胞凋亡，降低细胞数量，并抑制体内肿瘤特性。
