Cho Seung-Ju, Ryu Jae-Ha, Surh Young-Joon
Tumor Microenvironment Global Core Research Center, College of Pharmacy, Seoul National University, Seoul, Korea.
College of Pharmacy, Sookmyung Women's University, Seoul, Korea.
J Cancer Prev. 2019 Jun;24(2):112-122. doi: 10.15430/JCP.2019.24.2.112. Epub 2019 Jun 30.
NAD(P)H:quinone oxidoreductase-1 (NQO1) is a widely-distributed flavin adenine dinucleotide-dependent flavoprotein that promotes obligatory 2-electron reductions of quinones, quinoneimines, nitroaromatics, and azo dyes. This reduces quinone levels and thereby minimizes generation of excess reactive oxygen species (ROS) formed by redox cycling, and concurrent depletion of intracellular thiol pools. Ajoene is derived from crushed garlic. It is formed by a reaction involving two allicin molecules, and is composed of allyl sulfide and vinyl disulfide. Ajoene is present in two isomers, E- and Z-form.
Expression of antioxidant enzymes and nuclear factor E2-related factor-2 (Nrf2) was measured by Western blot analysis. NQO1 promoter activity was assessed by the luciferase reporter gene assay. ROS accumulation was monitored by using the fluorescence-generating probe 2',7'-dichlorofluorescein diacetate. The intracellular glutathione levels were measured by using a commercially available kit.
Z-ajoene significantly up-regulated the expression of representative antioxidant enzyme NQO1 in non-tumorigenic breast epithelial MCF-10A cells at non-toxic concentrations. Z-ajoene enhanced up-regulation and nuclear translocation of Nrf2, which plays a pivotal role in the induction of many genes encoding antioxidant enzymes and other cytoprotective proteins. Z-ajoene treatment also increased the activity of -promoter harboring antioxidant response element consensus sequences in MCF-10A cells. Silencing of Nrf2 by small interfering RNA abrogated ajoene-induced expression of NQO1. Z-ajoene activated extracellular signal-regulated kinase (ERK). Inhibition of ERK activation by U0126 abrogated ability of Z-ajoene to activate Nrf2 and to induce NQO1 expression. Intracellular ROS accumulation was observed after treatment with Z-ajoene, whereas the E-isoform was not effective. The inhibition of ROS by treatment with N-acetylcysteine, a radical scavenger, abrogated Z-ajoene-induced expression of NQO1 as well as activation of ERK and Nrf2, suggesting that Z-ajoene augments the Nrf2-dependent antioxidant defense via ROS generation and ERK activation.
Z-ajoene induces NQO1 expression in MCF-10A cells through ROS-mediated activation of Nrf2.
NAD(P)H：醌氧化还原酶-1（NQO1）是一种广泛分布的黄素腺嘌呤二核苷酸依赖性黄素蛋白，可促进醌、醌亚胺、硝基芳烃和偶氮染料的强制性双电子还原。这会降低醌水平，从而将氧化还原循环形成的过量活性氧（ROS）的产生以及细胞内硫醇池的同时消耗降至最低。阿霍烯源自碾碎的大蒜。它由涉及两个蒜素分子的反应形成，由烯丙基硫醚和乙烯基二硫醚组成。阿霍烯有两种异构体，即E型和Z型。
通过蛋白质免疫印迹分析测量抗氧化酶和核因子E2相关因子-2（Nrf2）的表达。通过荧光素酶报告基因测定评估NQO1启动子活性。使用产生荧光的探针2',7'-二氯荧光素二乙酸酯监测ROS积累。使用市售试剂盒测量细胞内谷胱甘肽水平。
在无毒浓度下，Z-阿霍烯显著上调了非致瘤性乳腺上皮MCF-10A细胞中代表性抗氧化酶NQO1的表达。Z-阿霍烯增强了Nrf2的上调和核转位，Nrf2在诱导许多编码抗氧化酶和其他细胞保护蛋白的基因中起关键作用。Z-阿霍烯处理还增加了MCF-10A细胞中含有抗氧化反应元件共有序列的启动子的活性。通过小干扰RNA使Nrf2沉默消除了阿霍烯诱导的NQO1表达。Z-阿霍烯激活细胞外信号调节激酶（ERK）。用U0126抑制ERK激活消除了Z-阿霍烯激活Nrf2和诱导NQO1表达的能力。用Z-阿霍烯处理后观察到细胞内ROS积累，而E异构体无效。用自由基清除剂N-乙酰半胱氨酸处理抑制ROS消除了Z-阿霍烯诱导的NQO1表达以及ERK和Nrf2的激活，表明Z-阿霍烯通过ROS生成和ERK激活增强了Nrf2依赖性抗氧化防御。
Z-阿霍烯通过ROS介导的Nrf2激活诱导MCF-10A细胞中NQO1的表达。
