Kim Sanghyun, Lee Hee-Geum, Park Sin-Aye, Kundu Joydeb Kumar, Keum Young-Sam, Cha Young-Nam, Na Hye-Kyung, Surh Young-Joon
Tumor Microenvironment Global Core Research Center, College of Pharmacy, Seoul National University, Seoul, South Korea.
College of Pharmacy, Keimyung University, Daegu, South Korea.
PLoS One. 2014 Jan 28;9(1):e85984. doi: 10.1371/journal.pone.0085984. eCollection 2014.
Diallyl sulfide, diallyl disulfide, and daillyl trisulfide (DATS) are major volatile components of garlic oil. In this study, we assessed their relative potency in inducing antioxidant enzyme expression. Among the three organosulfur compounds, DATS was found to be most potent in inducing heme oxygenase-1 (HO-1) and
NAD(P)H: quinone oxidoreductase-1 (NQO1) in human gastric epithelial (AGS) cells. Furthermore, DATS administration by gavage increased the expression of HO-1 and NQO1 in C57BL/6 mouse stomach. Treatment with DATS increased the accumulation of nuclear factor-erythroid-2-related factor-2 (Nrf2) in the nucleus of cultured AGS cells and in mouse stomach in vivo. The DATS-induced expression of HO-1 and NQO1 was abrogated in the cells transiently transfected with Nrf2-siRNA or in the embryonic fibroblasts from Nrf2-null mice, indicating that Nrf2 is a key mediator of the cytoprotective effects of DATS. Pretreatment of AGS cells with N-acetylcysteine or dithiothreitol attenuated DATS-induced nuclear localization of Nrf2 and the expression of HO-1 and NQO1. Cysteine-151, -273 and -288 of Kelch-like ECH-associated protein-1 (Keap1), a cytosolic repressor of Nrf2, have been considered to act as a redox sensor and play a role in Nrf2 activation. To determine whether DATS could inactivate Keap1 through thiol modification, we established cell lines constitutively expressing wild type-Keap1 or three different mutant constructs in which cysteine-151, -273, or -288 of Keap1 was replaced with serine by retroviral gene transfer. DATS failed to activate Nrf2, and to induce expression of HO-1 and NQO1 only in Keap1-C288S mutant cells. LC-ESI-MS/MS analysis of recombinant Keap1 treated with DATS revealed that the peptide fragment containing Cys288 gained a molecular mass of 72.1 Da equivalent to the molecular weight of mono-allyl mono-sulfide. Taken together, these findings suggest that DATS may directly interact with the Cys288 residue of Keap1, which partly accounts for its ability to induce Nrf2 activation and upregulate defensive gene expression.
二烯丙基硫醚、二烯丙基二硫醚和二烯丙基三硫醚（DATS）是大蒜油的主要挥发性成分。在本研究中，我们评估了它们在诱导抗氧化酶表达方面的相对效力。在这三种有机硫化合物中，发现DATS在诱导人胃上皮（AGS）细胞中的血红素加氧酶-1（HO-1）和烟酰胺腺嘌呤二核苷酸磷酸（NAD(P)H）：醌氧化还原酶-1（NQO1）方面效力最强。此外，通过灌胃给予DATS可增加C57BL/6小鼠胃中HO-1和NQO1的表达。用DATS处理可增加培养的AGS细胞细胞核以及体内小鼠胃中核因子红系2相关因子2（Nrf2）的积累。在瞬时转染Nrf2-siRNA的细胞或Nrf2基因敲除小鼠的胚胎成纤维细胞中，DATS诱导的HO-1和NQO1表达被消除，这表明Nrf2是DATS细胞保护作用的关键介质。用N-乙酰半胱氨酸或二硫苏糖醇预处理AGS细胞可减弱DATS诱导的Nrf2核定位以及HO-1和NQO1的表达。Kelch样ECH相关蛋白1（Keap1）是Nrf2的胞质阻遏物，其半胱氨酸-151、-273和-288被认为可作为氧化还原传感器并在Nrf2激活中发挥作用。为了确定DATS是否可通过硫醇修饰使Keap1失活，我们通过逆转录病毒基因转移建立了组成性表达野生型Keap1或三种不同突变体构建体的细胞系，其中Keap1的半胱氨酸-151、-273或-288被丝氨酸取代。DATS仅在Keap1-C288S突变体细胞中未能激活Nrf2，也未能诱导HO-1和NQO1的表达。对用DATS处理的重组Keap1进行液相色谱-电喷雾串联质谱（LC-ESI-MS/MS）分析表明，包含Cys288的肽片段分子量增加了72.1 Da，相当于单烯丙基单硫醚的分子量。综上所述，这些发现表明DATS可能直接与Keap1的Cys288残基相互作用，这部分解释了其诱导Nrf2激活和上调防御基因表达的能力。
