Department of Pharmaceutical Sciences, College of Pharmacy, Western University of Health Sciences, Pomona, CA 91766, United States.
Department of Pharmacology and Toxicology, Shanghai Institute for Food and Drug Control, Shanghai, China.
Phytomedicine. 2018 Feb 1;40:1-9. doi: 10.1016/j.phymed.2017.12.031. Epub 2017 Dec 27.
The nuclear factor erythroid 2-related factor 2 (Nrf2) is a potential molecular target for cancer chemoprevention. Si-Wu-Tang (SWT), a popular traditional Chinese medicine for women's health, was reported with a novel activity of cancer prevention.
The present study was aimed to identify the bioactive constituents in SWT responsible for the Nrf2 activating and cancer preventive activity and explore the pharmacological mechanisms.
Nine compounds detectable from various batches of SWT were ranked using in silico molecular docking based on their ability to interfere the forming of Nrf2-Keap1 complex. The predicted Nrf2 activating effect was validated using the antioxidant response element (ARE) luciferase reporter assay and quantitative RT-PCR analysis for select Nrf2 regulated genes Hmox1, Nqo1 and Slc7a11. The antimutagenic activity of the compounds were determined by the Ames test. The chemopreventive activity of these compounds were assessed on EGF-induced neoplastic transformation of JB6 P+ cells, an established non-cancerous murine epidermal model for studying tumor promotion and identifying cancer preventive agents. These compounds were further characterized using luciferase reporter assay on EGF-induced activation of AP-1, a known transcription factor mediating carcinogenesis.
Three of the nine compounds predicted as Nrf2 activators by molecular docking, gallic acid (GA), Z-liguistilide (LIG), and senkyunolide A (SA), were confirmed with highest potency of increasing the Nrf2/ARE promoter activity and upregulating the expression of Hmox1, Nqo1 and Slc7a11. In addition, GA, LIG and SA exhibited an antimutagenic activity against the direct mutagen 2-nitrofluorene while no mutagenic effects were observed at the same time in Ames test. At nontoxic concentrations, GA, LIG, and SA inhibited EGF-induced neoplastic transformation of JB6 P+ cells. Combined treatment of GA, LIG and SA, in the same ratio as detected in SWT, showed enhanced effect against JB6 transformation compared with that of the single compound alone. GA, LIG and SA, alone or in combination, suppressed EGF-induced activation of AP-1.
We identified three bioactive constituents in SWT responsible for the Nrf2 activating and cancer preventive activity. This study provides evidence supporting novel molecular basis of SWT in cancer prevention.
核因子红细胞 2 相关因子 2（Nrf2）是癌症化学预防的潜在分子靶标。四物汤（SWT）是一种用于女性健康的流行中药，据报道具有预防癌症的新活性。
本研究旨在鉴定 SWT 中负责 Nrf2 激活和癌症预防活性的生物活性成分，并探讨其药理机制。
基于其干扰 Nrf2-Keap1 复合物形成的能力，使用基于计算机的分子对接对来自不同批次 SWT 的九种化合物进行排序。使用抗氧化反应元件（ARE）荧光素酶报告基因测定和定量 RT-PCR 分析选择的 Nrf2 调节基因 Hmox1、Nqo1 和 Slc7a11 来验证预测的 Nrf2 激活作用。使用 Ames 试验测定化合物的抗突变活性。使用 EGF 诱导的 JB6 P+细胞癌变评估这些化合物的化学预防活性，JB6 P+细胞是一种用于研究肿瘤促进和鉴定癌症预防剂的成熟非致癌性小鼠表皮模型。使用 EGF 诱导的 AP-1 激活的荧光素酶报告基因测定进一步对这些化合物进行表征，AP-1 是一种已知的介导致癌作用的转录因子。
通过分子对接预测的九种 Nrf2 激活剂中的三种化合物，没食子酸（GA）、Z-当归酰基丁内酯（LIG）和升麻醇 A（SA），被证实具有最强的增加 Nrf2/ARE 启动子活性和上调 Hmox1、Nqo1 和 Slc7a11 表达的能力。此外，GA、LIG 和 SA 对直接诱变剂 2-硝基氟苯表现出抗突变活性，而在 Ames 试验中同时未观察到致突变作用。在非毒性浓度下，GA、LIG 和 SA 抑制 EGF 诱导的 JB6 P+细胞癌变。GA、LIG 和 SA 以与 SWT 中检测到的相同比例联合治疗，与单一化合物单独治疗相比，对 JB6 转化的抑制作用增强。GA、LIG 和 SA 单独或联合使用可抑制 EGF 诱导的 AP-1 激活。
我们鉴定了 SWT 中负责 Nrf2 激活和癌症预防活性的三种生物活性成分。本研究为 SWT 在癌症预防中的新分子基础提供了证据。
