RESUMEN
BACKGROUND: 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. PURPOSE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.
Asunto(s)
Anticarcinógenos/química , Anticarcinógenos/farmacología , Medicamentos Herbarios Chinos/química , Medicamentos Herbarios Chinos/farmacología , Factor 2 Relacionado con NF-E2/metabolismo , Animales , Elementos de Respuesta Antioxidante/efectos de los fármacos , Elementos de Respuesta Antioxidante/genética , Línea Celular , Transformación Celular Neoplásica/efectos de los fármacos , Transformación Celular Neoplásica/genética , Regulación de la Expresión Génica/efectos de los fármacos , Hemo-Oxigenasa 1 , Humanos , Medicina Tradicional China , Ratones , Simulación del Acoplamiento Molecular , NAD(P)H Deshidrogenasa (Quinona)/genética , NAD(P)H Deshidrogenasa (Quinona)/metabolismo , Transducción de Señal/efectos de los fármacos , Factor de Transcripción AP-1/genética , Factor de Transcripción AP-1/metabolismoRESUMEN
Exploring traditional medicines may lead to the development of low-cost and non-toxic cancer preventive agents. Si-Wu-Tang (SWT), comprising the combination of four herbs, Rehmanniae, Angelica, Chuanxiong, and Paeoniae, is one of the most popular traditional Chinese medicines for women's diseases. In our previous studies, the antioxidant Nrf2 pathways were strongly induced by SWT in vitro and in vivo. Since Nrf2 activation has been associated with anticarcinogenic effects, the purpose of this study is to evaluate SWT's activity of cancer prevention. In the Ames test, SWT demonstrated an antimutagenic activity against mutagenicity induced by the chemical carcinogen 7,12-dimethylbenz(a)anthracene (DMBA). In JB6 P+ cells, a non-cancerous murine epidermal model for studying tumor promotion, SWT inhibited epidermal growth factor (EGF)-induced neoplastic transformation. The luciferase reporter gene assays demonstrated that SWT suppressed EGF-induced AP-1 and TNF-α-induced NF-κB activation, which are essential factors involved in skin carcinogenesis. In a DMBA-induced skin hyperplasia assay in 'Sensitivity to Carcinogenesis' (SENCAR) mice, both topical and oral SWT inhibited DMBA-induced epidermal hyperplasia, expression of the proliferation marker Proliferating cell nuclear antigen (PCNA), and H-ras mutations. These findings demonstrate, for the first time, that SWT prevents tumor promoter and chemical-induced carcinogenesis in vitro and in vivo, partly by inhibiting DNA damage and blocking the activation of AP-1 and NF-κB.