Discovery of molecular mechanisms of traditional Chinese medicinal formula Si-Wu-Tang using gene expression microarray and connectivity map

Abstract

To pursue a systematic approach to discovery of mechanisms of action of traditional Chinese medicine (TCM), we used microarrays, bioinformatics and the "Connectivity Map" (CMAP) to examine TCM-induced changes in gene expression. We demonstrated that this approach can be used to elucidate new molecular targets using a model TCM herbal formula Si-Wu-Tang (SWT) which is widely used for women's health. The human breast cancer MCF-7 cells treated with 0.1 µM estradiol or 2.56 mg/ml of SWT showed dramatic gene expression changes, while no significant change was detected for ferulic acid, a known bioactive compound of SWT. Pathway analysis using differentially expressed genes related to the treatment effect identified that expression of genes in the nuclear factor erythroid 2-related factor 2 (Nrf2) cytoprotective pathway was most significantly affected by SWT, but not by estradiol or ferulic acid. The Nrf2-regulated genes HMOX1, GCLC, GCLM, SLC7A11 and NQO1 were upregulated by SWT in a dose-dependent manner, which was validated by real-time RT-PCR. Consistently, treatment with SWT and its four herbal ingredients resulted in an increased antioxidant response element (ARE)-luciferase reporter activity in MCF-7 and HEK293 cells. Furthermore, the gene expression profile of differentially expressed genes related to SWT treatment was used to compare with those of 1,309 compounds in the CMAP database. The CMAP profiles of estradiol-treated MCF-7 cells showed an excellent match with SWT treatment, consistent with SWT's widely claimed use for women's diseases and indicating a phytoestrogenic effect. The CMAP profiles of chemopreventive agents withaferin A and resveratrol also showed high similarity to the profiles of SWT. This study identified SWT as an Nrf2 activator and phytoestrogen, suggesting its use as a nontoxic chemopreventive agent, and demonstrated the feasibility of combining microarray gene expression profiling with CMAP mining to discover mechanisms of actions and to identify new health benefits of TCMs.

Figure 1. Experimental design of microarray gene expression profiling.

The 24 samples were obtained from MCF-7 cells which were divided into eight treatment groups. 0.001% DMSO was used as the vehicle control (C). The cells were treated with 0.1 µM estradiol, FA at three concentrations (0.1, 1.0, and 10 µM) and SWT at three concentrations (0.0256, 0.256, and 2.56 mg/ml). For each treatment group, 3 biological replicates were included.

Figure 2. The hierarchical clustering analysis and heatmap of the correlation coefficients between gene expression profiles.

(A) All 24 samples from the 8 treatment groups; (B) controls and ferulic acid treatments; and (C) controls and treatments by estradiol and SWT. There was good reproducibility between the three biological replicates in each treatment group. No clear treatment effect was observed for ferulic acid treatments. Low- and medium-concentration SWT treatments showed mild effects, while the strongest treatment effects were seen from estradiol and high-concentration SWT treatments.

Figure 3. The Nrf2-mediated oxidative stress response pathway in IPA database.

The red color and green color indicate the up- and down-regulated genes after treatment with high-concentration of SWT in this pathway, respectively.

Figure 4. Dose-responsive genes with the largest fold changes.

Among 15 dose-responsive up-regulated genes (probe sets) with the largest fold changes, ten are related to Nrf2 (highlighted in red box) according to PubMed literature search.

Figure 5. Luciferase assay results.

(A) Luciferase assay established using HEK-293 cells co-transfected with a plasmid containing an ARE-luciferase reporter gene (pGL4.22-ARE) or empty vector (pGL4.22) and a plasmid encoding renillar luciferase (pGL4.74). The transfected cells were treated with sulforaphane for 24 hr prior to measurement of firefly and renillar luciferase activities using the dual luciferase reporter gene assay. (B) SWT (three doses SL, SM and SH in 0.0256, 0.256 and 2.56 mg/ml) and four herbal components of SWT (2.56 mg/ml) activated the Nrf2/ARE signaling pathway in MCF-7 cells.