br Please cite this article in press
Please cite this article in press as: Huang et al., A Systems Pharmacology Approach Uncovers Wogonoside as an Angiogenesis Inhibitor of Triple-Negative Breast Cancer by Targeting Hedgehog Signaling, Cell Chemical Biology (2019), https://doi.org/10.1016/j.chembiol.2019.05.004
Wogonoside Inhibits the Expression of VEGF
To explore the mechanism-of-action of wogonoside in TNBC, MTT cell viability assay was performed. However, we found that wogonoside had no effect on the viability of both MDA-MB-231 and MDA-MB-468 Pifithrin-α (PFTα) after 24 h pretreatment (Fig-ure S1). To further investigate the potential molecular mecha-nism, we integrated 13 literature-reported TNBC gene-encoding proteins targeted potentially by wogonoside in breast-specific coexpressed human protein-protein interactome network (see the STAR Methods). The network was derived from RNA sequencing profiles across 32 human tissues in the Genotype-Tissue Expression (GTEx) database (GTEx Consortium, 2015). Network analysis (Cheng et al., 2018) shows that wogonoside potentially targets VEGFA in TNBC (Figure 2A) consistent with our previous study that wogonoside decreases the secretion of VEGF in MCF-7, an estrogen-positive breast cancer cell line (Huang et al., 2016). RNA sequencing data from the GTEx data-base (GTEx Consortium, 2015) suggests that VEGFA is highly expressed in breast tissue.
We next inspected whether wogonoside inhibits VEGF expression in TNBC using the transplanted MDA-MB-231 tu-mors. Western blot analysis (Figure 2B) reveals that wogonoside reduces VEGF expression of mouse tumor tissues, which is further confirmed by the immunohistochemical (IHC) staining assay (Figure 2C). We further evaluated the effect of wogonoside on VEGF expression in TNBC cells by enzyme-linked immuno-sorbent assay. Figure 2D reveals that VEGF secretion in MDA-MB-231 and MDA-MB-468 cells are significantly reduced by wo-gonoside. Western blot assays show that wogonoside inhibits VEGF protein expression in a concentration-dependent manner (Figure 2E) as well. In addition, RT-PCR (Figure 2F) and luciferase reporter assays (Figure 2G) reveal that VEGF mRNA levels and VEGF promoter activity are suppressed by wogonoside in both MDA-MB-231 and MDA-MB-468 cell lines. Altogether, wogono-side inhibits VEGF expression in both mouse model (Figures 2B and 2C) and human MDA-MB-231 and MDA-MB-468 cell lines (Figures 2D–2G).
Wogonoside Inhibits the Hedgehog Signaling Pathway The activation of the Hedgehog/Gli signaling pathway is signifi-cantly increased in TNBC cells and Gli1 regulates the VEGFA gene promoter directly (Cao et al., 2012; Tao et al., 2011). We therefore examined the effects of wogonoside on Hedgehog/ Gli signaling. The IHC staining assay reveals that the nuclear translocation of Gli1 is inhibited by wogonoside (Figure 3A) in vivo. Western blot analysis further confirms that wogonoside reduces Gli1 expression in the nucleoplasm of mouse tumor tis-sues (Figure 3B).
Furthermore, the Gli luciferase reporter assay shows that the transcriptional activity of Gli signaling is inhibited by wogonoside (half maximal inhibitory concentration of 22.17 mM, Figure S2A) in both MDA-MB-231 and MDA-MB-468 cell lines. In addition, the mRNA levels of three main target genes of Gli1 (including Cyclin D2, HIP, and GAS1) are decreased by wogonoside (Figure 3C). As the cytoplasmic accumulation and intracellular localization of Gli1 are essential for activating the transcription of target genes (Briscoe and Therond, 2013), we tested Gli1 expression in cytosolic and nuclear lysates by western blot analysis. We found that wogonoside decreased Gli1 levels in both MDA-
MB-231 and MDA-MB-468 cell nucleus in a concentration-dependent manner (Figure 3D). In addition, immunofluorescence (Figure 3E) shows that the nuclear translocation of Gli1 in MDA-MB-231 and MDA-MB-468 cell lines is inhibited by wogonoside. Collectively, wogonoside inhibits Gli1 nuclear translocation and transcriptional activity of the Hedgehog signaling pathway.