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  • B Effect of wogonoside on VEGF expression in xenograft

    2020-03-17

    (B) Effect of wogonoside on VEGF expression in xenograft model of MDA-MB-231 cells in nude mice (n = 5) was detected by western blot analysis.
    (C) The expression of VEGF in xenograft model was detected by immunochemistry using specific antibody.
    (D) The concentration of VEGF in MDA-MB-231 and MDA-MB-468 CM were measured by enzyme-linked immunosorbent assay kits.
    (E) VEGF expression was detected by western blot analysis using specific antibodies.
    (F) The mRNA level of VEGF was investigated by RT-PCR.
    (G) VEGF transcriptional activity was tested by Dual-Luciferase reporter assay.
    The comparisons were made relative to the control group and the significance of the difference is indicated as *p < 0.05 and **p < 0.01. See also Figures S1 and S4.
    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
    Figure 3. Effects of Wogonoside on Hedgehog Signaling Pathway
    (A) Effect of wogonoside on Gli1 expression in xenograft tissue of MDA-MB-231 cells in nude mice was detected by immunochemistry.
    (B) Gli1 expression in nucleoplasm of MDA-MB-231 cell xenograft tissue was detected by western blot analysis using specific Angeli’s Salt and lamin A was used as nuclear marker.
    (legend continued on next page)
    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
    These observations suggest that Cullin4 is a potential E3 ligase for human SMO, consistent with ubiquitination regulation of SMO in Drosophila melanogaster.
    Small-molecule modulators inhibit the Hedgehog signaling pathway by binding to Gli directly, or by targeting SMO to sup-press Gli indirectly (Hui et al., 2013; Wang et al., 2013). To explore how wogonoside promotes the proteasome degradation of SMO, we first performed molecular docking simulation to investigate potential interactions of wogonoside with SMO or Gli1. This suggested a strong interaction between Angeli’s Salt wogonoside and SMO, including hydrogen bonds involving Arg515 and Ser456 (Figure 5C), and aromatic ring docking into the hydro-phobic pocket consisting of Glu380, Cys381, Ala382, Trp383, Leu384, Glu385, Ser456, Gly457, Tyr459, Thr460, Arg515, Ser518, Asn519, and Met522 (Figure 5D), while a weak interac-tion between wogonoside and Gli1 (Figure S2C).
    To investigate the interaction between wogonoside and SMO/ Gli further, SMO binding and cellular thermal shift assays (CET-SAs) were performed. BODIPY-cyclopamine, a fluorescent derivative of cyclopamine, specifically binding SMO-expres-sing cells, was used for SMO binding assay (Chen et al., 2002). We found that wogonoside significantly reduced BODIPY-cyclopamine binding to human SMO in a dose-dependent manner (Figure 5E), indicating a direct interaction between wo-gonoside and SMO by binding with the cyclopamine pocket. The CETSA assay monitors and quantifies the extent to which a drug reaches and directly binds to a protein of interest within cells by quantifying the changes in the thermal stability of pro-teins upon ligand binding (Jafari et al., 2014; Martinez Molina et al., 2013). In the CETSA, the heat challenge results in Gli protein degradation due to unfolding, while wogonoside has no effect on Gli protein stability in MDA-MB-231 cells (Figure 5F). Taken together, wogonoside is a potential, direct SMO inhibitor in TNBC cells, by specifically promoting ubiquitination-depen-dent degradation of SMO.
    Wogonoside Inhibits Angiogenesis Induced by TNBC Cells
    It has been reported that SMO overexpression is involved in the development of TNBC, and that the Hedgehog/Gli signaling pathway regulates the expression of VEGF (Cao et al., 2012; Tao et al., 2011) by accelerating the proliferation, migration, and invasion of vascular endothelial cells (Ferrara et al., 2003). We therefore examined the effects of wogonoside on angiogen-esis induced by TNBC cells. We incubated human umbilical vein endothelial cells (HUVECs) in TNBC cell-conditioned medium (MDA-MB-231 CM or MDA-MB-468 CM). The Matrigel invasion assay indicates that CM collected from MDA-MB-231 or MDA-MB-468 cells without wogonoside treatment stimulated apparent migration and invasion of HUVECs (Figure S3A). Upon exposure to CM collected from TNBC cells treated with wogonoside (25, 50, and 100 mM), the number of invasive HUVECs decreased in a concentration-dependent manner.