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  • br Wogonoside Inhibits the Expression of


    Wogonoside Inhibits the Expression of SMO via Promoting Its Ubiquitination r> SMO is a key upstream activator involved in the Hedgehog signaling pathway (Cannonier and Sterling, 2015; Stanton and Peng, 2010). To explore how wogonoside affects Gli1 nuclear translocation, we tested protein expression of SMO in whole-cell lysate. In vivo assay reveals that wogonoside reduces the SMO expression, confirmed by IHC staining (Figure 4A) and western blot assay (Figure 4B). Moreover, we found that wogo-noside downregulated the expression of SMO in a concentra-tion-dependent manner (Figure 4C), without affecting Gli1 in both MDA-MB-231 and MDA-MB-468 Z-Guggulsterone cell lines. To explore the mechanism of the downregulating of SMO expression by wogo-noside in depth, we examined the transcription level of SMO. Interestingly, the mRNA levels of SMO in both MDA-MB-231 and MDA-MB-468 cell lines are not changed by wogonoside (Figure S2B). We therefore turned to inspect wogonoside’s ef-fects by focusing on SMO protein expression.
    It has been reported that Drosophila SMO is degraded via both lysosome- and proteasome-dependent mechanisms (Li et al., 2012; Zhou et al., 2017). As expected, the protein level of SMO is significantly increased when both MDA-MB-468 and MDA-MB-231 cell lines were treated with either MG132, a proteasome inhibitor, or NH4Cl, a lysosome inhibitor, in a time-dependent manner (Figure 4D). These observations suggest that SMO is also degraded in both lysosome- and proteasome-dependent manner in TNBC cells. We then treated TNBC Z-Guggulsterone by wogono-side with MG132 (10 mM) or NH4Cl (25 mM) at the same time. Figure 4E shows that the SMO reduction by wogonoside in MDA-MB-231 and MDA-MB-468 cell lines were both abrogated by adding MG-132. However, the expression of SMO still decreased at different wogonoside concentrations (25, 50, and 100 mM), when NH4Cl was added. In addition, wogonoside (100 mM) promotes the ubiquitination of SMO in both MDA-MB-231 and MDA-MB-468 cell lines, as indicated by multiple coim-munoprecipitation experiments followed by western blot assays (Figure 4F). In summary, wogonoside downregulates SMO expression by promoting its proteasome degradation.
    Wogonoside Binding to SMO Promotes Its Ubiquitination It has been reported that the Smurf-family of E3 ubiquitin ligases and Cul4-DDB1 are involved in the ubiquitination of SMO in Drosophila melanogaster (Li et al., 2018a, 2018b). We therefore investigated whether Cul4-DDB1 is involved in human SMO degradation triggered by wogonoside. Firstly, we silenced the expression of Cul4A in MDA-MB-231 cells by specific small interfering RNAs (siRNAs) (Figure 5A). Endogenous SMO protein was then immunoprecipitated and detected by ubiquitin anti-body using western blotting. We found that wogonoside signifi-cantly increased the SMO ubiquitination (Figure 5B). Notably, siRNA-mediated downregulation of Cul4A effectively impairs wogonoside-induced SMO ubiquitination in MDA-MB-231 cells.
    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),
    Figure 2. Effects of Wogonoside on VEGF Secretion, Protein and mRNA Expression, and Transcriptional Activity in MDA-MB-231 and MDA-MB-468 Cell Lines
    (A) Network analysis highlighting the inferred mechanism-of-action for wogonoside in TNBC. The potential molecular mechanisms of wogonoside against TNBC were investigated via integration of known drug targets and experimentally validated TNBC genes into tissue-specific coexpressed protein-protein interactome network (see the STAR Methods). Node size indicates the protein-coding gene expression level in breast compared with other 31 tissues from GTEx database (GTEx Con-sortium, 2015). Larger size highlighting the high expression level in breast compared with other tissues. Coexpression denotes the coexpressed gene pairs (p < 0.05, F statistics) encoding protein-protein interactions in TNBC RNA sequencing data from The Cancer Genome Atlas database (Cancer Genome Atlas Network, 2012).