br Thus wogonoside downregulates the expression
Thus, wogonoside downregulates the expression and transcrip-tion of VEGF in TNBC cells, as well as the migration and invasion ability of HUVECs induced by TNBC cell CM.
We next examined the effect of wogonoside on angiogenesis in TNBC cells. The tube formation assay shows that elongated and robust tube-like structures and the number of tubes of HUVECs are markedly inhibited by wogonoside compared with MDA-MB-231 CM- or MDA-MB-468 CM-stimulated groups (Fig-ure 6A). In addition, the rat aortic ring assay, which could mimic several key stages of angiogenesis, including endothelial cell proliferation, migration, and tube formation, was also performed. We found that the vessel sprouting of the rat aortic ring was significantly stimulated by MDA-MB-231 CM or MDA-MB-468 CM compared with the control group (Figure 6B). As a result, the new microvessel growth from aortic rings was inhibited by wogonoside in both MDA-MB-231 and MDA-MB-468 cell lines. After injecting MDA-MB-231 or MDA-MB-468 AS1517499 in the chicken embryos, the chorioallantoic membrane (CAM) assay indicated that more new blood vessels formed in the TNBC cell group compared with the control group (Figure 6C). We found that wogonoside (50, 100, and 200 ng/CAM) inhibited the growth of the newly formed blood vessels (Figure 6C). In summary, wo-gonoside inhibits angiogenesis induced by MDA-MB-231 and MDA-MB-468 cells.
Wogonoside Inhibits Angiogenesis of TNBC In Vivo
We further investigated the effect of wogonoside on TNBC cell-induced angiogenesis in vivo. The Matrigel plug assay shows that wogonoside (80 mg/kg) suppresses the formation of blood vessels compared with the MDA-MB-231 or MDA-MB-468 cell-injected groups (Figure 7A). Furthermore, the whole mount of CD31 staining shows that wogonoside significantly reduces the vascular density in Matrigel plugs compared with MDA-MB-231 or MDA-MB-468 cell-injected groups as well (Figure 7B). Specifically, the hemoglobin concentration in Matrigel plugs is reduced by 66.3% ± 1.3% and 57.3% ± 5.3% with the treatment of wogonoside in MDA-MB-231 and MDA-MB-468 cell-injected groups, respectively (Figure 7C). In addition, histological analysis of sections stained with CD31 (an endothelial-specific marker) shows that wogonoside reduces the vascular density in tumors compared with the control group (Figure 7D). Taken together, wogonoside inhibits angiogenesis induced by MDA-MB-231 and MDA-MB-468 cells in vivo.
We next used VEGF-neutralizing antibody (VEGF mAb) to deter-mine whether the VEGF inhibition by wogonoside contributes to suppressing angiogenesis in TNBC. VEGF mAb (10 mg/mL) markedly reduces the number of HUVEC tubes stimulated by MDA-MB-231 CM or MDA-MB-468 CM (Figure 7E) compared with the CM-treated group. In addition, the newly formed blood vessels in CAM decreased after adding VEGF mAb as well (Fig-ure 7F). VEGF is primarily secreted by tumor cells and specifically activates VEGFR2 (vascular endothelial growth factor receptor 2), a membrane receptor mainly expressed on endothelial cells
(C) The mRNA level of Cyclin D2, HIP, and GAS1 was detected by RT-PCR.
(D) Western blot analysis of Gli1 expression in cytosolic and nuclear lysates. Lamin A and b-tubulin were used as nuclear and cytoplasmic markers, respectively.
(E) Gli1 nuclear translocation was analyzed by immunofluorescence confocal microscopy.
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 Figure S2.
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