Ligustilide Inhibits Tumor Angiogenesis by Downregulating VEGFA Secretion from Cancer-Associated Fibroblasts in Prostate Cancer via TLR4

SIMPLE SUMMARY: Inhibiting the production of vascular endothelial growth factor A (VEGFA) can inhibit angiogenesis, thereby inhibiting tumor growth. We found that ligustilide can inhibit the secretion of VEGFA from prostate cancer-related fibroblasts (CAFs), and this signaling pathway is related to Toll-like receptor 4 (TLR4)-ERK/JNK/p38. Using the above receptors or signals pathway molecule blockers can block the effect of ligustilide to downregulate the secretion of VEGFA from CAFs. The concentration of ligustilide used in this study does not directly inhibit the growth of CAFs, but changes its function, and this study is different from the direct blocking effect of the existing VEGFA antibodies, but cuts off the source of VEGFA, which is expected to become a novel therapeutic strategy for oncology. ABSTRACT: CAFs secrete VEGFA in the tumor microenvironment to induce angiogenesis and promote tumor growth. The downregulation of VEGFA secretion from CAFs helps block angiogenesis and exerts an anti-tumor effect. In vivo experiments showed that the angiogenesis of the tumor-bearing mice in the ligustilide group was significantly reduced. The results of MTT, tube formation, Transwell and scratch experiments showed that ligustilide did not affect the proliferation of HUVECs in a certain concentration range (<60 μM), but it inhibited the proliferation, tube formation and migration of HUVECs induced by CAFs. At this concentration, ligustilide did not inhibit CAF proliferation. The qPCR and WB results revealed that ligustilide downregulated the level of VEGFA in CAFs via the TLR4-ERK/JNK/p38 signaling pathway, and the effect was attenuated by blockers of the above molecules. Ligustilide also downregulated the autocrine VEGFA of HUVECs induced by CAFs, which inhibited angiogenesis more effectively. In addition, ligustilide inhibited glycolysis and HIF-1 expression in CAFs. Overall, ligustilide downregulated the VEGFA level in CAFs via the TLR4-ERK/JNK/p38 signaling pathway and inhibited the promotion of angiogenesis. This study provides a new strategy for the anti-tumor effect of natural active molecules, namely, blockade of angiogenesis, and provides a new candidate molecule for blocking angiogenesis in the tumor microenvironment.