Ursolic acid inhibits glioblastoma through suppressing TGFβ-mediated epithelial-mesenchymal transition (EMT) and angiogenesis

Abstract

Found in many fruits and plants, Ursolic acid (UA), a pentacyclic triterpene that occurs naturally, is recognized for its anti-cancer effects, especially in combating glioblastoma. However, the intricate molecular mechanisms underpinning its anti-tumor actions are still not fully understood, despite the recognition of these effects. By examining the functions of epithelial-mesenchymal transition (EMT) and angiogenesis, crucial for glioblastoma progression, and their regulation through Transforming Growth Factor Beta (TGFβ) - a key marker for glioblastoma, our research aims to fill this knowledge gap. This study explores how ursolic acid can block the progression of glioblastoma by precisely targeting TGFβ-triggered EMT and angiogenesis. The findings show that UA successfully blocks the spread, movement, and invasion of glioblastoma cells. Accompanying this, there is a significant reduction in the expression of TGFβ and crucial EMT indicators like snail and vimentin. Furthermore, UA shows a reduction in angiogenesis that depends on the dosage, highlighted by decreased vascular endothelial growth factor (VEGF) in human umbilical vein endothelial cells (HUVECs). Interestingly, increased TGFβ expression in U87 and U251 glioblastoma cell lines was found to weaken UA's anti-tumor properties, shedding more light on TGFβ's critical function in glioblastoma's pathology. Supporting these laboratory results, UA also showed considerable inhibition of tumor growth in a glioblastoma xenograft mouse model. Overall, our research emphasizes Ursolic acid's promise as a new treatment for glioblastoma and clarifies its action mechanism, mainly by inhibiting TGFβ signaling and thereby EMT and angiogenesis.

Keywords: Angiogenesis; Epithelial-mesenchymal transition (EMT); Glioblastoma; TGFβ; Ursolic acid.

Fig. 1

UA inhibited cell proliferation, invasion and downregulated TGFβ and EMT markers in the glioblastoma cells. Glioblastoma cells U87, U251, and HA1800 were exposed to varying UA concentrations for 24 h. (A–C) The CCK8 assay assessed cell viability. (D–G) Transwell assays measured the migration and invasion of U87, U251, and HA1800 cells post 24-h UA treatment (scale bar = 100 μm). (H) Western blot analysis investigated the protein quantities of TGFβ and EMT markers. *P < 0.05, compared with control (0 μM).

Fig. 2

Over-expression of TGFβ abolished UA-mediated cell proliferation, invasion and EMT. U87 and U251 cells, transfected with either TGFβ or a control vector for 18 h, were collected. (A–B) 5000 cells were placed in a 96-well plate and then treated with either 10 μM UA or a control vehicle for 24 h. The CCK-8 assay determined cell growth rates. (C–F) 10,000 cells were introduced into a transwell chamber, and their ability to invade was assessed using transwell migration and invasion assays (scale bar = 100 μm). (G) Protein levels were analyzed through Western blotting. Control: transfection vector; ursolic acid: transfection vector + ursolic acid; oe-TGFβ: transfection TGFβ; Both: transfection TGFβ + ursolic acid. *P < 0.05 vs control. ^#P < 0.05, compared with either ursolic acid treatment or TGFβ transfection alone.

Fig. 3

The effect of UA on angiogenesis in HUVECs. HUVEC were treated with the indicated concentration of UA (A) Cell proliferation under various UA treatments was evaluated using the CCK8 assay. (B, C) UA's influence on HUVEC angiogenesis was quantified through tube formation assays. (D) VEGF protein levels were identified via Western blot analysis. (E) Post-TGFβ overexpression, TGFβ protein levels in HUVECs were measured. (F–I) TGFβ overexpression counteracted the anti-angiogenic effects of UA in HUVECs. HUVECs, after 18 h of TGFβ or control plasmid transfection, were treated with 10 μM UA or left untreated for 24 h. Tube counts (F, G) and VEGF levels (H, I) were determined through Western blot and ELISA. Control: transfection vector; ursolic acid: transfection vector + ursolic acid; oe-TGFβ: transfection TGFβ; Both: transfection TGFβ + ursolic acid. *P < 0.05 vs control. #P < 0.05, compared with either ursolic acid treatment or TGFβ transfection alone.

Fig. 4

UA decreased glioblastoma growth in xenograft mouse model. 7 days post-inoculation, mice were randomly assigned to two groups, each comprising five mice. Mice received oral administration of either a vehicle (normal saline) or UA (50 mg/kg/day). (A) The reduction in xenografted U87 tumor sizes was documented. Tumor volumes (B) and body weights (C) were recorded every five days. At the end of the experiments, tumor tissues were harvested for analysis, with protein lysates examined via Western blot (D) and CD31 levels assessed through immunohistochemistry (E) (scale bar = 50 μm). Each western blotting image represented at least three independent results*P < 0.05, compared with control.

(A-B) Immunoblot showing the amount of TGFβ protein in U87 and U251 cells.