Fig 1.
Lupeol, β-sitosterol, and stigmasterol exhibited cytotoxic effects on HUVECs.
Structures and cell viability of lupeol (A), β-sitosterol (B), and stigmasterol (C). Cell viability of HUVECs, which were exposed for 48 hours to different doses of lupeol, β-sitosterol, and stigmasterol, as determined by MTT assays. Means ± S.D. *P value < 0.05 vs. control. n = 3. (D) The IC50 values of bevacizumab, lupeol, β-sitosterol, and stigmasterol in HUVECs. Means ± S.D.
Fig 2.
Lupeol and stigmasterol inhibited HUVEC migration and capillary network formation in vitro.
(A) Lupeol and stigmasterol inhibited HUVEC migration. The effects of the compounds on HUVEC migration abilities were determined using 2-dimensional wound healing assays. HUVECs were seeded at 100% confluency and scratched at the middle of each well, then each compound was added to the culture at 5 μM concentration. Scale bar, 50 μm. (B) Lupeol and stigmasterol also significantly disrupted HUVEC network formation. Endothelial network formation assays were performed by seeding HUVECs between two type I collagen gel layers and cultured for 4 days. Scale bar, 200 μm.
Fig 3.
Lupeol and stigmasterol downregulated TNF-α and inhibited VEGF signaling.
The expression of inflammatory cytokines and components of the VEGF pathway was examined by qRT-PCR in HUVECs treated with the compounds at 5 μM. (A-C) Lupeol and stigmasterol suppressed expression levels of TNF-α but showed no impact on IL-6 and CXCL-8. (D-F) Expression of VEGFR-2, but not VEGF-A or VEGFR-1, significantly decreased upon lupeol and stigmasterol treatment. (G) Western blotting showed a decrease in the p-FAK level at both concentrations and a slight decrease in levels of p-Src, p-PCL, p-Akt only at low concentration. L, lupeol; S; stigmasterol. (H) Densitometry calculations for TNF-αWestern blot data in (G) using ImageJ normalized to beta-actin. (I) ELISA for secreted TNF-alpha in the conditioned media from HUVECs treated with lupeol or stigmasterol or combination. Means ± S.D. *P value < 0.05 vs. control. n = 3. BV, bevacizumab; L, lupeol; S, stigmasterol; (1), 1 μM; (5), 5 μM.
Fig 4.
TNF-α suppression is necessary for the inhibitory effects of lupeol and stigmasterol on VEGFR-2 signaling and HUVEC network formation.
(A-B) Endothelial network formation assays were performed upon the treatments of each compound at 5 μM with or without TNF-α. Addition of TNF-α drastically improved the ability of HUVEC to form endothelial network, similar to control. (C-D) Quantification of mean network area. (E) Lupeol or stigmasterol treatment suppressed VEGFR-2 expression, which could be rescued by the addition of recombinant TNF-α. Data presented ± S.D. *P value < 0.05, ** P value < 0.01 (n = 4–5).
Fig 5.
Lupeol and stigmasterol did not exhibit significant toxic effects in mice.
(A) Treatment timeline for the toxicity studies. (B) No significant difference in weight change was observed between the control and treatment groups. Data presented as percent weight mean change ± S.D. (n = 5). (C) H&E staining of the livers showed no difference between the control and treatment groups. (D) Sera were also collected from mice for blood chemistry analyses. No significant differences were observed in ALT, AST, RBC, Hemoglobin, WBC, Hematocrit using one-way ANOVA. Group 1, control; Group 2, lupeol (1 mg/kg); Group 3, lupeol (10 mg/kg); Group 4, stigmasterol (1 mg/kg); Group 5, stigmasterol (10 mg/kg); Group 6, lupeol + stigmasterol (1 mg/kg); Group 7, lupeol + stigmasterol (10 mg/kg).
Fig 6.
Treatment of lupeol and stigmasterol reduced tumor angiogenesis, tumor growth, and macrophage recruitment in cholangiocarcinoma xenograft models.
(A) KKU-M213 and (B) RMCCA-1 tumors weighed less in the lupeol or stigmasterol, or combination treatment groups compared with control. Data, as mean tumor weight ± SD; *P value < 0.05 (n = 4–5). (C) CD31 staining of compound-treated KKU-M213 tumors; scale bar, 200 μm. (D) Quantification of CD31-positive areas. Data, mean percentage of the CD31-positive area ± SD; *P < 0.003 (n = 4–5). (E) H&E, Masson’s trichrome, and (F) F4/80 staining of KKU-M213 tumors. Scale bar, 200 μm. L, lupeol; S; stigmasterol.