Lupeol and stigmasterol suppress tumor angiogenesis and inhibit cholangiocarcinoma growth in mice via downregulation of tumor necrosis factor-α

<div><p>Lupeol and stigmasterol, major phytosterols in various herbal plants, possess anti-inflammatory activities and have been proposed as candidates for anti-cancer agents, but their molecular mechanisms are still unclear. Here, we investigated the effects of lupeol and stigmasterol on tumor and endothelial cells in vitro and their anti-cancer activities in vivo. Our results demonstrated that lupeol and stigmasterol suppressed cell viability, migration, and morphogenesis of human umbilical vein endothelial cells (HUVECs) but not cholangiocarcinoma (CCA) cells. Expression analyses showed that the treatment of both compounds significantly reduced the transcript level of tumor necrosis factor-α (TNF-α), and Western blot analyses further revealed a decrease in downstream effector levels of VEGFR-2 signaling, including phosphorylated forms of Src, Akt, PCL, and FAK, which were rescued by TNF-α treatment. In vivo, lupeol and stigmasterol disrupted tumor angiogenesis and reduced the growth of CCA tumor xenografts. Immunohistochemical analyses confirmed a decrease in CD31-positive vessel content and macrophage recruitment upon treatment. These findings indicate that lupeol and stigmasterol effectively target tumor endothelial cells and suppress CCA tumor growth by their anti-inflammatory activities and are attractive candidates for anti-cancer treatment of CCA tumors.</p></div

Treatment of lupeol and stigmasterol reduced tumor angiogenesis, tumor growth, and macrophage recruitment in cholangiocarcinoma xenograft models.

<p>(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.</p

Lupeol and stigmasterol did not exhibit significant toxic effects in mice.

<p>(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).</p

Lupeol and stigmasterol inhibited HUVEC migration and capillary network formation in vitro.

<p>(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.</p

Lupeol, β-sitosterol, and stigmasterol exhibited cytotoxic effects on HUVECs.

<p>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.</p

Lupeol and stigmasterol downregulated TNF-α and inhibited VEGF signaling.

<p>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.</p