In vivo and In vitro effects of ethanolic extract of Trigonella foenum-graecum L. seeds on proliferation, angiogenesis and tube formation of endothelial cells

Abstract

The role of angiogenesis in tumor progression and metastasis formation has been well recognized. Recent studies have reported that Trigonella foenum-graecum L. (fenugreek) seed extracts have potential anticancer properties. The current study was planned to investigate the anti-angiogenic activity of hydroalcoholic extract of fenugreek (HAEF) in vitro and in vivo. Effect of HAEF (50-3000 µg/mL) and thalidomide (200-3000 µmol/L), as a positive control, on the viability of human umbilical vein endothelial cells (HUVECs) and 3T3 fibroblast cells was assessed by thiazolyl blue tetrazolium bromide (MTT) assay. Effect of HAEF on vessel-like tube formation by HUVECs was examined in the matrigel-based assay. Furthermore, the chick chorioallantoic membrane (CAM) was used as in vivo model to study the anti-angiogenic effect of HAEF. HAEF, similar to thalidomide, significantly inhibited the viability of HUVECs and 3T3 cells dose-dependently after 24 h. Moreover, both HAEF and thalidomide significantly reduced tube formation by HUVECs in cell culture condition. In CAM model, HAEF and thalidomide caused a significant decline in the number of neovascular points and in the amount of grades 1 and 2 vessels. These findings revealed that fenugreek has cytotoxic and anti-angiogenic effects in vitro and in vivo. Therefore, this medicinal plant can be subjected to further investigations as antitumor agents.

Keywords: Angiogenesis; Cancer; Chorioallantoic membrane; Human umbilical vein endothelial cells; Thalidomide; Trigonella foenum-graecum.

Fig. 1

Effects of the hydroalcoholic extract of fenugreek (HAEF) on the viability of 3T3 fibroblast cells and human umbilical vein endothelial cells (HUVECs). The cells were treated for 24 h with various concentrations of HAEF and the percentage of viable cells was measured by MTT assay. Data are presented as mean ± SEM (n = 6).

Fig. 2

Effects of thalidomide on the viability of 3T3 fibroblast cells and human umbilical vein endothelial cells (HUVECs). The cells were treated for 24 h with various concentrations of thalidomide and the percentage of viable cells was measured by MTT assay. Data are presented as mean ± SEM (n = 6).

Fig. 3

Effects of the hydroalcoholic extract of fenugreek (HAEF) and thalidomide on angiogenesis in the chicken chorioallantoic membrane (CAM) model. Representative photographs of the CAM treated with (A) vehicle (PBS), (B) 1000 μM of thalidomide, (C) 500 μg/mL of HAEF, and (D) 1000 μg/mL of HAEF on day 12. Magnification: × 20.

Fig. 4

Effects of a hydroalcoholic extract of fenugreek (HAEF) and thalidomide on the number of vessels in the chicken chorioallantoic membrane model. Data are presented as mean ± SEM (n = 8). ***P < 0.001 versus related grade in control group (0 µM).

Fig. 5

Effects of a hydroalcoholic extract of fenugreek (HAEF) and thalidomide on neovascular formation in the chicken chorioallantoic membrane model. Data are presented as mean ± SEM (n = 8). ***P < 0.001 versus control group (0 µM).

Fig. 6

Effects of the hydroalcoholic extract of fenugreek (HAEF) and thalidomide on tube formation by human umbilical vein endothelial cells (HUVECs). Representative photographs of the HUVECs treated with (A) vehicle, (B) 1000 μM of thalidomide, (C) 500 μg/mL of HAEF, and (D) 1000 μg/mL of HAEF. Magnification × 40.

Fig. 7

Effects of a hydro-alcoholic extract of fenugreek (HAEF) and thalidomide on the length of the vascular tubes formed by human umbilical vein endothelial cells (HUVECs) in collagen gels. Data are presented as mean ± SEM and calculated from three independent experiments. **P < 0.01 and ***P < 0.001 versus control group (0 µM).