Fenugreek inhibits cathepsin G activity and suppresses the progression of malignant phenotypes in MCF-7 cells

Abstract

Spices and herbs, which are derived from natural botanical sources, contain many bioactive compounds and play an important role in human health. The general and specific health benefits of these spices and herbs include anti-inflammatory, antioxidant, and anti-tumorigenic activities. Previously, we showed that cathepsin G, which is a neutrophil-derived serine protease localized in human breast cancer tissues, promotes cancer metastasis via induction of platelet-activating factor acetylhydrolase 1B2 (PAFAH1B2) expression in MCF-7 human breast cancer cells. Therefore, although regulation of cathepsin G activity is thought to be important in human breast cancer progression, no compounds that inhibit the activity have been identified for therapeutic purposes. In this study, we screened 50 spice and herb extracts. Peppermint, clove, Sichuan pepper, and fenugreek exhibited strong inhibitory effects on cathepsin G activity and suppressed cathepsin G-induced MCF-7 cell aggregation.; importantly, fenugreek suppressed the increase in PAFAH1B2 expression. The IC₅₀ of 37.38 μg/mL of fenugreek extract that showed inhibitory effect on cathepsin G-induced malignant progression was 5.87 times lower than the concentration that exerted cytotoxic effect. Interestingly, quercetin and trigonelline contained in fenugreek inhibited cathepsin G activity and suppressed the induction of cell aggregation and PAFAH1B2 expression in human breast cancer cells. These results suggest that quercetin and trigonelline are partly responsible for the inhibitory effect of fenugreek on cathepsin G-induced malignant progression of human breast cancer cells. Our findings provide a new breast cancer treatment strategy targeting cathepsin G, and fenugreek may have synergistic effects when combined with therapeutic drugs.

Keywords: Breast cancer; Cathepsin G; Fenugreek extracts; MCF-7; Platelet-activating factor acetylhydrorase 1B2; Quercetin; Trigonelline.

Graphical abstract

Fig. 1

(A) Cathepsin G (8 nM) was incubated with spice and herb extracts (5 μg/mL) for 30 min, followed by incubation with the substrate for another 60 min. Cathepsin G activity after treatment with the spice and herb extracts was analyzed relative to that in DMSO-treated samples as a control. The fluorescence intensity values are expressed as means ± SD (n = 4). Statistical significance was determined by one-way ANOVA followed by Dunnett's test. ∗p < 0.05; ∗∗p < 0.01.

Fig. 2

(A) Representative images of the morphology of MCF-7 cells incubated concurrently with cathepsin G (8 nM) and spice/herb extracts (peppermint, clove, Sichuan pepper, or fenugreek; 50 μg/mL each) for 24 h. Scale bar = 200 μm. (B) Cell aggregation assay in MCF-7 cells treated with spice/herb extracts (50 μg/mL) and cathepsin G (8 nM). The results are expressed as means ± SD (n = 4). Statistical significance was determined by one-way ANOVA followed by Dunnett's test. ∗∗p < 0.01.

Fig. 3

(A) Western blot analysis of PAFAH1B2 in MCF-7 cells treated with cathepsin G (8 nM) and the indicated spice and herb extracts (50 μg/mL) for 24 h (upper panels). Densitometry analysis of Western blot using image analysis software Fiji. Data are shown as mean ± SD (N = 3). Analyses were performed three independent experiments. Represent the relative protein levels were normalized against β-actin levels as fold changes relative to the cathepsin G treatment. (B) Western blot analysis of PAFAH1B2 in MCF-7 cells treated with cathepsin G (8 nM) and fenugreek extract (0, 25, 50, and 100 μg/mL) for 24 h. (C) MCF-7 cell aggregation assay using cathepsin G (8 nM) and fenugreek extract (0, 12.5, 25, 50, 100, 200, and 400 μg/mL). The results are expressed as means ± SD (n = 4). IC₅₀ nonlinear regression curve fit of cell aggregation (%) versus logarithm (con. μg/mL) graph was created using the GraphPad prism software. Statistical significance was determined by one-way ANOVA followed by Dunnett's test. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.

Fig. 4

MCF-7 cells were treated with various concentrations of fenugreek for 24 h. Cell viability was measured using the Cell Counting Kit-8 assay. The results are expressed as means ± SD (n = 4). LC₅₀ nonlinear regression curve fit of cell viability (%) versus logarithm (con. μg/mL) graph was created using the GraphPad prism software. Statistical significance was determined by one-way ANOVA followed by Dunnett's test. ∗∗p < 0.01.

Fig. 5

(A) Cathepsin G (8 nM) was incubated with fenugreek extracts (5 μg/mL), quercetin, scopoletin, trigonelline, 4-HIL, and diosgenin (2 mM), respectively, for 30 min, followed by incubation with the substrate for another 60 min. Cathepsin G activity after treatment with the spice and herb extracts was analyzed relative to that in DMSO-treated samples as a control. The fluorescence intensity values are expressed as means ± SD (n = 4). Statistical significance was determined by one-way ANOVA followed by Dunnett's test. ∗∗p < 0.01. (B) Representative images of the morphology of MCF-7 cells incubated concurrently with cathepsin G (8 nM) and fenugreek extracts (5 μg/mL), quercetin, scopoletin, trigonelline, 4-HIL, and diosgenin (2 mM), respectively, for 24 h. Scale bar = 200 μm. (C) Total protein extracted from the cells was analyzed by western blotting.