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.

Graphical abstract

Highlights

• •Peppermint, clove, Chinese cabbage, and fenugreek extracts inhibited cathepsin G activity.
• •Fenugreek extracts inhibited MCF-7 cell aggregation induced by cathepsin G.
• •Fenugreek extracts inhibited the increase in PAFAH1B2 expression by cathepsin G.
• •Quercetin and trigonelline contained in fenugreek extracts inhibit cathepsin G activity and suppress the progression of malignant phenotype in MCF-7 cells.

1. Introduction

Breast cancer is a common cancer worldwide and a leading cause of cancer mortality in women [1]. In 2022, there were an estimated 20 million new cases of breast cancer and 9.7 million deaths [2]. The incidence of breast cancer has decreased recently with the advancement of technology and new diagnostic and therapeutic methods; however, the high cost and long treatment duration of breast cancer drugs can lead to financial problems for many patients; thus, effective treatment options, including new drugs, are needed.

The neutrophil-derived serine protease cathepsin G is highly localized in approximately 60 % of breast cancer patients, especially those with invasive ductal carcinoma, suggesting that its expression is associated with malignant tumors [3]. Previously, we confirmed the presence of high levels of cathepsin G in tissue sections from breast cancer compared with normal mammary glands [4]. Cathepsin G is expressed in polymorphonuclear neutrophils, which are the first to be recruited to sites of infection or inflammation, and this molecule is secreted extracellularly by cell activation [5]. Cathepsin G controls inflammatory responses by stimulating the production of cytokines and chemokines involved in the activation and recruitment of immune cells to sites of pathogen and/or tissue damage [5,6]. On the other hand, previously reported effects of cathepsin G on human breast cancer cells showed that MCF-7, classified as a non-invasive human breast cancer cell line, acquired metastatic abilities such as aggregation, invasion, and migration [4,[7], [8], [9]]. Furthermore, we revealed that cathepsin G increases the expression of the oncogene platelet-activating factor acetylhydrolase 1B2 (PAFAH1B2) at the RNA and protein levels, and that siRNA-mediated knockdown of PAFAH1B2 suppresses malignant progression. In highly malignant MDA-MB-231 cells, cathepsin G-dependent cell aggregation and motility were not observed [10], while in T47D cells, which are of a similar subtype to MCF-7 cells, aggregates formed via the same mechanism as in MCF-7 cells [11]. These results suggest that the action of cathepsin G is not affected by differences in the malignancy of breast cancer.

PAFAH1B2 is involved in anaphylaxis and inflammation and is a driver of various carcinogenic activities. In human breast cancer, PAFAH1B2 has been identified as a key metabolic driver of pathogenesis and is highly expressed in breast cancer cell lines and primary human breast tumors [12]. Similarly, we observed higher expression of PAFAH1B2 in human breast cancer cells compared with normal breast ductal epithelial cells by immunohistological staining [4]. Inactivation of PAFAH1B2 using selective inhibitors impaired cancer cell survival by promoting tumor-suppressive signaling lipids [13]. These reports suggest that increased expression of PAFAH1B2 is correlated with the malignancy of human breast cancer tissues.

Spices and herbs are an integral part of culinary culture worldwide and have long been used for flavor, aroma, coloring, and preservatives. In addition, many spices and herbs have properties associated with a reduced risk of developing chronic diseases due to potential protection against cardiovascular diseases, neurodegenerative conditions, chronic inflammation, cancer, obesity, and diabetes, as well as antimicrobial properties [[14], [15], [16], [17], [18]]. Such effects are caused by various biologically active substances contained in spices and herbs, and many research reports are currently being published.

In this study, we searched for spice and herb extracts that suppress cathepsin G activity and investigated their effects on cell aggregation and PAFAH1B2 expression in MCF-7 cells. Our finding is a study that focused on a molecular mechanism that has not been targeted as an anticancer drug.

2. Materials and methods

2.1. Cell culture and materials

The human breast cancer cell line MCF-7 and human leukemia cell line HL-60 were maintained in our laboratory. Both cell lines were cultured in RPMI-1640 medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10 % heat-inactivated fetal bovine serum and 50 mg/mL penicillin/streptomycin in 10-cm tissue culture dishes at 37 °C in 5 % CO₂. MCF-7 cells were harvested using a trypsin–EDTA solution (Thermo Fisher Scientific). Cathepsin G, purified from human neutrophils (95 % purity), was purchased from BioCentrum (Krakow, Poland). Elastase, human neutrophils, human pancreas chymotrypsin, and human pancreas trypsin were purchased from Sigma-Aldrich (St Louis, MO, USA). Trigonelline, scopoletin, 4-hydroxyisoleucine (4-HIL), diosgenin and quercetin were obtained from Selleck (Shanghai, China).

2.2. Spice and herb extracts

Spices and herbs were purchased from a local spice shop in Japan. The sample preparation was performed as described previously with slight modifications [19]. Briefly, crude drugs (10 g) were subjected to reflux in 100 mL 70 % ethanol for 1 h, and the extracts were dried by evaporation. Subsequently, the samples were dissolved in dimethyl sulfoxide to attain a concentration of 10 mg/mL and were stored at −30 or −80 °C before use. In total, 50 spice and herb extracts were tested for their effects against cathepsin G activity.

2.3. Measurement of cathepsin G activity

Measurement of cathepsin G activity using fluorescence resonance energy transfer (FRET) was performed according to the method described by Korkmaz et al. [20]. The fluorescence-quenching substrate for human cathepsin G (Abz (o-aminobenzoic acid)-Glu-Pro-Phe-Trp-Glu-Asp-Gln-EDDnp (N-(2, 4-dinitrophenyl-)ethylenediamine) was purchased from Peptide Institute (Osaka, Japan). Cathepsin G (8 nM) and spice and herb extracts (5 μg/mL) were added to Milli-Q water to a total volume of 100 μL and then incubated at 37 °C for 30 min. After adding 1 μL 1 mM FRET substrate to the mixture, it was suspended and incubated in a 96-well plate at 37 °C for 60 min in the dark. Fluorescence intensity was measured using a fluorescent microplate reader (SpectraMax i3x, Molecular Devices, Sunnyvale, CA, USA) at an excitation wavelength of 320 nm and emission wavelength of 420 nm.

2.4. Cell aggregation assay

Aggregation of MCF-7 cells was assessed as described previously [4,9]. MCF-7 cells (2 × 10⁴) were seeded in 96-well plates and cultured for 24 h. The cells were then washed with serum-free RPMI-1640 medium for three times, and the medium was replaced with RPMI-1640 containing 1 % BSA and cathepsin G (8 or 32 nM). After cultivation for another 24 h, the culture medium was discarded, and the plate was vigorously tapped on paper towels for 10 times to eliminate the loosely attached cell spheroids. The remaining cells were dried at room temperature and stained with 0.1 % crystal violet in PBS for 10 min. Subsequently, the plate was extensively washed with tap water for three times. The plate was dried at room temperature, and the crystal violet in the residual cells was solubilized using 100 μL 0.5 % SDS. The optical density at 595 nm was measured using the Powerscan HT plate reader (DS Pharma Biomedical, Osaka, Japan). The absorbance derived from cells without cathepsin G was calculated assuming an adhesion rate of 100 %, and its reciprocal was taken as the aggregation rate.

2.5. Protein preparation and Western blot analysis

Cellular proteins were extracted and analyzed as described previously [21,22]. Briefly, cells were washed three times with ice-cold PBS and lysed in RIPA buffer (Cell Signaling Technology, Danvers, MA) for 1 h. Cells were then transferred to a 1.5 mL tube and centrifuged. The supernatant was transferred to a new tube, and the protein concentration was quantified using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Proteins (10 μg) were heated in SDS sample loading buffer at 70 °C for 10 min and loaded onto NuPage 4–12 % Bis-Tris gels (Thermo Fisher Scientific). After electrophoresis, proteins were transferred to a PVDF membrane using the iBlot 2 transfer system (Thermo Fisher Scientific). The membrane was washed with PBST (PBS with 0.1 % Tween 20), blocked overnight with ImmunoBlock solution (KAC Co., Ltd., Kyoto, Japan), and then incubated with a rabbit anti-human PAFAH1B2 (Abcam, Cambridge, UK; 1:2000 dilution) or rabbit anti-human β-actin (CST; 1:2000 dilution) antibody. After washing with PBST, the membrane was incubated for 1 h with HRP-conjugated goat anti-rabbit IgG secondary antibody (Thermo Fisher Scientific; 1:1500 dilution). Signals were developed using Amersham ECL Prime (GE Healthcare, Buckinghamshire, UK), and the images were scanned using the Amersham Imager 680 RGB (GE Healthcare). Densitometric analysis of specific bands was performed with Image J/Fiji software.

2.6. Cell viability assay

MCF-7 cells (2 × 10⁴) were seeded in 96-well plates and incubated for 24 h. Then, the culture medium was replaced with medium containing various concentrations of the spice and herb extracts, and the cells were incubated for another 24 h. Cell viability was assessed by the Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Inc, Kumamoto Japan). After adding 10 μL CCK-8 reagent per well, the cells were incubated for 1 h at 37 °C in a CO₂ incubator. Cell viability was quantified spectrophotometrically at a wavelength of 450 nm using a microplate reader (Powerscan HT plate reader, DS Pharma Biomedical).

2.7. Statistical analysis

Statistical analyses were performed using GraphPad PRISM 9 (GraphPad Software). For comparisons, one-way analysis of variance (ANOVA) followed by Dunnett's test were performed. The results are expressed as means ± SD. For nonlinear regression analysis (least squares method), the concentrations used were transformed to logarithmic scale to determine the IC₅₀ values and LC₅₀ value.

3. Results

3.1. Screening assay for spice and herb extracts that inhibit cathepsin G activity

To identify spice(s) and herb(s) that inhibit cathepsin G activity, we examined 50 spice and herb extracts kept in our laboratory. Since it has been reported that HL-60 human promyelocytic leukemia cells express cathepsin G [23], we evaluated the effect of spice and herb extracts on cathepsin G activity using HL-60 cell lysates. To measure cathepsin G activity, we used a FRET-based probe containing Abz fluorescence and EDDnp quenchers tethered by the cathepsin G substrate EPFWEDQ. Therefore, when cathepsin G cleaves the peptide region, fluorescence is emitted.

As a result, many of the spices and herbs suppressed cathepsin G activity derived from HL-60 cells, and nine samples were found to inhibit the activity by 40 % or more (Supplementary Fig. 1). Since HL-60 cells express various enzymes in addition to cathepsin G, the FRET substrate may have been affected by other enzymes. Therefore, we evaluated the inhibitory effect of the spice and herb extracts on pure cathepsin G; we found that four of the nine samples (peppermint, cloves, Sichuan pepper, and fenugreek extracts) inhibited cathepsin G activity by more than 30 % (Fig. 1). These results suggest that the difference in the inhibitory effect of the samples on cathepsin G activity is due to the presence of a factor in HL-60 lysates that cleaves the FRET substrate. To confirm the specificity of the cathepsin G substrate used in this experiment, we treated the cells with intracellular proteases including trypsin, chymotrypsin, and neutrophil elastase at the same concentration as that of cathepsin G. We confirmed that cathepsin G degraded the substrate in a concentration-dependent manner, but the other enzymes did not (Supplementary Fig. 2).

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.

3.2. Effect of peppermint, clove, Sichuan pepper, and fenugreek extracts on cathepsin G-induced MCF-7 cell aggregation

Cathepsin G contributes to the formation of three-dimensional (3D) multicellular spheroids in MCF-7 cells by inducing aggregation [7,9]. In the vascular endothelium, adhesion of homotypic tumor cells leads to formation of multicellular malignant cell aggregates. Cancer cells with high metastatic potential exhibit a greater ability to form homotypic aggregates compared with their low metastatic counterparts. Since cathepsin G activity is essential for the formation of MCF-7 cell aggregates, we investigated whether spice and herb extracts have an inhibitory effect on cell aggregation. The cathepsin G concentration used in this experiment was the concentration that showed the highest aggregation rate in MCF-7 cells in our previous study [4]. We first evaluated morphological changes in MCF-7 cells following the addition of cathepsin G. Cathepsin G alone induced significant aggregation of MCF-7 cells compared with DMSO-treated control cells at 24 h (Fig. 2A). It has been confirmed that the DMSO concentration (1 %) used in the experiment does not induce aggregation of MCF-7 cells. However, cathepsin G-induced aggregate formation was abolished when cells were treated with extracts of peppermint, clove, Sichuan pepper, and fenugreek for 24 h (Fig. 2A).

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.

To quantitate the extent of MCF-7 cell aggregation, we performed a cell aggregation assay. Consistent with microscopic observations, extracts of peppermint, clove, Sichuan pepper, and fenugreek almost completely inhibited cathepsin G-induced cell aggregation (Fig. 2B). Taken together, these results suggest that some spices and herbs inhibit cathepsin G activity and thereby suppress the aggregation of MCF-7 breast cancer cells induced by cathepsin G.

3.3. Effect of peppermint, clove, Sichuan pepper, and fenugreek extracts on cathepsin G-induced PAFAH1B2 expression in MCF-7 cells

PAFAH1B2 is highly expressed in human breast cancer and is an important indicator of cancer malignancy. To further clarify the effect of spice and herb extracts on cathepsin G activity in MCF-7 cells, we investigated the effect of peppermint, clove, Sichuan pepper, and fenugreek extracts on PAFAH1B2 expression. Western blot analysis confirmed that cathepsin G increased PAFAH1B2 expression by 24 h (Fig. 3A). Among the four extracts, only fenugreek suppressed the cathepsin G-induced increase in PAFAH1B2 expression, while peppermint, clove, and Sichuan pepper extracts had no effect (Fig. 3A). These results suggest that the suppression of cathepsin G-induced MCF-7 cell aggregation by fenugreek was due, at least in part, to its inhibitory effect on PAFAH1B2 expression. On the other hand, inhibition of cathepsin G-induced MCF-7 cell aggregation by peppermint, clove, and Sichuan pepper extracts is thought to be mediated by different mechanisms. The inhibitory effect of fenugreek on cathepsin G-induced PAFAH1B2 expression was evident at 50 μg/mL and higher concentrations (Fig. 3B).

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.

When MCF-7 cells were cultured with cathepsin G in the presence of various fenugreek extract concentrations, cell aggregation was inhibited in a concentration-dependent manner starting at 25 μg/mL, with an IC₅₀ of 37.38 μg/mL (Fig. 3C). According to these results, the inhibitory effect of fenugreek extract on cathepsin G-induced MCF-7 cell aggregation is due, at least in part, to inhibition of PAFAH1B2 expression.

We further investigated the potential cytotoxic effects of fenugreek extract on MCF-7 cells. MCF-7 cells were incubated with fenugreek extract for 24 h, and the final concentration of fenugreek was 0–400 μg/mL. The cytotoxic effect of fenugreek extract was not evident at concentrations of 100 μg/mL or lower; however, concentrations of 200 μg/mL and above showed significant cytotoxicity, with an LC₅₀ of 219.4 μg/mL (Fig. 4). Thus, 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.

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.

3.4. Quercetin and trigonelline contained in fenugreek extracts inhibit cathepsin G activity and suppress the progression of malignant phenotype in MCF-7 cells

Fenugreek extracts have been reported to contain a variety of bioactive substances [24]. Therefore, we attempted to identify components in fenugreek extract that exhibit inhibition of cathepsin G activity. In the experiments, quercetin, scopoletin, trigonelline, 4-HIL, and diosgenin were used as the main compounds in fenugreek extract. Results of cathepsin G activity assays showed that quercetin and trigonelline inhibited cathepsin G activity, as well as fenugreek, while scopoletin, 4-HIL and diosgenin had no such effect (Fig. 5A). Then, we evaluated the inhibitory effect of each compound on cathepsin G-induced MCF-7 cell aggregation. Quercetin and trigonelline inhibited cathepsin G-induced MCF-7 cell aggregation to the same degree as fenugreek, but scopoletin, 4-HIL, and diosgenin had no such effect (Fig. 5B). Western blotting analysis also revealed that quercetin and trigonelline completely suppressed cathepsin G-induced PAFAH1B2 expression consistent with fenugreek (Fig. 5C). These results suggest that quercetin and trigonelline in fenugreek extract may be responsible for the inhibitory effect of cathepsin G activity and the suppression of cathepsin G-induced malignancy in human breast cancer cells.

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.

4. Discussion

We previously reported that cathepsin G increases metastatic ability in MCF-7 cells by promoting aggregation, migration, and invasion along with elevated expression of PAFAH1B2, an oncogene [4]. In this study, we demonstrated that fenugreek extract suppresses PAFAH1B2 expression by inhibiting cathepsin G activity. Furthermore, fenugreek suppressed cathepsin G-induced malignant progression of MCF-7 cells. Fenugreek is a medicinal herb that has garnered considerable interest in the pharmaceutical, nutraceutical and functional food industries due to its potential medicinal properties and abundance bioactive compounds. These compounds are effective against lifestyle-related diseases such as diabetes and arteriosclerosis because they stimulate the pancreas to secrete insulin [25], as well as inhibit major carbohydrate hydrolases [26]. Fenugreek is also rich in antioxidants such as vitamin C, phenolic compounds, and essential omega-6 fatty acids (linoleic acid) and is thus recognized as an excellent antioxidant [27,28]. In addition, the antioxidant effect of fenugreek is one of the factors that suppresses the malignant progression of breast cancer. In a rat model of breast cancer induced by N-methyl-N-nitrosourea, a mammary gland-specific carcinogen, cell membranes were destroyed by increased serum lipid peroxidation via decreased concentrations of antioxidants such as superoxide dismutase and catalase. Fenugreek extract exerts strong antioxidant properties and inhibits lactate dehydrogenase activity, preventing loss of the functional integrity of cell membranes [29]. Fenugreek extract promotes antioxidant effects and functional decline due to mitochondrial DNA damage and inhibits MCF-7 cell proliferation and metastasis [30,31]. These results are consistent with our findings that fenugreek protects against the malignant progression of breast cancer cells.

On the other hand, fenugreek concentrations above 200 μg/mL showed cytotoxicity in MCF-7 cells. In this regard, there are multiple reports that fenugreek defends against malignant progression by inducing apoptosis. In vivo, in a rat model of 7,12-dimethylbenz [15]anthracene-induced breast cancer, feeding fenugreek extract dissolved in olive oil induced nuclear fragmentation and vacuolation, which are hallmarks of alternative death processes in mammary cells [32]. Real-time PCR and TUNEL assay showed that detection of fragmented DNA and expression of pro-apoptotic genes, such as caspases 3, 8, and 9, p53, Fas, FADD, Bax, and Bak, increased in time- and dose-dependent manners in human breast cancer cells [[33], [34], [35]]. Since these effects were observed in the triple-negative breast cancer cell line MDA-MB-231, one of the most aggressive, therapy-resistant, and metastatic tumor cell lines, as well as in non-metastatic MCF-7 cells and metastatic SK-BR3 cells, they are thought to be independent of breast cancer malignancy. In MTT and neutral red uptake (NRU) assays, 500 μg/mL fenugreek showed cytotoxicity due to apoptosis in Hep-2, MCF-7, WISH, and MDA-MB231 cancer cells, but not in MCF10A and Vero normal breast cells [36,37]. Importantly, fenugreek extract-induced apoptosis occurs more strongly in cancer cells than in normal cells. Thus, the cytotoxicity induced by fenugreek observed in this study could be due to induction of apoptosis in MCF-7 cells. Further study is needed to confirm this.

The suppression of cathepsin G-induced malignancy by fenugreek extract in MCF-7 cells is thought to be due to its strong inhibitory effect on cathepsin G activity. Our previous report showed that cell aggregation, an indicator of malignancy in human breast cancer cells, is E-cadherin-dependent 3D multicellular spheroid formation, which requires cathepsin G activity [9,10]. Such cell aggregation is inhibited by treatment with phenylmethylsulfonyl fluoride, an irreversible serine protease inhibitor [10]. Additionally, cell lysates derived from RBL-2H3 cells constitutively expressing cathepsin G induced MCF-7 cell aggregation, whereas lysates derived from S195G cathepsin G-expressing cells (enzymatically inactive mutant) did not [38]. Although the mechanism of cathepsin G inhibition by fenugreek is unknown, fenugreek has been reported to have inhibitory effects on several proteases. The main protease (M^pro) of SARS-CoV-2 plays a crucial role in host cell entry, viral replication, and transcription processes. Sen et al. found that compounds derived from fenugreek form a complex with M^pro to inhibit its activity [39]. A compound with a Kunitz-type domain isolated from fenugreek seeds showed high trypsin and chymotrypsin inhibitory activity [40]. Furthermore, in human glioblastoma T98G cells, fenugreek extract inhibited the activity of matrix metalloproteinases 2 and 9, thereby suppressing selective degradation of the basement membrane [41]. These results suggest that fenugreek extract contains compounds that may inhibit cathepsin G activity.

Fenugreek extract is particularly rich in bioactive compounds such as galactomannans, flavonoids, sapogenin, saponins, alkaloids, phenolic compounds, and lipids, and they have various pharmacological properties [42]. Therefore, we performed further experiments to confirm the effects of fenugreek using its main compounds: quercetin, scopoletin, trigonelline, 4-HIL, and diosgenin (Fig. 5). The results showed that quercetin and trigonelline may act defensively against the malignant progression of human breast cancer cells via inhibition of cathepsin G activity. Quercetin, a phenolic compound, has been reported to have potent anti-cancer effects, inhibiting the invasion and angiogenesis of esophageal cancer cells [43], inducing autophagy and apoptosis in lung cancer cells [44], and preventing the metastasis of triple-negative breast cancer [45]. In vivo experiments showed that quercetin reduced breast tumor volume and weight in a mouse 4T1 breast cancer model [46]. Trigonelline has been shown to have anti-cancer effects partially via inhibition of the hyperactivation of nuclear factor erythroid 2-related factor 2 (Nrf2) [47]. These results suggest that quercetin and trigonelline may be involved in the antitumor activity of fenugreek, and that at least part via, at least in part, inhibition of cathepsin G activity.

In this study, we demonstrated that fenugreek extract suppresses the expression of PAFAH1B2 and inhibits cathepsin G activity. Inhibition of cathepsin G activity was found for not only fenugreek but also peppermint, clove, and Sichuan pepper. Although these four spice/herb extracts similarly suppressed cathepsin G-induced MCF-7 cell aggregation, only fenugreek extract suppressed PAFAH1B2 expression. In our previous report, PAFAH1B2-knockdown MCF-7 cells exhibited suppressed cathepsin G-induced cell aggregation, but no such effect was observed with high concentrations of cathepsin G [4]. Additionally, CV-3988, a PAF receptor antagonist, induced invasion of MCF-7 cells, but the effect was weaker compared with cathepsin G addition. These results indicate that cathepsin G-mediated aggregation of MCF-7 cells may be due to other factors in addition to induction of PAFAH1B2 expression. Future studies elucidating the mechanism by which cathepsin G regulates PAFAH1B2 expression in MCF-7 cells are required.

5. Conclusion

In conclusion, we demonstrated that quercetin and trigonelline contained in fenugreek extract have a protective effect against cathepsin G-induced promotion of malignancy in MCF-7 cells. The mechanism involves suppression of PAFAH1B2 expression via inhibition of cathepsin G activity. Therefore, combining fenugreek extracts with breast cancer medications may enhance anticancer effects and reduce the treatment period and side effects. In the future, we would like to investigate the structure-activity relationship not only quercetin and trigonelline, but also of their analogous.

CRediT authorship contribution statement

Kazunari Tanigawa: Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Resources, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization. Takashi Tanikawa: Writing – review & editing, Resources, Methodology, Investigation, Conceptualization. Masashi Kitamura: Writing – review & editing, Resources. Yasuhiro Hayashi: Writing – review & editing. Mitsuo Kiriya: Writing – review & editing. Yasuhiro Nakamura: Writing – review & editing. Akira Kawashima: Writing – review & editing. Yoko Fujiwara: Writing – review & editing. Riyo Morimoto-Kamata: Writing – review & editing. Naoki Ohkura: Writing – review & editing. Satoru Yui: Writing – review & editing. Ken Karasawa: Writing – review & editing. Ryosuke Nakamura: Writing – review & editing, Supervision, Funding acquisition. Koichi Suzuki: Writing – review & editing, Supervision, Funding acquisition.

Data availability

All data are contained within the manuscript or supplemental information.

Declaration of generative AI and AI-assisted technologies in the writing process

During the development of this manuscript, no generative AI or AI-assisted technologies were used in the writing or editing process.

Funding sources

This study was supported by Grant-in-Aid for Scientific Research C (KAKENHI) (Grant Number: 21K07012 to K.T.) and Incubation grants for Teikyo university (Grant Number: 23-91 to K.T.).

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

The following is/are the supplementary data to this article.