Methanolic Fenugreek Seed Extract Induces p53-Dependent Mitotic Catastrophe in Breast Cancer Cells, Leading to Apoptosis

Abstract

Purpose: The plant Trigonella foenum-graecum, well-known as fenugreek, has been shown to control type-2 diabetes, the level of cholesterol, inflammation of wounds, disorders related to gastrointestinal tracts, and cancer as well. The present study aimed to evaluate the anti-cancer potential of methanolic fenugreek seed extract (FSE) and its possible molecular mechanism of action in breast cancer cells.

Methods: The anticancer potential of FSE was evaluated in MCF-7 and SK-BR3 breast cancer cells through various cellular assays after selecting the IC₁₀, IC₂₅, IC₃₅, and IC₅₀ doses by the cell cytotoxicity assay. Furthermore, the oral acute toxicity of FSE was examined in mice, according to the guidelines of the Organization for Economic Co-operation and Development (OECD).

Results: FSE exhibited dose-dependent cytotoxicity, as the IC₅₀ was found to be 150 and 40 μg/mL for MCF-7 and SK-BR3 breast cancer cells, respectively. The cytological observations showed the typical apoptotic morphology in both of the breast cancer cells upon treatment with FSE, as it inhibited the migration and adhesion, in a dose-dependent manner. The flow cytometry analysis revealed that FSE induced a significant shift from G₂/M, and polyploidy (>G) at higher concentrations that suggested the activation of p53-mediated mitotic catastrophe, consequently leading to apoptosis. FSE induced a significant increase in the mitochondrial depolarization, ROS as well as a Bax/Bcl-2 ratio, and also exhibited the mitochondrial associated p53 signaling pathway. The in vivo acute toxicity data revealed that the oral administration of FSE did not induce any toxic effect in mice.

Conclusion: This study, for the first time, reports the mechanistic details of the anti-cancer potential of FSE. It requires a detailed analysis to understand the effect of FSE to induce the apoptosis through the multiple signaling pathways at varying concentrations. The nontoxic effect of FSE in mice suggests to utilize it safely for pharmaceutical formulations in different cancer systems.

Keywords: apoptosis; breast cancer cells; fenugreek seed extract; mitochondria-associated pathway; mitotic catastrophe; oral acute toxicity; p53 signaling.

Figure 1

A typical chromatogram of the bioactive compounds present in FSE.

Figure 2

Concentration of polyphenols in FSE and antioxidant activity.

Figure 3

The analysis of cell viability by cell cytotoxicity assay.

Figure 4

FSE induced morphological changes in breast cancer cells.

Figure 5

FSE inhibited metastatic properties of breast cancer cells by wound healing/scratch motility assay.

Figure 6

Effect of FSE on adhesion of breast cancer cells by crystal violet staining assay.

Figure 7

FSE induced apoptosis using Annexin V-FITC/PI by flow cytometry.

Figure 8

Effect of FSE on cell cycle distribution using PI by flow cytometry.

Figure 9

Effect of FSE on mitochondrial membrane potential (MMP or ΔΨm). (A) MCF-7 and (B) SK-BR3 cells (2.5 × 10⁵ cells for flow cytometry and 5 × 10⁴ cells for confocal microscopy) were grown, followed by the treatment with the specified concentrations for 48 hours in 6 well and 24 well plates, respectively. The ΔΨm of the cells was measured quantitatively and qualitatively using TMRE by the flow cytometry and confocal microscopy (Magnification, 300X), correspondingly. The representative images and analyses of ΔΨm as ± SEM of three independent experiments. Statistical differences were analyzed by Ordinary one-way ANOVA, Tukey’s multiple comparison test. ^#Significant difference between each treated group. The FCCP (20 µM) was added 10 minutes prior to staining with TMRE in FSE 0 (+ve Ctrl) treated cells.

Figure 10

Effect of FSE on the generation of reactive oxygen species (ROS).

Figure 11

Effect of FSE on p53 signaling using the Western blotting.

Figure 12

Effect of FSE on body weight and relative organs weight (ROW).

Figure 13

Effect of FSE on biochemical parameters.

Figure 14

Schematic illustration of FSE induced dose-dependent signaling pathways.