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Journal of Inflammation Research Volume 14, 2021 - Issue
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Original Research

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

Faris A Alrumaihi1 Department of Medical Laboratories, College of Applied Medical Sciences, Qassim University, Buraydah, Saudi ArabiaORCID Iconhttps://orcid.org/0000-0002-0850-5500
ORCID Icon,
Masood A Khan2 Department of Basic Health Sciences, College of Applied Medical Sciences, Qassim University, Buraydah, Saudi ArabiaORCID Iconhttps://orcid.org/0000-0003-4031-4189
ORCID Icon,
Khaled S Allemailem1 Department of Medical Laboratories, College of Applied Medical Sciences, Qassim University, Buraydah, Saudi ArabiaORCID Iconhttps://orcid.org/0000-0002-6486-9835
ORCID Icon,
Mohammed A Alsahli1 Department of Medical Laboratories, College of Applied Medical Sciences, Qassim University, Buraydah, Saudi Arabia
,
Ahmad Almatroudi1 Department of Medical Laboratories, College of Applied Medical Sciences, Qassim University, Buraydah, Saudi ArabiaORCID Iconhttps://orcid.org/0000-0002-1491-6402
ORCID Icon,
Hina Younus3 Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh, IndiaORCID Iconhttps://orcid.org/0000-0003-3066-1312
ORCID Icon,
Sultan A Alsuhaibani1 Department of Medical Laboratories, College of Applied Medical Sciences, Qassim University, Buraydah, Saudi Arabia
,
Mohammad Algahtani4 Department of Laboratory Medicine, Security Forces Hospital, Mecca, Saudi Arabia
&
Arif Khan2 Department of Basic Health Sciences, College of Applied Medical Sciences, Qassim University, Buraydah, Saudi ArabiaCorrespondence[email protected]
ORCID Iconhttps://orcid.org/0000-0002-4528-2668
ORCID Icon show all
Pages 1511-1535 | Published online: 16 Apr 2021

Figures & data

Table 1 Biologically Active Chemical Compounds of FSE by GC-MS Analysis

Figure 1 A typical chromatogram of the bioactive compounds present in FSE.

Figure 1 A typical chromatogram of the bioactive compounds present in FSE.

Figure 2 Concentration of polyphenols in FSE and antioxidant activity.

Notes: (A) TPC and TFC. (B) The antioxidant (DPPH scavenging) activity as percentage of DPPH radical inhibition and IC50 value (μg/mL). The data represented mean value cells ± SEM of three independent experiments.
Abbreviations: FSE, fenugreek seed extract; TPC, total phenolic contents; TFC, total flavonoid contents; DPPH, 2,2-diphenyl-1-picrylhydrazyl; SEM, standard error mean.
Figure 2 Concentration of polyphenols in FSE and antioxidant activity.

Figure 3 The analysis of cell viability by cell cytotoxicity assay.

Notes: (A) MCF-7 and (B) SK–BR3 cells (1 × 104) were treated with a range of FSE doses (0-250 μg/mL) for 48 hours in 96 well plates, and the percentage of cell viability was measured by cell cytotoxicity assay kit. Data represented mean percentages of viable cells ± SEM of three independent experiments.
Figure 3 The analysis of cell viability by cell cytotoxicity assay.

Figure 4 FSE induced morphological changes in breast cancer cells.

Notes: (A) MCF-7 and (B) SK-BR3 cells (1 × 105) were treated with indicated concentrations of FSE for IC10, IC25, IC35, and IC50 received from cell cytotoxicity assay for 48 hours in 12 well plates. The representative photomicrographs were obtained after 48 hours of the treatment using Optika inverted microscope (Magnification, 100X) and recorded using TBS-3 software. The arrows indicate the morphological changes in the cells as 1. Cell shrinkage, 2. Membrane blebbing, 3. Apoptotic bodies, 4. Echinoid spikes, and 5. Pyknotic body.
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.

Notes: (A) MCF-7 and (B) SK-BR3 cells (1 × 105) were grown for 24 hours, followed by the treatment with selected concentrations as mentioned above for 48 hours in a 12 well plate. Photomicrographs were obtained after 0 and 48 hours of treatment using Optika inverted microscope (Magnification, 40X) and recorded using TBS-3 software. The representative images and analyses of wound healing assay 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.
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.

Notes: (A) MCF-7 and (B) SK-BR3 cells (1 × 105) were grown, followed by the treatment with selected concentrations, as mentioned above, for 48 hours in a 12 well plate. The representative images and density index of viable cells after being stained with 0.2% crystal violet 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.
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.

Notes: (A) MCF-7 and (B) SK-BR3 cells (2.5 × 105) were grown followed by the treatment with selected concentrations FSE, as mentioned above, for 48 hours in a 6 well plate. The representative changes in the proportion of cells were analyzed after staining the cells with Annexin V-FITC/PI by flow cytometry. The percentage of the viable (lower left; Annexin V–/PI–) necrotic (upper left; Annexin V–/PI+), early apoptosis (lower right; Annexin V+/PI–), and late apoptosis (upper right; Annexin V+/PI+) stages of FSE treated cells were evaluated by FlowJo software v10.7. The representative analyses of apoptosis as ± SEM of three independent experiments. Statistical differences were analyzed by Two-way ANOVA, Tukey’s multiple comparison test. #Significant difference between each treated group, *significant difference within the indicated groups.
Abbreviations: FITC, fluorescein isothiocyanate; PI, propidium iodide.
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.

Notes: (A) MCF-7 and (B) SK-BR3 cells (2.5 × 105) were grown, followed by the treatment with the indicated concentrations for 48 hours in a 6 well plate. The changes in the cell cycle distribution using PI were analyzed by flow cytometry. The percentage of G1, S, G2/M, and >G2 phase distribution of FSE exposed cells was measured by FlowJo software v10.7. The representative analyses of the distribution of cells in the phases as ± SEM of three independent experiments. Statistical differences were analyzed by Two-way ANOVA, Tukey’s multiple comparison test. #Significant difference between each treated group, *significant difference within the indicated groups.
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.

Abbreviations: TMRE, tetramethylrhodamine, ethyl ester; FCCP, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone.
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).

Notes: (A) MCF-7 and (B) SK-BR3 cells (2.5 × 105 cells for flow cytometry and 5 X 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 induction of ROS by FSE in the cells was measured quantitatively and qualitatively using DCFDA by flow cytometry and confocal microscopy (Magnification, 300X), correspondingly. The representative images and analyses of ROS generation as ± SEM of three independent experiments. Statistical differences were analyzed by Ordinary one-way ANOVA, Tukey’s multiple comparison test. *Significant difference vs FSE (+ve Ctrl), **significant difference vs FSE 10 and 40, ***significant difference vs FSE 80, #significant difference between each treated group. The TBHP (50 µM) was added 4 hours prior to staining with DCFDA in FSE 0 (+ve Ctrl) treated cells.
Abbreviations: DCFDA, 2ʹ,7ʹ-dichlorofluorescin diacetate; TBHP, tert-butyl hydrogen peroxide.
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.

Notes: (A) MCF-7 and (B) SK-BR3 cells (1 × 106) were treated with indicated concentrations of FSE for 48 hours in 100 mm plates. The cells were lysed to isolate the whole-cell protein for immunoblotting using antibodies specific for p53, p21, Bax, Bcl-2, and β-actin. The data are shown as the mean ± SEM of triplicate experiments. Statistical differences were analyzed by Two-way ANOVA, Tukey’s multiple comparison test. *Significant difference vs FSE 0, 40, and 80, #significant difference between each treated group.
Figure 11 Effect of FSE on p53 signaling using the Western blotting.

Table 2 General Appearance and Behavioral Observations of Oral Acute Toxicity Study for Vehicle and FSE Treated Groups

Figure 12 Effect of FSE on body weight and relative organs weight (ROW).

Notes: (A) The mice were weighed at the start of the experiment followed by every other day and before sacrifice, (B) RLW, (C) RHW, (D) RLvW, (E) RKW and (F) RSW. The relative organs weight were measured using the formula as described in materials and methods after sacrificing the mice at the end of the experiment. The values are expressed as ± SEM of six mice for each group.
Abbreviations: RLW, relative lung weight; RHW, relative heart weight; RLvW, relative liver weight; RKW, relative kidney weight; RSW, relative spleen weight.
Figure 12 Effect of FSE on body weight and relative organs weight (ROW).

Figure 13 Effect of FSE on biochemical parameters.

Notes: (A) ALT, (B) AST, (C) ALP, and (D) TBIL. The level of the enzymes was detected colorimetrically in the serum using the kits. The values are expressed as ± SEM of three independent experiments.
Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; ALP, alkaline phosphatase; TBIL, total bilirubin levels.
Figure 13 Effect of FSE on biochemical parameters.

Figure 14 Schematic illustration of FSE induced dose-dependent signaling pathways.

Figure 14 Schematic illustration of FSE induced dose-dependent signaling pathways.

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