Exploring the ROS-mediated anti-cancer potential in human triple-negative breast cancer by garlic bulb extract: A source of therapeutically active compounds

Abstract

Background and aim

Allium sativum L. has been used medicinally and traditionally since antiquity. This study sought to examine the Allium sativum ethanolic extract (ASEE) in inducing apoptosis in human triple-negative breast cancer (TNBC) MDA-MB-231 cells and the molecular interactions of the identified components with cell death markers using in silico method.

Experimental procedure

Cytotoxicity of ASEE was tested on MCF-7, MDA-MB-231, and Normal Vero cells. The ROS production, apoptosis, MMP, and cell cycle study were conducted utilizing flow cytometer, and western blot was also performed for protein expression analysis. ASEE was phytochemically characterized by the HPLC while AutoDock Vina and iGEMDOCK tools investigated in-silico binding interactions.

Results

The HPLC method identified two active organosulfur chemicals, allicin and alliin, in ASEE. MTT test revealed significant (p < 0.05) inhibition of breast cancer cells proliferation. The inhibitory effect of ASEE was more pronounced in MDA-MB-231 cells than in MCF-7 cells, however, no substantial cytotoxicity was seen in normal Vero cells. TNBC cells treated with high concentrations of ASEE were found in the late apoptotic stage and exhibited an increase in ROS level and a reduction in MMP. ASEE exposure increased the percentage of cells in the G2/M phase. ASEE upregulated the p53 and Bax proteins while downregulated the Bcl-2, p-Akt, and p-p38 proteins. Allicin and alliin compounds had strong binding affinity with targeted proteins of breast cancer, and both compounds also showed good pharmacokinetics and druglikeness properties.

Conclusion

ASEE could be useful in the treatment of human triple-negative breast cancer without any safety risks.

Graphical abstract

Highlights of the findings and novelties

• •HPLC examination revealed the presence of the organosulfur compounds allicin and alliin in garlic extract.
• •Dose dependent apoptotic cell death in MDA-MB-231 cells while no toxicity on normal Vero cell.
• •Upregulation of p53 and Bax while downregulated Bcl-2 protein, p-Akt and p-p38 proteins.
• •In silico analysis revealed structure-based strong binding affinity in ligand-protein complex.
• •ASEE could be used in a therapeutic and/or preventive drug development in breast cancer.

List of abbreviations

ASEE	Allium sativum ethanolic extract
MTT	3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide
TNBC	Triple-negative breast cancer
ROS	Reactive oxygen species
HPLC	High performance liquid chromatography
ER	Estrogen receptor
PR	Progesterone receptor
HER2	Human epidermal growth factor receptor 2
DCFH-DA	Dichloro-dihydro-fluorescein diacetate
Rh-123	Rhodamine 123
HB	Hydrogen bond
VDW	van der Waals forces
EI	Electrostatic interactions
PASS	Prediction of activity spectra for substances
BAS	Bioactivity score
ADMET	Absorption, distribution, metabolism, excretion, and toxicity
BBB	Blood -brain barrier
P-gp	P-glycoprotein
FBS	Feta bovine serum

1. Introduction

Cancer is a swiftly expanding and still a global health issue because of its deadly aggressiveness and low recovery rate.¹ Amongst cancers, breast cancer is the main factor of cancer-related deaths among women across the globe. In India, breast cancer is the most common cancer of females accounting for 25–32 % of the cases with a fatality rate of 12.7 % per 100,000 women.² In the USA, about 13 % of women are diagnosed with invasive breast cancer at some point in their lives, while over 30 % of new breast cancer are detected among females in 2023 (https://www.breastcancer.org/facts-statistics). Over 60 % of breast cancers are estrogen receptor (ER)⁺, the remaining 20 % lack expression of ER, progesterone (PR), and human epidermal growth factor receptor 2 (HER2) which are called triple-negative breast cancer (TNBC) and typically have a poor prognosis.³ One-third of women with breast cancer develop metastatic disease that spreads to other organs such as the bone, liver, and lungs, causing the patient's demise. The available therapies range from surgical excision and chemotherapy to radiation therapy and hormone replacement therapy, as well as synthetic lethalities. However, chemotherapy and radiation therapy may harm healthy cells, and surgical excision at the stage of metastasis can reduce the efficiency of the treatment.⁴ TNBC is not treatable utilizing the medicines that are presently on the market, even with the enormous progress that has been made in diagnosing and treating breast cancer. Therefore, plant-derived substances that are toxic to cancer cells but less so to healthy cells may offer a promising strategy for enhancing breast cancer treatment. Compounds found in nature are being utilized as a yardstick to evaluate the efficacy of novel drugs, particularly those used to combat cancer and infectious disorders. Numerous pieces of research have demonstrated that cancer can be prevented or treated with a variety of medicinal plants, their products, and gut microbiota.⁵,49, 50, 51, 52; In the context of medicinal plants, Garlic (Allium sativum), could be a promising alternative for breast cancer therapy.

Garlic has been used in many forms of medicine since ancient times due to its medicinal characteristics.⁶ A previous study has reported that bulbs of A. sativum contain various phytochemicals including sulfur-containing compounds that account for 82 % of the overall garlic sulfur content.⁷ The sulfur component alliin, a pungent liquid, is converted into allicin by activating the enzyme alliinase when the whole bulb of garlic is pulverized.⁸ A growing body of research has discovered that DADS has anticancer action against various tumor cell types, such as colon, breast, and gastrointestinal cancer cell lines.⁵ White garlic bulbs have demonstrated a variety of pharmacological and biological activities including anti-inflammatory, anti-microbial, anti-tumor, and anticancer.⁹ However, no study has been conducted to examine the effect of crude Allium sativum ethanolic extract (ASEE) on apoptotic cell death in human triple-negative breast cancer (TNBC) MDA-MB-231 cells through the mechanisms of reactive oxygen species (ROS) formation, cell cycle arrest, Akt, and p38 MAPK pathways. Due to the significant impact of metastasis on mortality rates, MDA-MB-231 cells were selected as the breast cancer model system in the current study. TNBC is a more aggressive type of cancer. The MDA-MB-231 cell line lacks ER, PR, and E-cadherin and contains a mutant p53 gene. Moreover, MDA-MB-231 cell suspensions have the characteristics to generate orthotopic murine model.^10,11

The present study investigated the effect of ASEE against MDA-MB-231 cells by MTT assay, fluorescence microscopy, and flow cytometry for ROS generation, MMP analysis, and cell cycle arrest as well as western blotting analysis. HPLC testing was done to check the presence of the main bioactive component in ASEE. Moreover, the binding interaction of allicin and alliin molecules with apoptotic proteins was investigated through Autodock v1.5.6 and Autodock Vina software.

2. Materials and methods

2.1. Reagents and chemicals

DMEM/F-12 media, FBS, penicillin, streptomycin solution, DCFH-DA and Rh-123 were procured from Sigma Aldrich, USA. Hoechst 33342 dye and MTT were purchased from Himedia, India. Annexin V FITC apoptosis kit (Biovision, USA), Hoechst 33258 (ThermoFisher Scientific, USA), RIPA and ECL chemiluminescence substrate kit (G Biosciences, USA) were procured. Antibodies (β-actin, cat no. 5125S; Bcl-2, cat no. 4223S; Cleaved caspase-3, cat no. 9661S; Akt, cat no. 9272S; p-Akt, cat no. 4060S; p38, cat no. 9212S; P-p38, cat no. 9211S) were purchased from CST, USA. p53, cat no. 10442-1-AP and Bax, cat no. 50599-2-Ig were purchased from Proteinteck, USA. Alliin (Item No. 14002, CAS No. 556-27-4) and Allicin (Item No. 15570, CAS No. 539-86-6) were purchased from Cayman, USA.

All the reagents utilized were of analytical grade.

2.2. Garlic extracts preparation

White garlic bulbs were purchased from a local vegetable market of Lucknow, India. In January 2020, white garlic bulbs were harvested from the adjacent place of Lucknow. The plant sample was deposited for authentication (specimen no. IU/PHAR/HRB/20/04) by Dr. Shazia Usmani and Dr. Muhammad Arif, Associate Professor, at the Faculty of Pharmacy, Integral University, Lucknow, India. The 95 % ethanolic extract of garlic bulb was prepared according to Siddiqui et al., 2019 and the obtained semisolid extract was stored at 4 °C until further use in experiments.

2.3. HPLC analysis of ASEE

ASEE was characterized using a Waters 515 HPLC Pump system (Milford, USA) outfitted with a W2998 PDA detector, a pump control module, a Waters column temperature controller, and an empowered chromatography workstation. Chromatographic analysis was performed using a reverse-phase, 4.6250 mm X BRIDGE C18 column, and a mobile phase consisting of a gradient. Water (Solvent A) and acetonitrile (Solvent B) were used in a gradient to form the mobile phase. Allicin and alliin were used as the standards. The standard and ASEE were analyzed in real-time HPLC at 254 nm to get chromatograms.¹²

2.4. MTT assay

The MTT assay was used to assess the inhibition of cell growth by garlic extract against human breast cancer cells MCF-7, MDA-MB-231, and the normal kidney epithelial cell line Vero, using a standard procedure.¹² ASEE stock was prepared in a culture medium (DMEM: F12) and then diluted in the same media to 5, 10, 15, 18, and 20 mg/mL concentrations to treat cultured cells in a 96-well plate for 24 h. MTT dye was used for crystal formation and absorbance was measured through an ELISA plate reader (Bio-Rad PW41, USA) at 550 nm. The IC₅₀ values were determined using GraphPad Prism 5.1. Inverted phase contrast microscopy (Nikon Eclipse TS100, Japan) was used to detect structural alterations.

2.5. Nuclear condensation assay

The apoptosis-inducing effect of ASEE was examined at three effective doses of 12 (low dose; LD), 13.69 (IC₅₀ dose), and 17 (high dose; HD) mg/mL. Hoechst 33258 staining was used to assess nuclear condensation, as described previously.¹² Images of stained cells were taken using an inverted fluorescent phase contrast microscope (Zeiss AxioVert 135, US).

2.6. Acridine orange-ethidium bromide (AO/EtBr) assay

The cytotoxic effects of ASEE on MDA-MB-231 cells at three effective doses were tested in a manner consistent with an earlier report.¹³ Cells were examined using an inverted fluorescence phase contrast microscope.

2.7. Annexin V-FITC double staining for apoptosis investigation

Percent apoptotic and necrotic cells at different doses were counted through flow cytometry (FACS Lyric, BD Biosciences, USA) as per instruction of Annexin V-FITC Apoptosis Kit.

2.8. Analysis of intracellular ROS level

DCFH-DA stain was used to measure intracellular ROS levels through fluorescence microscopy and flow cytometry, as described previously.¹⁴

2.9. Evaluation of mitochondrial membrane potential (MMP,ΔΨm)

As previously reported, MMP alterations were evaluated using the fluorescent probe Rh-123 through fluorescence microscopy and flow cytometer.¹²

2.10. Analysis of cellular DNA content

PI staining dye was used to examine the various stages of the cell cycle as well as the DNA content of the cells through a flow cytometer, as explained previously.¹⁴

2.11. Western blot analysis

Following a published protocol,¹⁵ western blotting was performed on ASEE-treated and untreated TNBC cells. In brief, a frozen RIPA lysis solution with a protease and phosphatase inhibitor cocktail was used to obtain cell lysates. Protein samples (40 μg each) were separated by electrophoresis on a 10 %–12 % SDS-PAGE gel and then transferred to a PVDF transfer membrane (Thermo Fisher Scientific, USA). Following this, the ECL chemiluminescence substrate reagent kit was used for immunodetection, as per the manufacturer's protocol. To assess the quantity of each band in comparison to the housekeeping β-actin protein, Image J software (version 1.43, NIH, USA) was used.

2.12. Prediction of activity spectra for substances (PASS) analysis, using Lipinski's rule of five

Based on canonical SMILES obtained from PubChem, the online Molinspiration chemoinformatics tool was used for the calculation of druglikeness of both identified phytoconstituents of garlic extract using Lipinski's rule of five.¹⁶

2.13. PASS analysis using toxicity potential assessment

OSIRIS Data Warrior version 5.2.1 was used for drug-toxicity parameter predictions such as druglikeness, mutagenic, tumorigenic, reproductive and irritant effects to determine the probable side effects of A. sativum constituents.¹⁷

2.14. Pharmacokinetic (PK) parameters prediction

Web-based SwissADME tool was used to calculate the ADMET (Absorption, Distribution, Metabolism, Excretion, and Toxicity) properties of the components.

2.15. Preparation of ligand and protein

The 3D SDF format of the identified compounds was downloaded from the PubChem database. ChemBio3D Ultra 14.0 was used to perform energy minimization of ligands, with MM2 Force Field, and saved in.pdb format.¹⁸ Protein Data Bank was used to access the 3-D crystal structures of selected proteins involved in apoptosis cell death process viz. pro-apoptotic protein Bax (PDB ID: 5W6O), anti-apoptotic protein Bcl-2 (PDB ID: 6QGG), effector caspase-3 (PDB ID: 1GFW), tumor suppressor protein p53 (PDB ID: 8HLN), mitogen-activated protein kinase (MAPK) protein p38 (PDB ID:4MYG), and apoptotic inhibiting protein Akt (PDB ID:6NPZ). All 3D protein structures were subjected to refinements and energy minimizations before docking analyses. Targeted proteins were selected based on their major role in the apoptosis signaling pathway.¹⁹

2.16. Molecular docking analysis

The molecular docking was conducted using AutoDock v1.5.6.¹⁸ The AutoDock Vina software was employed to execute additional testing of the binding affinity to specific targeted proteins associated with breast cancer.²⁰

2.17. Analysis and visualization of docked ligand-protein complexes

The software program Accelrys Biovia Discovery Studio version 2017 R2 was employed to figure out the finest structure of the ligand-protein interaction. The selection of the best orientation was made based on the criteria of achieving the lowest binding energy (B.E.) and the value of the dissociation constant (Kd).

2.18. Statistical analysis

The biological data were expressed as the mean ± SD from three independent experiments. Statistical evaluation was determined by one-way ANOVA followed by the Tukey Multiple Comparison Test using GraphPad Prism software (Version 5.1). A p-value of less than 0.05 was considered statistically significant.

3. Results

3.1. HPLC characterization of ASEE extract

Fig. 1 displays the chromatograms of reference standards allicin & alliin and crude ASEE. A clear separation of allicin with a Rt value of 9.50 min and alliin at a Rt value of 20.48 min at 254 nm wavelength was achieved using HPLC chromatographic analysis utilizing a reverse phase column and water (A) and methanol-acetonitrile (B) solvents as mobile phase (Fig. 1A and B). Under the same experimental circumstances, the HPLC analytical peak for ASEE corresponded at a Rt value of 9.50 min and 20.50 min (Fig. 1C). This chromatographic analysis showed the presence of allicin and alliin as active ingredients in ASEE.

Fig. 1.

HPLC profile of two standards allicin and alliin, and ASEE (A) HPLC chromatogram of allicin standard (Rt = 9.50 min) (B) HPLC chromatogram of standard alliin (Rt = 20.48 min) (C) chromatogram of ASEE. The optimum peak had an Rt of 9.50 min and 20.50 min in the ASEE sample which was similar to that of standard allicin and alliin.

3.2. Effect of garlic extract on cellular morphology and cell viability

Fig. 2A and S1A demonstrate that after 24 h incubation with crude ASEE, the unexposed cells displayed the usual characteristics, such as an adherent, homogenous, and even cell surface. A large number of both MCF-7 and MDA-MB-231 cells exhibited a non-adherent, unattached, and spherical shape after being exposed to 5–20 mg/mL ASEE. However, ASEE showed more prominent effect in MDA-MB-231 cells than in MCF-7 cells. As indicated in Fig. 2B and S1B, crude ASEE reduced the cell viability of both types of breast cancer cells in a dose-dependent manner. The cell viability investigation revealed that exposure to ASEE significantly suppressed the growth of MDA-MB-231 cells in comparison to MCF-7 cells. The inhibitory concentration (IC₅₀) of ASEE for the reduction in MCF-7 and MDA-MB-231 cell count was -15.35 mg/mL and 13.69 mg/mL, respectively. On the other hand, the exposed Vero normal cells did not display the unusual morphological alteration and reduction in cell viability (Fig. 2C and D). Few numbers of normal cells exhibited a non-adherent, unattached after being exposed to higher ASEE doses. MDA-MB-231 cells were selected for further investigation due to the substantial toxic effects of ASEE with their three effective doses: a low dose (LD = 12 mg/mL), an IC₅₀ dose (13.69 mg/mL), and a high dose (HD = 15 mg/mL).

Fig. 2.

Microscopic observation and cytotoxic potential of different concentrations (5–20 mg/mL) of Crude ASEE after 24 h incubation. (A), and (C) Photomicrographs of untreated and treated MDA-MB-231 and normal Vero cells, respectively at various concentrations of Crude ASEE under an inverted phase contrast microscope, Scale bar = 100 μm. (B) and (D) Percent cell viability of MDA-MB-231 and normal Vero cells, respectively at various concentrations of Crude ASEE. Values are expressed as mean ± SD of three independent experiments. *p < 0.05 as compared to control and ^#p < 0.05 as compared to low dose 5 mg/mL.

3.3. Apoptotic body formation and chromatin condensation assay

A microscopic investigation of chromatin condensation demonstrates that ASEE-treated TNBC cells induced chromatin condensation in comparison to control cells. ASEE increased the condensation of chromatin in a dose-dependent manner. A high level of nuclear condensation was seen at HD (Fig. 3A). In addition, the AO/EtBr double stain demonstrated that healthy control cells had uniformly stained green nuclei. Condensed nuclei with a green cytoplasm indicated early apoptosis, whereas condensed nuclei and an orange cytoplasm indicated late apoptosis in treated cells. At LD of the ASEE, cancer cells exhibited early apoptotic characteristics, but at higher concentrations, late apoptotic features were detected (Fig. 3B).

Fig. 3.

(A) Photomicrographs show the chromatic condensation in TNBC-treated cells at a low dose (LD), IC_50, and high dose (HD) of ASEE after 24 h (A) Fluorescent photomicrographs of AO/EtBr-double stained MDA-MB-231 cells at LD, IC₅₀, and HD of ASEE after 24 h. Control TNBC cells depict a healthy structure while ASEE-exposure showed chromatin condensation and membrane blebbing. VC: Viable cells; CC: Chromatin condensation; LA: Late apoptosis and SN: Secondary necrosis. Scale bar = 100 μm.

3.4. Apoptosis quantification at the early and late stage

To verify the statistical effectiveness of apoptosis induction, TNBC cells were subjected to additional testing using an Annexin V-FITC double stain. Cells that had not been treated exhibited a survival rate of 96.67 % and were considered healthy and viable. Treated TNBC cells showed an increase in cell death to 7.6 % early apoptotic and 19.93 % late apoptotic at LD, 7.6 % early apoptotic and 21.41 % late apoptotic at IC₅₀ dose, and 3.6 % early apoptotic and 24.43 % late apoptotic at HD of ASEE (Fig. 4A).

Fig. 4.

ASEE mediated induction of apoptosis and intracellular ROS generation in MDA-MB-231 cells. (A) Flow cytometer analysis after 24 h of treatment using annexin V/FITC & PI double stain. Representative quadrants showing the population of viable (annexin V– PI-), early apoptotic (annexin V + PI), late apoptotic (annexin V + PI+), and necrotic (annexin V– PI+) cells. (B) Photomicrographs showing intracellular ROS generation induced by three effective concentrations of ASEE after 12 h incubation. Photomicrographs were taken with a fluorescence microscope. Scale bar = 100 μm (C) The fluorescence intensity in the cells is represented as the percentage of ROS production analyzed using a flow cytometer.

3.5. ASEE induces intracellular ROS generation

As shown in Fig. 4B, the photomicrograph reveals that, in contrast to untreated cells, ROS intensity was increased significantly in treated TNBC cells. Flow cytometer evaluation of ROS production revealed a 1.21 % ROS level only in control cells that is typically seen in healthy cells. However, ASEE induced ROS levels significantly to 17.07, 50.36, and 64.11 % at LD, IC₅₀, and HD, respectively (Fig. 4C).

3.6. Analysis of MMP loss by ASEE

Flow cytometry results and fluorescence microscopic photomicrographs revealed higher MMP loss in treated TNBC in contrast with untreated cells. Microscopic photographs (Fig. 5A) show loss of MMP at an increased dose of ASEE as shown by diminished green fluorescence of Rh 123 dye. The flow cytometry result as a percent loss in MMP is shown in Fig. 5B. Findings indicated that the percentage loss of MMP in treated TNBC cells was increased in a dose-dependent manner. The untreated breast cancer cells exhibited 11.36 % green fluorescence loss, while at LD, IC₅₀, and HD, they exhibited 63.06 %, 75.63 %, and 80.40 % fluorescence, respectively. In addition, a reduction in the ratio of aggregate (red fluorescence) to monomer (green fluorescence) was found, suggesting a loss in mitochondrial membrane potential.

Fig. 5.

MMP and Cell cycle analysis of MDA-MB-231 cells treated with three effective concentrations of ASEE (A) Photomicrographs indicate a decrease in MMP (an early event in apoptosis) in MDA-MB-231 cells. Cells were stained with Rh 123 dye. Scale bar = 100 μm. (B) Representative profile of flow cytometer analysis after Rhodamine 123 staining in MDA-MB-231 cells. The number shown in each panel indicates the percentage of cells with mitochondrial potential loss (C) Flow cytometer graph indicates the proportion of cells in different phases of the cell cycle treated with three effective concentrations of ASEE for 24 h.

3.7. ASEE induces G2/M phase arrest

Exposure to ASEE greatly increased the proportion of MDA-MB-231 cells in the G2/M phase while concurrently reducing the number of cells in the G0/G1 phase. As shown in Fig. 5C, the cell population in the G2/M phase was found to be 24.93 %, while LD, IC₅₀ dose, and HD arrested the cells at 34.77, 39.76 and 51.28 %, respectively in the G2/M phase. These results imply that the G2/M checkpoint of the cell cycle is triggered by the ASEE in TNBC cells.

3.8. Western blotting analysis

Western blotting method was used to examine the expression of key apoptotic proteins for underlying processes of cell death. The band intensity of each blot (Fig. 6A) was calculated through Image J software and graphs were plotted as shown in Fig. 6B. Results demonstrated that in ASEE-treated cells, p53, Bax, and cleaved caspase-3 expression level were elevated whereas level of Bcl-2 was downregulated. Following 24 h administration, the expression of phosphorylated-AKT and phosphorylated-P38 was decreased in breast cancer cell line (Fig. 5).

Fig. 6.

Western blot analysis of apoptotic proteins and signaling molecules (A) Western blot showing the expression levels of p53, Bax, Bcl2, cleaved Caspase-3, AKT, p-AKT, p38 and p-p38. MDA-MB-231 cells were treated at two effective concentrations of ASEE (LD = 12 mg/mL and HD = 15 mg/mL)) for 24 h. Equal amounts of protein samples (40 μg/lane) were resolved on SDS-PAGE gel and transferred to the PVDF membrane. Expression of all proteins and β-actin were detected using specific antibodies. Protein markers p53, Bax, Bcl2, cleaved Caspase-3, and one of the β-actin proteins were cropped from different parts of the same blot, while AKT, p-AKT, P38 and p-P38 and other β-actin proteins were cropped from different blots. β-actin was used as a loading control. The full-length blots were cut before antibody hybridization and each section was incubated with primary antibody individually. (B) Graph showing the relative intensity of protein expression. Data represents the mean ± SD of three independent experiments. *, ^@p < 0.05 compared to the control group.

3.9. PASS analysis using RO5, druglikeness, toxicity potential and ADMET properties

Table 1 shows the PASS analysis of major phytocomponents of A. sativum in terms of their physicochemical properties by applying LRO5. Allicin and alliin from A. sativum revealed no Lipinski violation. OSIRIS data warrior was used to calculate the druglikeness and toxicity risk assessment of identified compounds (Table 2). The obtained results are represented in green, yellow and red predicting no toxicity, mild toxicity and high-risk effects, respectively. None of the identified phytoconstituents had any mutagenic, tumorigenic, reproductive or irritant side effects. As is evident from Table 3 for ADMET analysis based on the calculated LogP value, both molecules were found to be lipid soluble (lipophilic) which suggests that they can easily diffuse across cell membranes. None of phytoconstituents were found to behave as P-gp substrates, they may be expected to persist in the cells and show their intracellular pharmacological effect. Compounds that inhibit the five classes of CYPs viz. CYP3A4, CYP1A2, CYP2C9, CYP2C19 and CYP2D6 would cause an increase in their plasma concentrations, thus contributing to improved bioavailability. None of the molecules acted as inhibitors of any of the five classes of CYPs (Table 3). Interestingly, both phytocomponents showed negative value for skin permeability (Kp) which indicates less ability of topical absorption of these phytoconstituents. Skin permeability (Kp) is widely used to quantitatively describe the rate of chemical permeation through the outermost layer (epidermis) of the skin.

Table 1.

PASS Analysis of A. sativum phytoconstituents.

S.No.	Phytoconstituent	% Absorption (>50 %)a	Topological Polar Surface Area (Å) (TPSA)b (160 Å)	MW (<500)	c logP (<5)c	Heavy atom coun (natoms)	Hydrogen Bond Donors (nOHNH) (≤5)	Hydrogen Bond Acceptors (nON) (≤10)	Number of Rotatable bonds (≤10)	Lipinski's violation
1.	Allicin	103.11	17.07	162.28	2.06	9	0	1	5	0
2.	Alliin	81.26	80.39	177.22	−3.39	11	3	4	5	0

Table 2.

Druglikeness and toxicity calculation of A. sativum phytoconstituents.

Table 3.

Calculated ADMET properties of A. sativum phytoconstituents.

S. No.	Phytoconstituents	Lipophilicity (Consensus Log Po/w)	GI absorbtion	BBB permeant	P-gp substrate	CYP1A2 inhibitor	CYP2C19 inhibitor	CYP2C9 inhibitor	CYP2D6 inhibitor	CYP3A4 inhibitor	Log Kp (skin permeation)
1.	Allicin	1.61	High	Yes	No	No	No	No	No	No	−6.36 b cm/s
2.	Alliin	1.33	High	No	No	No	No	No	No	No	−9.89 b cm/s

3.10. Molecular docking investigation of identified components in ASEE against targeted proteins

In this study, the docking tools AutoDock v1.5.6 and Autodock Vina were employed to investigate the binding affinity of allicin and alliin compounds with targeted proteins associated with breast cancer cell death. The visualization of the binding interaction acquired through both docking programs was performed using the BIOVIA Discovery Studio program. AutoDock Vina utilizes a statistical scoring function as a replacement for the semi-empirical free energy force field employed in AutoDock 4.2. Tables S1 and S2 represent the active constituent allicin and alliin of ASEE with their chemical structure, binding energy, dissociation constant, interaction types, and best docking poses with amino acid residues contributing to the binding pocket of the selected proteins. As shown in Tables S1 and S2, allicin and alliin phytoconstituents from ASEE exhibited potent binding interaction with different target proteins associated with breast cancer. It is evident from Table S1 that both compounds showed promising binding kinetics to the various therapeutic targets of TNBC. AutoDock v1.5.6 analyses showed that binding affinities of the targeted proteins with allicin decreased in the order: 6QGG (Bcl-2) > 5W6O (Bax) > 8HLN (p53) > 6NPZ (AKT) > 4MYG (p38) > 1GFW (Caspase-3). The binding affinity of allicin (BE = −4.01 kcal/mol, Kd = 1.15 mM) was found to be highest with Bcl-2. On the other hand, the binding affinity of alliin decreased in the order: 5W6O (Bax) > 8HLN (p53) > 4MYG (P38), 6NPZ (AKT) > 1GFW (caspase-3) > 6QGG (Bcl-2) (Table S1). Alliin showed the highest binding interaction (BE = −5.33 kcal/mol, Kd = 124.36 μM) with Bax (Table S1). AutoDock Vina data showed that binding affinities of the breast cancer proteins with phytoconstituent allicin decreased in the order: 6QGG (Bcl-2) > 4MYG (P38) > 6NPZ (AKT) > 1GFW (caspase-3) > 5W6O (Bax) > 8HLN (p53). Allicin showed highest binding interaction (BE = −4.5 kcal/mol, Kd = 563.89 μM) with Bcl-2 (Table S2). The Binding affinities of the breast cancer targeted proteins with phytoconstituents alliin decreased in the order: 4MYG (P38) 6NPZ (AKT) > 6QGG (Bcl-2) > 5W6O (Bax), 1GFW (caspase-3), 8HLN (p53). Alliin displayed the best interaction (BE = −4.3 kcal/mol, Kd = 954.05 μM) with P38 (Table S2).

4. Discussion

Breast cancer affected around 2.3 million women and caused 67000 casualties in 2022 and is responsible for the highest percentage of fatalities resulting from cancer in the female population [WHO, 2024;²¹]. In addition to available chemotherapeutic medicines, herbal remedies, and phytocompounds in particular, are now receiving a lot of interest for their potential use in the treatment and prevention of cancer disorders.²² The ethno-traditional use of medicinal herbs has formed an important part of the research that has led to the possible identification of cancer-fighting drugs.²³ According to the findings of prior research, raw garlic extract has been shown to possess the most efficient and highly specific anticancer treatment after contrasting with 33 natural extracts against various cancer cells without damaging the benign cells.²⁵ The finding of the current study also showed the anticancer potential of ASEE against MDA-MD-231 cells while leaving healthy cells unaffected. Since cancer cells proliferate and expand far more quickly than other body cells, this extract from garlic bulbs may help in reducing the growth of cancer cells. High-performance liquid chromatography (HPLC) analysis of ASEE showed the presence of alliin and allicin of garlic as the active organosulfur compounds. Structural analysis of ASEE-treated TNBC cells indicated the cells became more spherical, shrank in clustered size, and detached from their surface (Fig. 1A). This finding provided early evidence for an indication of apoptotic cell death in breast cancer. The presence of sulfur compounds in ASEE is believed to be the primary factor causing the demise of breast cancer cells. Interestingly, a growing body of evidence has suggested that diallyl disulfide of garlic can impede the progression of gastric, hepatic, prostate, lung, and breast cancer. This inhibition is achieved through the induction of apoptosis and the prevention of cell cycle arrest.8, 54, 55, 56 Furthermore, a prior investigation has documented that allicin can trigger apoptosis in glioma cells via Bcl-2-associated proteins and Fas/FasL-mediated pathways.⁵³ Furthermore, Ravindra et al.²⁶ have reported the antiproliferative, apoptotic, and anti-angiogenic effects of allicin in zebrafish.

The current research examined the primary apoptotic processes using fluorescent microscopy in ASEE-treated MDA-MB-231 cells to verify the qualitative efficiency of cell death. Nuclear condensation results showed the typical characteristics of apoptosis, whereas early and late apoptosis in ASEE-treated MDA-MB-231 cells were shown by AO/EtBr staining. Annexin-V/PI dual staining was additionally analyzed by flow cytometer to provide statistical support for these findings. As shown by the results, a moderate change in early apoptosis and an upsurge in late apoptosis were seen, along with a reduction in the proportion of viable cells. Cells underwent early apoptosis in response to a low dosage of ASEE and late apoptosis in response to an increased dose of ASEE (Fig. 3A). These numerical results showed that ASEE caused cancer cell death by pushing most cells towards the terminal apoptotic phase. DATS, a compound found in cloves of garlic, has been shown to cause programmed cell death in breast cancer cells MCF-7.³⁹ To provide more evidence about cell death process initiated through signaling molecules or stimulants, the production of intracellular ROS in ASEE-treated MDA-MB-231 was investigated. Enhanced ROS formation as evidenced by flow cytometer showed that TNBC cells were indeed subjected to oxidative stress, mitochondrial destabilization, and death after being exposed to ASEE (Fig. 3B and C). ROS have been considered a significant modulator of the extrinsic as well as the intrinsic pathways of cell viability and apoptotic processes.^12,27 Excessive generation of ROS results in a disorganized plasma membrane and cytoskeleton, which ultimately results in chromosomal destruction.²⁸ The current investigation revealed the destruction of mitochondrial membrane structure and loss of MMP in ASEE-treated cells which showed the induction of intrinsic apoptosis (Fig. 4A and B). Mitochondria are involved in many cellular processes beyond producing ATP, including control of membrane capacity, apoptotic cell death, calcium signaling, and metabolic processes.¹⁵ Encouraging MMP reduction may boost the effectiveness of cancer treatment.²⁹ Cell cycle investigation indicated an increased proportion of cells in the G2/M phases equated to control cells, while the proportion of cell in the G0/G1 phases was reduced (Fig. 4C). This current finding is consistent with a recently published study that showed exposing HCC cells to ajwa dates pulp extract raised the proportion of cells in the S and G2/M phases relative to control cells, while decreasing the number of cells in the G0/G1 phase.¹²

Moreover, immunoblot was carried out to analyze the anticancer mechanism of cell death of ASEE against MDA-MB-231 cells. Results revealed that ASEE induced the expression level of tumor suppressor p53, pro-apoptotic Bax, and effector caspase-3 while reducing the anti-apoptotic Bcl-2 protein level as compared to untreated cells. Moreover, the expression of p-AKT and p-p38 was decreased in the MDA-MB-231 cell as compared to control (Fig. 5). These findings show that ASEE induces apoptosis in breast cancer cells through the activation of p53 pathway which initiates caspases-3 activation and Bcl-2 family by opening the mitochondrial permeability transition pore. p38 MAPK plays a dual role which under physiological conditions, p38 can function as a mediator of ROS signaling and either activate or suppress cell cycle progression depending on the activation stimulus. Several reports have shown that p38 functions as an antitumorigenic factor while some other reports have shown as a tumor promoter.^30,31 In the current study, ASEE reduced the level of p38 and caused cellular apoptosis which indicated that p38 MAPK acts as tumor promoter in MDA-MB-231 cell death. In addition, Akt, also known as protein kinase B, is a pivotal protein that acts as an anti-apoptotic in many different cell death paradigms.³² The inhibition of Akt phosphorylation has the potential to decrease the anti-apoptotic impact of Akt, leading to an elevation in the Bax/Bcl-2 ratio, the release of cytochrome-c, and the activation of caspase-3. Interestingly, our study has shown that ASEE reduced the level of AKT expression thereby leading to apoptosis of breast cancer cells.

Furthermore, an attempt was made to validate the anti-TNBC potential of identified components using chemoinformatic tools. Both alliin and allicin organosulphure compounds showed potential binding to selected TNBC proteins/receptors as evident by AutoDock v1.5.6 and AutoDock Vina analysis (Tables S1 and S2). These compounds were checked for their druglikeness using LRO5. Generally, an orally active compound should have no more than one Lipinski's violation otherwise its bioavailability is compromised.¹⁶ Interestingly, both allicin and alliin compounds exhibited no Lipinski's violation (Table 1). Toxicity risk assessment is an important consideration in preventing further drug screening of unwanted compounds with adverse effects. The software estimates a compound's toxicity based on similarities between the compound being analyzed and those present in its database [Siddiqui et al., 2020]. Interestingly, both phytocomponents were found to be safe completely with no predicted toxicity (Table 3). All phytocomponents exhibited good ADMET properties including transport kinetics suggesting that they can easily diffuse across the cell membranes and through the gut (Table 2).

5. Conclusion

In this study, we investigated the cell death induced by ASEE in MDA-MB-231 cells, as well as the in silico investigation to analyze the binding interaction of identified components alliin and allicin, through HPLC analysis, with effective cancer markers of breast cancer cells. We observed that ASEE induced mitochondrion-mediated apoptosis through the inhibition of Akt phosphorylation leading to an elevation in the Bax/Bcl-2 ratio and p53 signaling that resulted in activation of caspase-3 and DNA degradation. ASEE reduced the level of p38 and caused cellular apoptosis which indicates that p38 MAPK acts as tumor promoter in MDA-MB-231 cell death. More importantly, chemoinformatics tools showed good binding characteristics and druglikeness properties of both compounds. Based on both in vitro data and in silico findings, ASEE could be developed as an alternative or complementary therapeutic agent for breast cancer treatment. A recent study revealed that MDA-MB-231 cells exhibited less genetic similarity to the basal-like metastatic breast cancer sample of a human patient.³³ While, organoid, 3-D cell culture model, more closely resembles the transcriptome of metastatic breast cancer samples compared to cell lines.³⁴ Some previous studies have shown that MDA-MB-231, a more aggressive cancer cell line, lacks ER, PR, and E-cadherin and has a mutant p53 gene and can develop orthotopic mouse models.^10,11 Based on these reports and their similarity to the architectures of human organs, organoid culture is more advantageous than cancer cell lines. Thus, further organoid culture, in vivo, and clinical studies are needed to understand how a drug candidate may affect cancer cells.

Author contribution

SS, RA and SU conceived and designed the study. SU and SS helped in data curation. SU performed the methodology. SS and RA supervised the study. SU and SS drafted the original manuscript. RA, SU, RK, SG, AS, KA, IH, MAB, MZH, YIA, and SS critically reviewed and suggested modifications. All authors read and approved the final manuscript.

Data availability statement

All data are available in the manuscript and supplementary files.

Declaration of competing interest

The authors declare no conflicts of interest.

Appendix A. Supplementary data

The following are the Supplementary data to this article:

Fig. S1.

Microscopic observation and cytotoxic potential of different concentrations (5–20 mg/mL) of Crude ASEE after 24 h incubation. (A) Photomicrographs of untreated and treated MCF-7 cells at various concentrations of Crude ASEE under an inverted phase contrast microscope, Scale bar = 100 μm. (B) Percent cell viability of MCF-7 cells, at various concentrations of Crude ASEE. Values are expressed as mean ± SD of three independent experiments. *p < 0.05 as compared to control.