Reagents

This investigation employed only molecular-grade reagents. S-allyl 2-propene-1-thiosulfinate, propidium iodide (PI), 4′,6-diamidino-2-phenylindole (DAPI), ′,7′-dichlorodihydrofluorescein diacetate (DCF-DA), dihydrorhodamine123,3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich Chemical Co., USA. USA-based BD Biosciences supplied fluorescein isothiocyanate (FITC) Annexin V Apoptosis kit. Thermo Fisher Scientific supplied fetal bovine serum (FBS), Dulbecco's modified Eagle medium (DMEM), and other chemicals.

Cell culture and MTT assay

Stocked HeLa cells were cultured in DMEM supplemented with 10% FBS and 1% penicillin–streptomycin antibiotic–antimycotic solution in a humidified atmosphere of 5% CO2 at 37 °C until confluent. HeLa cells were seeded in a 96-well plate to assess the inhibitory effects of allicin at different doses by MTT assay according to the protocol [20]. After treating HeLa cells with 0, 5, 10, 20, 30, and 40 µM of allicin for 24 and 48 h, 10 µL (5 mg/mL) MTT reagent was added in each well and incubated for 4 h. Followed by adding 100 µL DMSO to dissolve formazan crystals with gentle shaking, the absorbance was taken 570 nm and cell viability was represented as percentage of control group.

Analysis of cellular morphological changes of cervical cancer cells by phase contrast microscopy

In phase contrast microscopy, allicin-treated HeLa cells were evaluated for morphological changes [21]. HeLa cells (50,000/well) were seeded to a 12-well plate in triplicate and incubated at 37 °C for 48 h. The cervical cancer cells were then treated to various allicin concentrations for 48 h. The treated cells were compared to the untreated group using a phase contrast microscope.

Analysis of migration inhibition of cervical cancer cells by wound healing assay

A wound healing assay was used to evaluate HeLa cell migratory potential [22]. We planted 5 × 104 HeLa cells per well in six-well plates and cultivated till confluence. A sterile scraper then made a linear scratch in the confluent cell monolayer. Following a culture media wash to remove debris, the cells were treated with various allicin concentrations for 24 h. HeLa cell movement was tracked using a phase contrast microscope. After that, Image J examined the photographs. Cell distance into the cell-free area was used to calculate migration inhibition efficiency.

Fluorescent microscopic analysis of nuclear morphology by DAPI staining

After DAPI labeling, fluorescence microscopy revealed nuclear morphological changes that indicated apoptosis [23]. After exposing HeLa cells to various doses, cells were washed twice with cold PBS and fixed in ice-cold 70% methanol for 10 min. Next, cells were treated with 1 µg/mL DAPI at 37 °C for 15 min in darkness. After another PBS rinse, the cells were evaluated for nuclear morphological changes under a fluorescent microscope.

Fluorescent microscopic analysis of apoptotic HeLa cells stained with Annexin V FITC and propidium iodide

Lipids, proteins, and carbohydrate moieties are asymmetrically distributed across plasma membrane leaflets. Phosphatidylserine (PS) is a negatively charged phospholipid, mainly located on the inner leaflet of the plasma membrane. Phospholipid-binding protein Annexin V has excellent PS affinity and specificity. PS distribution across plasma membrane leaflets changes during apoptosis. According to the manufacturer's procedure (Apoptosis Kit, Invitrogen, USA), staining with Alexa Fluor 488-Annexin V and propidium iodide (PI) helps in the measurement of early apoptosis by detecting PS which show green fluorescence and red fluorescence for late apoptotic cells. After treating with different concentrations of allicin for 24 h, cells were washed and fixed for 10 min in methanol/acetic acid (3:1 v/v). Thereafter, cells were treated with Alexa Fluor 488-Annexin V and PI for 30 min at room temperature in the dark. After incubation, cells were washed with 1× PBS and were examined under the fluorescence microscope.

Analysis of caspase-3 and caspase-9 activities in cervical cancer cells

In order to induce apoptosis in cancer cells, anticancer drugs must activate caspases. Thus, caspase activation was measured in normal and allicin-treated cervical cancer cells. In summary, cells were grown for 24 h and seeded in triplicate onto 96-well plates. Cells were treated with different allicin concentrations after seeding. Allicin-treated and untreated HeLa cells were tested for caspase activity using a colorimetric technique. After 10-min incubation on ice, cells were lysed in 50 µL of ice-cold cell lysis solution and centrifuged at 8000×g for 3 min to obtain the supernatant. After transferring 50 µL of cell lysate to a 96-well plate, 50 µL of reaction buffer with 10 mM dithiothreitol (DTT) was added. Next, add 5 µL of 4 mM DEVD-pNA substrate to each well and incubate at 37 °C for 1 h. Finally, a microplate reader measured absorbance at 405 nm.

Analysis of cellular caspase activity in cervical cancer cells in presence of caspase inhibitors

To test caspases' role in apoptosis, cell viability can be measured with caspase inhibitors. To evaluate the impact of allicin, HeLa cells were pretreated with 50 µM Z-DEVD-FMK (caspase-3 inhibitor) and 50 µM Z-LEHD-FMK (caspase-9 inhibitor) for 2 h. After pretreatment, HeLa cells were exposed to allicin at various doses for 24 h. As previously described, the MTT assay measured cell survival. To create formazan crystals, MTT was applied to each well and the cells were incubated. To test cell viability, DMSO solubilized the crystals and measured absorbance at 570 nm. To understand allicin's effect on caspase inhibitor-treated cells, cell viability was compared to untreated controls. This method determines whether caspase-3 and caspase-9 pathways mediate allicin-induced apoptosis in HeLa cells.

Fluorescent microscopic analysis of mitochondrial reactive oxygen species (mROS) in cervical cancer cells

Oxidative stress causes ROS overproduction, which can cause mitochondrial mutations and membrane potential disruption. DHR123, a cell-permeable fluorogenic sensor, detects peroxynitrite production to assess mitochondrial oxidative stress. After interacting with peroxynitrite, non-fluorescent DHR123 becomes fluorescent rhodamine 123, which localizes in mitochondria and produces green fluorescence [24]. Cultured HeLa cells were treated with various concentrations of allicin for 24 h. After adding 1.25 µM DHR123, the cells were incubated for 30 min at 37 °C in the dark. Before fluorescence microscopy, allicin-treated HeLa cells were fixed and washed twice with PBS.

Fluorescent microscopic analysis of intracellular reactive oxygen species (iROS) level in cervical cancer cells

The cell-permeable dye DCF-DA is widely used to detect ROS. Non-specific cellular esterases hydrolyze it and cellular peroxidases oxidize it to 2,7-dichlorofluorescin. DCF-DA approach was used to quantify intracellular reactive oxygen species [25]. HeLa cells were sown into a 12-well plate and treated for 24 h to test allicin. Allicin was then applied to the cells for 24 h at various doses. After treatment, cells were fixed and treated with 10 µM DCF-DA at 37 °C for 30 min, protected from light with aluminum foil. After washing the cells with PBS to eliminate excess DCF-DA, fluorescence microscopy images were taken.

Analysis of cell viability of cervical cancer cells in presence of a ROS inhibitor, N-acetylcysteine (NAC)

Use ROS inhibitors to confirm intracellular ROS production. N-acetylcysteine (NAC) was employed to confirm ROS production in allicin-treated HeLa cells. After 24-h allicin treatment, cultured cells were pre-incubated with 10 mM NAC for 2 h. As noted, MTT was employed to measure NAC cell viability.

Allicin docking

Grid-Based Ligand Docking with Energetics (GLIDE) is widely utilized molecular docking software developed by Schrödinger. The LigPrep application was employed to process Allicin_CID_65036, involving the addition of hydrogen atoms, removal of ionic species, and incorporation of ions within a pH range of 7 ± 2.0. The protein preparation module was used to generate the three-dimensional structures of both native and mutant protein variants, ensuring accuracy and structural integrity for subsequent docking studies.

This process included the completion of missing side chains, incorporation of terminal caps, formation of disulfide bonds, addition of hydrogen atoms, and establishment of proper bond orders. Energy minimization was carried out using the OPLS3e force field to ensure optimal conformational stability and accuracy.

A grid box was constructed using the known binding sites of the proteins. The receptor grid generation module's default settings for the van der Waals scaling factor (1.0) and charge cutoff (0.25) were used to define the grid dimensions. GLIDE docking was performed using the Ligand Docking program with GLIDE XP parameters. Before analyzing the medicinal compounds, the van der Waals radius was adjusted with a scaling factor of 0.80, and a scoring threshold of 0.15 was employed as a standard criterion.

Molecular dynamics (MD) simulation and principal component analysis

Root-mean-square deviation (RMSD) is a metric that quantifies the distance between frames. It is determined for every profile frame. The root-mean-square deviation of frame x is calculated.

$$}_=\sqrt_^_\left(_\right)\right)-_\left(_}\right)}^}$$

The chosen set contains a total of N atoms. The reference time, tref, is typically set to the first frame, t = 0. The position of the chosen atoms in frame x, after reference frame alignment, is denoted as r′. Frame x is associated with capture time tx. The MD trajectory parameters were analyzed using XmGrace. The root-mean-square fluctuation (RMSF) is useful for characterizing local changes along the protein chain. The RMSF for residue i is:

$$}_=\sqrt_^_\left(t\right)\right)-_\left(_}\right)}^>}$$

RMSF is determined over trajectory time T. The reference time is tref. The position of residue i is ri, and its atoms are r′ after superposition on the reference. The angle bracketed average square distance is calculated over residue atom selection. Protein regions with the highest simulated fluctuation are shown by the peaks on this map. N- and C-terminal protein tails vary more than other regions. Protein and protein–ligand complex microscopic stability is assessed using molecular dynamics, an advanced automated simulation method. Structure, function, fluctuation, interaction, and behavior are demonstrated to do this. The behavior of the four complexes was investigated using GROMACS version 2023.1 for MD calculations. CHARMM general force field parameterized protein content. The SwissParam server implemented ligand topologies. The structures were vacuum-minimized 2500 times using steepest descent to address steric issues. After adding Na+ and Cl− ions, the gmxgenion instrument balanced the system. This was done to ensure system electrical neutrality. After minimization, MD simulations went into production, NVT (constant number of particles, volume and temperature), and NPT (constant number of particles, pressure and temperature). Two phases balanced the systems. First, a 100 picosecond NVT equilibration was done to maintain particle number, volume, and temperature. The procedure aimed to raise system temperature to 300 Kelvin. The second stage involved a precise 100 picosecond NPT equilibration to achieve temperature, pressure, and particle number homogeneity. It was essential to maintain system density and pressure. The protein group's location was limited by bond limitations on all bonds during simulations. The system entropy reduced because NVT and NPT restricted water molecules around the protein, relaxing them. Parrinello–Rahman barostat method and v-rescale thermostat were used for molecular dynamics. The thermostat and barostat were adjusted for 100 picoseconds. To constrain covalent bonding, the linear constraint solver application was used. Chemical bond interactions were handled using the sophisticated (Particle-Mesh Ewald) or PME approach. Every system has a 100-ns production run after equilibrium.

Statistical analysis

The statistical study used one-way and two-way ANOVA and Dunnett's, Tukey's, and Sidak's multiple comparison tests for group comparisons. We calculated statistics with GraphPad Prism 6.0. Data from three independent experiments were provided as mean ± SD. A 0.05 p-value was significant.