2.1. Chemicals and reagents
All chemicals were purchased from Sigma-Aldrich (USA) and Abcam (UK). Antibodies against TNFa (ab46087), VEGFa (ab193555), MMP-2 (ab92536), COX-2 (ab38898), PI3K and phosphorylated (p)-PI3K 154 (ab191606), as well as ELISA kits for AKT and p-AKT (ab179463) were purchased from Abcam.
2.2. Obtaining Ag NPs in the presence of RJ
Raw royal jelly was supplied by the local Armenian beekeeping factory ‘’Royal Jelly’’ LTD (Province Kotayk, Armenia) and stored at – 20 °C. Silver nanoparticles were obtained by green synthesis in the presence of RJ. The NP synthesis method was adapted from earlier reports (Gevorgyan et al., 2021; Mendoza-Reséndez et al., 2014). RJ solution at 0.5 mg mL-1 concentration was prepared in double-distilled water and mixed with 0.01 M silver nitrate solution at a 1:1 ratio. Afterward, the solution was kept under constant stirring at room temperature for ~6 hours.
2.3. Characterization of obtained Ag NPs
AgNP characterization was performed by applying UV-Vis spectroscopy, dynamic light scattering (DLS), and transmission electron microscopy (TEM) in combination with selected area electron diffraction (SAED). UV-Vis absorption spectra were recorded in the wavelength range of 280 and 720 nm (GENESYS 10S UV–Vis, Thermo Scientific, USA). The hydrodynamic radius and polydispersity level of the AgNP sample were studied using DLS. Measurements were performed as described earlier with some modifications (Gevorgyan et al., 2022). For DLS characterization, 20-fold dilutions of AgNP samples were applied and monitored for ~30 min (Spectrosize 300, XtalConcepts, Germany; laser wavelength: 660 nm). Software supporting the CONTIN algorithm was used to analyze collected autocorrelation functions. For TEM and SAED analysis, a drop of AgNP sample was dried on a copper grid and a 200 kV acceleration voltage was applied (Gevorgyan et al., 2021). These experiments and data collection were performed in the XBI Biolab (European XFEL).
2․4. Cell culture
The cytotoxicity properties of the extracts were tested on cervical cancer (HeLa, CCL-2) and lung cancer (A549, CCL-185) cells. Cells were maintained in the appropriate Dulbecco's Modified Essential Medium (DMEM) supplemented with 10% bovine serum and a mixture of penicillin and streptomycin. Cells were grown in tissue culture multiwell microplates and maintained at 37°C in 5% CO2 air in a CO2 incubator (Ginovyan et al., 2022).
2․5.MTT cytotoxicity assay
The MTT (3[(4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) colorimetric method was used to evaluate the cytotoxicity of the investigated substances, which allows for determining the viability of the investigated cells. The method is based on mitochondrial dehydrogenases of living cells, on the transformation of MTT into formazan crystals. The cultured cells were treated with silver nanoparticles for 4, 24, and 72 hours (at a maximum concentration of 23 µg/ml). Sterile water, serving as the solvent in the nanoparticle solution, was used as a negative control. The medium was then replaced with a fresh medium containing a 10% MTT solution. The plates were incubated for an additional 4 hours at 37°C. After incubation, the medium was removed, and the cells were treated with DMSO to dissolve the formed formazan crystals. Optical density was then measured at a wavelength of 570nm using a SPECTROstar® Nano (Ginovyan et al., 2024).
2․6.Cell staining with hematoxylin and eosin
They were stained with hematoxylin and eosin to visualize treated cells. The cells were fixed with ice-cold methanol and subsequently washed with PBS (phosphate buffer, pH 7.4). Following this, the cells were counterstained with hematoxylin, immersed in 1% ammonia, and stained with eosin, before being rewashed with PBS. Images were then obtained using a light microscope (Optika IM3, Italy) (Javrushyan et al., 2022).
2․7.The preparation of the samples for determination of arginase activity, and changes in NO and MDA quantity
Arginase and NO activity, as well as changes in MDA quantity, were analyzed in HeLa and A549 cells treated with AgNPs. Cells were seeded into 24-well plates (5 × 104 cells per well) and incubated for 24 hours. After incubation, 450 μl of the medium in the wells was refreshed. Cells were treated with 50 μl of either control or test compounds at the following final concentrations: distilled water (control for Hela and A549 cells), RJAgNP (2.875 μg/ml and 5.75 μg/ml), and RJ (0.5 mg/ml). After 24 hours of incubation, the cell-free supernatant was discarded. Cells from each group were harvested (trypsinized, neutralized, centrifuged), and then lysed on ice with lysis buffer. The lysates were collected in centrifuge tubes and further lysed for 10 min. After centrifugation at 13,000 × g for 10 min at 4°C, the supernatant was collected. The levels of nitrite anions, MDA, and arginase activity were determined using the methods described below [Avtandilyan et al., 2018a; Ginovyan et al., 2023]. Each test sample (50 μL) was added to five different wells, each set up in triplicate.
2․8.NO quantity measurement
NO levels in the cell culture medium were determined as nitrite anions. For this purpose, Griess analysis was used (Ginovyan et al., 2023). 100 μL of Griess reagent was added to 100 μL of each sample. The supernatants were transferred to tubes containing cadmium beads and incubated at room temperature for 12 h to convert the nitrate to nitrite. The absorbance of the samples was measured at λ=550 nm, and the amount of NO was calculated based on the standard curve generated using NaNO2.
2․9. MDA analysis
Lipid peroxidation was assessed by spectrophotometric determination of malondialdehyde thiobarbiturate with some modifications using the Okawa method (Zeb and Ullah, 2016).
2․10.Arginase activity
To assess arginase activity in cell lysates, a modified diacetyl monoxime colorimetric method was used (Avtandilyan et al., 2022).
2.11. Phospho-PI 3 kinase p85 + Total In-Cell ELISA assay
A549 cells (1.5x104 cells per well) were seeded in the 96-well plates treated for tissue culture. After 24 h incubation, the cell medium (180 𝜇L) was refreshed and the cells were treated with 20𝜇L control or test compounds with the following final concentrations: distilled water (Control, A549C),) RJAgNP (5.75µg/mL), and 5FU (40µM). The calculations during the seeding of the cells were done in a way to reach approximately 80% confluency at fixation time. After 24 h exposure, the medium was discarded and cells were fixed with 100 µL of 4% formaldehyde in PBS. Crystal Violet staining was used to stain the cells, normalizing the readings at 450 nm for Phospho-PI 3 kinase p85 + Total. The measured OD450 readings were corrected for cell number by dividing the OD450 reading for a given well by the OD595 reading from the same well. This relative cell number was then used to normalize each reading. Total and phospho-PI 3 kinase p85 were each assayed in triplicate using the phospho- and total PI 3 Kinase p85 antibodies included in the PI 3 Kinase Kit. Levels of Phospho-PI 3 kinase p85 and Total PI3K were measured using an In-Cell ELISA kit (ab207484), according to the manufacturer's instructions.
2.12. MMP-2 ELISA assay
The assay was performed in A549 cell culture supernatant using the MMP-2 ELISA kit according to the manufacturer's instructions (protocol number ab267646). Test samples included the control group (distilled water) and GSAg Np (5.75 µg/mL).
2.13. COX-2 ELISA assay
The assay was performed in A549 cell culture lysate. The lysates were prepared as described in Material and Methods 2.7. section. Standards (100 μL) and samples (control distilled water (100 μL), and GSAg Np (5.75 µg/mL, 100 μL) were added to the 96-well plates and incubated for 2.5 hours at room temperature. After incubation, the solutions were removed, and the wells were washed. Following the wash process, 100 μL of 1x Biotinylated Antibody was added to each well and then incubated for one hour at room temperature. After repeating the washing process, 100 μL of Streptavidin solution was added to each well and incubated for 45 minutes. Following the final wash, 100 μL of TMB One-Step Substrate Reagent was added to each well and incubated for 30 minutes. In the last stage of the experiment, 50 μL of Stop Solution was added to each well, and the absorbance was measured at 450 nm using the manufacturer's instructions (ab267646).
2.14. TNF alpha ELISA assay
The assay was performed in A549 cell culture lysates. The lysates were prepared as described in the Material and Methods 2.7 section. Test samples included the control group (distilled water) and GSAg Np (5.75 µg/mL). Experiments have been performed according to the manufacturer's protocol (ab46087).
2.15. VEGFA ELISA assay
The assay was performed in A549 cell culture lysates. The lysates were prepared as described in Material and Methods 2.7. section. Test samples included the control group (distilled water) and GSAg Np (5.75 µg/mL). The assay was performed according to the manufacturer's instructions (ab119566).
2.16. Statistical analysis
The obtained results were presented as the mean values with standard errors (M±SD). Statistical analyses were performed using GraphPad Prism 8 software (San Diego, CA, USA), and a significance level of p<0.05 was deemed statistically significant.