Royal Jelly-Mediated Silver Nanoparticles Show Promising Anti-Cancer Effect on HeLa and A549 Cells Through Modulation of the VEGFa/PI3K/Akt/MMP-2 Pathway

Royal Jelly-Mediated Silver Nanoparticles Show Promising Anti-Cancer Effect on HeLa and A549 Cells Through Modulation of the VEGFa/PI3K/Akt/MMP-2 Pathway | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Royal Jelly-Mediated Silver Nanoparticles Show Promising Anti-Cancer Effect on HeLa and A549 Cells Through Modulation of the VEGFa/PI3K/Akt/MMP-2 Pathway Meri Kocharyan, Syuzan Marutyan, Edita Nadiryan, Mikayel Ginovyan, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4045087/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Cancer poses a significant challenge in the medical field, requiring thorough investigation into its mechanisms and the development of effective treatments. Recently, there has been increasing interest in integrating drugs with metal nanoparticles, which are notable for their unique size and physicochemical properties, aiming to enhance anticancer efficacy. Silver nanoparticles (AgNPs), especially those obtained through environmentally friendly methods known as green synthesis, have garnered attention. Royal jelly (RJ), a substance produced by bees recognized for its antioxidant, anti-inflammatory, and antibacterial properties, is particularly interesting. This study focuses on the green synthesis of AgNPs using royal jelly and its bioactivity against cancer cells. It provides a detailed characterization of the nanoparticles and examines their effects on cancer cells, specifically Hela cervical cancer and A549 lung cancer cell lines. The results highlight the cytotoxicity induced by AgNPs in HeLa and A549 cells, which is mediated through apoptosis via the PI3K/AKT signaling pathway. Our research findings demonstrate that one of the mechanisms underlying cell death involves increased concentration of ROS/RNS and downregulation of the VEGFa/MMP-2/COX-2 pathway. This study is among the few to elucidate the mechanism of the anticancer effects of nanoparticles synthesized through this method. Overall, our research contributes to the ongoing exploration of cancer biology and offers insights into potential therapeutic approaches by harnessing the capabilities of green-synthesized nanoparticles. silver nanoparticles royal jelly cytotoxicity PI3K/AKT pathway oxidative stress. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Over the last few years, nanotechnology has been introduced into our daily routine. This revolutionary technology has been applied in multiple fields through an integrated approach. The application of nanotechnology for medical purposes has been termed nanomedicine and is defined as the use of nanomaterials for the diagnosis, monitoring, control, prevention, and treatment of disease (Ahamed et al., 2015; Soares et al., 2018). The flexibility of nanoparticles in terms of size, shape, selective binding ability, high permeability and retention effect, surface modification, and other aspects play a very important role in microbial infections and cancer treatment, especially in cases of ovarian, breast, and lung cancer (Sun et al., 2014; Yılmaz et al., 2023). Cancer is the second leading cause of death in the world and one of the major public health problems. Despite the great advances in cancer therapy, the incidence and mortality rates of cancer remain high (Tomeh et al., 2019). The noble metal nanoparticles (Ag, Au, Pt) have attracted significant interest due to their photothermal and optical properties (Yaqoob et al., 2020). Silver nanoparticles have been proven to be most useful because they have excellent antimicrobial properties against lethal viruses, and microbes, as well as having cytotoxic effects against many cancer cells (Qing et al., 2018). These nanoparticles are certainly the most extensively utilized material of all (Yaqoob et al., 2020). The most common method that has been used to produce AgNPs is chemical synthesis, recruiting reagents whose function is to reduce the silver ions and stabilize the nanoparticles (Ratan et al., 2020). Despite this the AgNPs synthesized by chemical methods are highly harmful to the environment, they have toxic effects on various organs and tissues in the body. Biological methods of synthesis of silver nanoparticles in contrast involve using organisms or their derivatives, such as bacteria, fungi, or plant extracts, to reduce silver ions into nanoparticles in an eco-friendly and sustainable manner. They provide several advantages, such as low cost and ease of implementation (Waktole, 2023). In addition, the protective materials do not damage tissues and organs in the body. The process is considered green, environmentally and eco-friendly (Hovhannisyan et al., 2022). One of these biomaterials is Royal jelly (RJ), a milky secretion substance produced by worker honeybees. It is used in the nutrition of larvae, as well as adult queens. It contains a large amount of essential nutrients such as proteins, carbohydrates, and lipids, and has stronger and more specific biological activity compared to other bee products (Miyata and Sakai, 2018; Yuksel and Akyol, 2016). Many studies have validated the outstanding anticancer potential of green-synthesized AgNPs, as they can target cancer tissue both passively and actively (Alduraihem et al., 2023). AgNPs penetrate the cancer tissues, resulting in cell death through activating many signaling pathways related to mitochondrial dysfunction, oxidative stress, autophagy, and endoplasmic reticulum stress (Alduraihem et al., 2023). But still, despite the general data, it is not known what exact mechanism silver nanoparticles can act on human cancer cells. Drug interactions with GNPs of different shapes, sizes, and surface chemistry for anticancer activity have been widely reported in the literature (Yafout et al., 2021). However, most published studies do not fully discuss the exact mechanisms of action at the molecular level. In this study, we explored the potent anticancer activity of the nanocomplex against A459 and HeLa the cancer cell lines providing insight into the mechanisms of its cytotoxicity at the molecular level. Our goal was to elucidate the effect of RJ-synthesized AgNPs on the PI3Kα/Akt pathway. This pathway was considered as the PI3Kα/AKT/protein kinase B (AKT)/target of rapamycin (mTOR) signaling pathway in mammals as one of the most important molecular pathways controlling proliferation and cell apoptosis (Miricescu et al., 2020). 2. Materials and methods 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 × 10 4 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 NaNO 2 . 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 (1.5x10 4 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. 3. Results 3.1. Characteristics of Ag NPs In terms of heterogeneity and dimensions of NPs, a polydispersity index (PDI) of ~46.2% and an average hydrodynamic radius of 58.25 ± 9.7 nm were determined for particles in aqueous solution (Fig. 1, A). Further, the TEM analysis (Fig. 1, B) demonstrated the round shape of Ag NPs, but still with some variance in morphology. RJ-mediated green synthesized AgNPs’ absorbance peak was observed at ~430 nm (Fig. 1, C), consistent with an earlier study (Gevorgyan et al., 2021). 3.2. MTT cytotoxicity assay The growth-inhibiting properties of both royal jelly and silver nanoparticles synthesized in the presence of royal jelly were assessed on the HeLa and A549 cancer cell lines over different exposure times. Cancer cells were cultured for 4, 24, and 72 hours with various concentrations of silver nanoparticles and royal jelly. Based on the obtained data, royal jelly does not exhibit any inhibitory effect on either of the tested cancer cell lines at any of the tested concentrations and exposure times. Meanwhile, RJ-synthesized silver nanoparticles demonstrate a significant inhibitory effect on the growth of HeLa and A549 cancer cells in a dose-dependent manner (Fig. 2). The exposure time did not significantly affect the inhibitory properties of RJ-AgNPs, indicating that a 4-hour exposure is sufficient to achieve optimal growth-inhibiting properties. At the next stage of the work, the growth-inhibiting properties of silver nanoparticles obtained in the presence of an RJ were determined on the A549 lung cancer cell line (Fig. 2, A). As the concentration of silver nanoparticles increases, the viability of cell growth decreases. Thus, as can be seen from the results, the highest observed concentration of nanoparticles, 23 μg/ml, showed almost the same effect at 4, 24, and 72 hours of growth, showing a decrease in cell viability by 90-95% (Fig. 2, A). This concentration is also cytotoxic to the Hela cell line (Fig. 2, B). At a silver nanoparticle concentration of 11.5 μg/ml, cell viability decreased to 45%. Concentrations of 5.75 μg/ml, 2.845 μg/ml, and 1.4 μg/ml of silver nanoparticles at 4 and 24 hours do not significantly inhibit cell growth. Regarding the effect of royal jelly on the A549 lung cancer cell line, unlike silver nanoparticles, royal jelly again did not inhibit the proliferation of cancer cells. Unlike synthesized nanoparticles, RJ stimulates the proliferation of cancer cells. When HeLa cancer cells are exposed to silver nanoparticles at a concentration of 23 μg/ml, cell growth is inhibited by 90%. A concentration of nanoparticles of 11.5 μg/ml inhibited cell growth by up to 50% after 4 hours of exposure, and up to 60% at 24 and 72 exposures, and a concentration of 5.75 μg/ml inhibited cell growth after 72 hours, by 50%. At lower concentrations, 2.875 μg/ml, and 1.4 μg/ml, cell viability levels decrease by less than 50% (Fig. 2, B). To exclude the possibility that the observed anticancer effect was caused not by silver nanoparticles, but by royal jelly itself, in the next stage of work we determined the cytotoxic effect of royal jelly on the HeLa cell line. As shown in (Fig. 2, B), unlike silver nanoparticles, RJ did not have a cytotoxic effect on the HeLa cancer cell line at any concentration. 3.3. Assessment of morphological and quantitative changes in A549 and HeLa cells Furthermore, the morphological alterations, which were observed in A549 lung cancer cells and HeLa cervical cancer cells following exposure to silver nanoparticles and royal jelly were investigated. Notably, concentrations of 2.845 μg/m and 5.75 μg/ml were selected for silver nanoparticles, while a concentration of 0.5 mg/ml was utilized for royal jelly. A549 and HeLa cells were cultured in the presence of royal jelly and silver nanoparticles for 24 hours, following which potential morphological changes were assessed via staining with hematoxylin and eosin (Fig. 2, D, and E). Relative to the control group, no discernible morphological variations were observed in cancer cells subjected to royal jelly. Conversely, exposure to silver nanoparticles resulted in a reduction in cell count (Fig. 2, C). Furthermore, in comparison to the control group, pronounced nuclear damage and cellular wall edema were evident in this instance, indicative of potential apoptosis induction in cancer cells by silver nanoparticles (Fig. 2, D, and E). 3.4. The changes in arginase activity, and the quantities of nitrite ions and MDA following exposure to RJAgNP Given the role of arginase in the regulation of polyamine metabolism, arginase is naturally associated with tumorigenesis. Therefore, it was interesting to observe changes in arginase activity under the influence of RJAgNP. Hela and A549 cells were cultured for 24 hours under the influence of RJ (0.5 mg/mL) and RJAgNP (2.87 μg/mL and 5.75 μg/mL). As can be seen from Fig. 3A, RJ increases arginase activity in the A549 cell line compared to control. RJAgNP at a concentration of 5.75 μg/ml reduces arginase activity up to 1.6 times (p < 0.05). As indicated in Fig. 3D, the activity of arginase in chela cell lines under exposure to royal jelly at a concentration of 0.5 mg/ml decreases 1.6 times (p<0.05). And under the influence of 5.75 μg/ml silver nanoparticles, arginase activity decreases by almost two times (p<0.001). According to the obtained results, after 24 h of incubation of cells with RJAgNP at concentrations of 2.87 μg/ml and 5.75 μg/ml reduced the quantity of NO in the medium of A549 cells by nearly sixfold (p < 0.05). The same reduction rate was observed with the influence of royal jelly alone (p < 0.01) (Fig. 3, B). A similar result is observed in Hela cells, where under the influence of royal jelly and silver nanoparticles the NO level decreases by 2 times (Figure 3, E). At the next stage of research, we recorded a change in the quantity of malonaldehyde in the A549 and HeLa cell culture medium (Fig. 3, C, and F). Cell lines were cultured for 24 hours. According to the results, RJ (0.5 mg/mL), and all tested concentrations of silver nanoparticles, led to a significant change in the quantity of malonaldehyde in A549 cells, increasing the quantity from 2 to 4 times (Fig. 3, C). A significant change in the amount of MDA was also observed in HeLa cells. Silver nanoparticles at 2.87 μg/ml and 5.75 μg/ml concentrations stimulated the increase of the amount of MDA 3 times compared to the control group (p<0.05). Royal jelly increased the amount of malondialdehyde 5 times (p<0.01) ( Fig. 3, F ). 3.5 Effects of silver nanoparticles on the PI3K/AKT pathway Following the demonstration of the high cytotoxic properties of silver nanoparticles on cancer cells, exploration of the underlying mechanisms contributing to the reduction of cancer proved to be insightful. Findings revealed a significant reduction in TNF alpha levels within A549 cells exposed to silver nanoparticles at concentrations of 5.75 μg/ml compared to the control group (p≤0.01, Fig. 4, A). Exposure to RJNPAg for 24 hours resulted in a pronounced quantitative decrease in VEGF alpha (Figure 4, B, p≤0.001), with values of approximately 5 pg/ml compared to the control group's 25 pg/mg. Similarly, the assessment of COX-2 quantity in the test group yielded a nearly threefold decrease compared to the control group (p≤0.0001, Fig. 4, C). Additional experiments provided insights into the changes in PI3K, Akt, and MMP-2 levels. Notably, incubation of A549 cells with silver nanoparticles at a concentration of 5.75 μg/ml for 24 hours led to a reduction in MMP-2 levels to 325 pg/mg compared to the control group's 475 pg/mg (p≤0.001, Fig. 4, D). Furthermore, as demonstrated in Figure 4E, there was a 2.3-fold decrease in phosphoinositide 3-kinase (PI3K) levels (p < 0.01) and a 1.6-fold decrease in phosphorylated PI3K (p < 0.05) in the RJNPAg treatment group compared to the control group. These results suggest that silver nanoparticles, synthesized with royal jelly, significantly reduce the levels of phosphorylated PI3K in lung cancer cells. Additionally, the total quantity of AKT decreased threefold after 24 hours of incubation (p < 0.0001), and a twofold decrease in phospho-AKT (Ser473) levels was observed following treatment with silver nanoparticles (p < 0.01), indicating a reduction in protein kinase B (AKT) and its activated form (Fig. 4, F). 4. Discussion Metal nanoparticles have attracted the scientific community's interest due to their various applications in biology, materials science, medicine, etc. Within this group, silver nanoparticles have particularly attracted attention due to their unique physicochemical properties, such as chemical stability and electrical conductivity, and their biological activities, including antibacterial, antifungal, anti-inflammatory, antiviral, and antiangiogenic effects (Al-Sheddi et al., 2018). Green synthesis of silver nanoparticles is regarded as safer compared to chemical synthesis due to the potential toxicity and health hazards associated with the chemicals used in traditional synthesis methods. Green synthesis typically employs natural sources, which significantly reduces the likelihood of introducing harmful substances into the nanoparticles. This aspect is particularly crucial for medical applications, where nanoparticles may interact with the human body. Accordingly, in our study, we used royal jelly for the green synthesis of silver nanoparticles. The obtained nanoparticles had an average hydrodynamic radius of 58.25 ± 9.7 nm. In previous studies, the characteristics of RJ silver nanoparticles were detailed, and their antimicrobial effects were highlighted (Gevorgyan et al., 2022). In this research, we focused on exploring their anticancer properties and their potential mechanisms of action. In previous works, it was shown, that royal jelly can have an anticancer effect in vitro at high concentrations (Miyata and Sakai, 2018). However, during our experiments, no anticancer effect was detected at any of the tested concentrations (the highest tested concentration was 5 mg/mL). An analysis of the viability of RJNPAg-treated cells demonstrates selective cytotoxicity towards the cancer cell lines HeLa and A549 at concentrations ranging from 2.874 μg/ml to 23 μg/ml. At the maximum tested concentration, the nanoparticles exhibited 98% cytotoxicity against both cancer lines. Notably, nanoparticles synthesized with royal jelly displayed enhanced cytotoxicity at lower concentrations compared to other silver nanoparticles, with the potency of the effect varying according to the size of the nanoparticles. In the HeLa cell line, the inhibitory concentration (IC50) was 6.788 μg/mL after 24 hours, while for the A549 cell line, the IC50 was 13.37 μg/mL (Abdellatif et al., 2021). The mechanism of cytotoxicity may include interaction with cellular structures, induction of oxidative stress, and disruption of cellular processes ( Fig.5 ). Studies have confirmed that the PI3K/AKT/mTOR signaling pathway is one of the classic ways to inhibit apoptosis in which PI3K, a type of lipid kinase, can be directly or indirectly activated by various growth factors and signal transduction complexes through the focal adhesion kinase pathway AKT, a protein located downstream of PI3K plays a certain vital role in this (Wu et al., 2020). RJAgNP reduces the amounts of both Total PI3K and Akt, as well as their phosphorylated forms (Fig. 4, E, and F). This result indicates that RJAgNP affects both the expression of the gene for the synthesis of these proteins and the enzymes themselves, inhibiting the phosphorylation to the active form. This circumstance indicates the widespread and multi-target effect of RJAgNP. Previous studies have highlighted the critical role of arginase in the metabolism of L-arginine, which is involved in cancer growth and control. This involves mediating polyamine biosynthesis by arginase, nitric oxide production by NOS, and the immune response (Avtandilyan et al., 2022, 2019; Ginovyan et al., 2023). These metabolites play key functions in cell physiology and the health of the human body (Chen et al., 2021). Additionally, both basic and clinical studies revealed that arginase is highly expressed in various types of cancer, including breast, lung, gastric, colorectal, and liver cancer (Avtandilyan et al., 2018). This is significant since arginine is the most consumed amino acid in the inner necrotic core of the tumor mass (Niu et al., 2022). Arginine also stimulates various cellular mechanisms, including the PI3K/Akt/mTOR pathway, which in turn stimulates cancer growth (Chen et al., 2021). In our study, RJ green synthesized silver nanoparticles showed a decrease in arginase activity, which in turn means that nanoparticles have an antiproliferative effect. As previously mentioned, arginine serves as a precursor to NO under the action of NOS. Recent data highlight diverse roles of NO in tumorigenesis, including promoting angiogenesis, metastasis, anti-apoptotic processes, and modulating the immune response (Avtandilyan et al., 2022). However, it is important to note that the NOS-PI3K-AKT signaling pathway also mediates cell invasion (Ghafouri-Fard et al., 2022). In our research, we demonstrated that silver nanoparticles reduce the level of NO in vitro and decrease the levels of PI3K and AKT, leading to the activation of anti-invasion and anti-angiogenesis mechanisms. NO produced by NOS, whether located within the tumor or in the surrounding stroma, may promote the formation of new blood vessels by upregulating vascular endothelial growth factor (VEGF) (Secondini et al., 2017; Timoshenko et al., 2006; Vahora et al., 2016). Given the complexity of the process of angiogenesis, VEGF (VEGF-A), plays a prominent role in signaling through the VEGF-2 receptor, which induces angiogenesis in both healthy and diseased processes (Quintero-Fabián et al., 2019). It was clear that silver nanoparticles also reduce the amount of VEGF, thereby influencing cell metastasis. Moreover, AgNPs significantly reduced the expression of MMP-2, which is closely associated with angiogenesis, invasiveness, and metastasis. In addition to PI3K/Akt, VEGF, and MMP-2, we analyzed the production of COX-2 due to its critical role in the pathogenesis of several inflammatory diseases and cancers; COX-2 is typically expressed at low levels in some tissues and cells; therefore, it can be strongly induced by certain types of cytokines such as TNF-α (St-Germain et al., 2004; Yang et al., 2020). On the other hand, TNF-α is involved in systemic inflammation by stimulating the acute phase response. The anticancer properties of TNF-α are mainly achieved by inducing cancer cell death. Genes that are regulated by NF-kB have been shown to suppress apoptosis. Thus, cytokines such as TNFa activate apoptotic and antiapoptotic pathways (Gaur and Aggarwal, 2003). NF-κB activation usually precedes the apoptotic effects of TNF. Although TNF activates NF-κB in all cell types, it very rarely induces apoptosis. This may be due to the ability of NF-kB to suppress apoptosis. Interestingly, however, both the apoptotic and anti-apoptotic effects of TNFa are mediated by the formation of reactive oxygen intermediates (ROIs) (Gaur and Aggarwal, 2003). It is generally accepted that DOX alters DNA chains and gene expression of various enzymes during the stages of transcription or translation, either directly or indirectly through the free radicals it generates, thereby causing changes in the activity of antioxidant enzymes (Erbaş et al., 2024). In some studies, free radicals responsible for pathogenesis include superoxide, hydroxyl radicals, and NO (He et al., 2021). Free radical-induced lipid peroxidation products such as MDA have also been shown to promote this event and cause cellular damage by reducing antioxidant enzymes (Erbaş et al., 2024). Our results showed that nanoparticles increased MDA concentration several times in both cell lines. The increase in the amount of MDA and the concomitant decrease in the amount of Akt (responsible for survival) are important prerequisites for promoting apoptosis. In this way, our obtained results and assumptions also contribute to the understanding of the molecular mechanisms underlying cell death ( Fig.5 ). Summarizing the obtained results, we can note that the inhibition of the VEGF/PI3K/Akt pathway and the quantitative increase of MDA (therefore ROS increase) are the basis of RJAgNP cytotoxicity. In addition, these nanoparticles show promise and great potential for positive regulation of anti-inflammatory, anti-angiogenic, and anti-metastatic pathways by reducing TNFa, COX-2, and MMP-2 levels. 5․ Conclusion In conclusion, our study highlights the promising anticancer potential of silver nanoparticles synthesized via green methods using royal jelly. These nanoparticles demonstrate significant cytotoxic effects against HeLa and A549 cancer cell lines. Underlying molecular mechanisms contributing to their anticancer activity were elucidated. Our findings revealed a significant decrease in arginase activity upon exposure to silver nanoparticles, accompanied by reductions in PI3K and phosphorylated and total Akt levels, indicative of pathway inhibition. Additionally, RJAgNP demonstrated a capacity to reduce nitric oxide levels and suppress angiogenesis-related factors like VEGF and MMP-2, and inflammation-related factors like TNFa and COX-2, thus impeding angiogenesis and metastasis. Moreover, our investigation shed light on the involvement of reactive oxygen intermediates (ROIs) in mediating apoptotic pathways, as evidenced by the increase in malondialdehyde (MDA) concentration and the corresponding decrease in Akt levels, ultimately promoting death in cancer cells. Our research contributes to the expanding field of nanoparticle-based therapeutics and opens new avenues for cancer treatment strategies. Abbreviations AKT - Protein kinase B; AgNP - Silver nanoparticles; COX-2 - Cyclooxygenase-2; DLS -Dynamic light scattering; DMEM - Dulbecco's Modified Eagle Medium; DMSO - Dimethyl sulfoxide; ELISA - Enzyme-linked immunosorbent assay; FU – Fluorouracil; GNPs - Green synthesis nanoparticles; H&E - Haematoxylin and Eosin; MDA – Malondialdehyde; MMP-2 - Matrix metalloproteinase-2; mTOR - mammalian target of rapamycin; MTT - 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide; NF-κB - Nuclear factor kappa-light-chain-enhancer of activated B cells; NO - Nitric oxide; PBS - Phosphate buffered saline; PDI - Protein disulfide isomerase; PI3K - Phosphoinositide 3-kinase; RJ - Royal Jelly; RJAgNP- Silver nanoparticles synthesized in the presence of royal jelly; ROS - Reactive oxygen species; SAED - Selected area (electron) diffraction; TEM - Transmission electron microscopy; TNF alpha - Tumor necrosis factor-alpha; VEGF - Vascular endothelial growth factor․ Declarations Acknowledgments This work was supported by the Science Committee of MESCS RA through research projects numbered 21T-1F283, 21T-1F300, and 23LCG-1F010. Many thanks to Zaruhi Karabekian, Head of the Laboratory of Immunology and Tissue Engineering at the L. A. Orbeli Institute of Physiology NAS RA, for providing the HeLa and A549 cell lines, which were purchased from the ATCC collection. Thank you Dr. Robin Schubert from European XFEL GmbH for the TEM analysis. Conflict of interest The authors declare no conflict of interest. Author contributions The study's conception and design were the results of collective contributions from all authors. The investigations and analysis of results were carried out by MK, EN, and SM. MK, MG, HJ, and NA wrote the manuscript. NA and SM directed the project, and corrected, and edited the manuscript. All authors participated in the revision and approval of the final version of the manuscript. References Abdellatif, Ah.A.H., Alsharidah, M., Rugaie, O. Al, Tawfeek, H.M., Tolba, N.S., 2021. Silver nanoparticle-coated ethyl cellulose inhibits tumor necrosis factor-α of breast cancer cells. 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Int J Mol Sci. https://doi.org/10.3390/ijms20051033 Vahora, H., Khan, M.A., Alalami, U., Hussain, A., 2016. The Potential Role of Nitric Oxide in Halting Cancer Progression Through Chemoprevention. J Cancer Prev 21, 1–12. https://doi.org/10.15430/JCP.2016.21.1.1 Waktole, G., 2023. Toxicity and Molecular Mechanisms of Actions of Silver Nanoparticles. J Biomater Nanobiotechnol 14, 53–70. https://doi.org/10.4236/jbnb.2023.143005 Wu, Y., Ma, J., Sun, Y., Tang, M., Kong, L., 2020. Effect and mechanism of PI3K/AKT/mTOR signaling pathway in the apoptosis of GC-1 cells induced by nickel nanoparticles. Chemosphere 255. https://doi.org/10.1016/j.chemosphere.2020.126913 Yafout, M., Ousaid, A., Khayati, Y., El Otmani, I.S., 2021. Gold nanoparticles as a drug delivery system for standard chemotherapeutics: A new lead for targeted pharmacological cancer treatments. Sci Afr. https://doi.org/10.1016/j.sciaf.2020.e00685 Yang, J., Wang, X., Gao, Y., Fang, C., Ye, F., Huang, B., Li, L., 2020. Inhibition of PI3K-AKT signaling blocks PGE2-induced COX-2 expression in lung adenocarcinoma. Onco Targets Ther 13, 8197–8208. https://doi.org/10.2147/OTT.S263977 Yaqoob, A.A., Ahmad, H., Parveen, T., Ahmad, A., Oves, M., Ismail, I.M.I., Qari, H.A., Umar, K., Mohamad Ibrahim, M.N., 2020. Recent Advances in Metal Decorated Nanomaterials and Their Various Biological Applications: A Review. Front Chem. https://doi.org/10.3389/fchem.2020.00341 Yılmaz, G.E., Göktürk, I., Ovezova, M., Yılmaz, F., Kılıç, S., Denizli, A., 2023. Antimicrobial Nanomaterials: A Review. Hygiene 3, 269–290. https://doi.org/10.3390/hygiene3030020 Yuksel, S., Akyol, S., 2016. The consumption of propolis and royal jelly in preventing upper respiratory tract infections and as dietary supplementation in children. J Intercult Ethnopharmacol. https://doi.org/10.5455/jice.20160331064836 Zeb, A., Ullah, F., 2016. A Simple Spectrophotometric Method for the Determination of Thiobarbituric Acid Reactive Substances in Fried Fast Foods. J Anal Methods Chem 2016. https://doi.org/10.1155/2016/9412767 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4045087","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":277932794,"identity":"29e19463-9850-4d58-8e53-d570596fcbe7","order_by":0,"name":"Meri Kocharyan","email":"","orcid":"","institution":"Yerevan State University","correspondingAuthor":false,"prefix":"","firstName":"Meri","middleName":"","lastName":"Kocharyan","suffix":""},{"id":277932795,"identity":"4f4a9c0b-699f-4e94-9101-d43e11796f6b","order_by":1,"name":"Syuzan Marutyan","email":"","orcid":"","institution":"Yerevan State University","correspondingAuthor":false,"prefix":"","firstName":"Syuzan","middleName":"","lastName":"Marutyan","suffix":""},{"id":277932796,"identity":"1c360164-0f01-4e02-8efc-bf97d39f469d","order_by":2,"name":"Edita Nadiryan","email":"","orcid":"","institution":"Yerevan State University","correspondingAuthor":false,"prefix":"","firstName":"Edita","middleName":"","lastName":"Nadiryan","suffix":""},{"id":277932797,"identity":"79651ca7-28bd-4dad-b275-5a1579619af1","order_by":3,"name":"Mikayel Ginovyan","email":"","orcid":"","institution":"Yerevan State University","correspondingAuthor":false,"prefix":"","firstName":"Mikayel","middleName":"","lastName":"Ginovyan","suffix":""},{"id":277932798,"identity":"47d29082-1e5e-485b-92cd-41ce8b4825b9","order_by":4,"name":"Hayarpi Javrushyan","email":"","orcid":"","institution":"Yerevan State University","correspondingAuthor":false,"prefix":"","firstName":"Hayarpi","middleName":"","lastName":"Javrushyan","suffix":""},{"id":277932799,"identity":"639e4470-9344-46b4-93f0-ca24f7d74428","order_by":5,"name":"Seda Marutyan","email":"","orcid":"","institution":"Yerevan State University","correspondingAuthor":false,"prefix":"","firstName":"Seda","middleName":"","lastName":"Marutyan","suffix":""},{"id":277932800,"identity":"43a601a9-92e2-4ba0-b264-57dcef90e75e","order_by":6,"name":"Nikolay Avtandilyan","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA60lEQVRIiWNgGAWjYFCDw8wNQNKGWOUJIC2MIC1ppGg5ANZymLBic4kExs+8P+zy+I4ztkn83HE+cf6MBMZPN/BosZyRwCzNk5BcLHmYsU2y98ztxA03gCI5eLQYnDnAIAnUlrgBqEWCtw2oRSKBgZAW5p8zEurBWiT/tp0DOYz5N14txxvYJD4kHAZrkeZtO5DYcCOBDb8txxvbLD6kHU+ceZix2Vq2Ldl4w5mHbdZ4tRxmPnwjwaY6se/84YM337bZyc5vTz58G58WBgZwdIABiwSQcGxAEiEImD8ACXuilY+CUTAKRsGIAQAi01UFl8ZOAQAAAABJRU5ErkJggg==","orcid":"","institution":"Yerevan State University","correspondingAuthor":true,"prefix":"","firstName":"Nikolay","middleName":"","lastName":"Avtandilyan","suffix":""}],"badges":[],"createdAt":"2024-03-08 14:07:43","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4045087/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4045087/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":52394901,"identity":"be58bd20-f239-4e51-8c80-2c908d7f596b","added_by":"auto","created_at":"2024-03-11 04:52:21","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":330326,"visible":true,"origin":"","legend":"\u003cp\u003eThe characteristics of RJ-mediated Ag NPs: A – hydrodynamic radii evolution, B –TEM (dilution: 1:90), C – UV-Vis absorption spectra, and D – SAED pattern.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4045087/v1/8c488ab3a6f1504a13647a8a.png"},{"id":52394905,"identity":"3bbeb975-94bc-4701-bed7-be95327e765d","added_by":"auto","created_at":"2024-03-11 04:52:21","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":827747,"visible":true,"origin":"","legend":"\u003cp\u003eInhibition of A549 (A) and HeLa (B) cell growth by royal jelly and silver nanoparticles obtained from royal jelly was determined by the MTT test (n=3, p≤0.05). The observation of quantitative changes of cells in 3 different passages, in 10 fields of view of each, was carried out using the ImageJ program (C, * - p≤0.05, ** - p≤0.01, **** - p≤0.0001, The scale bar is 100 μm). Morphological and quantitative changes in the A549 (D) and HeLa (E) cells under the influence of various concentrations of silver nanoparticles and royal jelly (stain: H\u0026amp;E, magnification: x100). RJ - royal jelly with a concentration of 0.5 mg/ml, RJAgNP2.8 - silver nanoparticles with a concentration of 2.875 μg/ml, RJAgNP5.7 - nanoparticles with a silver concentration of 5.75 μg/ml.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4045087/v1/4741be8d4b0313c62041f2d4.png"},{"id":52394902,"identity":"6c21685b-ca1a-4ab5-bebf-e1b41e4e7f60","added_by":"auto","created_at":"2024-03-11 04:52:21","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":108673,"visible":true,"origin":"","legend":"\u003cp\u003eChange in arginase activity (A, D), the quantity of nitrite ions (B, E), and malondialdehyde (C, F) in A549 and Hela cells under the influence of silver nanoparticles and royal jelly (n=5, * - p≤0.05, ** - p≤0.01, *** - p≤0.001, **** - p≤0.0001).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4045087/v1/2af2e17c9cba3c555ea43d58.png"},{"id":52395005,"identity":"40d8a0dc-c365-465c-a668-6b68a4a6aafa","added_by":"auto","created_at":"2024-03-11 05:00:21","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":92272,"visible":true,"origin":"","legend":"\u003cp\u003eLevels of TNFa (A), VEGFa (B), Cox-2 (C), MMP-2 (D), PI3K (E), and AKT (F) in A549 after 24 hours of exposure to AgNP. Each value represents the mean ± SE of three experiments (n=3, * - p≤0.05, ** - p≤0.01, *** - p≤0.001, **** - p≤0.0001).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4045087/v1/f74e3fbe20f751a0fcb0a9c7.png"},{"id":52395006,"identity":"deba506d-dfb5-4d28-958a-b10613a69417","added_by":"auto","created_at":"2024-03-11 05:00:21","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":340254,"visible":true,"origin":"","legend":"\u003cp\u003ePossible molecular mechanism of RJAgNP cytotoxicity effect on cancer cells.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4045087/v1/a8c8498cfe83d97196263f18.png"},{"id":52395668,"identity":"2b7001bb-3950-474e-99ba-655587008c1e","added_by":"auto","created_at":"2024-03-11 05:08:22","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1896541,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4045087/v1/ad9f69ff-4730-4d20-97ad-1b5e74982d93.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Royal Jelly-Mediated Silver Nanoparticles Show Promising Anti-Cancer Effect on HeLa and A549 Cells Through Modulation of the VEGFa/PI3K/Akt/MMP-2 Pathway","fulltext":[{"header":"1. Introduction ","content":"\u003cp\u003eOver the last few years, nanotechnology has been introduced into our daily routine. This revolutionary technology has been applied in multiple fields through an integrated approach. The application of nanotechnology for medical purposes has been termed nanomedicine and is defined as the use of nanomaterials for the diagnosis, monitoring, control, prevention, and treatment of disease (Ahamed et al., 2015; Soares et al., 2018). The flexibility of nanoparticles in terms of size, shape, selective binding ability, high permeability and retention effect, surface modification, and other aspects play a very important role in microbial infections and cancer treatment, especially in cases of ovarian, breast, and lung cancer (Sun et al., 2014; Yılmaz et al., 2023). Cancer is the second leading cause of death in the world and one of the major public health problems. Despite the great advances in cancer therapy, the incidence and mortality rates of cancer remain high (Tomeh et al., 2019). The noble metal nanoparticles (Ag, Au, Pt) have attracted significant interest due to their photothermal and optical properties (Yaqoob et al., 2020). Silver nanoparticles have been proven to be most useful because they have excellent antimicrobial properties against lethal viruses, and microbes, as well as having cytotoxic effects against many cancer cells (Qing et al., 2018). These nanoparticles are certainly the most extensively utilized material of all (Yaqoob et al., 2020). The most common method that has been used to produce AgNPs is chemical synthesis, recruiting reagents whose function is to reduce the silver ions and stabilize the nanoparticles (Ratan et al., 2020). Despite this the AgNPs synthesized by chemical methods are highly harmful to the environment, they have toxic effects on various organs and tissues in the body. Biological methods of synthesis of silver nanoparticles in contrast involve using organisms or their derivatives, such as bacteria, fungi, or plant extracts, to reduce silver ions into nanoparticles in an eco-friendly and sustainable manner. They provide several advantages, such as low cost and ease of implementation (Waktole, 2023). In addition, the protective materials do not damage tissues and organs in the body. The process is considered green, environmentally and eco-friendly (Hovhannisyan et al., 2022). One of these biomaterials is Royal jelly (RJ), a milky secretion substance produced by worker honeybees. It is used in the nutrition of larvae, as well as adult queens. It contains a large amount of essential nutrients such as proteins, carbohydrates, and lipids, and has stronger and more specific biological activity compared to other bee products (Miyata and Sakai, 2018; Yuksel and Akyol, 2016). Many studies have validated the outstanding anticancer potential of green-synthesized AgNPs, as they can target cancer tissue both passively and actively (Alduraihem et al., 2023). AgNPs penetrate the cancer tissues, resulting in cell death through activating many signaling pathways related to mitochondrial dysfunction, oxidative stress, autophagy, and endoplasmic reticulum stress (Alduraihem et al., 2023). But still, despite the general data, it is not known what exact mechanism silver nanoparticles can act on human cancer cells. Drug interactions with GNPs of different shapes, sizes, and surface chemistry for anticancer activity have been widely reported in the literature (Yafout et al., 2021). However, most published studies do not fully discuss the exact mechanisms of action at the molecular level. In this study, we explored the potent anticancer activity of the nanocomplex against A459 and HeLa the cancer cell lines providing insight into the mechanisms of its cytotoxicity at the molecular level. Our goal was to elucidate the effect of RJ-synthesized AgNPs on the PI3K\u0026alpha;/Akt pathway. This pathway was considered as the PI3K\u0026alpha;/AKT/protein kinase B (AKT)/target of rapamycin (mTOR) signaling pathway in mammals as one of the most important molecular pathways controlling proliferation and cell apoptosis (Miricescu et al., 2020).\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cp\u003e\u003cstrong\u003e2.1. Chemicals and reagents\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll 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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2. Obtaining Ag NPs in the presence of RJ\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRaw royal jelly was supplied by the local Armenian beekeeping factory ‘’Royal Jelly’’ LTD (Province Kotayk, Armenia) and stored at – 20 °C.\u0026nbsp;Silver nanoparticles were obtained by green synthesis in the presence of RJ. The NP synthesis method was adapted from earlier reports\u0026nbsp;(Gevorgyan et al., 2021; Mendoza-Reséndez et al., 2014).\u0026nbsp;RJ solution at 0.5 mg mL\u003csup\u003e-1\u003c/sup\u003e 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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3. Characterization of obtained Ag NPs\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAgNP 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).\u0026nbsp;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\u0026nbsp;(Gevorgyan et al., 2022).\u0026nbsp;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).\u0026nbsp;These experiments and data collection were performed in the XBI Biolab (European XFEL).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2․4.\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eCell culture\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe 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\u0026nbsp;and\u0026nbsp;maintained at 37°C in 5% CO2 air in a CO2 incubator\u0026nbsp;(Ginovyan et al., 2022).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2․\u003c/strong\u003e\u003cstrong\u003e5.\u003c/strong\u003e\u003cstrong\u003eMTT cytotoxicity assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe 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\u0026nbsp;(Ginovyan et al., 2024).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2․\u003c/strong\u003e\u003cstrong\u003e6.\u003c/strong\u003e\u003cstrong\u003eCell staining with hematoxylin and eosin\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThey 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)\u0026nbsp;(Javrushyan et al., 2022).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2․\u003c/strong\u003e\u003cstrong\u003e7.\u003c/strong\u003e\u003cstrong\u003eThe preparation of the samples for determination of arginase activity, and changes in NO and MDA quantity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eArginase 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 × 10\u003csup\u003e4\u003c/sup\u003e 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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2․\u003c/strong\u003e\u003cstrong\u003e8.\u003c/strong\u003e\u003cstrong\u003eNO quantity measurement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNO levels in the cell culture medium were determined as nitrite anions. For this purpose, Griess analysis was used\u0026nbsp;(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 NaNO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2․\u003c/strong\u003e\u003cstrong\u003e9. MDA analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLipid peroxidation was assessed by spectrophotometric determination of malondialdehyde thiobarbiturate with some modifications using the Okawa method\u0026nbsp;(Zeb and Ullah, 2016).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2․\u003c/strong\u003e\u003cstrong\u003e10.\u003c/strong\u003e\u003cstrong\u003eArginase activity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo assess arginase activity in cell lysates, a modified diacetyl monoxime colorimetric method was used\u0026nbsp;(Avtandilyan et al., 2022).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.11. Phospho-PI 3 kinase p85 + Total In-Cell ELISA assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA549 cells (1.5x10\u003csup\u003e4\u003c/sup\u003e cells per well) were seeded in the 96-well plates treated for tissue culture. After 24 h incubation, the cell medium (180\u0026nbsp;𝜇L) was refreshed and the cells were treated with 20𝜇L\u0026nbsp;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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.12.\u003c/strong\u003e \u003cstrong\u003eMMP\u003c/strong\u003e\u003cstrong\u003e-\u003c/strong\u003e\u003cstrong\u003e2 ELISA assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe 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).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.13. COX-2\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;ELISA assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;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\u0026nbsp;distilled water\u0026nbsp;(100 μL), and GSAg Np\u0026nbsp;(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).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.14\u003c/strong\u003e. \u003cstrong\u003eTNF alpha ELISA assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe 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).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.15. VEGFA ELISA assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe 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).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.16. Statistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe 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\u0026lt;0.05 was deemed statistically significant.\u003c/p\u003e"},{"header":"3. Results","content":"\u003cp\u003e\u003cstrong\u003e3.1. Characteristics of Ag NPs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn terms of heterogeneity and dimensions of NPs, a polydispersity index (PDI) of ~46.2% and an average hydrodynamic radius of 58.25 ± 9.7 nm were determined for particles in aqueous solution (Fig. 1,\u0026nbsp;A). Further, the TEM analysis (Fig. 1, B) demonstrated the round shape of Ag NPs, but still with some variance in morphology. RJ-mediated green synthesized AgNPs’ absorbance peak was observed at ~430 nm (Fig. 1, C), consistent with an earlier study\u0026nbsp;(Gevorgyan et al., 2021).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2. MTT cytotoxicity assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe growth-inhibiting properties of both royal jelly and silver nanoparticles synthesized in the presence of royal jelly were assessed on the HeLa and A549 cancer cell lines over different exposure times. Cancer cells were cultured for 4, 24, and 72 hours with various concentrations of silver nanoparticles and royal jelly. Based on the obtained data, royal jelly does not exhibit any inhibitory effect on either of the tested cancer cell lines at any of the tested concentrations and exposure times. Meanwhile, RJ-synthesized silver nanoparticles demonstrate a significant inhibitory effect on the growth of HeLa and A549 cancer cells in a dose-dependent manner (Fig. 2). The exposure time did not significantly affect the inhibitory properties of RJ-AgNPs, indicating that a 4-hour exposure is sufficient to achieve optimal growth-inhibiting properties.\u003c/p\u003e\n\u003cp\u003eAt the next stage of the work, the growth-inhibiting properties of silver nanoparticles obtained in the presence of an RJ were determined on the A549 lung cancer cell line (Fig. 2, A). As the concentration of silver nanoparticles increases, the viability of cell growth decreases. Thus, as can be seen from the results, the highest observed concentration of nanoparticles, 23 μg/ml, showed almost the same effect at 4, 24, and 72 hours of growth, showing a decrease in cell viability by 90-95% (Fig. 2, A). This concentration is also cytotoxic to the Hela cell line (Fig. 2, B). At a silver nanoparticle concentration of 11.5 μg/ml, cell viability decreased to 45%. Concentrations of 5.75 μg/ml, 2.845 μg/ml, and 1.4 μg/ml of silver nanoparticles at 4 and 24 hours do not significantly inhibit cell growth. Regarding the effect of royal jelly on the A549 lung cancer cell line, unlike silver nanoparticles, royal jelly again did not inhibit the proliferation of cancer cells. Unlike synthesized nanoparticles, RJ stimulates the proliferation of cancer cells.\u003c/p\u003e\n\u003cp\u003eWhen HeLa cancer cells are exposed to silver nanoparticles at a concentration of 23 μg/ml, cell growth is inhibited by 90%. A concentration of nanoparticles of 11.5 μg/ml inhibited cell growth by up to 50% after 4 hours of exposure, and up to 60% at 24 and 72 exposures, and a concentration of 5.75 μg/ml inhibited cell growth after 72 hours, by 50%. At lower concentrations, 2.875 μg/ml, and 1.4 μg/ml, cell viability levels decrease by less than 50% (Fig. 2, B). To exclude the possibility that the observed anticancer effect was caused not by silver nanoparticles, but by royal jelly itself, in the next stage of work we determined the cytotoxic effect of royal jelly on the HeLa cell line. As shown in (Fig. 2, B), unlike silver nanoparticles, RJ did not have a cytotoxic effect on the HeLa cancer cell line at any concentration.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3. Assessment of morphological and quantitative changes in A549 and HeLa cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFurthermore, the morphological alterations, which were observed in A549 lung cancer cells and HeLa cervical cancer cells following exposure to silver nanoparticles and royal jelly were investigated. Notably, concentrations of 2.845 μg/m and 5.75 μg/ml were selected for silver nanoparticles, while a concentration of 0.5 mg/ml was utilized for royal jelly. A549 and HeLa cells were cultured in the presence of royal jelly and silver nanoparticles for 24 hours, following which potential morphological changes were assessed via staining with hematoxylin and eosin (Fig. 2, D, and E). Relative to the control group, no discernible morphological variations were observed in cancer cells subjected to royal jelly. Conversely, exposure to silver nanoparticles resulted in a reduction in cell count (Fig. 2, C). Furthermore, in comparison to the control group, pronounced nuclear damage and cellular wall edema were evident in this instance, indicative of potential apoptosis induction in cancer cells by silver nanoparticles (Fig. 2, D, and E).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4.\u003c/strong\u003e\u003cstrong\u003eThe changes in arginase activity, and the quantities of nitrite ions and MDA following exposure to RJAgNP\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGiven the role of arginase in the regulation of polyamine metabolism, arginase is naturally associated with tumorigenesis. Therefore, it was interesting to observe changes in arginase activity under the influence of\u0026nbsp;RJAgNP. Hela and A549 cells were cultured for 24 hours under the influence of RJ (0.5 mg/mL) and\u0026nbsp;RJAgNP\u0026nbsp;(2.87 μg/mL and 5.75 μg/mL). As can be seen from Fig. 3A, RJ increases arginase activity in the A549 cell line compared to control.\u0026nbsp;RJAgNP\u0026nbsp;at a concentration of 5.75 μg/ml reduces arginase activity up to 1.6 times (p \u0026lt; 0.05). As indicated in Fig. 3D, the activity of arginase in chela cell lines under exposure to royal jelly at a concentration of 0.5 mg/ml decreases 1.6 times (p\u0026lt;0.05). And under the influence of 5.75 μg/ml silver nanoparticles, arginase activity decreases by almost two times (p\u0026lt;0.001).\u003c/p\u003e\n\u003cp\u003eAccording to the obtained results, after 24 h of incubation of cells with\u0026nbsp;RJAgNP\u0026nbsp;at concentrations of 2.87 μg/ml and 5.75 μg/ml reduced the quantity of NO in the medium of A549 cells by nearly sixfold (p \u0026lt; 0.05). The same reduction rate was observed with the influence of royal jelly alone (p \u0026lt; 0.01) (Fig. 3, B). A similar result is observed in Hela cells, where under the influence of royal jelly and silver nanoparticles the NO level decreases by 2 times (Figure 3, E).\u003c/p\u003e\n\u003cp\u003eAt the next stage of research, we recorded a change in the quantity of malonaldehyde in the A549 and HeLa cell culture medium (Fig. 3, C, and F). Cell lines were cultured for 24 hours. According to the results, RJ (0.5 mg/mL), and all tested concentrations of silver nanoparticles, led to a significant change in the quantity of malonaldehyde in A549 cells, increasing the quantity from 2 to 4 times (Fig. 3, C). A significant change in the amount of MDA was also observed in HeLa cells. Silver nanoparticles at 2.87 μg/ml and 5.75 μg/ml concentrations stimulated the increase of the amount of MDA 3 times compared to the control group (p\u0026lt;0.05). Royal jelly increased the amount of malondialdehyde 5 times (p\u0026lt;0.01) (\u003cstrong\u003eFig. 3, F\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.5 Effects of silver nanoparticles on the PI3K/AKT pathway\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFollowing the demonstration of the high cytotoxic properties of silver nanoparticles on cancer cells, exploration of the underlying mechanisms contributing to the reduction of cancer proved to be insightful. Findings revealed a significant reduction in TNF alpha levels within A549 cells exposed to silver nanoparticles at concentrations of 5.75 μg/ml compared to the control group (p≤0.01, Fig. 4, A). Exposure to RJNPAg for 24 hours resulted in a pronounced quantitative decrease in VEGF alpha (Figure 4, B, p≤0.001), with values of approximately 5 pg/ml compared to the control group's 25 pg/mg. Similarly, the assessment of COX-2 quantity in the test group yielded a nearly threefold decrease compared to the control group (p≤0.0001, Fig. 4, C). Additional experiments provided insights into the changes in PI3K, Akt, and MMP-2 levels. Notably, incubation of A549 cells with silver nanoparticles at a concentration of 5.75 μg/ml for 24 hours led to a reduction in MMP-2 levels to 325 pg/mg compared to the control group's 475 pg/mg (p≤0.001, Fig. 4, D).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFurthermore, as demonstrated in Figure 4E, there was a 2.3-fold decrease in phosphoinositide 3-kinase (PI3K) levels (p \u0026lt; 0.01) and a 1.6-fold decrease in phosphorylated PI3K (p \u0026lt; 0.05) in the RJNPAg treatment group compared to the control group. These results suggest that silver nanoparticles, synthesized with royal jelly, significantly reduce the levels of phosphorylated PI3K in lung cancer cells. Additionally, the total quantity of AKT decreased threefold after 24 hours of incubation (p \u0026lt; 0.0001), and a twofold decrease in phospho-AKT (Ser473) levels was observed following treatment with silver nanoparticles (p \u0026lt; 0.01), indicating a reduction in protein kinase B (AKT) and its activated form (Fig. 4, F).\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eMetal nanoparticles have attracted the scientific community's interest due to their various applications in biology, materials science, medicine, etc. Within this group, silver nanoparticles have particularly attracted attention due to their unique physicochemical properties, such as chemical stability and electrical conductivity, and their biological activities, including antibacterial, antifungal, anti-inflammatory, antiviral, and antiangiogenic effects\u0026nbsp;(Al-Sheddi et al., 2018).\u003c/p\u003e\n\u003cp\u003eGreen synthesis of silver nanoparticles is regarded as safer compared to chemical synthesis due to the potential toxicity and health hazards associated with the chemicals used in traditional synthesis methods. Green synthesis typically employs natural sources, which significantly reduces the likelihood of introducing harmful substances into the nanoparticles. This aspect is particularly crucial for medical applications, where nanoparticles may interact with the human body. Accordingly, in our study, we used royal jelly for the green synthesis of silver nanoparticles. The obtained nanoparticles had an average hydrodynamic radius of 58.25 ± 9.7 nm. In previous studies, the characteristics of RJ silver nanoparticles were detailed, and their antimicrobial effects were highlighted\u0026nbsp;(Gevorgyan et al., 2022). In this research, we focused on exploring their anticancer properties and their potential mechanisms of action. In previous works, it was shown, that royal jelly can have an anticancer effect \u003cem\u003ein vitro\u003c/em\u003e at high concentrations (Miyata and Sakai, 2018). However, during our experiments, no anticancer effect was detected at any of the tested concentrations (the highest tested concentration was 5 mg/mL). An analysis of the viability of RJNPAg-treated cells demonstrates selective cytotoxicity towards the cancer cell lines HeLa and A549 at concentrations ranging from 2.874 μg/ml to 23 μg/ml. At the maximum tested concentration, the nanoparticles exhibited 98% cytotoxicity against both cancer lines. Notably, nanoparticles synthesized with royal jelly displayed enhanced cytotoxicity at lower concentrations compared to other silver nanoparticles, with the potency of the effect varying according to the size of the nanoparticles. In the HeLa cell line, the inhibitory concentration (IC50) was 6.788 μg/mL after 24 hours, while for the A549 cell line, the IC50 was 13.37 μg/mL\u0026nbsp;(Abdellatif et al., 2021). The mechanism of cytotoxicity may include interaction with cellular structures, induction of oxidative stress, and disruption of cellular processes (\u003cstrong\u003eFig.5\u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eStudies have confirmed that the PI3K/AKT/mTOR signaling pathway is one of the classic ways to inhibit apoptosis in which PI3K, a type of lipid kinase, can be directly or indirectly activated by various growth factors and signal transduction complexes through the focal adhesion kinase pathway AKT, a protein located downstream of PI3K plays a certain vital role in this\u0026nbsp;(Wu et al., 2020). RJAgNP reduces the amounts of both Total PI3K and Akt, as well as their phosphorylated forms (Fig. 4, E, and F). This result indicates that RJAgNP affects both the expression of the gene for the synthesis of these proteins and the enzymes themselves, inhibiting the phosphorylation to the active form. This circumstance indicates the widespread and multi-target effect of RJAgNP.\u003c/p\u003e\n\u003cp\u003ePrevious studies have highlighted the critical role of arginase in the metabolism of L-arginine, which is involved in cancer growth and control. This involves mediating polyamine biosynthesis by arginase, nitric oxide production by NOS, and the immune response (Avtandilyan et al., 2022, 2019; Ginovyan et al., 2023). These metabolites play key functions in cell physiology and the health of the human body\u0026nbsp;(Chen et al., 2021). Additionally, both basic and clinical studies revealed that arginase is highly expressed in various types of cancer, including breast, lung, gastric, colorectal, and liver cancer (Avtandilyan et al., 2018). This is significant since arginine is the most consumed amino acid in the inner necrotic core of the tumor mass\u0026nbsp;(Niu et al., 2022). Arginine also stimulates various cellular mechanisms, including the PI3K/Akt/mTOR pathway, which in turn stimulates cancer growth (Chen et al., 2021). In our study, RJ green synthesized silver nanoparticles showed a decrease in arginase activity, which in turn means that nanoparticles have an antiproliferative effect. As previously mentioned, arginine serves as a precursor to NO under the action of NOS. Recent data highlight diverse roles of NO in tumorigenesis, including promoting angiogenesis, metastasis, anti-apoptotic processes, and modulating the immune response (Avtandilyan et al., 2022). However, it is important to note that the NOS-PI3K-AKT signaling pathway also mediates cell invasion\u0026nbsp;(Ghafouri-Fard et al., 2022). In our research, we demonstrated that silver nanoparticles reduce the level of NO \u003cem\u003ein vitro\u003c/em\u003e and decrease the levels of PI3K and AKT, leading to the activation of anti-invasion and anti-angiogenesis mechanisms. NO produced by NOS, whether located within the tumor or in the surrounding stroma, may promote the formation of new blood vessels by upregulating vascular endothelial growth factor (VEGF) (Secondini et al., 2017; Timoshenko et al., 2006; Vahora et al., 2016). Given the complexity of the process of angiogenesis, VEGF (VEGF-A), plays a prominent role in signaling through the VEGF-2 receptor, which induces angiogenesis in both healthy and diseased processes\u0026nbsp;(Quintero-Fabián et al., 2019). It was clear that silver nanoparticles also reduce the amount of VEGF, thereby influencing cell metastasis. Moreover, AgNPs significantly reduced the expression of MMP-2, which is closely associated with angiogenesis, invasiveness, and metastasis. In addition to PI3K/Akt, VEGF, and MMP-2, we analyzed the production of COX-2 due to its critical role in the pathogenesis of several inflammatory diseases and cancers; COX-2 is typically expressed at low levels in some tissues and cells; therefore, it can be strongly induced by certain types of cytokines such as TNF-α\u0026nbsp;(St-Germain et al., 2004; Yang et al., 2020). On the other hand, TNF-α\u0026nbsp;is involved in systemic inflammation by stimulating the acute phase response. The anticancer properties of TNF-α\u0026nbsp;are mainly achieved by inducing cancer cell death. Genes that are regulated by NF-kB have been shown to suppress apoptosis. Thus, cytokines such as TNFa activate apoptotic and antiapoptotic pathways (Gaur and Aggarwal, 2003). NF-κB activation usually precedes the apoptotic effects of TNF. Although TNF activates NF-κB in all cell types, it very rarely induces apoptosis. This may be due to the ability of NF-kB to suppress apoptosis. Interestingly, however, both the apoptotic and anti-apoptotic effects of TNFa are mediated by the formation of reactive oxygen intermediates (ROIs)\u0026nbsp;(Gaur and Aggarwal, 2003).\u003c/p\u003e\n\u003cp\u003eIt is generally accepted that DOX alters DNA chains and gene expression of various enzymes during the stages of transcription or translation, either directly or indirectly through the free radicals it generates, thereby causing changes in the activity of antioxidant enzymes (Erbaş et al., 2024). In some studies, free radicals responsible for pathogenesis include superoxide, hydroxyl radicals, and NO (He et al., 2021). Free radical-induced lipid peroxidation products such as MDA have also been shown to promote this event and cause cellular damage by reducing antioxidant enzymes\u0026nbsp;(Erbaş et al., 2024). Our results showed that nanoparticles increased MDA concentration several times in both cell lines. The increase in the amount of MDA and the concomitant decrease in the amount of Akt (responsible for survival) are important prerequisites for promoting apoptosis. In this way, our obtained results and assumptions also contribute to the understanding of the molecular mechanisms underlying cell death (\u003cstrong\u003eFig.5\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eSummarizing the obtained results, we can note that the inhibition of the VEGF/PI3K/Akt pathway and the quantitative increase of MDA (therefore ROS increase) are the basis of RJAgNP cytotoxicity. In addition, these nanoparticles show promise and great potential for positive regulation of anti-inflammatory, anti-angiogenic, and anti-metastatic pathways by reducing TNFa, COX-2, and MMP-2 levels.\u003c/p\u003e"},{"header":"5․ Conclusion","content":"\u003cp\u003eIn conclusion, our study highlights the promising anticancer potential of silver nanoparticles synthesized via green methods using royal jelly. These nanoparticles demonstrate significant cytotoxic effects against HeLa and A549 cancer cell lines. Underlying molecular mechanisms contributing to their anticancer activity were elucidated. Our findings revealed a significant decrease in arginase activity upon exposure to silver nanoparticles, accompanied by reductions in PI3K and phosphorylated and total Akt levels, indicative of pathway inhibition. Additionally, RJAgNP demonstrated a capacity to reduce nitric oxide levels and suppress angiogenesis-related factors like VEGF and MMP-2, and inflammation-related factors like TNFa and COX-2, thus impeding angiogenesis and metastasis. Moreover, our investigation shed light on the involvement of reactive oxygen intermediates (ROIs) in mediating apoptotic pathways, as evidenced by the increase in malondialdehyde (MDA) concentration and the corresponding decrease in Akt levels, ultimately promoting death in cancer cells. Our research contributes to the expanding field of nanoparticle-based therapeutics and opens new avenues for cancer treatment strategies.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eAKT - Protein kinase B; AgNP - Silver nanoparticles; COX-2 - Cyclooxygenase-2; DLS -Dynamic light scattering; DMEM - Dulbecco\u0026apos;s Modified Eagle Medium; DMSO - Dimethyl sulfoxide; ELISA - Enzyme-linked immunosorbent assay; FU \u0026ndash; Fluorouracil; GNPs - Green synthesis nanoparticles; H\u0026amp;E - Haematoxylin and Eosin; MDA \u0026ndash; Malondialdehyde; MMP-2 - Matrix metalloproteinase-2; mTOR - mammalian target of rapamycin; MTT - 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide; NF-\u0026kappa;B - Nuclear factor kappa-light-chain-enhancer of activated B cells; NO - Nitric oxide; PBS - Phosphate buffered saline; PDI - Protein disulfide isomerase; PI3K - Phosphoinositide 3-kinase; RJ - Royal Jelly; RJAgNP- Silver nanoparticles synthesized in the presence of royal jelly; ROS - Reactive oxygen species; SAED - Selected area (electron) diffraction; TEM - Transmission electron microscopy; TNF alpha - Tumor necrosis factor-alpha; VEGF - Vascular endothelial growth factor․\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Science Committee of MESCS RA through research projects numbered 21T-1F283, 21T-1F300,\u0026nbsp;and 23LCG-1F010. Many thanks to Zaruhi Karabekian, Head of the Laboratory of Immunology and Tissue Engineering at the L. A. Orbeli Institute of Physiology NAS RA, for providing the HeLa and A549 cell lines, which were purchased from the ATCC collection. Thank you Dr. Robin Schubert from European XFEL GmbH for the TEM analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study\u0026apos;s conception and design were the results of collective contributions from all authors. The investigations and analysis of results were carried out by MK, EN, and SM. MK, MG, HJ, and NA wrote the manuscript. NA and SM directed the project, and corrected, and edited the manuscript. All authors participated in the revision and approval of the final version of the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAbdellatif, Ah.A.H., Alsharidah, M., Rugaie, O. Al, Tawfeek, H.M., Tolba, N.S., 2021. Silver nanoparticle-coated ethyl cellulose inhibits tumor necrosis factor-\u0026alpha; of breast cancer cells. Drug Des Devel Ther 15, 2035\u0026ndash;2046. https://doi.org/10.2147/DDDT.S310760\u003c/li\u003e\n \u003cli\u003eAhamed, M., Akhtar, M.J., Alhadlaq, H.A., Khan, M.A.M., Alrokayan, S.A., 2015. Comparative cytotoxic response of nickel ferrite nanoparticles in human liver HepG2 and breast MFC-7 cancer cells. Chemosphere 135, 278\u0026ndash;288. https://doi.org/10.1016/j.chemosphere.2015.03.079\u003c/li\u003e\n \u003cli\u003eAlduraihem, N.S., Bhat, R.S., Al-Zahrani, S.A., Elnagar, D.M., Alobaid, H.M., Daghestani, M.H., 2023. 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J Anal Methods Chem 2016. https://doi.org/10.1155/2016/9412767\u003cstrong\u003e\u003cbr\u003e\u003c/strong\u003e\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"silver nanoparticles, royal jelly, cytotoxicity, PI3K/AKT pathway, oxidative stress.","lastPublishedDoi":"10.21203/rs.3.rs-4045087/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4045087/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Cancer poses a significant challenge in the medical field, requiring thorough investigation into its mechanisms and the development of effective treatments. Recently, there has been increasing interest in integrating drugs with metal nanoparticles, which are notable for their unique size and physicochemical properties, aiming to enhance anticancer efficacy. Silver nanoparticles (AgNPs), especially those obtained through environmentally friendly methods known as green synthesis, have garnered attention. Royal jelly (RJ), a substance produced by bees recognized for its antioxidant, anti-inflammatory, and antibacterial properties, is particularly interesting. This study focuses on the green synthesis of AgNPs using royal jelly and its bioactivity against cancer cells. It provides a detailed characterization of the nanoparticles and examines their effects on cancer cells, specifically Hela cervical cancer and A549 lung cancer cell lines. The results highlight the cytotoxicity induced by AgNPs in HeLa and A549 cells, which is mediated through apoptosis via the PI3K/AKT signaling pathway. Our research findings demonstrate that one of the mechanisms underlying cell death involves increased concentration of ROS/RNS and downregulation of the VEGFa/MMP-2/COX-2 pathway. This study is among the few to elucidate the mechanism of the anticancer effects of nanoparticles synthesized through this method. Overall, our research contributes to the ongoing exploration of cancer biology and offers insights into potential therapeutic approaches by harnessing the capabilities of green-synthesized nanoparticles.","manuscriptTitle":"Royal Jelly-Mediated Silver Nanoparticles Show Promising Anti-Cancer Effect on HeLa and A549 Cells Through Modulation of the VEGFa/PI3K/Akt/MMP-2 Pathway","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-11 04:52:16","doi":"10.21203/rs.3.rs-4045087/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"61003b28-e75d-465d-84ab-bc575919fa23","owner":[],"postedDate":"March 11th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-03-11T04:52:16+00:00","versionOfRecord":[],"versionCreatedAt":"2024-03-11 04:52:16","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4045087","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4045087","identity":"rs-4045087","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}