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

Authors:
Meri Kocharyan at Yerevan State University
Meri Kocharyan
Syuzan Marutyan at Yerevan State University
Syuzan Marutyan
Edita Nadiryan at Yerevan State University Research Institute of Biology
Edita Nadiryan
  • Yerevan State University Research Institute of Biology
Mikayel Ginovyan at Yerevan State University
Mikayel Ginovyan
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Abstract and Figures

In recent years, nanotechnology has revolutionized various sectors, particularly in nanomedicine, where nanomaterials are used for diagnosis, monitoring, control, prevention, and treatment. Among these, silver nanoparticles (AgNPs) stand out due to their remarkable antimicrobial and cytotoxic properties. Traditional chemical synthesis of AgNPs poses significant environmental and health risks. This study introduces a novel, eco‐friendly synthesis method using royal jelly (RJ), a nutrient‐rich secretion from honeybees, to produce AgNPs with potent anticancer effects. Our research provides a detailed investigation into RJ‐mediated AgNPs’ modulation of the VEGFa/PI3K/Akt/MMP‐2 pathway in HeLa and A549 cancer cell lines. 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). Cell signaling system components were determined by ELISA analysis. The cells were subjected to staining with hematoxylin and eosin to visualize the treatment’s effects. This study focuses on the green synthesis of AgNPs using RJ 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. Green synthesized AgNPs demonstrate significant cytotoxic effects against HeLa and A549 cancer cell lines. The underlying molecular mechanisms contributing to their anticancer activity were elucidated. Our findings revealed a significant decrease in arginase activity upon exposure to AgNPs, 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 TNF‐α and COX‐2, thus impeding angiogenesis and metastasis. Moreover, our results 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 study contributes significantly to the field of nanomedicine and cancer therapy by introducing the use of green synthesis AgNPs using RJ and providing in‐depth insights into their molecular mechanisms of anticancer action. Our research findings demonstrate the anticancer potential of RJ‐AgNPs by targeting the VEGFa/PI3K/Akt/MMP‐2 pathway and modulation of ROS/RNS in cancer cells.
The characteristics of RJ‐mediated Ag NPs: (A) hydrodynamic radii evolution, (B) TEM (dilution: 1:90), (C) UV–Vis absorption spectra, and (D) SAED pattern.
The characteristics of RJ‐mediated Ag NPs: (A) hydrodynamic radii evolution, (B) TEM (dilution: 1:90), (C) UV–Vis absorption spectra, and (D) SAED pattern.
… 
Inhibition 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 three 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&E, magnification: 100×). 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.
Inhibition 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 three 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&E, magnification: 100×). 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.
… 
Change 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. Each condition was added to five different passages in triplicate (n = 5, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, **** p ≤ 0.0001).
Change 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. Each condition was added to five different passages in triplicate (n = 5, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, **** p ≤ 0.0001).
… 
Levels of TNF‐α (A), VEGFa (B), Cox‐2 (C), MMP‐2 (D), PI3K (E), and AKT (F) in A549 after 24 h of exposure to AgNP. Each test sample was added to three different passages in triplicate. Each value represents the mean ± SE of three experiments (n = 3, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001).
Levels of TNF‐α (A), VEGFa (B), Cox‐2 (C), MMP‐2 (D), PI3K (E), and AKT (F) in A549 after 24 h of exposure to AgNP. Each test sample was added to three different passages in triplicate. Each value represents the mean ± SE of three experiments (n = 3, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001).
… 
Possible molecular mechanism of RJAgNP cytotoxicity effect on cancer cells.
Possible molecular mechanism of RJAgNP cytotoxicity effect on cancer cells.
… 
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Applied Organometallic Chemistry, 2024; 38:e7726
https://doi.org/10.1002/aoc.7726
1 of 11
Applied Organometallic Chemistry
Royal Jelly–Mediated Silver Nanoparticles Show Promising
Anticancer Effect on HeLa and A549 Cells Through
Modulation of the VEGFa/PI3K/Akt/MMP- 2 Pathway
MeriKocharyan1 | SyuzanMarutyan1 | EditaNadiryan1 | MikayelGinovyan1 | HayarpiJavrushyan1 | SedaMarutyan2 |
NikolayAvtandilyan1
1Research Institute of Biology, Yerevan State University, Yerevan, Armenia | 2Department of Biochemistry, Microbiology, and Biotechnology, Yerevan State
University, Yerevan, A rmenia
Correspondence: Nikolay Avtandilyan (nv.avtandilyan@ysu.am)
Received: 23 March 202 4 | Revised: 19 July 2024 | Accepted: 14 August 2024
Funding: This work was supported by the Science Committee of MESCS RA through research projects numbered 21T- 1F283, 21T- 1F300, and
23LCG- 1F010.
Keywords: cytotoxicity | oxidative stress | PI3K/AKT pathway | royal jelly | silver nanoparticles
ABSTR ACT
In recent years, nanotechnology has revolutionized various sectors, particularly in nanomedicine, where nanomaterials are used
for diagnosis, monitoring, control, prevention, and treatment. Among these, silver nanoparticles (AgNPs) stand out due to their
remarkable antimicrobial a nd cytotoxic properties. Traditional chemical synthesis of AgN Ps poses significant environmental and
health risks. This study introduces a novel, eco- friendly synthesis method using royal jelly (RJ), a nutrient- rich secretion from
honeybees, to produce AgNPs with potent anticancer effects. Our research provides a detailed investigation into RJ- mediated
AgNPs’ modulation of the VEGFa/PI3K/Akt/MMP- 2 pathway in HeLa and A549 cancer cell lines. 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). Cell signaling system components were determined by ELISA anal-
ysis. The cells were subjected to staining with hematoxylin and eosin to visualize the treatment’s effects. This study focuses on
the green synthesis of AgNPs using RJ and its bioactivity against cancer cells. It provides a detailed characterization of the na-
noparticles and examines their effects on cancer cells, specifically HeLa cervical cancer and A549 lung cancer cell lines. Green
synthesized AgNPs demonstrate significant cytotoxic effects against HeLa and A549 cancer cell lines. The underlying molecular
mechanisms contributing to their anticancer activity were elucidated. Our findings revealed a significant decrease in arginase
activity upon exposure to AgNPs, 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 TNF- α and COX- 2, thus impeding angiogenesis and
metastasis. Moreover, our results 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 study contributes significantly to the field of nanomedicine and cancer therapy
© 2024 J ohn Wiley & Sons, Lt d.
Abbreviations: AKT, protein k inase B; AgN P, silver nanop articles; COX- 2 , cyclooxygena se- 2; DLS , dynamic light s cattering; DM EM, Dulbecc o’s Modified Eag le Medium; DMSO, di methyl
sulfox ide; ELISA, en zyme- linked immu nosorbent assay ; FU, fluorourac il; GNPs, gre en synthesis na noparticles; H& E, hematoxyli n and eosin; MDA, m alondialdehyde; MM P- 2, matri x
metallopr oteinase- 2; mTOR, mam malian targe t of rapamycin; MT T, 3- (4,5 - dimeth ylthiazol- 2 - yl)- 2 ,5- d iphenyltetrazoliu m bromide; NF- κB, nuclear f actor kappa- light- chain- enhancer of
activat ed B cells; NO, nitr ic oxide; PBS, phosph ate buffered sa line; PDI, protein d isulfide isomer ase; PI3K, phosph oinositide 3- k inase; RJ, r oyal jelly; R JAgNP, silver nanopa rticles synthe sized in
the presenc e of royal jelly; ROS , reactive ox ygen species; SA ED, selected are a (electron) di ffraction; T EM, transmi ssion electron micr oscopy; TNF a lpha, tumor necro sis factor- a lpha; VEGF,
vascular endothelial growth factor.
RESEARCH ARTICLE
... Metal nanoparticles have become integral to advancements in nanotechnology, driven by their unique physical, chemical, and biological properties [1]. Among these, silver nanoparticles (Ag-NPs) are particularly renowned for their potent antibacterial and antifungal activities, making them highly valuable in various applications [2]. Metals such as gold, silver, copper, and zinc are commonly used to synthesize nanoparticles. ...
... The cytotoxicity is possibly due to the ability of Ag-NPs to induce oxidative stress, thus leading to apoptosis through mitochondrial damage and the activation of apoptotic pathways. The anticancer potential of green-synthesized Ag-NPs was also shown in other studies [2]. ...
... Among all noble metal NPs, Ag-NPs occupy a special place due to their unique properties, such as chemical stability, good conductivity, catalytic potential, and, most importantly, remarkable antimicrobial, anticancer, and anti-inflammatory activities. Hence, our findings align with previous reports which highlighted the dual antimicrobial and anticancer properties of green-synthesized Ag-NPs [2,21,22]. ...
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