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Comparative assessment of the apoptotic potential of silver nanoparticles synthesized by Bacillus tequilensis and Calocybe indica in MDA-MB-231 human breast cancer cells: targeting p53 for anticancer therapy.

Gurunathan S, Park JH, Han JW, Kim JH - Int J Nanomedicine (2015)

Bottom Line: This is especially true in the area of nanomedicine, due to physicochemical properties, such as mechanical, chemical, magnetic, optical, and electrical properties, compared with bulk materials.The first goal of this study was to produce silver nanoparticles (AgNPs) using two different biological resources as reducing agents, Bacillus tequilensis and Calocybe indica.Cells pretreated with pifithrin-alpha were protected from p53-mediated AgNPs-induced toxicity.

View Article: PubMed Central - PubMed

Affiliation: Department of Animal Biotechnology, Konkuk University, Seoul, Republic of Korea.

ABSTRACT

Background: Recently, the use of nanotechnology has been expanding very rapidly in diverse areas of research, such as consumer products, energy, materials, and medicine. This is especially true in the area of nanomedicine, due to physicochemical properties, such as mechanical, chemical, magnetic, optical, and electrical properties, compared with bulk materials. The first goal of this study was to produce silver nanoparticles (AgNPs) using two different biological resources as reducing agents, Bacillus tequilensis and Calocybe indica. The second goal was to investigate the apoptotic potential of the as-prepared AgNPs in breast cancer cells. The final goal was to investigate the role of p53 in the cellular response elicited by AgNPs.

Methods: The synthesis and characterization of AgNPs were assessed by various analytical techniques, including ultraviolet-visible (UV-vis) spectroscopy, X-ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy, dynamic light scattering (DLS), and transmission electron microscopy (TEM). The apoptotic efficiency of AgNPs was confirmed using a series of assays, including cell viability, leakage of lactate dehydrogenase (LDH), production of reactive oxygen species (ROS), DNA fragmentation, mitochondrial membrane potential, and Western blot.

Results: The absorption spectrum of the yellow AgNPs showed the presence of nanoparticles. XRD and FTIR spectroscopy results confirmed the crystal structure and biomolecules involved in the synthesis of AgNPs. The AgNPs derived from bacteria and fungi showed distinguishable shapes, with an average size of 20 nm. Cell viability assays suggested a dose-dependent toxic effect of AgNPs, which was confirmed by leakage of LDH, activation of ROS, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells in MDA-MB-231 breast cancer cells. Western blot analyses revealed that AgNPs induce cellular apoptosis via activation of p53, p-Erk1/2, and caspase-3 signaling, and downregulation of Bcl-2. Cells pretreated with pifithrin-alpha were protected from p53-mediated AgNPs-induced toxicity.

Conclusion: We have demonstrated a simple approach for the synthesis of AgNPs using the novel strains B. tequilensis and C. indica, as well as their mechanism of cell death in a p53-dependent manner in MDA-MB-231 human breast cancer cells. The present findings could provide insight for the future development of a suitable anticancer drug, which may lead to the development of novel nanotherapeutic molecules for the treatment of cancers.

No MeSH data available.


Related in: MedlinePlus

B-AgNPs and F-AgNPs induce ROS production in MDA-MB-231 cells.Notes: Fluorescence images of MDA-MB-231 cells treated with respective IC50 concentrations of B-AgNPs or F-AgNPs incubated for 24 hours. The image shows significant formation of H2O2 inside the MDA-MB-231 cells, whereas no fluorescence was observed in control cells.Abbreviations: B-AgNPs, bacterium-derived AgNPs; BF, bright field; DAPI, 4′,6-diamidino-2-phenylindole; DCF, 2′,7′-dichlorofluorescein; F-AgNPs, fungus-derived AgNPs; IC50, half-maximal inhibitory concentration; ROS, reactive oxygen species.
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f8-ijn-10-4203: B-AgNPs and F-AgNPs induce ROS production in MDA-MB-231 cells.Notes: Fluorescence images of MDA-MB-231 cells treated with respective IC50 concentrations of B-AgNPs or F-AgNPs incubated for 24 hours. The image shows significant formation of H2O2 inside the MDA-MB-231 cells, whereas no fluorescence was observed in control cells.Abbreviations: B-AgNPs, bacterium-derived AgNPs; BF, bright field; DAPI, 4′,6-diamidino-2-phenylindole; DCF, 2′,7′-dichlorofluorescein; F-AgNPs, fungus-derived AgNPs; IC50, half-maximal inhibitory concentration; ROS, reactive oxygen species.

Mentions: ROS are chemical species produced as byproducts of cellular oxygen metabolism, which occurs via mitochondrial respiration in eukaryotic cells, and are general mediators of nanoparticle-induced cytotoxicity.8,74 Therefore, we were interested in evaluating whether AgNPs could generate intracellular ROS in MDA-MB-231 cells. We examined the ability of AgNPs to induce oxidative stress by measuring ROS with carboxy-H2DCFDA in AgNP-treated MDA-MB-231 cells. The cells were treated with 10 μg/mL and 2.0 μg/mL B-AgNPs and F-AgNPs, respectively, for 24 hours. Figure 8 shows the fluorescence images of untreated MDA-MB-231 cells and cells treated with AgNPs. The control sample showed no green fluorescence, indicating a lack of H2O2 formation, whereas B-AgNP- and F-AgNP-treated cells showed bright green fluorescence (Figure 8) similar to H2O2-treated cells. The AgNP-treated cells showed significantly greater fluorescence intensity than untreated cells. Thus, the increase in fluorescence intensity is directly proportional to the generation of ROS. ROS target mitochondria and cause DNA damage and apoptosis.75 Earlier studies have suggested that AgNPs are inducers of ROS in a variety of cells.10,20,65,76 The exposure of MDA-MB-231 cells to AgNPs could result in the production of ROS, which could explain the outcomes of metabolic and toxicological problems (Figure 8). Kang et al reported that AgNPs stimulate ROS-dependent apoptosis and the inhibition of mitochondrial function.75 Finkel and Holbrook reported that ROS can induce various cellular events, such as cell growth and cell signaling.77 The production of intracellular ROS has been implicated as a mediator of nanotoxicity, including toxicity of AgNPs in human hepatoma cells.78 Several studies have reported AgNPs-mediated generation of ROS, which induced cell death, in different cell types.29,67,79 AgNPs with an average size of 25 nm produced a significant increase in ROS production in vitro and in vivo.29 The induction of apoptosis by exposure to AgNPs was mediated by oxidative stress in fibroblasts, muscle, and colon cells.67,79 The production of ROS was detected in both the MC3T3-E1 and PC12 cell lines in a particle size- and concentration-dependent manner.80 The results from the present study and previous studies provide strong evidence for a link between AgNPs-mediated production of ROS and subsequent generation of oxidative stress and cytotoxicity.65 Oxidative stress, in turn, plays an important role in many types of cellular injury that can result in DNA damage and apoptotic cell death.65


Comparative assessment of the apoptotic potential of silver nanoparticles synthesized by Bacillus tequilensis and Calocybe indica in MDA-MB-231 human breast cancer cells: targeting p53 for anticancer therapy.

Gurunathan S, Park JH, Han JW, Kim JH - Int J Nanomedicine (2015)

B-AgNPs and F-AgNPs induce ROS production in MDA-MB-231 cells.Notes: Fluorescence images of MDA-MB-231 cells treated with respective IC50 concentrations of B-AgNPs or F-AgNPs incubated for 24 hours. The image shows significant formation of H2O2 inside the MDA-MB-231 cells, whereas no fluorescence was observed in control cells.Abbreviations: B-AgNPs, bacterium-derived AgNPs; BF, bright field; DAPI, 4′,6-diamidino-2-phenylindole; DCF, 2′,7′-dichlorofluorescein; F-AgNPs, fungus-derived AgNPs; IC50, half-maximal inhibitory concentration; ROS, reactive oxygen species.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4494182&req=5

f8-ijn-10-4203: B-AgNPs and F-AgNPs induce ROS production in MDA-MB-231 cells.Notes: Fluorescence images of MDA-MB-231 cells treated with respective IC50 concentrations of B-AgNPs or F-AgNPs incubated for 24 hours. The image shows significant formation of H2O2 inside the MDA-MB-231 cells, whereas no fluorescence was observed in control cells.Abbreviations: B-AgNPs, bacterium-derived AgNPs; BF, bright field; DAPI, 4′,6-diamidino-2-phenylindole; DCF, 2′,7′-dichlorofluorescein; F-AgNPs, fungus-derived AgNPs; IC50, half-maximal inhibitory concentration; ROS, reactive oxygen species.
Mentions: ROS are chemical species produced as byproducts of cellular oxygen metabolism, which occurs via mitochondrial respiration in eukaryotic cells, and are general mediators of nanoparticle-induced cytotoxicity.8,74 Therefore, we were interested in evaluating whether AgNPs could generate intracellular ROS in MDA-MB-231 cells. We examined the ability of AgNPs to induce oxidative stress by measuring ROS with carboxy-H2DCFDA in AgNP-treated MDA-MB-231 cells. The cells were treated with 10 μg/mL and 2.0 μg/mL B-AgNPs and F-AgNPs, respectively, for 24 hours. Figure 8 shows the fluorescence images of untreated MDA-MB-231 cells and cells treated with AgNPs. The control sample showed no green fluorescence, indicating a lack of H2O2 formation, whereas B-AgNP- and F-AgNP-treated cells showed bright green fluorescence (Figure 8) similar to H2O2-treated cells. The AgNP-treated cells showed significantly greater fluorescence intensity than untreated cells. Thus, the increase in fluorescence intensity is directly proportional to the generation of ROS. ROS target mitochondria and cause DNA damage and apoptosis.75 Earlier studies have suggested that AgNPs are inducers of ROS in a variety of cells.10,20,65,76 The exposure of MDA-MB-231 cells to AgNPs could result in the production of ROS, which could explain the outcomes of metabolic and toxicological problems (Figure 8). Kang et al reported that AgNPs stimulate ROS-dependent apoptosis and the inhibition of mitochondrial function.75 Finkel and Holbrook reported that ROS can induce various cellular events, such as cell growth and cell signaling.77 The production of intracellular ROS has been implicated as a mediator of nanotoxicity, including toxicity of AgNPs in human hepatoma cells.78 Several studies have reported AgNPs-mediated generation of ROS, which induced cell death, in different cell types.29,67,79 AgNPs with an average size of 25 nm produced a significant increase in ROS production in vitro and in vivo.29 The induction of apoptosis by exposure to AgNPs was mediated by oxidative stress in fibroblasts, muscle, and colon cells.67,79 The production of ROS was detected in both the MC3T3-E1 and PC12 cell lines in a particle size- and concentration-dependent manner.80 The results from the present study and previous studies provide strong evidence for a link between AgNPs-mediated production of ROS and subsequent generation of oxidative stress and cytotoxicity.65 Oxidative stress, in turn, plays an important role in many types of cellular injury that can result in DNA damage and apoptotic cell death.65

Bottom Line: This is especially true in the area of nanomedicine, due to physicochemical properties, such as mechanical, chemical, magnetic, optical, and electrical properties, compared with bulk materials.The first goal of this study was to produce silver nanoparticles (AgNPs) using two different biological resources as reducing agents, Bacillus tequilensis and Calocybe indica.Cells pretreated with pifithrin-alpha were protected from p53-mediated AgNPs-induced toxicity.

View Article: PubMed Central - PubMed

Affiliation: Department of Animal Biotechnology, Konkuk University, Seoul, Republic of Korea.

ABSTRACT

Background: Recently, the use of nanotechnology has been expanding very rapidly in diverse areas of research, such as consumer products, energy, materials, and medicine. This is especially true in the area of nanomedicine, due to physicochemical properties, such as mechanical, chemical, magnetic, optical, and electrical properties, compared with bulk materials. The first goal of this study was to produce silver nanoparticles (AgNPs) using two different biological resources as reducing agents, Bacillus tequilensis and Calocybe indica. The second goal was to investigate the apoptotic potential of the as-prepared AgNPs in breast cancer cells. The final goal was to investigate the role of p53 in the cellular response elicited by AgNPs.

Methods: The synthesis and characterization of AgNPs were assessed by various analytical techniques, including ultraviolet-visible (UV-vis) spectroscopy, X-ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy, dynamic light scattering (DLS), and transmission electron microscopy (TEM). The apoptotic efficiency of AgNPs was confirmed using a series of assays, including cell viability, leakage of lactate dehydrogenase (LDH), production of reactive oxygen species (ROS), DNA fragmentation, mitochondrial membrane potential, and Western blot.

Results: The absorption spectrum of the yellow AgNPs showed the presence of nanoparticles. XRD and FTIR spectroscopy results confirmed the crystal structure and biomolecules involved in the synthesis of AgNPs. The AgNPs derived from bacteria and fungi showed distinguishable shapes, with an average size of 20 nm. Cell viability assays suggested a dose-dependent toxic effect of AgNPs, which was confirmed by leakage of LDH, activation of ROS, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells in MDA-MB-231 breast cancer cells. Western blot analyses revealed that AgNPs induce cellular apoptosis via activation of p53, p-Erk1/2, and caspase-3 signaling, and downregulation of Bcl-2. Cells pretreated with pifithrin-alpha were protected from p53-mediated AgNPs-induced toxicity.

Conclusion: We have demonstrated a simple approach for the synthesis of AgNPs using the novel strains B. tequilensis and C. indica, as well as their mechanism of cell death in a p53-dependent manner in MDA-MB-231 human breast cancer cells. The present findings could provide insight for the future development of a suitable anticancer drug, which may lead to the development of novel nanotherapeutic molecules for the treatment of cancers.

No MeSH data available.


Related in: MedlinePlus