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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

Effect of B-AgNPs and F-AgNPs on membrane integrity of MDA-MB-231 cells.Notes: The cells were treated with respective IC50 concentrations of B-AgNPs or F-AgNPs, and LDH leakage was measured by changes in optical density due to NAD+ reduction, monitored at 490 nm, as described in “Materials and methods”. The results are expressed as the mean ± SD of three independent experiments, each of which contained three replicates. Treated groups showed statistically significant differences from the control group by the Student’s t-test (P<0.05).Abbreviations: B-AgNPs, bacterium-derived AgNPs; Con, control; DOX, doxorubicin; F-AgNPs, fungus-derived AgNPs; IC50, half-maximal inhibitory concentration; LDH, lactate dehydrogenase; NAD, nicotinamide adenine dinucleotide; SD, standard deviation.
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f7-ijn-10-4203: Effect of B-AgNPs and F-AgNPs on membrane integrity of MDA-MB-231 cells.Notes: The cells were treated with respective IC50 concentrations of B-AgNPs or F-AgNPs, and LDH leakage was measured by changes in optical density due to NAD+ reduction, monitored at 490 nm, as described in “Materials and methods”. The results are expressed as the mean ± SD of three independent experiments, each of which contained three replicates. Treated groups showed statistically significant differences from the control group by the Student’s t-test (P<0.05).Abbreviations: B-AgNPs, bacterium-derived AgNPs; Con, control; DOX, doxorubicin; F-AgNPs, fungus-derived AgNPs; IC50, half-maximal inhibitory concentration; LDH, lactate dehydrogenase; NAD, nicotinamide adenine dinucleotide; SD, standard deviation.

Mentions: Membrane integrity is an important factor for cell survival. LDH is a cytosolic enzyme present in all mammalian cells that can be easily utilized for determining the extent of cell death.71 The plasma membrane is impermeable to LDH, and loss of its integrity is detectable by the release of LDH into the supernatant.72 In vitro release of LDH from cells provides an accurate measure of cell membrane integrity and cell viability, irrespective of the type of cell death.71 Therefore, we examined the effect of B-AgNPs and F-AgNPs on membrane integrity. Cells were treated with IC50 concentrations of AgNPs for 24 hours, with DOX used as a positive control, and these induced LDH leakage significantly more than did the control. The results suggest that cell-membrane leakage was significantly affected (Figure 7). The results from the LDH assay were consistent with cell viability; both AgNPs induced cytotoxicity in cancer cells. BRL 3A rat liver cells exposed to AgNPs for 24 hours resulted in a concentration-dependent increase in LDH leakage and significant cytotoxicity at 10–50 μg/mL.21 Park et al demonstrated size-dependent toxicity in L929 fibroblasts by measuring LDH leakage.19 The LDH level increased in a concentration- and time-dependent manner when cells were cultured with AgNPs at 100 μg/mL for 48 hours. Further, polyvinylpyrrolidine-coated AgNPs inhibited cell proliferation and viability, and increased LDH leakage, in a time- and concentration-dependent manner in the murine dendritic DC2.4 cell line (Kang et al).75 The results from our experiments and the previous literature suggest that AgNPs can reduce the membrane potential of MDA-MB-231 cells.73


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)

Effect of B-AgNPs and F-AgNPs on membrane integrity of MDA-MB-231 cells.Notes: The cells were treated with respective IC50 concentrations of B-AgNPs or F-AgNPs, and LDH leakage was measured by changes in optical density due to NAD+ reduction, monitored at 490 nm, as described in “Materials and methods”. The results are expressed as the mean ± SD of three independent experiments, each of which contained three replicates. Treated groups showed statistically significant differences from the control group by the Student’s t-test (P<0.05).Abbreviations: B-AgNPs, bacterium-derived AgNPs; Con, control; DOX, doxorubicin; F-AgNPs, fungus-derived AgNPs; IC50, half-maximal inhibitory concentration; LDH, lactate dehydrogenase; NAD, nicotinamide adenine dinucleotide; SD, standard deviation.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4494182&req=5

f7-ijn-10-4203: Effect of B-AgNPs and F-AgNPs on membrane integrity of MDA-MB-231 cells.Notes: The cells were treated with respective IC50 concentrations of B-AgNPs or F-AgNPs, and LDH leakage was measured by changes in optical density due to NAD+ reduction, monitored at 490 nm, as described in “Materials and methods”. The results are expressed as the mean ± SD of three independent experiments, each of which contained three replicates. Treated groups showed statistically significant differences from the control group by the Student’s t-test (P<0.05).Abbreviations: B-AgNPs, bacterium-derived AgNPs; Con, control; DOX, doxorubicin; F-AgNPs, fungus-derived AgNPs; IC50, half-maximal inhibitory concentration; LDH, lactate dehydrogenase; NAD, nicotinamide adenine dinucleotide; SD, standard deviation.
Mentions: Membrane integrity is an important factor for cell survival. LDH is a cytosolic enzyme present in all mammalian cells that can be easily utilized for determining the extent of cell death.71 The plasma membrane is impermeable to LDH, and loss of its integrity is detectable by the release of LDH into the supernatant.72 In vitro release of LDH from cells provides an accurate measure of cell membrane integrity and cell viability, irrespective of the type of cell death.71 Therefore, we examined the effect of B-AgNPs and F-AgNPs on membrane integrity. Cells were treated with IC50 concentrations of AgNPs for 24 hours, with DOX used as a positive control, and these induced LDH leakage significantly more than did the control. The results suggest that cell-membrane leakage was significantly affected (Figure 7). The results from the LDH assay were consistent with cell viability; both AgNPs induced cytotoxicity in cancer cells. BRL 3A rat liver cells exposed to AgNPs for 24 hours resulted in a concentration-dependent increase in LDH leakage and significant cytotoxicity at 10–50 μg/mL.21 Park et al demonstrated size-dependent toxicity in L929 fibroblasts by measuring LDH leakage.19 The LDH level increased in a concentration- and time-dependent manner when cells were cultured with AgNPs at 100 μg/mL for 48 hours. Further, polyvinylpyrrolidine-coated AgNPs inhibited cell proliferation and viability, and increased LDH leakage, in a time- and concentration-dependent manner in the murine dendritic DC2.4 cell line (Kang et al).75 The results from our experiments and the previous literature suggest that AgNPs can reduce the membrane potential of MDA-MB-231 cells.73

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