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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

PFT-α inhibits B-AgNPs- and F-AgNPs-induced increase in MTP in a p53-independent manner.Notes: Cells were pretreated with PFT-α (10 μM) for 1 hour and then incubated with respective IC50 concentrations of B-AgNPs or F-AgNPs for 24 hours. Changes in MTP were determined using the cationic fluorescent indicator JC-1. Fluorescence images of control and treated cells were recorded using fluorescence microscopy. JC-1 formed red fluorescent J-aggregates in healthy control cells with high MTP, whereas cells exposed to B-AgNPs or F-AgNPs had low MTP, and JC-1 existed as a monomer showing green fluorescence.Abbreviations: B-AgNPs, bacterium-derived AgNPs; F-AgNPs, fungus-derived AgNPs; IC50, half-maximal inhibitory concentration; MTP, mitochondrial membrane potential; PFT-α, pifithrin-alpha.
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f15-ijn-10-4203: PFT-α inhibits B-AgNPs- and F-AgNPs-induced increase in MTP in a p53-independent manner.Notes: Cells were pretreated with PFT-α (10 μM) for 1 hour and then incubated with respective IC50 concentrations of B-AgNPs or F-AgNPs for 24 hours. Changes in MTP were determined using the cationic fluorescent indicator JC-1. Fluorescence images of control and treated cells were recorded using fluorescence microscopy. JC-1 formed red fluorescent J-aggregates in healthy control cells with high MTP, whereas cells exposed to B-AgNPs or F-AgNPs had low MTP, and JC-1 existed as a monomer showing green fluorescence.Abbreviations: B-AgNPs, bacterium-derived AgNPs; F-AgNPs, fungus-derived AgNPs; IC50, half-maximal inhibitory concentration; MTP, mitochondrial membrane potential; PFT-α, pifithrin-alpha.

Mentions: Mitochondria play a key role in the apoptotic pathway of cell death, and changes in mitochondrial membrane permeability comprise the early events of apoptosis, via depolarization of the mitochondrial membrane. Depolarized mitochondria result from the formation of mitochondrial permeability transition pores.108–111 Mitochondrial permeability transition has been associated with various metabolic consequences, such as halted functioning of the electron transport chain with associated elevation in ROS and decreased production of ATP.112 Neema et al suggested that p53-dependent neuronal death involves a drop in MTP, which reflects both a loss in the integrity of mitochondria and an increase in mitochondrial membrane permeability.113 As described from an earlier study, the inner MTP decreases during apoptosis. Thus, the effect of AgNPs exposure on the MTP of MDA-MB-231 cells in the present study was further investigated using JC-1 staining. To test whether loss of MTP occurred in AgNPs-treated cells, we used JC-1, a cationic dye that aggregates in mitochondria, giving rise to red, punctate fluorescence in healthy cells (Figure 15). The cells were exposed to both B-AgNPs and F-AgNPs at their respective IC50 concentrations. Fluorescence microscopic observation of control cells (Figure 15) showed completely polarized mitochondria, which formed J-aggregates as red dots. In contrast, treatment with B-AgNPs and F-AgNPs resulted in depolarization of the mitochondrial membrane in MDA-MB-231 cells, as evident from the loss of the red dots and simultaneous increase of green fluorescence (Figure 15). This change in fluorescence pattern indicates a loss of mitochondrial membrane integrity (Figure 15). The cells pretreated with PFT-α showed no loss of MTP. These findings suggest that AgNPs induced increases in mitochondrial membrane permeability, resulting in part from p53 action. Sanpui et al reported that mitochondrial dysfunction due to nanoparticle immobilization may lead to oxidative stress, and the mitochondrial membrane is among the most susceptible targets of the deleterious effects associated with intracellular ROS.109 Govender et al demonstrated that a significant increase in mitochondria depolarization after AgNPs treatment, with an accompanying decrease in ATP concentration, induces cellular apoptosis in cancerous lung cells via the intrinsic apoptosis pathway.111 The present results indicate that adverse changes in mitochondrial function due to AgNPs, with possible association of intracellular ROS production, trigger apoptosis. Taking the literature and the present study into account, results suggest that an increase in ROS generation after exposure to B-AgNPs and F-AgNPs can result in disruption of the mitochondrial membrane and apoptosis.


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)

PFT-α inhibits B-AgNPs- and F-AgNPs-induced increase in MTP in a p53-independent manner.Notes: Cells were pretreated with PFT-α (10 μM) for 1 hour and then incubated with respective IC50 concentrations of B-AgNPs or F-AgNPs for 24 hours. Changes in MTP were determined using the cationic fluorescent indicator JC-1. Fluorescence images of control and treated cells were recorded using fluorescence microscopy. JC-1 formed red fluorescent J-aggregates in healthy control cells with high MTP, whereas cells exposed to B-AgNPs or F-AgNPs had low MTP, and JC-1 existed as a monomer showing green fluorescence.Abbreviations: B-AgNPs, bacterium-derived AgNPs; F-AgNPs, fungus-derived AgNPs; IC50, half-maximal inhibitory concentration; MTP, mitochondrial membrane potential; PFT-α, pifithrin-alpha.
© Copyright Policy
Related In: Results  -  Collection

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

f15-ijn-10-4203: PFT-α inhibits B-AgNPs- and F-AgNPs-induced increase in MTP in a p53-independent manner.Notes: Cells were pretreated with PFT-α (10 μM) for 1 hour and then incubated with respective IC50 concentrations of B-AgNPs or F-AgNPs for 24 hours. Changes in MTP were determined using the cationic fluorescent indicator JC-1. Fluorescence images of control and treated cells were recorded using fluorescence microscopy. JC-1 formed red fluorescent J-aggregates in healthy control cells with high MTP, whereas cells exposed to B-AgNPs or F-AgNPs had low MTP, and JC-1 existed as a monomer showing green fluorescence.Abbreviations: B-AgNPs, bacterium-derived AgNPs; F-AgNPs, fungus-derived AgNPs; IC50, half-maximal inhibitory concentration; MTP, mitochondrial membrane potential; PFT-α, pifithrin-alpha.
Mentions: Mitochondria play a key role in the apoptotic pathway of cell death, and changes in mitochondrial membrane permeability comprise the early events of apoptosis, via depolarization of the mitochondrial membrane. Depolarized mitochondria result from the formation of mitochondrial permeability transition pores.108–111 Mitochondrial permeability transition has been associated with various metabolic consequences, such as halted functioning of the electron transport chain with associated elevation in ROS and decreased production of ATP.112 Neema et al suggested that p53-dependent neuronal death involves a drop in MTP, which reflects both a loss in the integrity of mitochondria and an increase in mitochondrial membrane permeability.113 As described from an earlier study, the inner MTP decreases during apoptosis. Thus, the effect of AgNPs exposure on the MTP of MDA-MB-231 cells in the present study was further investigated using JC-1 staining. To test whether loss of MTP occurred in AgNPs-treated cells, we used JC-1, a cationic dye that aggregates in mitochondria, giving rise to red, punctate fluorescence in healthy cells (Figure 15). The cells were exposed to both B-AgNPs and F-AgNPs at their respective IC50 concentrations. Fluorescence microscopic observation of control cells (Figure 15) showed completely polarized mitochondria, which formed J-aggregates as red dots. In contrast, treatment with B-AgNPs and F-AgNPs resulted in depolarization of the mitochondrial membrane in MDA-MB-231 cells, as evident from the loss of the red dots and simultaneous increase of green fluorescence (Figure 15). This change in fluorescence pattern indicates a loss of mitochondrial membrane integrity (Figure 15). The cells pretreated with PFT-α showed no loss of MTP. These findings suggest that AgNPs induced increases in mitochondrial membrane permeability, resulting in part from p53 action. Sanpui et al reported that mitochondrial dysfunction due to nanoparticle immobilization may lead to oxidative stress, and the mitochondrial membrane is among the most susceptible targets of the deleterious effects associated with intracellular ROS.109 Govender et al demonstrated that a significant increase in mitochondria depolarization after AgNPs treatment, with an accompanying decrease in ATP concentration, induces cellular apoptosis in cancerous lung cells via the intrinsic apoptosis pathway.111 The present results indicate that adverse changes in mitochondrial function due to AgNPs, with possible association of intracellular ROS production, trigger apoptosis. Taking the literature and the present study into account, results suggest that an increase in ROS generation after exposure to B-AgNPs and F-AgNPs can result in disruption of the mitochondrial membrane and apoptosis.

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