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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

Western blot analysis of p-p53, p-Erk1/2, p-c-Jun, Bcl-2, procaspase-3, and actin expression in MDA-MB-231 cells exposed to B-AgNPs or F-AgNPs.Notes: MDA-MB-231 cells were treated with respective IC50 concentrations of B-AgNPs or F-AgNPs for 24 hours. Expression of p-p53, p-Erk1/2, p-c-Jun, Bcl-2, and procaspase-3 protein levels were determined by Western blot analysis. Both B-AgNPs and F-AgNPs led to increased levels of p-p53, p-Erk1/2, and decreased levels of procaspase-3, whereas no alteration in expression was observed for p-c-Jun. Bcl-2 expressions significantly reduced. Equal protein loading was confirmed by analysis of β-actin protein levels. The results are representative of three independent experiments.Abbreviations: B-AgNPs, bacterium-derived AgNPs; Con, control; F-AgNPs, fungus-derived AgNPs; IC50, half-maximal inhibitory concentration.
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f10-ijn-10-4203: Western blot analysis of p-p53, p-Erk1/2, p-c-Jun, Bcl-2, procaspase-3, and actin expression in MDA-MB-231 cells exposed to B-AgNPs or F-AgNPs.Notes: MDA-MB-231 cells were treated with respective IC50 concentrations of B-AgNPs or F-AgNPs for 24 hours. Expression of p-p53, p-Erk1/2, p-c-Jun, Bcl-2, and procaspase-3 protein levels were determined by Western blot analysis. Both B-AgNPs and F-AgNPs led to increased levels of p-p53, p-Erk1/2, and decreased levels of procaspase-3, whereas no alteration in expression was observed for p-c-Jun. Bcl-2 expressions significantly reduced. Equal protein loading was confirmed by analysis of β-actin protein levels. The results are representative of three independent experiments.Abbreviations: B-AgNPs, bacterium-derived AgNPs; Con, control; F-AgNPs, fungus-derived AgNPs; IC50, half-maximal inhibitory concentration.

Mentions: Apoptosis is characterized by typical morphological and biochemical hallmarks, including cell shrinkage, nuclear DNA fragmentation, and membrane blebbing.83 The underlying mechanisms for initiation of an apoptotic response upon cytotoxic therapy may depend on the individual stimulus.84 To investigate whether both B-AgNPs and F-AgNPs activate ROS-mediated intracellular signaling, Western blot analysis was performed in MDA-MB-231 cells to analyze the activation or deactivation of various signaling molecules. The cells were exposed to the IC50 concentration of both B-AgNPs and F-AgNPs for 24 hours. The results indicate that AgNPs-treated cells showed a significant increase in the phosphorylation of both p53 and ERK1/2. Recently, AshaRani et al observed a concentration-dependent increase in phosphorylated p53 accompanied by the cleavage of caspase-3 and poly (ADP-ribose) polymerase (PARP) in cancer cells, but phosphorylation was not detected in normal cells.85 Recent studies have suggested that p53 plays a critical role in the cellular response to DNA damage and apoptosis induced by ROS.86,87 Our Western blotting data (Figure 10) suggest that both B-AgNPs and F-AgNPs activated p53, which mediated apoptosis. This result is consistent with previous studies by Lu et al and Mroz et al, which showed that nanoparticles and ROS can induce DNA damage, activate p53, and mimic irradiation-related carcinogenesis pathways.88,89 Similarly, several studies reported that polyvinylpyrrolidone (PVP)-coated AgNPs induced p53 in the larval tissue of Drosophila melanogaster.75,90 Taken together, the induction of the apoptotic pathway is indicated by the phosphorylation of p53 and ERK1/2.


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)

Western blot analysis of p-p53, p-Erk1/2, p-c-Jun, Bcl-2, procaspase-3, and actin expression in MDA-MB-231 cells exposed to B-AgNPs or F-AgNPs.Notes: MDA-MB-231 cells were treated with respective IC50 concentrations of B-AgNPs or F-AgNPs for 24 hours. Expression of p-p53, p-Erk1/2, p-c-Jun, Bcl-2, and procaspase-3 protein levels were determined by Western blot analysis. Both B-AgNPs and F-AgNPs led to increased levels of p-p53, p-Erk1/2, and decreased levels of procaspase-3, whereas no alteration in expression was observed for p-c-Jun. Bcl-2 expressions significantly reduced. Equal protein loading was confirmed by analysis of β-actin protein levels. The results are representative of three independent experiments.Abbreviations: B-AgNPs, bacterium-derived AgNPs; Con, control; F-AgNPs, fungus-derived AgNPs; IC50, half-maximal inhibitory concentration.
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f10-ijn-10-4203: Western blot analysis of p-p53, p-Erk1/2, p-c-Jun, Bcl-2, procaspase-3, and actin expression in MDA-MB-231 cells exposed to B-AgNPs or F-AgNPs.Notes: MDA-MB-231 cells were treated with respective IC50 concentrations of B-AgNPs or F-AgNPs for 24 hours. Expression of p-p53, p-Erk1/2, p-c-Jun, Bcl-2, and procaspase-3 protein levels were determined by Western blot analysis. Both B-AgNPs and F-AgNPs led to increased levels of p-p53, p-Erk1/2, and decreased levels of procaspase-3, whereas no alteration in expression was observed for p-c-Jun. Bcl-2 expressions significantly reduced. Equal protein loading was confirmed by analysis of β-actin protein levels. The results are representative of three independent experiments.Abbreviations: B-AgNPs, bacterium-derived AgNPs; Con, control; F-AgNPs, fungus-derived AgNPs; IC50, half-maximal inhibitory concentration.
Mentions: Apoptosis is characterized by typical morphological and biochemical hallmarks, including cell shrinkage, nuclear DNA fragmentation, and membrane blebbing.83 The underlying mechanisms for initiation of an apoptotic response upon cytotoxic therapy may depend on the individual stimulus.84 To investigate whether both B-AgNPs and F-AgNPs activate ROS-mediated intracellular signaling, Western blot analysis was performed in MDA-MB-231 cells to analyze the activation or deactivation of various signaling molecules. The cells were exposed to the IC50 concentration of both B-AgNPs and F-AgNPs for 24 hours. The results indicate that AgNPs-treated cells showed a significant increase in the phosphorylation of both p53 and ERK1/2. Recently, AshaRani et al observed a concentration-dependent increase in phosphorylated p53 accompanied by the cleavage of caspase-3 and poly (ADP-ribose) polymerase (PARP) in cancer cells, but phosphorylation was not detected in normal cells.85 Recent studies have suggested that p53 plays a critical role in the cellular response to DNA damage and apoptosis induced by ROS.86,87 Our Western blotting data (Figure 10) suggest that both B-AgNPs and F-AgNPs activated p53, which mediated apoptosis. This result is consistent with previous studies by Lu et al and Mroz et al, which showed that nanoparticles and ROS can induce DNA damage, activate p53, and mimic irradiation-related carcinogenesis pathways.88,89 Similarly, several studies reported that polyvinylpyrrolidone (PVP)-coated AgNPs induced p53 in the larval tissue of Drosophila melanogaster.75,90 Taken together, the induction of the apoptotic pathway is indicated by the phosphorylation of p53 and ERK1/2.

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