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Braz. Arch. Biol. Technol. v.59: e16150529, Jan/Dec 2016
Vol.59: e16150529, January-December 2016
http://dx.doi.org/10.1590/1678-4324-2016150529
ISSN 1678-4324 Online Edition
BRAZILIAN ARCHIVES OF
BIOLOGY AND TECHNOLOGY
A N I N T E R N A T I O N A L J O U R N A L
Cytotoxic Effect on Cancerous Cell Lines by Biologically
Synthesized Silver Nanoparticles
Kulandaivelu, Balaji
1
; Gothandam, K M
1
*
1
VTI University, School of Bio Sciences and Technology Vellore, Tamil Nadu, India
ABSTRACT
The biosynthesis of nanoparticles has been proposed as an environmental friendly and cost effective
alternative to chemical and physical methods. Silver nanoparticles are biologically synthesized and
characterized were used in the study. The invitro cytotoxic effect of biologically synthesized silver
nanoparticles against MCF-7 cancer cell lines were assessed. The cytotoxic effects of the silver nanoparticles
could significantly inhibited MCF-7 cancer cell lines proliferation in a time and concentration-dependent
manner by MTT assay. Acridine orange, ethidium bromide (AO/EB) dual staining, caspase-3 and DNA
fragmentation assays were carried out using various concentrations of silver nanoparticles ranging from 1 to
100 μg/mL. At 100 μg/mL concentration, the silver nanoparticles exhibited significant cytotoxic effects and the
apoptotic features were confirmed through caspase-3 activation and DNA fragmentation assays. Western blot
analysis has revealed that nanoparticle was able to induce cytochrome c release from the mitochondria, which
was initiated by the inhibition of Bcl-2 and activation of Bax. Thus, the results of the present study indicate
that biologically synthesized silver nanoparticles might be used to treat breast cancer. The present studies
suggest that these nanoparticles could be a new potential adjuvant chemotherapeutic and chemo preventive
agent against cytotoxic cells. However, it necessitates clinical studies to ascertain their potential as anticancer
agents.
Key words: Silver nanoparticle; nanotechnology; nanobiotechnology; nanoparticle; cytotoxicity
*
Author for correspondence: gothandam@gmail.com
Human & Animal Health
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Gothandam, K M et al.
Braz. Arch. Biol. Technol. v.59: e16150529, Jan/Dec 2016
INTRODUCTION
Nanotechnology is an escalating field of
modern research with desired applications in
electronic and medicine and has been
expanding rapidly in recent years, impacting
on diverse areas such as the economy and the
environment (Badri Narayanan et al. 2010). In
this context, the number of commercial
products comprising nanomaterials is
increasing. Among the commercially
available nano-sized materials, silver
nanoparticles are by far the most used nano-
compounds (Ahmed et al. 2008) currently,
sustainability initiatives that use green
chemistry to improve and/or protect our
global environment are focal issues in many
fields of research. The biogenic synthesis of
metal NPs reduces these hazards through the
elimination / minimization of generated waste
and the implementation of sustainable
processes. Biological methods of NPs
synthesis using microorganisms (Klaus et al.
1999; Konishi et al. 2007), enzymes (Willner
et al. 2006) have been studied as possible eco-
friendly alternatives to chemical and physical
methods. Among all the noble metals, silver
has attracted major attention due to its
disinfecting nature and tremendous medicinal
value to culinary items as well as showing
enormous effectiveness as an anticancer
agent. Moreover, several salts of silver and
their derivatives are commercially
manufactured as antimicrobial agents
(Krutyakov et al. 2008).Breast cancer is the
second leading cause of cancer death among
women in the U.S. An estimated 39,620
breast cancer deaths and 232,340 new cases
are expected among women in 2013 (AMS,
2014). The existing cytotoxic agents used for
the breast cancer treatment are found to be
expensive and inefficient because they induce
severe side effects due to their toxicity in
noncancerous tissues (Kim et al. 2007;
Yeruva et al. 2008) Earlier studies show that
the cytotoxicity of synthesized Ag NPs is
related to the involvement of the level of
cellular reactive oxygen species (ROS) and
mitochondrial membrane disruption (Asha
Rani et al. 2009; Sanpui et al. 2011). The
present study also tried to validate the
possible invitro anti-proliferative effects of
green synthesized Ag NPs against the breast
cancer cell lines.
MATERIALS AND METHODS
Biological synthesis of
AgNPs
AgNPs using fungus Trichoderma viride was
procured, synthesized and characterised. In
the present investigation the use of the fungus
Trichoderma viride for the extracellular
biosynthesis of AgNPs from silver nitrate
solution is reported (Fayaz et al. 2009). It was
observed that the aqueous silver (Ag+) ions,
when exposed to a filtrate of T. viride, were
reduced in solution, thereby leading to
formation of extremely stable AgNPs. These
AgNPs were characterized by means of
several techniques. The nanoparticles show
maximum absorbance at 420 nm on
ultraviolet-visible spectra. The presence of
proteins was identified by Fourier transform–
infrared spectroscopy. The reduction of Ag+
ions to elemental silver was characterized by
x-ray photoelectron spectrophotometry.
Electrokinetic measurements (zeta potential)
of AgNPs as a function of pH in 1 × 10−3 mol
dm−3 aqueous solution were evaluated. The
transmission electron micrograph revealed the
formation of poly-dispersed nanoparticles of
5–40 nm, and the presence of elemental silver
was confirmed by energy-dispersed
spectroscopy analysis (Fayaz et al. 2009).
Cell lines and culture conditions
All MCF-7 cell lines were grown in RPMI-
1640 supplemented with 10% heat inactivated
fetal bovine serum (FBS), 100 IU/ml
penicillin, 100 mg/ml streptomycin and 2
mm-glutamine. Cultures were maintained in a
humidified atmosphere with 5% CO
2
at 37 °C.
The cells were sub-cultured twice each week,
seeding at a density of about 2× 10
3
cells/ml.
Before the analysis of the
AgNPs, cells were
washed with PBS and fresh medium was
added. For final analysis, exponentially
growing cells were collected and re-
suspended in fresh culture medium with 10%
FBS.
Silver nanoparticles mediated anti-cancerous effect
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Braz. Arch. Biol. Technol. v.59: e16150529, Jan/Dec 2016
MTT assay
The silver nanoparticles (BK1) were
evaluated for cytotoxic activity against MCF-
7 breast cancer cell line. The cells were plated
separately in 96 well plates at a concentration
of 1 × 10
5
cells/well. After 24 h, cells were
washed twice with 100 µL of serum-free
medium and starved for an hour at 37
o
C. After
starvation, cells were treated with the test
material for 24 h. At the end of the treatment
period the medium was aspirated and serum
free medium containing MTT (0.5 mg/mL)
was added and incubated for 4 h at 37ºC in a
CO
2
incubator. The MTT containing medium
was then discarded and the cells were washed
with PBS (200 µL). The crystals were then
dissolved by adding 100 µL of DMSO and
this was mixed properly by pipetting up and
down. Spectrophotometrical absorbance of the
purple blue formazan dye was measured in a
microplate reader at 570 nm (Biorad 680).The
experiment was carried out in triplicates and
the average of the viable cells was calculated.
A graph was plotted between the percentage
cell viability and dilution.
Caspase-3 activation in fold by Enzyme
linked immunosorbent assay (ELISA)
Caspase-3 is an intracellular cysteine protease
that exists as a proenzyme, becoming
activated during the cascade of events
associated with apoptosis. Caspases activities
were determined by chromogenic assays using
caspase - 3 activation kits according to the
manufacturer’s protocol (Calbiochem,
Merck). After treating with test compound,
the cells were lysed using Lysis buffer (50mM
HEPES, 100 mM NaCl, 0.1% CHAPS, 1mM
DTT, 100 mM EDTA). Lysates were
centrifuged at 10,000 rpm for 1 min. The
supernatants (cytosolic extract) were collected
and protein concentration was determined by
the Lowry’s method (Lowry 1951) using BSA
as a standard. 100 μg protein (cellular
extracts) was diluted in 50 μL cell lysis buffer
for each assay. Cellular extracts were then
incubated in 96 well microtiter plates with 5
μL of the 4 mM p -nitroanilide (pNA)
substrates, DEVD--pNA (caspase-3 activity)
for 2 h at 37°C. Caspase activity was
measured by cleavage of the above substrates
to free pNA. Free pNA (cleaved substrates)
was measured at 405 nm in a microtiter plate
reader. Relative caspase-3 activity was
calculated as a ratio of the absorbance of
treated cells to untreated cells.
Acridine orange/ethidium bromide dual
staining
In this study, we used acridine
orange/ethidium bromide (AO/EB) double
staining assay (Popovic et al. 2006). Acridine
orange is taken up by both viable and
nonviable cells and emits green fluorescence
if interrelated into double stranded nucleic
acid (DNA) or red fluorescence if bound to
single stranded nucleic acid (RNA). Ethidium
bromide is taken up only by nonviable cells
and emits red fluorescence by intercalation
into DNA. We distinguished four types of
cells according to the fluorescence emission
and the morphological aspect of chromatin
condensation in the stained nuclei. Viable
cells have uniform bright green nuclei with
organized structure. Apoptotic cells have
orange to red nuclei with condensed or
fragmented chromatin was examined by a
fluorescence microscope at 400X
magnification. Necrotic cells have a
uniformly orange to red nuclei with
condensed structure.
DNA fragmentation assay
In 24 flat-wells plate, incubated 2x10
5
MCF 7
in DMEM medium for 24 h (triplicate wells
of 10
5
per well) with different concentration
of BK1. Cells were harvested and re-suspend
with 0.5 mL PBS and 55 µL of lysis buffer
(40 mL of 0.5 M EDTA 5 mL of 1 M Tris-Cl
buffer pH 8.0 5 mL of 100% Triton X-100 50
mL of H2O) incubated for 20 min on ice. Cell
suspension was centrifuged at 12,000 g for 30
min at 4˚C. The supernatant was extracted
with 1:1 mixture of phenol:chloroform and
precipitated in two volume of cold ethanol
and one-tenth volume of sodium acetate.
Pellet was re-suspended in 30 µL of deionized
water-RNase solution. The DNA were
resolved in 1.2% agarose gel.
Western blotting
A modified version of (Cavalieri et al. 2004)
was employed for estimation of Caspases,
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Gothandam, K M et al.
Braz. Arch. Biol. Technol. v.59: e16150529, Jan/Dec 2016
Bax and Bcl-2. Exponentially growing cells (1
x 10
6
cells/mL) were plated in six well plates
and after the addition of nanoparticles at 1000
µg were incubated for different time points
(0–24). Cells incubated for different periods
of time were harvested by centrifugation at
500g for 5 min, washed twice with PBS and
homogenized in 200 ll of lysis buffer
containing 10 mM NaCl, 1.5 mM MgCl2, 10
mM Tris–HCl, 1 mM sodium orthovanadate,
0.3% NP40 and protease inhibitor cocktail
(EDTA free). The lysates thus obtained were
centrifuged at 12,000g for 30 min at 4˚C.
Fifty micrograms of the total protein, as
determined by Bradford’s assay, was resolved
into 10% SDS–PAGE and then transferred to
nitrocellulose membrane using a semi dry
electroblotter for 2 h at RT. The membrane
was blocked over night in 5% BSA. In all
cases, antibodies raised against active forms
of p53, Bcl-2, Bax and b-actin as internal
control (Calbiochem, Merck, Germany) were
incubated for 2 h with gentle agitation. After
washing three times with Tris buffered saline
(TBS; 50 mM Tris/HCl, pH 7.5, and 0.15 M
NaCl) containing 0.1% Triton X-100, the
membrane was incubated with alkaline
phosphatase-conjugated secondary antibodies
for 2 h with agitation. The probed
immunoblots were visualized with the
NBT/BCIP chromogenic substrate and
documented. The optical density of the
immunoblots was quantified by densitometric
scanning and data were analyzed by Biorad
Quantity One software.
RESULTS
MTT assay
The anticancer activity of the synthesized
silver nanoparticles were evaluated invitro
against MCF-7 breast cancer cell lines after
24 h exposure and their IC50 values were
determined from a graph of cell viability
measured over a range of concentrations
between 1 and 100 µg/mL . The IC50 was
determined at a broad range of concentrations
specifically 1, 10, 25, 50 & 100 µg/mL
against the cell lines. For this data, a line
graph was plotted between concentrations (X-
axis) versus % inhibition (Y-axis) and then an
intersection drawn at 50% inhibition on Y-
axis and then correlated to the concentration
value on X-axis. From the data it is revealed
that silver nanoparticle exhibited different
range of significant cytotoxic activities
varying from 94.91 µg/mL to 34.51 µg/mL
due to structural differences. Their bioactivity
data is presented in the graphical
representation also (Fig. 1).
A B
Figure 1 - A. Dose dependent cytotoxicity effect of SNp over cell viability. B. Morphological changes of
MCF 7cells cell lines treated with SNp .Cells were visualized under a inverted light microscope. Detachment
of cells from substratum, cell shrinkage, nuclear condensation and fragmentation were evident in cells treated
(a) Normal MCF 7cells(b) Low toxicity 1 μg/mL (c) toxicity 10 μg/mL (d) toxicity 25 μg/ML, (e) 50 μg/mL,
(f) 100 μg/mL.
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Braz. Arch. Biol. Technol. v.59: e16150529, Jan/Dec 2016
Caspase-3
To investigate whether apoptosis is mediated
by caspase-3, cell lysates treated with, silver
nanoparticles was analysed. Levels of
caspase-3 were found to be elevated in the
silver nanoparticles treated tumour cells (Fig.
2). The increased levels of caspase-3
activation suggest that silver nanoparticles
induced apoptosis in MCF 7 breast cancer
cells in a caspase-3-dependent manner.
Figure 2- Metal nanoparticles induced apoptosis in MCF 7 breast cancer cells via caspase-3 (Caspase assay).
Morphological evidence of apoptosis by
dual staining
Apoptotic morphological changes caused by
silver nanoparticles were studied using
acridine orange/ethidium bromide differential
staining method. The stained cells were
characterized to viable (light green), early
apoptotic (bright green fluorescence and
condensed chromatin), late apoptotic (orange
fluorescence) and nonviable cells (red
coloured fluorescence) (Fig. 3 a–c). Silver
nanoparticles treated cells showed condensed
nuclei, membrane blebbing and apoptotic
bodies. In contrast, the control cells showed
intact nuclear architecture.
A B C
Figure 3- Morphological evidence of apoptosis by AO/EB dual staining (A) 10 μg/mL (B) 20 μg/mL (C) 40
μg/Ml
DNA fragmentation assay
The evaluation of apoptosis was further
carried out by determining the DNA
fragmentation patterns, MCF-7 cells treated
with the extracts showed characteristics of
DNA laddering that had apoptotic cells higher
when treated even at low concentration
(20µg/mL). The difference in effect towards
cancer cells could be targeting a particular
molecular event exclusively in tumor cells