Rapeseed flower pollen bio-green synthesized silver nanoparticles: a promising antioxidant, anticancer and antiangiogenic compound

Abstract

Based on recent researches, bio synthesized silver nanoparticles (Ag-NPs) seem to have the potential in declining angiogenesis and oxidative stress. In the current study, rapeseed flower pollen (RFP) water extract was triggered to synthesize RFP–silver nanoparticles (RFP/Ag-NPs). Moreover, antioxidant, antiangiogenesis and cytotoxicity of the RFP/Ag-NPs against MDA-MB-231, MCF7 and carcinoma cell lines and normal human skin fibroblast HDF were compared. Results indicated that RFP/Ag-NPs have a peak at 430 nm, spherical shape and an average size of 24 nm. According to the results of FTIR, rapeseed pollen capped Ag-NPs. RFP/Ag-NPs have cytotoxicity on MDA-MB-231 and MCF7 cells and decrease cancerous cell viability (IC50 = 3 µg/ml and 2 µg/ml, respectively) in a dose- and time-dependent manner. The morphological data showed that the RFP/Ag-NPs increase the percentage of apoptotic cells compared to the control group and normal cells (human skin fibroblast cells). The apoptotic morphological change was also confirmed with a flow cytometric analysis. RFP/ Ag-NPs’ antioxidant activity was evaluated by measuring their ability to scavenge ABTS and DPPH free radicals. The IC50 values were determined at 800 and 830 μg/ml for ABTS and DPPH tests, respectively. According to the results, green-synthesized RFP/Ag-NPs as a safe efficient apoptosis inducer and strong antioxidant compound have the potential to suppress breast cancer carcinogenesis by VEGF down-regulatiion and thus sensitizing them against apoptosis. However, further researches are required to clarify RFP/Ag-NPs’ cell specificity and therapeutic doses in in vivo conditions.

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Vol.:(0123456789)
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JBIC Journal of Biological Inorganic Chemistry
https://doi.org/10.1007/s00775-019-01655-4
ORIGINAL PAPER
Rapeseed ower pollen bio‑green synthesized silver nanoparticles:
apromising antioxidant, anticancer andantiangiogenic compound
SaharHajebi1· MasoudHomayouniTabrizi1· MahboobehNakhaeiMoghaddam1· FarzanehShahraki1·
SoheylaYadamani1
Received: 18 January 2019 / Accepted: 25 March 2019
© Society for Biological Inorganic Chemistry (SBIC) 2019
Abstract
Based on recent researches, bio synthesized silver nanoparticles (Ag-NPs) seem to have the potential in declining angio-
genesis and oxidative stress. In the current study, rapeseed flower pollen (RFP) water extract was triggered to synthesize
RFP–silver nanoparticles (RFP/Ag-NPs). Moreover, antioxidant, antiangiogenesis and cytotoxicity of the RFP/Ag-NPs
against MDA-MB-231, MCF7 and carcinoma cell lines and normal human skin fibroblast HDF were compared. Results
indicated that RFP/Ag-NPs have a peak at 430 nm, spherical shape and an average size of 24 nm. According to the results
of FTIR, rapeseed pollen capped Ag-NPs. RFP/Ag-NPs have cytotoxicity on MDA-MB-231 and MCF7 cells and decrease
cancerous cell viability (IC50 = 3µg/ml and 2µg/ml, respectively) in a dose- and time-dependent manner. The morphologi-
cal data showed that the RFP/Ag-NPs increase the percentage of apoptotic cells compared to the control group and normal
cells (human skin fibroblast cells). The apoptotic morphological change was also confirmed with a flow cytometric analysis.
RFP/ Ag-NPs’ antioxidant activity was evaluated by measuring their ability to scavenge ABTS and DPPH free radicals.
The IC50 values were determined at 800 and 830 μg/ml for ABTS and DPPH tests, respectively. According to the results,
green-synthesized RFP/Ag-NPs as a safe efficient apoptosis inducer and strong antioxidant compound have the potential to
suppress breast cancer carcinogenesis by VEGF down-regulatiion and thus sensitizing them against apoptosis. However,
further researches are required to clarify RFP/Ag-NPs’ cell specificity and therapeutic doses in invivo conditions.
Keywords Rapeseed flower pollen· Green synthesized silver nanoparticles· Breast cancerous cell line (MDA-MB-231)·
VEGF down-regulating· Apoptosis-inducer· Antioxidant
Introduction
Due to the unpaired electrons, intracellular free radi-
cals react with other biomolecules and affect apoptosis
[1]. High levels of reactive oxygen species (ROS) lead
to oxidative stress, which is capable of damaging pro-
teins and nucleic acids [2]. The antioxidant agents are
used for developing novel cancer therapy strategies [3].
On the other hand, the interest in the use of nanomateri-
als in biological and commercial applications has been
developing, continuously. Chemical methods for synthe-
sizing efficient nanomaterials in human health are still not
safe enough [4]. Therefore, researchers have used green
approaches to developing more efficient nanomaterials
[5]. Metal nanoparticles including, zinc, gold, copper and
silver have been more interesting among the other met-
als, because of their exclusive properties. Silver nano-
particles (Ag-NPs) have unique properties such as high
electrical conductivity. Therefore, they could be useful
in various fields including medical, pharmaceutical and
food industries [6]. Also, they could be applied as anti-
bacterial agents, medical device coatings, cosmetics, drug
delivery systems, and anticancer agents [7]. The biological
* Masoud Homayouni Tabrizi
mhomayouni6@gmail.com
Sahar Hajebi
hajebisahar@gmail.com
Mahboobeh Nakhaei Moghaddam
mahboobe_nak@yahoo.com
Farzaneh Shahraki
shahraki09@yahoo.com
Soheyla Yadamani
soheyla_yadamani@yahoo.com
1 Department ofBiology, Islamic Azad University, Mashhad
Branch, Mashhad, Iran

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activity of Ag-NPs depends on several factors including
size distribution, particle morphology, composition, type
of reducing agents, and reactivity in solution [8–10]. Vari-
ous biomasses have been used in synthesizing Ag-NPs [11,
12]. We used flower pollen (RFP) water extract to synthe-
size RFP–silver nanoparticles (RFP–Ag-NPs), which are
cost-effective enough for synthesizing metal nanoparticles
[4]. The enzymes and other organic biomolecules, which
exist in plant water extract, act as both capping and reduc-
ing factors for synthesizing nanoparticles [4, 13]. Rape-
seed, a member of the family Brassicaceae, is cultivated
for its oil-rich seed [14]. In the current study, firstly we
synthesized Ag-NPs by triggering rapeseed flowers pollen
(RFP) as a bio-platform. Lastly, we planned to evaluate the
RFP–Ag-NPs’ antioxidant, antiangiogenesis, and proap-
optotic activities.
Materials andmethods
Reagents andmedia
Silver nitrate (Merck) was of analytical grade and was used
as a metal ion precursor to synthesize Ag-NPs from RFP.
MDA-MB-231 cell line was purchased from Iran Pasteur
Institute (Tehran, Iran). Fetal bovine serum (FBS), trypsin,
antibiotic, and DMEM culture medium were purchased
from Sigma-Aldrich Company, Ltd. (Poole, UK). Acridine
orange dye was obtained from Sigma-Aldrich Company,
Ltd. (Poole, UK). MTT [3-(4, 5-dimethylthiazol-2-yl)-2,
5-diphenyltetrazolium bromide] was provided by Sigma-
Aldrich Company, Ltd. (Poole, UK). The high Pure RNA
Isolation kits were purchased from Roche (Mannheim,
Germany) and cDNA synthesis and SYBR Green kit
from Fermentas Inc. All the solutions were prepared with
double distilled water and other chemicals with analytical
grade.
Preparation ofrapeseed pollen extract
20 g of rapeseed pollen was weighed, powdered and then
mixed with 200 ml of deionized distilled water. The result-
ing mixture was placed on a hot plate stirrer for 40 min at
60 °C. After cooling, the mixture was filtered and stored at
4 °C (Fig.1).
RFP/Ag‑NPs synthesis andcharacterization
RFP/Ag-NPs were synthesized by composition; 50 ml
AgNO3 (1mM) were mixed with 1ml RFP (100 mM in
water) water extract. The mixture was left at room tempera-
ture until the color change took place. UV–visible spectra
were recorded at 300–700 nm using microplate reader spec-
trophotometer (Epoch, US). The shapes of Bi/Ag-NPs were
observed by TEM imaging (CM-120, Philips) and FESEM.
FTIR andXRD methods
FTIR was used to examine the conjugation of rapeseed pol-
len with Ag nanoparticles (Perkin Elmer, Walthman, MA,
USA). To remove the non-binding rapeseed pollen, the syn-
thesized RFP/Ag-NPs were centrifuged (8000 rpm, 30 min),
washed with distilled water and dried. FTIR spectra for RFP/
Ag-NPs and rapeseed pollen were separately recorded at
the wavelength of 400–4000 nm, and the two spectra were
compared as well. The synthesized RFP/Ag-NPs were cen-
trifuged (9000 rpm, 30 min) and placed on carbon film for
XRD analysis (GNR, Explorer).
Fig. 1 Extraction method of a rapeseed flower, b powder, c water extract

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DPPH procedure
The ability of nanoparticle to trap the DPPH free radicals
represents the antioxidant potential of RFP/Ag-NPs. For
this purpose, the first DPPH free radicals were prepared by
solving the DPPH powder in 96% ethanol. Then, an equal
volume of RFP/Ag-NPs with various concentrations was
mixed with the DPPH working solution and incubated at
room temperature for 30 min. BHA was used as a standard
antioxidant. Finally, the absorbance was recorded at 517 nm.
ABTS procedure
To investigate the ability of RFP/Ag-NPs to inhibit ABTS
free radicals, first, the ABTS free radicals were synthesized
by dissolving ABTS powder and potassium sulfate in deion-
ized distilled water. After 16 h in darkness, the resulting
solution was diluted 50 times with distilled water to obtain
the ABTS·+ working solution. Then, an equal volume of
RFP/Ag-NPs with various concentrations was mixed with
the ABTS working solution and incubated at 37 °C for 1 h.
BHA was used as a standard antioxidant. Finally, the absorb-
ance was recorded at 734 nm.
Cell culture andtreatment
MDA-MB-231, MCF7 (breast cancer) and normal human
skin fibroblast cells were cultured in DMEM (10% FBS) and
incubated. Normal human skin fibroblast cells were chosen
as normal cells to examine the cytotoxicity of RFP/Ag-NPs
on normal cells versus MDA-MB-231 and MCF7 cancerous
cell lines.
Catalase gene expression assay
In the current study, the catalase gene expression was
evaluated in MDA-MB-231 cells treated with 1.5, 3µg/ml
concentrations of RFP/Ag-NPs at 24, 48, 72 and 96 h after
treatment using molecular analysis. For this purpose, RNA
was extracted and transcribed in reverse to cDNA using the
producer’s protocol. cDNA was amplified using a real-time
technique by using SYBR Green kit. Specific primers for this
research are presented in Table1.
Cytotoxicity eects andapoptosis induction
Cytotoxicity eects
MDA-MB-231, MCF7 and normal human skin fibroblast
(HDF) cells were seeded into a 96-well culture plate (5 × 104
cells/well) and treated with 0–50µg/ml of RFP/Ag-NPs for
24, 48 and 72 h. At the end of the incubations, MTT assay
was done and absorbance was recorded at 570 nm.
Acridine orange (AO) andpropidium iodide (PI)
double staining fordetermining type ofcell death
The apoptosis induction in MDA-MB-231 and normal
human skin fibroblast cell lines (HDF) which were treated
with RFP/Ag-NPs was determined by AO/PI staining. The
cells were treated with different concentrations of RFP/Ag-
NPs (0, 6, 12µg/ml) for 48 h. Then, the cell suspension was
prepared and an equal volume of it was mixed with the fluo-
rescent dye (1 AO: 1 PI) and detected under a fluorescence
microscope.
Analysis ofcell distribution byow cytometry
For cell cycle assessment, The MDA-MB-231 cells were
cultured, seeded and exposed with 0, 3, 6, 12µg/ml concen-
trations of RFP/Ag-NPs. Then the cells were isolated from
the flask floor and the cell suspension was prepared. The
resulting suspensions of RFP/Ag-NPs were mixed with PI
(Sigma) working solution and incubated (30 min, 37 °C in
the dark). Analysis of cell distribution was performed using
a FACScan laser flow cytometer (FACSCalibur, Becton-
Dickinson, USA).
Gene expression assay
VEGF gene expression were evaluated with real-time PCR.
For this purpose, the cancerous cells were seeded and
treated with 1.5, 3 and 6µg/ml concentrations of RFP/Ag-
NPs (0) for 48 h, After the treatment time, the whole RNA
was extracted and transcribed in reverse to cDNA using the
producer’s protocol. cDNA was amplified using a real-time
technique by using SYBR Green kit.
Table 1 The VEGF and CAT
genes primer sets. GAPDH:
Glyceraldehyde 3-phosphate
dehydrogenase
Gene Forward Reverse
GAPDH GCA GGG GGG AGC CAA AAC GGT TGG GTG GCA GTG ATG GCA TGG
VEGF CTG CTG TCT TGG GTG CAT TG TTC ACA TTT GTT GTG CTG TAG
CAT CGT GCT GAA TGA GGA ACA GA AGT CAG GGT GGA CCT CAG TG

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Results anddiscussion
Silver nanoparticle formation andcolor change
The results of the observations showed that biosynthesized
RFP/Ag-NP had a dark color. After addition of AgNO3
to the RFP extract and incubation, the color of the result-
ing mixture was altered from light yellowish to dark color.
Reduction of Ag+ to silver nanoparticles and alteration in
color are revealed in Fig.2.
UV–visible spectral analysis
The RFP/Ag-NPs absorbance peak was detected by UV–Vis
spectrophotometer at about 320 and 440 nm (Fig.3).
Transmission electron microscope (TEM) analysis
The TEM image and particle size histogram showed that
particle sizes range from 1 to 50 nm with an average of 23
nm (Fig.4). Dark shadows on the surface of the nanoparti-
cles in the TEM image may be the bioorganic molecules of
RFP water extract.
Field emission scanning electron microscope
analysis (FESEM)
The FESEM images of RFP/Ag-NPs are shown in (Fig.5).
The exact observation of the structure of the particles dem-
onstrates that the Ag nanoparticles are spherical in shape.
Chemical reaction mechanism ofRFP/Ag‑NPs
The amino acid functional groups in RFP water extract
react with silver nitrate to produce nanoparticles [21].
FTIR spectra were recorded to determine the interactions
of RFP water extract with silver nitrate (Fig.6). The FTIR
spectra represent the possible role of responsible biomol-
ecules in the reduction and stabilization of Ag-NPs. Absorp-
tion peaks at the range of 3416–2851 cm−1 were assigned
to O–H stretching of alcohol and phenol compounds and
aldehyde–C–H– stretching of alkanes. The absorption peaks
at 1646 indicate the C=C group. Also, the peaks ranging
at 1395–1024 indicate the amides’ N–H bond. Moreo-
ver, amines’ –C–N– stretching vibration bond, alcohols’
–C–O– stretching bonds, ethers, carboxylic acids, and
anhydrides were detected at 668 nm, which refer to O–H
(H-bonded) or broad CO variable weak bending vibrations
of alcohols and phenols (Fig.6).
Fig. 2 Color change from light yellowish to dark color
Fig. 3 UV–visible spectra of
RFP/Ag-NPs

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XRD results ofRFP/Ag‑NPs
To determine the element compositions, RFP/Ag-NPs were
subjected to XRD analysis. Figure7 shows the results of
XRD for RFP/Ag-NPs. The oxygen and carbon existence on
the elemental analysis indicates the RFP-conjugated silver
nanoparticles, as can be seen in Fig.7.
ABTS assay
Figure8 shows the antioxidant activity of RFP/Ag-NPs.
The level of antioxidant activity of RFP/Ag-NPs depends
on the concentration. In other words, increasing nanoparticle
concentration increases its antioxidant activity. The calcu-
lated IC50 in this test is about 0.3 μg/ml, which indicates that
about 50% of free radicals are inhibited in this concentration.
DPPH assay
As shown in Fig.9, the biosynthesized RFP/Ag-NPs were
able to remove the DPPH free radicals significantly. The
results show that by increasing the concentration of nano-
particles, the rate of inhibitory free radicals also increased
significantly with IC50 about 0.15µg/ml. RFP/Ag-NPs dis-
played good inhibitory effect on DPPH free radical.
Evaluation ofcatalase gene expression
Molecular analysis showed a significant increase
(P ≤ 0.001***) in the expression of catalase gene in treated
cells with increasing treatment time from 24 to 96 h
(Fig.10).
Eects ofRFP/Ag‑NPs onMDA‑MB‑231 cells
The cytotoxic eect ofRFP/Ag‑NPs onMDA‑MB‑231, MCF7
andHDF cells
The cytotoxic effect of RFP/Ag-NPs on breast carcinoma
cells was examined by MTT assay. The results show
(Figs.11, 12) that RFP/Ag-NPs can reduce the proliferation
of MDA-MB-231 and MCF7 cells. The calculated-IC50 for
treated cells was 3µg/ml and 2µg/ml (in 48 h), respectively.
Also, we demonstrated that green synthesized silver nano-
particles could decrease cell viability in a both dose- and
time-dependent manner. According to Fig.13, RFP/Ag-NPs
have no significant effect on HDF cells (IC50: above 12.5µg/
ml ( compared to cancerous cell lines.
Apoptosis induction assay results
The alterations of nuclear morphology in RFP/Ag-NPs-
treated cells, which were observed by AO/PI staining
method, indicate that the treated cells displayed apoptotic
morphology compared to untreated groups and also normal
cells (human skin fibroblast cells). The color changes, which
have been observed in the picture, show that the penetration
of acridine dye into the cells changes their color from green
Fig. 4 TEM image and particle size distribution histogram of RFP/
Ag-NPs
Fig. 5 FESEM image of RFP/Ag-NPs

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in untreated to red color in treated cells (Figs.14, 15). The
color changes in normal cells (human skin fibroblast cells)
show cell viability compared to MDA-MB-231 cells.
The apoptotic alteration was also confirmed with a flow
cytometric analysis. As shown in Fig.16, the sub-G1 popu-
lation, which indicates apoptotic cells, increased from 4.8%
at 0 μg/ml (control) to 23.7% at 1.5 and 80.5% at 6 μg/ml,
after exposure with RFP/Ag-NPs for 48 h.
Real‑time PCR results
The VEGF gene expression was assessed using real-time
PCR in treated cells with 1.5, 3 and 6µg/ml of RFP/Ag-NPs
in 48 h. Data show that the VEGF gene expression in treated
cells with different concentrations of RFP/Ag-NPs had a
significant decrease (P ≤ 0.001***) compared to untreated
cells. Figure17 shows about 80% reduction in the VEGF
Fig. 6 FTIR result of RFP/Ag-NPs
Fig. 7 XRD result of RFP/
Ag-NPs

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expression in cells treated with 6 μg/ml dose of RFP/Ag-NPs
compared to control cells.
The present study was designed in two sections: the first
part refers to generation and characterization of the nanopar-
ticles, and the second part focuses on their biological proper-
ties’ evaluation. Researches show that phenolic compounds
*** *** *** ***
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***
0
20
40
60
80
100
120
BHA 5 2.5 1.2 0.6 0.3 0.15
ABTS 10077706467.06 55 43
Concentraons (µg/ml)
% Inhibions of ABTS free radicals
Fig. 8 Radical inhibition activity of RFP/Ag-NPs. Treated groups
compared with the BHA group. Data are expressed as mean ± stand-
ard division
***
*** *** *** ***
***
0
20
40
60
80
100
120
BHA 5 2.5 1.2 0.6 0.3 0.15
DPPH 10082727275.08 74 54.02
Concentraons (µg/ml)
% Inhibions
Fig. 9 Radical inhibition activity of RFP/Ag-NPs. Treated groups
were compared with the BHA group. Data are expressed as
mean ± standard division
***
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***
0
10
20
30
40
50
60
70
24 48 72 96
1.5 microgaram/ml 1 0.90668 10.26166 41.97753
IC50 (3 microgaram/ml) 1 1.57538 12.23856 55.38669
Hours
Fold change relave to control
Fig. 10 The antioxidant capacity of RFP/Ag-NPs, by measuring
the expression of catalase gene in treated cells with 1.5 and 3µg/ml
of RFP/Ag-NPs compared to untreated cells at 24, 48, 72 and 96 h
after treatment (***P value < 0.001). Results were expressed as the
mean ± standard deviation
** ***
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*** *** ***
0
20
40
60
80
100
120
0 1.5 3612 24
24 100 92.4 84.833336.2533 31.073324.1667
48 100 85.1 55.566737.9333 28.463318.5867
72 100 88.8133 63 17.9618.3033 18.5
Concentraons (µg/ml)
% Viability of MDA cells
Fig. 11 Percentage of viable MDA-MB-231 cells treated with RFP/
Ag-NPs after 24, 48 and 72 h. Increasing concentrations of RFP/Ag-
NPs led to less percent of viable cell (***P value < 0.001)
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0
20
40
60
80
100
120
C 0.15 0.3 0.6 1.2
24h 100 72.4267 33.9 20.3133 10.4633
48h 100 71.1667 36.8933 21.59 7.0067
72h 100 65.8 36.2333 14.2333 5.5133
Concentraons (µg/ml)
% Viability of MCF7 cells
Fig. 12 The cytotoxic effect of RFP/Ag-NPs on MCF7 cells (24, 48
and 72 h). Increasing concentrations of RFP/Ag-NPs led to less per-
cent of viable cell (***P value < 0.001)
** ***
***
0
20
40
60
80
100
120
C 1.5 3.1 6.2 12.5
24H 100 98 89.4 83.4633 69.2533
Concentraons (µg/ml)
% Viability of HDF cells
Fig. 13 Percentage of viable HDF cells exposed with RFP/Ag-NPs
after 24 h (***P value < 0.001). The data were expressed as the
mean ± standard deviation

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Fig. 14 Fluorescent images of MDA-MB-231 cells stained by AO/PI. The cells treated with 1.5 and 3µg/ml of RFP/Ag-NPs for 48 h
Fig. 15 Fluorescent images of normal cells (human skin fibroblast cells) stained by AO/PI. The cells treated with 1.5 and 3µg/ml of RFP/Ag-
NPs for 48 h
Fig. 16 Cell distribution analysis of MDA-MB-231 cells exposure to various concentrations of RFP/Ag-NPs for 48 h

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and also proteins work as a reducing agent in the synthesis
of nanoparticles by triggering plant extracts [15–17].
In the current study, Ag-NPs were synthesized by RFP
as novel eco-friendly bio-platform. Ag-NPs formation
could easily be screened by detecting the color changes in
a mixture containing nanoparticles. The solution’s color
alterations might be due to the excitation of Ag-NPs sur-
face plasmon vibration [18]. The existence of bioactive
compounds such as amino acids in plant water extracts
acts as a key stabilizing and synthesizing factor to reach
Ag-NPs (Fig.2). Shankar etal. also synthesized Ag-NPs
using amino acids as reducing and capping agents [19].
The UV–Vis absorption curve of biosynthesized Ag-NPs
presents the absorbance edge at 320 and 440 nm, which
indicates the Ag-NPs formation (Fig.3). On the other
hand, these peaks confirm the absorbance peaks reported
by Thomber etal. (at 410–415 nm) and Tippayawat etal.
(at 420 nm) in synthesizing Ag-NPs [20]. The results of
TEM microscopy and particle size distribution histogram
of RFP/Ag NPs show that the RFP–Ag-NPs have an aver-
age particle size at 20 nm, which is similar to the other
reports for Ag-NPs production [21, 22]. The FESEM
images of RFP/Ag-NPs show the spherical shape, which
is comparable with the reported mages of Ag-NPs by
Shameli and Tippayawat etal. [23, 24]. The FTIR spec-
tra represent the possible role of biomolecules, which are
responsible for the reduction and stabilization of Ag-NPs
in the mixture. The results show that the nanoparticle spe-
cific bands (Fig.6) are similar to Koyyati etal.’s results.
We found that the XRD results were similar to other stud-
ies confirming the nanoparticles existence [18]. We also
evaluated the cytotoxic, proapoptotic, antiangiogenic and
antioxidant properties of RFP/Ag-NPs. Figures8 and 9,
refer to the antioxidant assays, ABTS and DPPH, and
determine the individual IC50 values for RFP/Ag-NPs
antioxidant activity. However, it has been reported that Ag/
NPs have an antioxidant effect [25]. In the previous survey,
the associated genes with antioxidant activity, such as the
catalase gene, has been examined [26], but in the present
study, for the first time, changes in the expression of this
gene have been investigated over a period of 96 h at a
specified time interval. The results show that on increasing
treatment time from 24 to 96 h, the expression of catalase
gene expression is significantly increased (Fig.10). There-
fore, in the current study, it was shown that the catalase
gene up-regulation as an antioxidant gene significantly
associates with increasing treatment time from 24 to 96h
in MDA-MB-231 cells. The cytotoxic results showed that
the biosynthesized nanoparticles suppress cancer cells
[IC50: 3µg/ml (MDA-MB-231) and 2µg/ml (MCF7)] and
have no inhibitory effect on normal cells (above 12.5µg/
ml) (Figs.11, 12, 13), which are similar with Sulaiman
etal.’s study that reported the green synthesis of silver
nanoparticles has cytotoxic effects on HL-60 cells [27].
There is a significant cross-talk between VEGF overex-
pression and BCL2, the most known antiapoptotic gene.
However, VEGF is considered a survival agent for both can-
cer and endothelial cells. Moreover, it acts as an apoptosis
suppressor through BCL2 up-regulation [28–30]. Several
studies confirm that VEGF gene expression could be up-reg-
ulated by increasing BCL2 expression in various tumor cells
[31–33]. According to the flow cytometry results, EGF over-
expression and cells’ fluorescent images, we investigated an
apoptotic death. Interestingly, we found that cancerous cells
trigger a significant antiapoptotic strategy by up-regulating
catalase gene expression to overcome the induced apoptotic
impacts of RFP/Ag-NPs after a long time incubation with
nontoxic lower concentrations. Therefore, the results suggest
a protective role for VEGF against apoptosis as explained
by Steven Le Gouill etal., for multiple myeloma cells. They
demonstrated that VEGF induces the myeloid cell leukemia
1 (Mcl-1) overexpression and thus prevents apoptosis indi-
rectly [34]. Mcl1 binding to Bak (proapoptotic gene) can
inactivate apoptosis [35, 36]. In this regard, we demonstrated
that RFP/Ag-NPs makes the cells more sensitive to apopto-
sis by down-regulating VEGF expression.
Conclusion
We found that green synthesized RFP/Ag-NPs have the
potential to suppress breast cancerous cell line (MDA-
MB-231) carcinogenesis by VEGF down-regulation and thus
sensitize the cells against apoptosis. Therefore, we suggest
RFP/Ag-NPs can be applied as a safe and efficient apoptosis
inducer and strong antioxidant compound in cancer therapy.
However, further researches are required to clarify RFP/Ag-
NPs’ cell specificity and pharmaceutical doses in invivo
conditions.
C 1.5 3 6
VEGF 1 0.0134 0.28583 0.20877
***
***
***
0
0.2
0.4
0.6
0.8
1
1.2
Fold change relave to contro
l
Concentraons (µg/ml)
Fig. 17 The expression of VEGF gene in MDA-MB231 cancerous
cells treated with RFP/Ag-NPs compared to untreated cells (***P
value < 0.001). Results were expressed as the mean ± standard devia-
tion

JBIC Journal of Biological Inorganic Chemistry
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Acknowledgements This work was supported by the Islamic Azad
University, Mashhad, Iran, which is appreciated by the authors.
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