Potential of colloidal or silver nanoparticles to reduce the growth of B16F10 melanoma tumors

Abstract

Previosuly, we reported the cytotoxic effect of colloidal silver (AgC) on MCF-7 breast cancer cell line. However, there is scarce information on its antitumor potential. The aim of this study was to evaluate the anti-tumoral activity of colloidal silver (AgC) or silver nanoparticles (AgNPs) in a B16F10 melanoma mice model. In vitro, B16F10 cells were treated with different concentrations of AgC or AgNPs and cell viability was evaluated by MTT method, both treatments had cytotoxic effects against B16F10 cell line. In vivo, B16F10 melanoma cells (5 × 10 5) were implanted in six weeks old C57BL/6 mice. About 8 days after cells injection, the subcutaneous treatments were started with AgC or AgNPs, tumor volume and tumor weight were evaluated and the difference of treated groups and control demonstrated that melanoma tumor growth was significantly decreased. Our results suggest that AgC or AgNPs could be useful as an antiproliferative drug, inducing an impairment of tumoral growth.

Full text

Vol. 7(22), pp. 2745-2750, 28 May, 2013
DOI: 10.5897/AJMR12.1968
ISSN 1996-0808 ©2013 Academic Journals
http://www.academicjournals.org/AJMR
African Journal of Microbiology Research
Full Length Research Paper
Potential of colloidal or silver nanoparticles to reduce
the growth of B16F10 melanoma tumors
Sierra-Rivera C. A., Franco-Molina M. A.*, Mendoza-Gamboa E., Zapata-Benavides P., Tamez-
Guerra R. S. and Rodríguez-Padilla C.
Laboratorio de Inmunología y Virología, Departamento de Microbiología e Inmunología, Facultad de Ciencias Biológicas
de la Universidad Autónoma de Nuevo León, San Nicolás de los Garza, N. L. México.
Accepted 26 March, 2013
Previosuly, we reported the cytotoxic effect of colloidal silver (AgC) on MCF-7 breast cancer cell line.
However, there is scarce information on its antitumor potential. The aim of this study was to evaluate
the anti-tumoral activity of colloidal silver (AgC) or silver nanoparticles (AgNPs) in a B16F10 melanoma
mice model. In vitro, B16F10 cells were treated with different concentrations of AgC or AgNPs and cell
viability was evaluated by MTT method, both treatments had cytotoxic effects against B16F10 cell line.
In vivo, B16F10 melanoma cells (5 × 10
5
) were implanted in six weeks old C57BL/6 mice. About 8 days
after cells injection, the subcutaneous treatments were started with AgC or AgNPs, tumor volume and
tumor weight were evaluated and the difference of treated groups and control demonstrated that
melanoma tumor growth was significantly decreased. Our results suggest that AgC or AgNPs could be
useful as an antiproliferative drug, inducing an impairment of tumoral growth.
Key words: Colloidal silver, silver nanoparticles, melanoma, cancer, tumor.
INTRODUCTION
The recent increase in the incidence of malignant
melanoma urges the development of more specific and
effective therapies. Historically, silver has been a major
therapeutic agent in medicine, especially in infectious
disease, including surgical infections (Alexander, 2009).
Since 1990, there has been a resurgence on the use of
AgC as an alternative medicine because of increased
resistance of bacteria to antibiotics, and the continuing
search for novel and affordable antimicrobial agents.
Previously, we reported that AgC has antitumor activity
through induction of apoptosis in MCF-7 breast cancer
cell line (Franco-Molina et al., 2010) and other studies
with AgNPs, by Sriram et al. (2010) demonstrated the
efficacy of biolo-synthesized AgNPs as an antitumor
agent against Dalton’s lymphoma ascites cell lines in vitro
and in vivo and the capacity to affect cellular viability of
human colon cancer cells (HT 29) (Sanpui et al., 2011).
Despite the fact that nanoparticles and colloidal particles
possess great potential for future clinical application
therapeutics, and this application generated interest in
exploring other metals for potential anti-cancer properties
(Bhattacharyya et al., 2011), there is scarce information
on AgC or AgNPs antitumor potential. This study is
relevant due to their use within health remedies. The aim
of the present study was to determine the effects of AgC
or AgNPs on murine melanoma tumorigenesis under in
vitro and in vivo conditions.
*Corresponding author. E-mail: moyfranco@gmail.com. Tel: 01152-81-83-29-41-15. Fax: 01152- 81-83-52-42-12.
Abbreviations: AgC, Colloidal silver; AgNPs, silver nanoparticles; MTT, thiazoly blue tetrazolium bromide.

2746 Afr. J. Microbiol. Res.
Figure 1. Schematic representation of the treatment protocols. Tumor time-line. 5x10
5
B16F10 cells were
inoculated subcutaneously into C57BL/6 mice on day 0, later beginning on day 8 after tumor cell
inoculation, the treatments with AgC (28 mg/kg) or AgNPs (1000 mg/kg) were administrated around tumor
with daily subcutaneous dose, the mice were monitored until 21 days post-cell inoculation and were finally
sacrificed by cervical dislocation.
MATERIALS AND METHODS
Animals
In vivo experiments were conducted in male C57BL/6 mice of six
weeks old (18 to 25 g), housed at 22 ± 2°C under a 12/12 h
light/dark cycle with free access to food and water until use. All
animal handling and experimental procedures were conducted with
prior approval of the ethics committee on animal use of the
Facultad de Ciencias Biológicas, Universidad Autónoma de Nuevo
León.
AgC or AgNPs
The grenetine-stabilized AgC (120 nm) was purchased from
MICRODYN (Mexico, D.F.) as a 0.35% stock solution. It was filtered
and diluted to a concentration of 17.5 µg/mL with DMEM/F-12
supplemented with 10% FBS. The AgNPs (10 nm) was purchased
from (Nanostructured & Amorphous Materials Inc. Houston, Tx.
USA); 5 mg/mL stock solution was prepared in DMEM-F12 supple-
mented with 10% FBS, previously sonicated during 15 min for its
homogenization and sterilized by filtration (0.2 µm filter, Millipore,
USA) in vitro for assays. For In vivo studies, 28 mg/kg (AgC) or
1000 mg/kg (AgNPs) per mice were daily administered subcuta-
neously around the tumor.
Cell viability
Cells (5 × 10
3
cells/well) were plated on 96 flat-bottom well plates,
and incubated 24 h at 37°C in 5% CO
2
atmosphere. After incuba-
tion, culture medium was removed, and AgC diluted in the same
medium was added at concentrations ranging from 0 to 17.5 µg/mL
and AgNPs at concentrations ranging from 0 to 5 mg/mL. The
plates were then incubated for 24 h at 37°C, and 5% CO
2
atmo-
sphere. Thereafter, the supernatant was removed and cells were
washed twice with DMEM/F-12 medium. Cell viability was deter-
mined by the MTT method. Quantification was obtained by the
absorbance reading at a wavelength of 570 nm and cellular viability
was expressed as percentage. Results were given as the mean ±
SD of three independent experiments.
Melanoma cells implant
All animal procedures were made in Laboratorio de Inmunología de
la Facultad de Ciencia Biológicas de la UANL, in accordance with
Facultad de Ciencias Biológicas of the UANL Ethics Committee.
Briefly, 5 × 10
5
B16F10 melanoma cells were injected subcuta-
neously into C57BL/6 mice. When tumors were palpable (around 8
days after implanted) mice were treated around tumor with subcuta-
neous daily dose of 28 mg/Kg of AgC or 1000 mg/Kg of AgNPs.
The mice were monitored until 21 days post-cell inocula-tion and
were sacrificed by cervical dislocation (Figure 1). The tumors were
surgically collected, and body weight, volume, tumor weight and
metastasis were determined (metastasis was deter-mined during
the necropsy by findings of tumor cells in muscle, peritoneal cavity,
bowel and liver). Tumor volume was recorded using a measuring
gauge (PRETUL, USA) using the formula: volume = length x
(width)
2
.
Statistical analysis
Data represent the mean ±SD of triplicates from three independent
experiments. Statistical differences were obtained using the analy-
sis of variance, and the Dunnett's tests (SPSS v. 17.0 program).
The results were considered statistically significant if the *p value
was <0.05.
RESULTS
Cytotoxic activity of AgC or AgNPs on B16F10
melanoma cell line
AgC induced dose-dependent cytotoxic effect (7 to 17.5
µg/mL) on B16F10 cells (Figure 2). AgNPs induced dose-
dependent cytotoxic effect (1.5 to 5 mg/mL) on B16F10
melanoma cells in 24 h of incubation (Figure 3).
Treatment of B16F10 melanoma tumor
The B16F10 melanoma model was used to demonstrate
the therapeutic value of AgC or AgNPs. After the tumor
appearance by the eight day, the group mice were daily
treated with 28 mg/kg of AgC or AgNPs 1000 mg/kg,
respectively, by subcutaneous route around tumor. The
mice were sacrificed at 21 days of treatment (Figure 1).
There was no difference regarding the body weight bet-
ween treatments (control (25.00 g ± 2.78), AgNPs (21.30
g ± 1.14) and AgC (25.81 g ± 0.39) (Table 1 and Figure
4). However, these treatments significantly (p<0.05)
decreased the tumor weight {(AgNPs (0.51 ± 0.22 g) and
AgC (0.85 ± 0.64 g)} when compared with the control
(4.97 g ± 0.31) (Table 1 and Figure 4); and the tumor

Sierra-Rivera et al. 2747
Figure 2. B16F10 cell viability treated with AgC. B16F10 cells (5 x 10
3
cells/well) were cultured into
96 well plates and incubated overnight. Thereafter, the plates were treated with AgC concentrations
ranging from 0 to 17.5 µg/mL, and incubated for 24 h at 37°C, and 5% CO
2
atmosphere. Thereafter, a
MTT assay was performed. The optical density was determined at 570 nm. Data represent means of
triplicate samples with ±SD indicated. *p<0.05 as compared with untreated cells.
Figure 3. B16F10 cell viability treated with AgNPs. B16F10 cells (5 x 10
3
cells/well) were cultured
into 96 well plates and incubated overnight. Thereafter, the plates were treated with AgNPs
concentrations ranging from 0 to 5 mg/mL, and incubated for 24 h at 37°C, and 5% CO
2
atmosphere. Thereafter, a MTT assay was performed. The optical density was determined at 570
nm. Data represent means of triplicate samples with ±SD indicated. *p<0.05 as compared with
untreated cells.
volume was significantly (p<0.05) decreased {(AgNPs
(1.21 mm
3
± 0.68) and AgC (1.30 mm
3
± 0.73)} when
compared with the control (11.01 mm
3
± 0.86) (Table 1
and Figure 4). Regarding to the necropsy findings; the
treatments of AgC or AgNPs prevented the metastasis
from muscle, peritoneal cavity, bowel, and liver when
compared with control (Table 2). None mice showed
tumoral erradication in both groups treated. The mice

2748 Afr. J. Microbiol. Res.
A
B
C
Figure 4. Tumor growth decrease in mice treated with AgC or AgNPs. C57BL/6 mice bearing B16F10 tumor,
were treated daily with AgNP´s or AgC at doses of 28 and 1000 mg/kg, respectively. A) Control, B) AgC and
C) AgNPs, these pictures are representative mice groups.
Table 1. Comparison of volume-weight of tumors in mice treated with AgC or AgNPs.
Treatment
Body weight (g ± SD)
Tumor weight (g ± SD)
Tumor volume (mm
3
± SD)
Control
25.00±2.78
4.97±0.31
11.01±0.86
AgC
25.81±0.39
0.85±0.64*
1.30±0.73*
AgNPs
21.30±1.14
0.5±0.22*
1.21±0.68*
C57BL/6 mice bearing B16F10 tumor were daily treated with AgNPs or AgC at doses of 1000 or 28 mg/kg, respectively. In
this study, the mice were sacrificed at 21 days. *p<0.05 as compared to the controls.
Table 2. Incidence of metastasis in mice treated with AgC or AgNPs
Treatment
Muscle
Peritoneal cavity
Bowel
Liver
Control
+
+
+
+
AgC
-
-
-
-
AgNPs
-
-
-
-
C57BL/6 mice bearing B16F10 tumor, were treated daily with AgNPs or AgC at doses of 1000 or 28
mg/kg, respectively. In this study, the mice were sacrificed by cervical dislocation at 21 days; and the
metastasis was determined during the necropsy in muscle, peritoneal cavity, bowel and liver. +:
Metastasis, - : without metastasis.
treated with AgC showed a skin induration like fibrosis in
the zone treated for five days, this condition was ob-
served until sacrifice; the mice treated with AgNPs were
not affected by this symptoms.
DISCUSSION
Skin cancer is the most common form of cancer in the
United States. More than 3.5 million skin cancers in over
two million people are diagnosed annually. Each year
there are more new cases of skin cancer than the com-
bined incidence of cancers of the breast, prostate, lung
and colon. An estimated 123,590 new cases of mela-
noma were diagnosed in the US in 2011: 53,360 noninva-
sive (in situ) and 70,230 invasive, with nearly 8,790
resulting in death (http://www.skincancer.org). Some
cytotoxic agents used for its treatment are costly and
known to induce several side effects such as myelosup-

pression, anemia and most importantly the generation of
cellular resistance. For this, it is important to find alterna-
tive therapies or drugs to overcome these drawbacks
(Kim et al., 2007). Since ancient times, the silver has
been used to treat numerous diseases, mainly used in
antimicrobial agents, in treating wounds, burns and
catherter related infections (AshaRani et al., 2009),
however; it was until only a few years ago that there was
boom in taking different silver particles due to the rate of
exposure increasing progresively over the years when
engineered nanomaterials were extensively used in a
variety of industries including medical applications
(AshaRani et al., 2012). The use of smaller particles have
a wider tissue distribution, penetrate further within the
skin and intestine, are internalised to a greater extent,
and have a larger toxic potency (Johnston et al., 2010).
So the study of different silver particles is vital to explore
their therapeutic potential. Previously, we reported the
antitumor activity of colloidal silver on MCF-7 human
breast cancer cells (Franco-Molina et al., 2010), and for
this reason we decided to continue with a tumor model In
vivo. In the present study, our In vitro results demon-
strated that AgC or AgNPs significantly decreased in a
dose-dependent manner (p<0.05), the growth of B16F10
melanoma cells. The effects of cytotoxicity are similar to
those shown by other studies (Hsin et al., 2008) and the
mechanism of cell death probably are due to decreased
mitochondrial membrane potential, inducing apoptotic
death (Franco-Molina et al., 2010). Although, the exact
mechanism of action of silver particles is unknown; some
reports showed that AgNP uptake occurs mainly through
endocytosis where clathrin mediated process and macro-
pinocytosis were involved, the nuclear deposition of
AgNP is unknown, but the AgNP treatment leads to
changes in the cell membrane permeability, facilitating
the entry of Ca
+
ions which activate enzymes like pro-
teases and endonuclease that increase toxicity, resulting
in mitochondrial membrane dysfunction and reactive
oxygen species production and oxidative stress, damage
to DNA can be induced through binding of DNA or via
oxidative to DNA, reducing DNA synthesis and producing
chromosomal aberrations, errors in chromosome segre-
gation and production of micronuclei, leading to cell death
(AshaRani et al., 2009), mainly observed in tumor cells
and not in normal cells (Franco-Molina et al., 2010). It is
important to notice that major doses were used to induce
cytotoxic effect by AgNPs (1.5 to 5 mg/mL) when com-
pared with AgC (7 to 17.5 µg/mL) on cancer cells. This
findings were probably due to the fact that AgNPs treated
cells have limited exposure to Ag ions because AgNPs
solution contained a minimun amount of free Ag ions.
However, it has been suggested that AgNPs and Ag can
induce cell death in vitro through a ROS production. It
has been demonstrated that there are some differences
in their mechanism of action, example is that Ag induced
metallothionein 1b (MT1b) and AgNPs did not. Ag also is
capable of inducing a major production of oxidative stress
Sierra-Rivera et al. 2749
related glutathione peroxidase and catalase expression
compared with AgNPs. This mechanism of action could
indicate that AgC appeared more toxic than AgNPs such
has been demonstrated by Hsin et al., 2008.
“In vivo” we found that AgC or AgNPs have the poten-
tial to impair the growth tumor, since AgNPs is better than
AgC treatment in reducing the tumoral volume and
weight. These finding could be correlated with another
study where the AgNPs had the potential of inhibiting the
VEGF in a model employing bovine retinal endothelial
cells in vitro and angiogenesis in a mice model in vivo
(Gurunathan et al., 2009). At the beginning of the study,
we observed that intraperitoneal injection of AgC (28
mg/kg) induced mice exhibited nervous symptoms after
15 s of administration, like jumping and disnea by 10 min
and lethargy during all day long (data not shown), by this
reason the use of subcutaneous administration at doses
of 28 mg/kg, without side effects was decided, avoiding
increases of the doses like that used with AgNPs. There
are reports that show adverse effects when doses higher
than 100 mg/kg of AgC were used (Faust, 1992). And the
adverse effects could be possible because the Ag can
enter through the blood-brain barrier and accumulate in
large motoneurones in the brain stem and spinal cord,
neurons in cerebellar nuclei and glia; and the toxic symp-
toms such as cerebral ataxia are associated with pro-
longed exposure to silver (Panyala et al., 2008), which is
different from the findings previously mentioned by us.
In this experiment, we used the highest doses reported
for AgNPs because in a study by Rahman et al. (2009)
on the effects of AgNPs (25 nm) on gene expression in
different regions of the mouse brain where the particles
were administered to adult male mice route intraperito-
neal injection at doses of 100, 500, or 1000 mg/kg for 24
h, array data indicated changes in the expression of
genes in the caudate nucleus, frontal cortex and hippo-
campus of mice treated with the AgNPs. Analysis of
these changes led the authors to suggest that AgNPs
may produce neurotoxicity by stimulating oxidative stress
generation and altered gene expression, leading to apop-
tosis (Rahman et al., 2009). In the present study, despite
the use of AgNPs daily for thirteen days, we did not find
adverse effects, like nervous symptoms associated at its
administration.
Although, the mechanism of the antitumoral action of
AgC or AgNPs is not properly understood, it has been
reported that heavy metals react with proteins by getting
attached to the thiol group and the proteins get inacti-
vated through a mechanism implicated in avoiding the
cellular proliferation of cancer cells (Liau et al., 1997).
With these results, we can confirm the potential of AgC
or AgNPs on this melanoma tumor model suggesting
them as a potential agent for use in cancer treatment.
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