Assessment of gold nanoparticle effect on prostate cancer LNCaP cells
In recent years gold nanoparticles (AuNPs) have received considerable attention for various biomedical applications including diagnostics and targeted drug delivery. However, more research is still needed to characterize such aspects of their use in clinical oncology as permeability, retention and f...
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Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України
2015
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| Cite this: | Assessment of gold nanoparticle effect on prostate cancer LNCaP cells / A.G. Reznikov, O.A. Salivonyk, A.G. Sotkis, Y.M. Shuba // Experimental Oncology. — 2015. — Т. 37, № 2. — С. 100-104. — Бібліогр.: 27 назв. — англ. |
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| author | Reznikov, A.G. Salivonyk, O.A. Sotkis, A.G. Shuba, Y.M. |
| author_facet | Reznikov, A.G. Salivonyk, O.A. Sotkis, A.G. Shuba, Y.M. |
| citation_txt | Assessment of gold nanoparticle effect on prostate cancer LNCaP cells / A.G. Reznikov, O.A. Salivonyk, A.G. Sotkis, Y.M. Shuba // Experimental Oncology. — 2015. — Т. 37, № 2. — С. 100-104. — Бібліогр.: 27 назв. — англ. |
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| container_title | Experimental Oncology |
| description | In recent years gold nanoparticles (AuNPs) have received considerable attention for various biomedical applications including diagnostics and targeted drug delivery. However, more research is still needed to characterize such aspects of their use in clinical oncology as permeability, retention and functional effect on tumor cells. Aims: This study was designed to describe the effect of non-functionalized AuNPs on LNCaP prostate cancer cells growth. Material and Methods: LNCaP cells were cultured in RPMI-1640 medium containing AuNPs covered by polyvinylpyrrolidone of average size 26.4 nm (10.0 μg/ml). Counts of cells were calculated and their morphology was examined. Results: AuNPs conglomerates have been visualized in cultured cells. After 4-day incubation in presence of AuNPs significant retardation of LNCaP cells growth was observed both in 5α-dihydrotestosterone stimulated and non-stimulated cultures. No morphological changes of live LNCaP cells were seen in any experiment. Conclusion: Given absence of morphological changes in live cells and dribble and relatively constant numbers of dead cells, it was concluded that inhibitory effect of AuNPs on LNCaP cells growth was caused by alterations of proliferation. Key Words: prostate cancer, LNCaP, gold nanoparticles, 5α-dihydrotestosterone.
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100 Experimental Oncology 37, 100–104, 2015 (June)
ASSESSMENT OF GOLD NANOPARTICLE EFFECT ON PROSTATE
CANCER LNCaP CELLS
A.G. Reznikov1*, O.A. Salivonyk1, A.G. Sotkis2, 3, Y.M. Shuba2, 3
1V.P. Komisarenko Institute of Endocrinology and Metabolism,
National Academy of Medical Sciences of Ukraine, Kyiv 01601, Ukraine
2O.O. Bogomolets Institute of Physiology, Kyiv 01024, Ukraine
3International Center of Molecular Physiology, NAS of Ukraine, Kyiv 01024, Ukraine
In recent years gold nanoparticles (AuNPs) have received considerable attention for various biomedical applications including di-
agnostics and targeted drug delivery. However, more research is still needed to characterize such aspects of their use in clinical
oncology as permeability, retention and functional effect on tumor cells. Aims: This study was designed to describe the effect
of non-functionalized AuNPs on LNCaP prostate cancer cells growth. Material and Methods: LNCaP cells were cultured
in RPMI-1640 medium containing AuNPs covered by polyvinylpyrrolidone of average size 26.4 nm (10.0 μg/ml). Counts of cells
were calculated and their morphology was examined. Results: AuNPs conglomerates have been visualized in cultured cells. After
4-day incubation in presence of AuNPs significant retardation of LNCaP cells growth was observed both in 5α-dihydrotestosterone
stimulated and non-stimulated cultures. No morphological changes of live LNCaP cells were seen in any experiment. Conclusion:
Given absence of morphological changes in live cells and dribble and relatively constant numbers of dead cells, it was concluded
that inhibitory effect of AuNPs on LNCaP cells growth was caused by alterations of proliferation.
Key Words: prostate cancer, LNCaP, gold nanoparticles, 5α-dihydrotestosterone.
Prostate adenocarcinoma (prostate cancer — PCa)
is one of the most common cancer that affects 12–17%
of male population in the developed countries [1, 2].
PCa case incidence and mortality increased during
past decades. In the year of 2012, PCa in Ukraine was
diagnosed as the second malignancy in human male
population (after lung cancer) and the most common
oncourological disease [3].
PCa represents a classical model of hormone-de-
pendent malignancy. In the most cases, growth of PCa
is promoted by testosterone and other androgens.
For this reason, advanced PCa is quite successfully
treated by surgical or pharmacological (androgen
receptor antagonists, hypothalamic luteinizing hor-
mone — releasing hormone analogues, estrogens and
other drugs) androgen ablation. However, castration
and hormonal therapy exert only palliative effect, and
usually after remission the tumor relapses due to re-
fractoriness to drugs. Some patients initially demon-
strate refractoriness to castration or other modalities
of systemic endocrine therapy. Possible mechanisms
of hormone resistance include androgen receptor (AR)
gene mutations and amplification, ligand-independent
AR signaling, changed interplay between AR and
growth factors, intratumoral conversion of adrenal
androgens to testosterone, and so on. Alternative
treatments are needed for aggressive localized PCa
and hormone-refractory metastatic disease.
Modern nanotechnologies provide the new pos-
sibilities in biology and medicine [4, 5]. In the recent
decades, nanotechnology has brought new hopes
in malignant tumors diagnosis and targeted therapy.
Among various nanoparticles, the gold ones (AuNPs)
have received considerable appreciation by investiga-
tors and clinicians due to their unique properties [6–8].
They have inert core, permeate through the cell mem-
brane, can be functionalized easily, and demonstrate
chemical stability and surface plasmon oscillation.
Because of advanced technology of chemical syn-
thesis, a large range of AuNPs with different size,
shape and optical properties are available. Currently,
the AuNPs are already applied or likely to be applied
in the foreseeable future for bio-nano-analytics, diag-
nostic, photothermal therapy and targeted drug de-
livery [9–13]. In the meantime, however, clinical trials
of AuNPs as antitumor agents are rather limited, and
much more basic research is still needed to facilitate
their transition to clinical oncology.
Earlier we observed suppressive effect of AuNPs
on human PCa xenograft growth in mouse model [14].
Exploration of their effects on PCa cells in vitro could
bring new information on the peculiarities of interac-
tion between AuNPs and tumor cells. There was shown
a cytotoxic influence of AuNPs on androgen-refractory
PCa PC-3 cell line [15]. Cytotoxic activity of AuNPs
conjugated with non-steroidal antiandrogens has been
demonstrated in vitro toward hormone-insensitive
PCa cell line [16]. Incorporation of AuNPs conjugated
with glutamate carboxypeptidase inhibitor, prostate-
specific membrane antigen, into LNCaP and PC-3 cells
was observed [17], but their effects on the cells
have not been studied. In this article, we represent
the results of exploration of the effects of simple,
non-functionalized AuNPs on androgen-sensitive
PCa LNCaP cells growing freely or stimulated with
5α-dihydrotestosterone (DHT).
Submitted: February 24, 2015.
*Correspondence: E-mail: reznikov39@gmail.com
Abbreviations used: AR — androgen receptor; DHT —
5α-dihydrotestosterone; AuNPs — gold nanoparticles; PVP — poly-
vinylpyrrolidone; PCa — prostate cancer.
Exp Oncol 2015
37, 2, 100–104
Experimental Oncology 37, 100–104, 2015 (June) 101
MATERIALS AND METHODS
AuNPs. AuNPs were prepared using citric tech-
niques of reduction of chloroauric acid [18], and kindly
provided to us by the Research Institute for Nano-
technological Industry, Open International University
of Human Development “Ukraine”.
There was used ethanol dispersion of spherical
AuNPs covered by polyvinylpyrrolidone (PVP) in order
to prevent aggregation. The estimation of AuNPs pa-
rameters was carried out with laser correlation spec-
troscopy (Zetasizer-3, Malvern Instruments Ltd, UK).
Cells and experimental protocol. Human PCa
cell line LNCaP was provided by R.E. Kavetsky Institute
of Experimental Pathology, Oncology and Radiobio-
logy, National Academy of Sciences of Ukraine, Kyiv.
Three series of experiments with similar design were
carried out. Cells were cultured on 24 × 24 mm square
glass cover slips placed in Petri dishes filled with
culture medium of the following composition: RPMI-
1640 medium (Sigma), 10% fetal bovine serum
(Sigma), 50.000 IU/l penicillin and 50 mg/l strepto-
mycin. Cell cultures were maintained in the thermostat
at 37 °C in a 95% air — 5% CO2 humidified atmosphere.
Culture medium was changed daily.
After cells’ density reached 50–60% of cover
slip’s surface, the cultures were separated onto
5 groups each consisting of 3–5 cover slips. The cul-
ture medium in the first group (control) contained
ethanol, and the second one (PVP group) contained
PVP-ethanol solution just to make sure it doesn’t af-
fect cells. The third group (AuNPs group) was exposed
to AuNPs (10 μg/ml) alone, the fourth group (DHT
group) contained DHT (10–4 M) alone, and the fifth
group (AuNPs + DHT group) was supplemented with
AuNPs (10 μg/ml) plus DHT (10–4 M). Ethanol con-
centration in culture medium of all experimental and
control groups did not exceed 1%. After 4-days of cul-
turing with daily replacement of respective medium,
cells from each group were detached from cover slips
using 0.25% trypsin-EDTA in 0.5 ml medium, sus-
pended in additional 0.5 ml medium and centrifuged
at 3200 rpm. The cells were resuspended in 1.0 ml me-
dium, stained with Trypan Blue in order to discriminate
live and dead cells, and the numbers of live and dead
cells were calculated in Goryaev chamber.
Morphological studies. Part of cover slips was
used for morphological studies. The cells were fixed
in 4% paraformaldehide, then stained with Ferrum
hematoxylin and underwent examination using the mi-
croscope Leіka DME (Leika Microsystems, Germany).
Data analysis and statistics. Each experiment
was repeated 4–6 times. Accretion percentage of cell
counts in relation to initial seeding was calculated,
and results were averaged and expressed as mean
± s.e.m. (standard error of the mean). The results
were compared with those of appropriate controls.
Student’s t-test was used for statistical analysis, with
p < 0.05 cons idered significant.
RESULTS AND DISCUSSION
AuNPs size measurements. It has been estima-
ted that the colloidal solution contained nanoparticles
with sizes ranging from 15.3 to 30.9 nm (90% of total
number) and the prevalence of 21.8 nm one (42% of to-
tal number) (Fig. 1). The average size of nanoparticles
was estimated as 26.4 nm.
Size distribution(s)
5 10 50 100 500 1000
Diameter (nm)
20
40
Nu
m
be
r i
n
cl
as
s,
%
Fig. 1. AuNPs size (diameter) and number distribution by laser
correlation spectroscopy
LNCaP cells growth. During 4-day-growth period,
a total count of control LNCaP cells (i.e., cultured
in the presence of ethanol and/or PVP) increased
7–21-fold. The relative numbers of dead cells against
initial seeding increased only 2-fold, and was similar
in all control and experimental groups. Given a total
numbers of cultivated cells and numbers of live and
dead cells in the end of the experiments, PVP in ap-
propriate amounts did not affect the culture growth
versus the vehicle control (p > 0.05) (Fig. 2).
100
600
1100
1600
2100
2600
Ethanol 1% PVP
*
*
AuNPs DHT DHT + AuNPs
%
Fig. 2. Effect of AuNPs on percentage of total counts of LNCaP
cells after 4-day cultivation in RPMI 1640 medium in relation
to initial seeding (M ± m). Footnotes: Each bar represents
an average data from 3 experiments. *р < 0.05 in comparison
with ethanol control
Our preliminary in vitro experiments with various
AuNPs concentrations of 0.1; 1.0 and 10.0 μg/ml had
shown that only 10.0 μg/ml of AuNPs was effective
in affecting LNCaP cells growth. In this study, after
4-day incubation of LNCaP cells in presence of AuNPs
(10.0 μg/ml) we observed significant suppression
of cells’ growth. AuNPs decreased the total cells
count on average by 40% and live cells count by 43%
compared to the control, and both decreases were
statistically significant (p < 0.02).
Supplementing culture medium with DHT stimulated
LNCaP cells growth by increasing total cells count
102 Experimental Oncology 37, 100–104, 2015 (June)
on average by 57% and live cells count by 61% compared
to the control. Addition of AuNPs in the presence of DHT
dramatically reduced stimulatory effect of DHT on LNCaP
cells growth by 80% and 77% when counted by the ac-
cretion of total and live cells, respectively, and brought
total cell counts to control level (p > 0.05).
Cell morphology. Dark violet increments, suppo-
sedly aggregates of nanoparticles, were clearly seen
in cells cultivated with AuNPs. No morphological changes
of live LNCaP cells were seen in any experiment (Fig. 3).
In summary, it must be emphasized that we used
androgen-sensitive LNCaP cell line derived from
the human PCa lymph node metastasis to study the ef-
fects of non-functionalized AuNPs on cells’ growth.
There is some discrepancy in published works con-
cerning cytotoxicity of AuNPs toward eukaryotic cells.
Cytotoxicity has been reported by a few authors [19,
20], meanwhile others did not observe it [21]. Cytoto-
xity of AuNPs both in vitro and in vivo strongly depends
on particles’ size, shape, dose, surface composition
a b
c d
e
Fig. 3. Appearance of DHT stimulated and non-stimulated LNCaP
cell cultures after 4 days growing with AuNPs (a — 96% ethanol;
b — PVP ethanol solution; c — AuNPs (10 μg/ml), visualization
of the nanoparticle aggregates; d — DHT (10–4 M); increased
mitotic activity; e — AuNPs (10 μg/ml) and DHT (10–4 M), visu-
alization of the nanoparticle aggregates). Ferrum hematoxylin,
× 300. Entire arrows indicate mitosis, dot arrows indicate apoptosis
Experimental Oncology 37, 100–104, 2015 (June) 103
and other features [15, 22–24]. Similarly to other
metallic nanoparticles, AuNPs interact with cellular
membrane, mitochondria, nucleus, resulting in dama-
ging DNA and organelles, oxidative stress, apoptosis.
In this study, we explored relatively homogenous
population of spherical AuNPs with average size
of about 26 nm. By being added to the culture medium
at concentration 10.0 μg/ml, these AuNPs inhibited
non-stimulated LNCaP cell line growth significantly. The
ratios of dead cells in the control and AuNPs-treated
cultures relative to the initially seeded cells were minor
and remained quite constant, whereas the relative num-
bers of live cells in the presence of AuNPs decreased
dramatically. Suppression of cell growth by AuNPs
in DHT stimulated culture was quite noticeable. Taken
into consideration that DHT binds to nuclear AR, which
interacts with specific regions of DNA followed by ex-
pression of growth factors and stimulation of mitosis,
it can be assumed that AuNPs interfere with androgen
and/or growth factor signaling in the affected cancerous
cells. Morphology of live cells did not change, and num-
bers of visualized apoptotic cells were similar in control
and experimental groups. From these observations,
we have concluded, that AuNPs suppressed cell prolif-
eration rather than caused apoptosis or necrosis.
AuNPs conglomerates have been visualized
in cultured cells, which is in line with reported data
on the ability of nanoparticles with 30 nm diameter
or less to be endocytosed by cells [15, 20, 25, 26].
It was shown in human histiocyte-rich lymphoma
U937 cell line that 20–30 nm AuNPs have a greater cell
accumulation than that of nanoparticles of other size
with prevalent localization in the lysosomes [11, 20].
3–5 nm AuNPs penetrate nucleus easily. AuNPs capa-
city to damage DNA deserves special attention in terms
of their safety. It was shown that even 10–20 nm AuNPs
can damage DNA structure in СНО-К1 cell line,
meanwhile 30 nm and 45 nm AuNPs were safe [27].
Genotoxity might be a cause of suppressive influence
of AuNPs on LNCaP cell growth in our experiments.
One possible mechanism of AuNPs induced inhibi-
tion of LNCaP cell growth is modulation of the cell mem-
brane enzyme activity. Though gold per se is known
to be inert metal, AuNPs possess high catalytic proper-
ties [5]. For instance, it had been reported their ability
to modulate in vitro the U937 tumor cell membrane Na+,
K+-ATPase activity [20]. Since Na+, K+ -ATPase acti vity
is required for adequate regulation of cell volume,
which undergoes significant disturbances during cell-
cycle progression, one might suggest that impaired
cell volume regulation may in part be responsible
for the anti-proliferative action of AuNPs. However,
biochemical mechanisms underlying effect of AuNPs
on cancerous cells are still poorly investigated and
understood.
There is a hope that in the future AuNPs could earn
a place in the treatment of PCa, however, much work
for this is to be addressed.
ACKNOWLEDGEMENTS
The authors are grateful to Dr. A.V. Usatenko, Di-
rector of the Research Institute of Nanotechnological
Industry, Open International University of Human De-
velopment “Ukraine”, for donating AuNPs for the study.
We would like to thank Dr. O.Yu. Chunikhin (O.V. Pal-
ladin Institute of Biochemistry, National Academy
of Sciences of Ukraine) for his kind help on carrying
out laser spectroscopy of the AuNPs preparation.
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Copyright © Experimental Oncology, 2015
|
| id | nasplib_isofts_kiev_ua-123456789-145463 |
| institution | Digital Library of Periodicals of National Academy of Sciences of Ukraine |
| issn | 1812-9269 |
| language | English |
| last_indexed | 2025-12-07T18:56:52Z |
| publishDate | 2015 |
| publisher | Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
| record_format | dspace |
| spelling | Reznikov, A.G. Salivonyk, O.A. Sotkis, A.G. Shuba, Y.M. 2019-01-22T11:04:12Z 2019-01-22T11:04:12Z 2015 Assessment of gold nanoparticle effect on prostate cancer LNCaP cells / A.G. Reznikov, O.A. Salivonyk, A.G. Sotkis, Y.M. Shuba // Experimental Oncology. — 2015. — Т. 37, № 2. — С. 100-104. — Бібліогр.: 27 назв. — англ. 1812-9269 https://nasplib.isofts.kiev.ua/handle/123456789/145463 In recent years gold nanoparticles (AuNPs) have received considerable attention for various biomedical applications including diagnostics and targeted drug delivery. However, more research is still needed to characterize such aspects of their use in clinical oncology as permeability, retention and functional effect on tumor cells. Aims: This study was designed to describe the effect of non-functionalized AuNPs on LNCaP prostate cancer cells growth. Material and Methods: LNCaP cells were cultured in RPMI-1640 medium containing AuNPs covered by polyvinylpyrrolidone of average size 26.4 nm (10.0 μg/ml). Counts of cells were calculated and their morphology was examined. Results: AuNPs conglomerates have been visualized in cultured cells. After 4-day incubation in presence of AuNPs significant retardation of LNCaP cells growth was observed both in 5α-dihydrotestosterone stimulated and non-stimulated cultures. No morphological changes of live LNCaP cells were seen in any experiment. Conclusion: Given absence of morphological changes in live cells and dribble and relatively constant numbers of dead cells, it was concluded that inhibitory effect of AuNPs on LNCaP cells growth was caused by alterations of proliferation. Key Words: prostate cancer, LNCaP, gold nanoparticles, 5α-dihydrotestosterone. The authors are grateful to Dr. A.V. Usatenko, Director of the Research Institute of Nanotechnological Industry, Open International University of Human Development “Ukraine”, for donating AuNPs for the study. We would like to thank Dr. O.Yu. Chunikhin (O.V. Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine) for his kind help on carrying out laser spectroscopy of the AuNPs preparation. en Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України Experimental Oncology Original contributions Assessment of gold nanoparticle effect on prostate cancer LNCaP cells Article published earlier |
| spellingShingle | Assessment of gold nanoparticle effect on prostate cancer LNCaP cells Reznikov, A.G. Salivonyk, O.A. Sotkis, A.G. Shuba, Y.M. Original contributions |
| title | Assessment of gold nanoparticle effect on prostate cancer LNCaP cells |
| title_full | Assessment of gold nanoparticle effect on prostate cancer LNCaP cells |
| title_fullStr | Assessment of gold nanoparticle effect on prostate cancer LNCaP cells |
| title_full_unstemmed | Assessment of gold nanoparticle effect on prostate cancer LNCaP cells |
| title_short | Assessment of gold nanoparticle effect on prostate cancer LNCaP cells |
| title_sort | assessment of gold nanoparticle effect on prostate cancer lncap cells |
| topic | Original contributions |
| topic_facet | Original contributions |
| url | https://nasplib.isofts.kiev.ua/handle/123456789/145463 |
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