Expression of human beta-defensins-1–4 in thyroid cancer cells and new insight on biologic activity of hBD-2 in vitro
The study was aimed on analysis of human beta-defensin-1–4 (hBDs) mRNA expression in cultured thyroid cancer cells and evaluation of effects of recombinant hBD-2 (rec-hBD-2) on growth patterns, migration properties and expression of E-cadherin and vimentin in these cells. Methods: The study was perf...
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| Zitieren: | Expression of human beta-defensins-1–4 in thyroid cancer cells and new insight on biologic activity of hBD-2 in vitro / O.V. Zhuravel, O.L. Gerashchenko, M.R. Khetsuriani, M.A. Soldatkina, P.V. Pogrebnoy // Experimental Oncology. — 2014. — Т. 36, № 3. — С. 174-178. — Бібліогр.: 22 назв. — англ. |
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Zhuravel, O.V. Gerashchenko, O.L. Khetsuriani, M.R. Soldatkina, M.A. Pogrebnoy, P.V. 2019-01-20T20:53:56Z 2019-01-20T20:53:56Z 2014 Expression of human beta-defensins-1–4 in thyroid cancer cells and new insight on biologic activity of hBD-2 in vitro / O.V. Zhuravel, O.L. Gerashchenko, M.R. Khetsuriani, M.A. Soldatkina, P.V. Pogrebnoy // Experimental Oncology. — 2014. — Т. 36, № 3. — С. 174-178. — Бібліогр.: 22 назв. — англ. 1812-9269 https://nasplib.isofts.kiev.ua/handle/123456789/145362 The study was aimed on analysis of human beta-defensin-1–4 (hBDs) mRNA expression in cultured thyroid cancer cells and evaluation of effects of recombinant hBD-2 (rec-hBD-2) on growth patterns, migration properties and expression of E-cadherin and vimentin in these cells. Methods: The study was performed on cultured follicular thyroid cancer WRO cells, papillary thyroid cancer TPC1 cells, and anaplastic thyroid cancer KTC-2 cells. For analysis of hBD-1–4 mRNA expression in thyroid cancer cells, semiquantitative RT-PCR was used. Effects of rec-hBD-2 on cell proliferation, viability, and migration were analyzed using direct cell counting, MTT test, and scratch assay respectively. Expression of vimentin and E-cadherin was evaluated by quantitative PCR (qPCR). Results: By the data of RT-PCR, all three studied thyroid cancer cell lines express hBD-1 and -4 mRNA, but not hBD-2 mRNA, while hBD-3 expression was detected in WRO and KTC-2 cells. The treatment of TPC-1, WRO, and KTC-2 cells with 100–1000 nM rec-hBD-2 resulted in significant concentration-dependent suppression of cell proliferation, viability, and migratory property. By the data of qPCR, significant up-regulation of vimentin expression was registered in KTC-2 and WRO cells treated with 500 nM rec-hBD-2. Significant down-regulation of E-cadherin expression (p < 0.05) was detected only in KTC-2 cells treated with the defensin. Also, it has been shown that TPC-1 cells treated with 500 nM rec-hBD-2 acquired more elongated morphology. Conclusion: The data demonstrate that hBD-2 in concentrations higher than 100 nM exerts significant concentration-dependent suppression of thyroid cancer cell growth and migration, and affects vimentin and E-cadherin expression dependent on histologic type of thyroid cancer cells. Key Words: thyroid cancer, human beta-defensin-2, E-cadherin, vimentin, proliferation, viability. This work was in part supported with grant 0110U005758 of National Academy of Sciences of Ukraine “Fundamental Basis of Molecular and Cellular Biotechnologies”. en Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України Experimental Oncology Original contributions Expression of human beta-defensins-1–4 in thyroid cancer cells and new insight on biologic activity of hBD-2 in vitro Article published earlier |
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Expression of human beta-defensins-1–4 in thyroid cancer cells and new insight on biologic activity of hBD-2 in vitro |
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Expression of human beta-defensins-1–4 in thyroid cancer cells and new insight on biologic activity of hBD-2 in vitro Zhuravel, O.V. Gerashchenko, O.L. Khetsuriani, M.R. Soldatkina, M.A. Pogrebnoy, P.V. Original contributions |
| title_short |
Expression of human beta-defensins-1–4 in thyroid cancer cells and new insight on biologic activity of hBD-2 in vitro |
| title_full |
Expression of human beta-defensins-1–4 in thyroid cancer cells and new insight on biologic activity of hBD-2 in vitro |
| title_fullStr |
Expression of human beta-defensins-1–4 in thyroid cancer cells and new insight on biologic activity of hBD-2 in vitro |
| title_full_unstemmed |
Expression of human beta-defensins-1–4 in thyroid cancer cells and new insight on biologic activity of hBD-2 in vitro |
| title_sort |
expression of human beta-defensins-1–4 in thyroid cancer cells and new insight on biologic activity of hbd-2 in vitro |
| author |
Zhuravel, O.V. Gerashchenko, O.L. Khetsuriani, M.R. Soldatkina, M.A. Pogrebnoy, P.V. |
| author_facet |
Zhuravel, O.V. Gerashchenko, O.L. Khetsuriani, M.R. Soldatkina, M.A. Pogrebnoy, P.V. |
| topic |
Original contributions |
| topic_facet |
Original contributions |
| publishDate |
2014 |
| language |
English |
| container_title |
Experimental Oncology |
| publisher |
Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
| format |
Article |
| description |
The study was aimed on analysis of human beta-defensin-1–4 (hBDs) mRNA expression in cultured thyroid cancer cells and evaluation of effects of recombinant hBD-2 (rec-hBD-2) on growth patterns, migration properties and expression of E-cadherin and vimentin in these cells. Methods: The study was performed on cultured follicular thyroid cancer WRO cells, papillary thyroid cancer TPC1 cells, and anaplastic thyroid cancer KTC-2 cells. For analysis of hBD-1–4 mRNA expression in thyroid cancer cells, semiquantitative RT-PCR was used. Effects of rec-hBD-2 on cell proliferation, viability, and migration were analyzed using direct cell counting, MTT test, and scratch assay respectively. Expression of vimentin and E-cadherin was evaluated by quantitative PCR (qPCR). Results: By the data of RT-PCR, all three studied thyroid cancer cell lines express hBD-1 and -4 mRNA, but not hBD-2 mRNA, while hBD-3 expression was detected in WRO and KTC-2 cells. The treatment of TPC-1, WRO, and KTC-2 cells with 100–1000 nM rec-hBD-2 resulted in significant concentration-dependent suppression of cell proliferation, viability, and migratory property. By the data of qPCR, significant up-regulation of vimentin expression was registered in KTC-2 and WRO cells treated with 500 nM rec-hBD-2. Significant down-regulation of E-cadherin expression (p < 0.05) was detected only in KTC-2 cells treated with the defensin. Also, it has been shown that TPC-1 cells treated with 500 nM rec-hBD-2 acquired more elongated morphology. Conclusion: The data demonstrate that hBD-2 in concentrations higher than 100 nM exerts significant concentration-dependent suppression of thyroid cancer cell growth and migration, and affects vimentin and E-cadherin expression dependent on histologic type of thyroid cancer cells. Key Words: thyroid cancer, human beta-defensin-2, E-cadherin, vimentin, proliferation, viability.
|
| issn |
1812-9269 |
| url |
https://nasplib.isofts.kiev.ua/handle/123456789/145362 |
| citation_txt |
Expression of human beta-defensins-1–4 in thyroid cancer cells and new insight on biologic activity of hBD-2 in vitro / O.V. Zhuravel, O.L. Gerashchenko, M.R. Khetsuriani, M.A. Soldatkina, P.V. Pogrebnoy // Experimental Oncology. — 2014. — Т. 36, № 3. — С. 174-178. — Бібліогр.: 22 назв. — англ. |
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| fulltext |
174 Experimental Oncology 36, 174–178, 2014 (September)
EXPRESSION OF HUMAN BETA-DEFENSINS-1‒4 IN THYROID
CANCER CELLS AND NEW INSIGHT ON BIOLOGIC ACTIVITY
OF hBD-2 IN VITRO
O.V. Zhuravel, O.L. Gerashchenko, M.R. Khetsuriani, M.A. Soldatkina, P.V. Pogrebnoy*
R.E. Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology
of NAS of Ukraine, Kyiv 03022, Ukraine
The study was aimed on analysis of human beta-defensin-1–4 (hBDs) mRNA expression in cultured thyroid cancer cells and evaluation
of effects of recombinant hBD-2 (rec-hBD-2) on growth patterns, migration properties and expression of E-cadherin and vimentin in these
cells. Methods: The study was performed on cultured follicular thyroid cancer WRO cells, papillary thyroid cancer TPC1 cells, and ana-
plastic thyroid cancer KTC-2 cells. For analysis of hBD-1–4 mRNA expression in thyroid cancer cells, semiquantitative RT-PCR was
used. Effects of rec-hBD-2 on cell proliferation, viability, and migration were analyzed using direct cell counting, MTT test, and scratch
assay respectively. Expression of vimentin and E-cadherin was evaluated by quantitative PCR (qPCR). Results: By the data of RT-PCR,
all three studied thyroid cancer cell lines express hBD-1 and -4 mRNA, but not hBD-2 mRNA, while hBD-3 expression was detected
in WRO and KTC-2 cells. The treatment of TPC-1, WRO, and KTC-2 cells with 100–1000 nM rec-hBD-2 resulted in significant
concentration-dependent suppression of cell proliferation, viability, and migratory property. By the data of qPCR, significant up-regu-
lation of vimentin expression was registered in KTC-2 and WRO cells treated with 500 nM rec-hBD-2. Significant down-regulation
of E-cadherin expression (p < 0.05) was detected only in KTC-2 cells treated with the defensin. Also, it has been shown that TPC-1 cells
treated with 500 nM rec-hBD-2 acquired more elongated morphology. Conclusion: The data demonstrate that hBD-2 in concentrations
higher than 100 nM exerts significant concentration-dependent suppression of thyroid cancer cell growth and migration, and affects vi-
mentin and E-cadherin expression dependent on histologic type of thyroid cancer cells.
Key Words: thyroid cancer, human beta-defensin-2, E-cadherin, vimentin, proliferation, viability.
The incidence of thyroid cancer in Ukraine is con-
stantly rising and in 2012 it comprises 1.8% of all
cancers [1]. Thyroid carcinoma is represented by five
major histological types — papillary carcinoma (more
than 80% cases), follicular, medullary, poorly dif-
ferentiated, and anaplastic carcinoma. These types
of thyroid cancer have different prognosis, the course
of the disease and require different treatment.
At the same time, fundamental aspects of thyroid
tumorige nesis share a number of common underlying
molecular mechanisms, among which the process
of epithelial-mesenchymal transition (EMT) has been
recognized recently among important events which
determine metastatic progression [2, 3]. Thyroid
cancer cells are often used as model for investigation
of EMT mechanisms and elements of EMT machinery.
EMT and reverse event — mesenchymal-epithelial
transition (MET) represent recently discovered funda-
mental processes of epithelial cell plasticity control.
During EMT epithelial cells acquire the special proper-
ties of mesenchymal cells: cell morphology is changed
to a spindle shaped mesenchymal type, migratory
and invasive properties of the cells are enhanced, and
expression of some markers (in particular, vimentin
and E-cadherin) is altered. During EMT, expression
of E-cadherin, a cell-to-cell adhesion molecule,
is down-regulated, while expression of structural
protein vimentin is up-regulated.
It is accepted that EMT could occur in three diffe-
rent biologic settings — embryogenesis/organoge-
nesis (type 1 EMT), tissue regeneration /wound repair
(type 2 EMT), and cancer progression/metastasis
(type 3 EMT) [4, 5]. It is supposed that all mentioned
types of EMT represent different biologic processes
but may have a lot in common in regard to underly-
ing molecular events and elements. EMT machinery
is under close investigation at present time due to its
crucial role in tumor metastasis.
In the present study we have investigated a possible
effect of human beta-defensin-2 (hBD-2) on expres-
sion of special EMT markers in thyroid cancer cells.
hBD-2 is an inducible cationic peptide, a member
of hBD family of antimicrobial peptides, involved in an-
timicrobial protection of human body and in wound he-
aling processes [6–9]. In a number of studies it has been
demonstrated that some hBDs, especially hBD-2, play
an important role in wound repair affecting keratinocyte
migration and proliferation and accelerating wound he-
aling. Hypothetically, these antimicrobials could be in-
volved in II type EMT, however, this hypothesis has not
been analyzed yet. From the other hand, in in vitro set-
ting it has been shown that hBD-2 and hBD-4 may regu-
late the growth of cultured human cancer cells via cell
cycle control and affect their migratory properties and
malignant potential in a concentration-dependent man-
ner [10–12]. Also, deregulation of expression of some
defensins in human tumors of different histogenesis
has been shown, and possible implication of these
antimicrobials in tumor development has been stated
Received: May 14, 2014.
*Correspondence: E-mail: pogrebnoy@onconet.kiev.ua
Abbreviations used: EMT — epithelial-mesenchymal transition;
hBD — human beta-defensin; MET — mesenchymal-epithelial transition;
qPCR — quantitative PCR; rec-hBD-2 — recombinant human beta-
defensin-2; RT-PCR — reverse transcription polymerase chain reaction.
Exp Oncol 2014
36, 3, 174–178
Experimental Oncology 36, 174–178, 2014 (September) 175
[13, 14]. Moreover, the latest studies demonstrated
an association between expression of hBD-1 and in oral
squamous cell carcinoma and migratory and invasive
properties of OSCC cells, and suggested that hBD-1 ex-
pression in OSCC may be considered as prognostic
marker in this type of cancer [15]. However, in regard
to thyroid cancer, no data on expression and biological
effects of hBDs have been reported so far.
Therefore, in the present study we have analyzed
the profile of hBD-1–4 mRNA expression in three hu-
man thyroid cancer cell lines originating from papillary
carcinoma (TPC-1 cell line), follicular carcinoma (WRO
cell line), and anaplastic cancer (KTC-2 cell line). Also,
we have studied an influence of recombinant hBD-2 (rec-
hBD-2) on proliferation and viability of these cells, their
morphology and migratory properties, and expression
of important EMT markers — E-cadherin and vimentin.
Taken together, our data demonstrate that hBD-2 not
only controls the growth and migratory pro perties of hu-
man thyroid cancer cells but also significantly affects
expression levels of EMT markers and alters cell mor-
phology toward spindle-shaped phenotype.
MATERIALS AND METHODS
Cell lines. In the study, follicular thyroid cancer
WRO cell line, papillary thyroid cancer TPC1 cell line,
and anaplastic thyroid cancer KTC-2 cell line were
used. The cells were cultured in DMEM culture medium
supplemented with 10% fetal bovine serum (FBS),
100 units/mL penicillin G sodium, 100 μg/mL strepto-
mycin sulfate in humidified 5% CO2 atmosphere at 37 °C.
Preparation of rec-hBD-2. To study the effect
of exogenous defensin on cell growth, we used puri-
fied rec-hBD-2 [16]. In brief, E.coli BL21(DE3) cells
transformed with GST-hBD-2-recombinant plasmid
were induced with 1 mM IPTG for 6 h, pelleted by cen-
trifugation, resuspended in lysis buffer (50 mМ Tris-HCl,
pH 7.6; 250 mМ NaCl; 1% Triton Х-100 and cocktail
of protease and phosphatase inhibitors), and disrupted
using ultrasound disintegrator (UD-11 Automatic, Po-
land). Then cell lysate was applied to affinity chromatog-
raphy on glutathione-agarose co lumn (GE Healthcare,
Sweden) with following cleavage of the defensin from
fusion protein by thrombin digestion. hBD-2 peptide
was further purified by reverse phase chromatography
on Sep-Pack C18 cartridge (Waters, USA), vacuum-
dried, and re-dissolved in water. Protein concentration
was determined by UV absorbance at 280 nm using
spectrophotometer Nanodrop-1000 (USA).
Direct cell counting. To study the effect of rec-
hBD-2 on cell proliferation, KTC-2, TPC-1, and WRO
cells were routinely cultured in 24-well plates (5•104 cells
per well) to nearly 50% confluence, then culture medium
was replaced with fresh DMEM supplemented with 2.5%
FBS. Rec-hBD-2 was added into the medium in con-
centrations of 0.1; 1; 10; 100; 500; 1000 nM, followed
by culturing for 48 h. After the treatment, cells were
washed with PBS, detached with trypsin, and counted
in hemocytometer. The percentage of dead cells was
analyzed using trypan blue staining.
MTT assay. To evaluate the effect of rec-hBD-2 on cell
viability, MTT-test has been applied [17]. Thyroid cancer
cells were seeded into 96-well plates (7•103 cells per
well) and incubated with rec-hBD-2 at the concentration
range from 100 pM to 1 μM in DMEM supplemented with
2.5% FBS for 48 h. Then cells were routinely treated with
МТТ (3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetra-
zolium bromide) according to standard protocol, and
colorimetric reaction was evaluated with the use of ELISA
reader (Awareness Technology Inc, USA) at = 545.
Scratch wound healing assay. An effect of rec-
hBD-2 on mobility of cultured human thyroid can-
cer cells was performed by scratch wound healing
assay [18]. The cells were seeded in 6-well plates
(1.5•105 cells per well) and routinely grown till 80%
confluence, than a scratch was performed using a tip,
and the cells were grown for next 24 h in the presence
of 500 or 1000 nM rec-hBD-2 in serum free medium.
Then the cell migration into wound area was examined
using inverted microscope Axioplan (Zeiss, Germany).
RT-PCR analysis of hBD-1–4 mRNA expres-
sion in thyroid cancer cells. To evaluate the hBD-
1–4 mRNA expression in thyroid cancer cells, total RNA
was extracted from KTC-2, WRO and TPC-1 cell lysates
using Trizol RNA Isolation Kit (Neogene, Russian Fed-
eration) according to manufacturer’s protocol. For
detection of hBD-1–4 mRNA expression, semiquan-
titative RT-PCR analysis was performed using specific
primers (Table). The expression level of beta-actin (the
house-keeping gene) served as a loading control. The
products of RT-PCR were routinely analyzed by elec-
trophoresis in agarose gel.
Table. Primers for the genes of interest
Gene Primers
DEFB1 (hBD-1) F: TGTTGCCTGCCAGTCGCCATGAG
R: TCACTTGCAGCACTTGGCCTTCCC
DEFB4 (hBD-2) F: 5´-GAAGCTCCCAGCCATCAGCC
R: 5´-GTCGCACGTCTCTGATGAGGGA
DEFB103 (hBD-3) F: CCTGTTTTTGGTGCCTGTTCC
R: CTTTCTTCGGCAGCATTTTCG
DEFB104 (hBD-4) F: GAAGCTCCCAGCCATCAGCC
R: GTCGCACGTCTCTGATGAGGGA
Beta-actin F: CTGGAACGGTGAAGGTGACA
R: AAGGGACTTCTTGTAACAATGCA
Quantitative PCR analysis of vimentin and
E-cadherin expression in thyroid cancer cells. Ex-
pression of vimentin and E-cadherin in KTC-2, WRO and
TPC-1 cells treated with 500 nM rec-hBD-2 for 24 h, was
analyzed using qPCR. Total RNA was extracted from cell
lysates using Trizol RNA Isolation Kit (Neogene, Rus-
sian Federation) according to manufacturer’s protocol.
2 g of RNA were reverse transcribed using an M-MLV
Reverse Transcriptase, Ribonuclease Inhibitor Ribolock
(Thermo Scientific Inc., USA) and oligo(dT)18 primer.
Expression levels of vimentin and E-cadherin genes
were analyzed by qPCR on ABI 7500 Fast real-time PCR
system (Applied Biosystems, USA) using 0.2 μM of each
primer and SYBR Green master mix (Thermo Scientific
Inc., USA) and 1 l cDNA. The following conditions
of reaction were used: 10 min 95 °C initial denaturation;
40 cycles15 s 95 °C denatu ration, 60 s 60 °C primer
annealing/elongation. The fluorescence was recorded
176 Experimental Oncology 36, 174–178, 2014 (September)
during the annealing/elongation step in each cycle.
A melting curve analysis was performed at the end
of each PCR by gradually increasing the temperature
from 60 to 95 °C while recording the fluorescence. A sin-
gle peak at the melting temperature of the PCR-product
confirmed primer specificity. The β2-microglobulin
was used as a reference gene to standar dize the level
of other transcripts. Following primers were used for
vimentin: F — 5´-GTGAATACCAAGACCTGCTCAA-3´,
R — 5`-AGGGAGGAAAAGTTTGGAAGAG-3´; for
E-cadherin: F — 5´- CTGGTTCAGATCAAATCCAACA-3´,
R — 5´-CTTCAGCCATCCTGTTTCTCTT-3´ and
β2-microglobulin: F — 5´-CCGTGTGAACCATGT-
GACTTTGTC-3´, R — 5´-TGCGGCATCTTCAAACCTC-
CATGATG-3´. The relative amounts of the transcripts
were first normalized to the reference gene and then
normalized to the gene expression level in the untreated
samples according to the 2−ΔΔCt method [19], statistical
analysis was performed using statistical application
of Microsoft Excel software based on t-test with p <
0.05 repre senting significance.
Statistical analysis. Data on direct cell counting and
MTT data were reported as the mean ± SD of values ob-
tained from three independent experiments and analyzed
by Student’s t-test to assess the statistical significance
of the differences between the groups. A statistically
significant difference was considered at p < 0.05.
RESULTS AND DISCUSSION
Expression of hBD-1–4 mRNA in human thy-
roid cancer cells. Semiquantitative RT-PCR analysis
of expression of hBD-1–4 mRNA in human papillary
carcinoma TPC-1 cells, follicular carcinoma WRO cells,
and anaplastic carcinoma KTC-2 cells has revealed
the presence of mRNA for hBD-1 and -4 and no detec-
table hBD-2 expression in all cell lines. hBD-3 expres-
sion was registered in 2 from 3 cell lines (WRO and
KTC-2 cells). WRO cells were characterized by high
expression of hBD-3 while TPC1 — by high expression
of hBD-4 (Fig. 1). So, thyroid cancer cells of different
histological type express beta-defensins-1,-3 and -4,
but not hBD-2. To our knowledge, it is the first report
on hBDs expression in thyroid cancer cells. Up-to-
date, just a single study reported on low-level expres-
sion of hBD-4 mRNA in normal thyroid gland [20].
Effect of rec-hBD-2 on proliferation and viabi lity
of human thyroid cancer cells in vitro. We have analyzed
an effect of rec-hBD-2 on proliferation and viability of TPC-1,
WRO, and KTC-2 cells. As it has been shown using direct
cell counting technique (Fig. 2), in concentrations higher
than 100 nM rec-hBD-2 exerted concentration-dependent
growth suppression of the thyroid cancer cells, especially
KTC-2 cells, and in concentrations lower than 100 nM exerts
no significant influence on cell counts (see Fig. 2).
As it has been shown by MTT assay, viability
of cultured TPC-1, WRO, and KTC-2 cells is affected
by rec-hBD-2 in a similar concentration-dependent
manner (Fig. 3). Our data have shown that in con-
centrations lower than 100 nM rec-hBD-2 had no sig-
nificant effect on viability of TPC-1 and WRO cells and
stimulated viabi lity of KTC-2 cells at 100 pM concen-
tration (see Fig. 3). In all three cell lines significant
suppression of cell viability in the presence of rec-
hBD-2 in concentrations higher than 100 nM has been
registered. No viable cells have been revealed after
treatment of the cells with 1 μM rec-hBD-2.
1
hBD-1
hBD-3
hBD-4
β-actin
2 3
Fig. 1. RT-PCR analysis of hBDs mRNA expression in thyroid
cancer cells: 1 — WRO; 2 — KTC-2; 3 — TPC1. β-actin is used
as a house-keeping gene
0
50
100
150
200
250
300
350
Control 0,1 1 10 100 1000
hBD-2 concentration, nM
Ce
ll
nu
m
be
r,%
TPC-1
KTC-2
WRO
* * *
*
Fig. 2. A concentrat ion-dependent ef fect of rec-
hBD-2 on the number of viable cultured cells of TPC-1, KTC-2,
and WRO lines. The number of attached cells was evaluated by di-
rect cell co unting. The data of three independent experiments
are presented as the mean ± SD. *The difference is significant
as compared to appropriate control (p < 0.05)
0
0,05
0,1
0,15
0,2
0,25
0,3
Control 0,1 1 10 100 500 1000
hBD-2 concentration, nM
Ab
so
rb
an
ce
, A
U
KTC2
WRO
TPC1
*
*
**
*
*
Fig. 3. A concentration-dependent effect of rec-hBD-2 on vi-
ability of TPC-1, WRO, and KTC-2 cells (MTT analysis). The data
of three independent experiments are presented as the mean
± SD.*p < 0.05
The data on the influence of rec-hBD-2 on prolif-
eration and viability of thyroid cancer cells are in ac-
cordance with the reported earlier results on a concen-
tration-dependent growth suppressive effects of this
defensin toward cancer cells of other histological
types (such as carcinoma cells of A431 and A549 lines,
cultured human melanoma cells [10, 12]). As it was
reported, growth suppression caused by hBD-2 in vi-
Experimental Oncology 36, 174–178, 2014 (September) 177
tro is exerted via cell cycle arrest at G1/S checkpoint,
down-regulation of cyclin D1 expression and activa-
tion of pRB [10, 12]. In the present research we did
not study cell cycle distribution of thyroid cancer cells
treated with rec-hBD-2; we suppose that the mecha-
nism of hBD-2 dependent growth suppression in thy-
roid cancer cells may be similarly exerted via blockage
of cell cycle. Here, we have focused our attention
on other effects of rec-hBD-2 in growth-inhibitory
concentrations toward cultured thyroid cancer cells,
in particular, its effects on morphology and migration
ability of these cells, and on expression of common
EMT markers — vimentin and E-cadherin.
Effect of rec-hBD-2 on thyroid cancer cell
migration and morphology. To analyze a possible
effect of rec-hBD-2 on cancer cell migration, wound
healing assay has been applied. It was revealed that
100 nM and 500 nM of rec-hBD-2 significantly sup-
pressed migration of TPC-1 cells (Fig. 4) as well as WRO
and KTC-2 (data not shown). Also, we have noted
an altered morphology of TPC-1 cells after 48 h treat-
ment with 500 nM or 1000 nM rec-hBD-2 (Fig. 5).
TPC-1 cells treated with rec-hBD-2 acquire more elon-
gated fibroblast-like shape compared to the untreated
control cells. No notable morphological changes in WRO
and KTC-2 cells treated with 500 nM or 1000 nM rec-
hBD-2 were observed (data not shown).
From the reports of other authors it is known that
some human defensins may affect cancer cell migra-
tion, in particular, hBD-1 suppresses migration and
invasion of oral squamous cell carcinoma cells [15],
hBD-3 suppresses head and neck cancer cell migra-
tion [21], hBD-4 may stimulate or inhibit migration
of A431 and TPC-1 cells in scratch assay dependent
on its concentration [11]. However, little is known yet
about possible impact of defensins as peptides affect-
ing cell migratory capability in vivo as well as the mech-
anisms of defensin-dependent suppression of cell mi-
gratory properties. In our study, we detected not only
hBD-2 dependent suppression of thyroid cancer cell
migration, but also alterations in TPC-1 cell morphol-
ogy toward more elongated mesenchymal phenotype.
Therefore, in the last part of our study we have studied
the effects of rec-hBD-2 on expression levels of EMT
markers in thyroid cancer cells in vitro.
Expression of vimentin and E-cadherin in thyroid
cancer cells treated with rec-hBD-2. The study of ex-
pression of two common EMT markers — vimentin and
E-cadherin — has been carried out using qPCR analysis
in TPC-1, KTC-2 and WRO cells treated with 500 nM rec-
hBD-2 for 24 h. It has been found out that in KTC-2 cells
treated with the defensin, expression level of vimentin
was significantly higher, and E-cadherin — notably
lower than in untreated control cells by 3.39 ± 0.19 and
0.59 ± 0.034 times, respectively (p < 0.05) (Fig. 6).
In WRO cells, treatment with defensin led to significant
up-regulation of vimentin expression by 3.07 ± 0.13 times,
and had a tendency for down-regulation of E-cadherin
expression level (Fig. 6). In TPC-1 cells vimentin level was
unaffected by rec-hBD-2 treatment while E-cadherin
expression is undetectable in this cell line (Fig. 6).
To our knowledge, here we present the first evi-
dence on effects of hBD-2 on vimentin and E-cadherin
expression in human cancer cells. Up-to-date, a recent
study has reported on effects of synthetic human
alpha-defensin-5 (HD-5), antimicrobial peptide pro-
duced by the Paneth cells on expression of E-cadherin
in squamous cell line Het-1A in vitro [22]. Using immu-
nocytochemistry, flow cytometry, and Western blotting,
the authors showed that the E-cadherin expression was
down-regulated in HD-5 treated cells and suppose that
HD-5 produced by metaplastic Paneth cells may be in-
volved in the development of Barrett’s esophagus due
to its ability to decrease E-cadherin expression [22].
In conclusion, the data of present research de-
monstrate that hBD-2 in concentrations higher than
a b c
Fig. 4. Wound healing assay performed in TPC-1 cells treated with 100 nM (b) or 500 nM (c) rec-hBD-2 for 48 h. a — control un-
treated cells. The results of a typical experiment are presented. Magnification ×400
a b c
Fig. 5. Morphology of TPC1 cells treated with 500 nM rec-hBD2 (b) or 1000 nM rec-hBD2 (c) for 48 h. a — control cells. Magnification ×400
178 Experimental Oncology 36, 174–178, 2014 (September)
100 nM exerts an inhibitory effect on cell growth and
migration of three thyroid cancer cell lines. At the same
time effects of hBD-2 on expression of vimentin and
E-cadherin strongly depended on thyroid cancer cell
type. The most prominent up-regulation of vimentin
expression and down-regulation of E-cadherin expres-
sion were observed in anaplastic thyroid cancer cells,
and at lesser extent in follicular thyroid cancer cells
while in papillary thyroid cancer cells vimentin expres-
sion seems to be unaffected by hBD-2 treatment, and
acquired elongated morphology. Taken together, these
data point on effects of hBD-2 toward some important
EMT markers and gain new insight on the biological
activity of this defensin toward cancer cells.
ACKNOWLEDGEMENT
This work was in part supported with grant
0110U005758 of National Academy of Sciences
of Ukraine “Fundamental Basis of Molecular and Cel-
lular Biotechnologies”.
REFERENCES
1. Cancer in Ukraine. Bull Nat Cancer Register of Ukraine.
ZP Fedorenko et al., eds. Kyiv 2013: 62–3.
2. Hombach-Klonisch S, Natarajan S, Thanasupawat T,
et al. Mechanisms of therapeutic resistance in cancer (stem)
cells with emphasis on thyroid cancer cells. Front Endocrinol
(Lausanne) 2014; 5: 37.
3. Hardin H, Montemayor C, Lloyd RV. Thyrod cancer
stem-like cells and epithelial-mesenchymal transition in thy-
roid cancers. Hum Pathol 2013; 44: 1707–13.
4. Kalluri R, Weinberg RA. The basics of epithelial-
mesenchymal transition. J Clin Invest 2009; 119: 1420–28.
5. Talbot LJ, Bhattacharya SD, Kuo PC. Epithelial-
mesenchymal transition, the tumor microenvironment, and
metastatic behavior of epithelial malignancies. Int J Mol Biol
2012; 3: 117–36.
6. Niyonsaba F, Ushio H, Nakano N, et al. Antimicrobial
peptides human beta-defensins stimulate epidermal keratino-
cyte migration, proliferation and production of proinflam-
matory cytokines and chemokines. J Invest Dermatol 2007;
127: 594–604.
7. Dressel S, Harder J, Cordes J, et al. Differential expres-
sion of antimicrobial peptides in margins of chronic wounds.
Exp Dermatol 2010; 19: 628–32.
8. Hirsch T, Spielmann M, Zuhaili B, et al. Human
beta-defensin-3 promotes wound healing in infected diabetic
wounds. Gene Med 2009; 11: 220–8.
9. Kesting MR, Stoeckelhuber M, Hölzle F, et al. Expres-
sion of antimicrobial peptides in cutaneous infections after skin
surgery. Br J Dermatol 2010; 163: 121–7.
10. Zhuravel E, Shestakova T, Efanova O, et al. Human
beta-defensin-2 controls cell cycle in malignant epithelial
cells: in vitro study. Exp Oncol 2011; 33: 114–21.
11. Gerashchenko OL, Zhuravel EV, Skachkova OV, et al.
Biologic activities of recombinant human-beta-defensin-4 to-
ward cultured human cancer cells. Exp Oncol 2013; 35: 84–91.
12. Gerashchenko O, Zhuravel E, Skachkova O, et al.
Involvement of human beta-defensin-2 in regulation of malig-
nant potential of cultured human melanoma cells. Exp Oncol
2014; 36: 17–23.
13. Droin N, Hendrab J-B, Ducoroyb P, et al. Human
defensins as cancer biomarkers and antitumour molecules.
J Proteomics 2009; 72: 918–27.
14. Prado-Montes de Oca E. Human beta-defen-
sin 1: a restless warrior against allergies, infections and cancer.
Int J Biochem Cell Biol 2010; 42: 800–4.
15. Han Q, Wang R, Sun C, et al. Human beta-defen-
sin-1 suppresses tumor migration and invasion and is an inde-
pendent predictor for survival of oral squamous cell carcinoma
patients. PLoS One 2014; 9: e91867.
16. Lisovskiy IL, Markeeva NV, Shnitsar VM, et al.
Produсtion of recombinant of hBD-2 — human antimicrobial
peptide expressed in cervical and vulval cancer. Exp Oncol
2003; 25: 36–9.
17. Mosman T. Rapid colorimetric assay for cellular growth
and survival: application to proliferation and cytotoxicity as-
says. Immunol Methods 1983; 65: 55–63.
18. Lampugnani MG. Cell migration into a wounded area
in vitro. Methods Mol Biol 1999; 96: 177–82.
19. Pfaffl MW. A new mathematical model for relative
quantification in real-time RT–PCR. Nucl Acids Res Volume
2001; 29: e45.
20. García JR, Krause A, Schulz S, et al. Human beta-de-
fensin 4: a novel inducible peptide with a specific salt-sensitive
spectrum of antimicrobial activity. FASEB J 2001; 15: 1819–21.
21. Wang K, Wang JH, Baskaran H, et al. Effect of human
beta-defensin-3 on head and neck cancer cell migration using
micro-fabricated cell islands. Head Neck Oncol 2012; 4: 41.
22. Nomura Y, Tanabe H, Moriichi K, et al. Reduction
of E-cadherin by human defensin-5 in esophageal squamous
cells. Biochem Biophys Res Commun 2013; 439: 71–7.
Copyright © Experimental Oncology, 2014
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Fig. 6. Relative expression of vimentin and E-cadherin mRNA in KTC-2, WRO, TPC-1 cells treated with 500 nM rec-hBD-2 for 24 h (2)
(qPCR data) compared to untreated cells (1). The β2-microglobulin was used as a reference gene to standardize the level of other
transcripts. The relative amounts of the transcripts were first normalized to the reference gene and then normalized to the gene
expression level in the un-treated samples according to the 2−ΔΔCt method
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