Human beta-defensin-2 controlS cell cycle in malignant epithelial cells: in vitro study
Aim: In the present research we analyze the mechanism of human beta-defensin-2 (hBD-2) influence on cultured malignant epithelial cell growth. Materials and Methods: The analysis of a concentration-dependent effect of recombinant hBD-2 (rec-hBD-2) on cell growth patterns and cell cycle distribution...
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| Cite this: | Human beta-defensin-2 controlS cell cycle in malignant epithelial cells: in vitro study / E. Zhuravel, T. Shestakova, O. Efanova, Yu. Yusefovich, D. Lytvin, M. Soldatkina, P. Pogrebnoy // Experimental Oncology. — 2011. — Т. 33, № 3. — С. 114-120. — Бібліогр.: 29 назв. — англ. |
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Zhuravel, E. Shestakova, T. Efanova, O. Yusefovich, Yu. Kytvin, D. Soldatkina, M. Pogrebnoy, P. 2018-06-19T11:14:50Z 2018-06-19T11:14:50Z 2011 Human beta-defensin-2 controlS cell cycle in malignant epithelial cells: in vitro study / E. Zhuravel, T. Shestakova, O. Efanova, Yu. Yusefovich, D. Lytvin, M. Soldatkina, P. Pogrebnoy // Experimental Oncology. — 2011. — Т. 33, № 3. — С. 114-120. — Бібліогр.: 29 назв. — англ. 1812-9269 https://nasplib.isofts.kiev.ua/handle/123456789/138664 Aim: In the present research we analyze the mechanism of human beta-defensin-2 (hBD-2) influence on cultured malignant epithelial cell growth. Materials and Methods: The analysis of a concentration-dependent effect of recombinant hBD-2 (rec-hBD-2) on cell growth patterns and cell cycle distribution has been performed in vitro with 2 cell lines (human lung adenocarcinoma A549 cells and human epidermoid carcinoma A431 cells) using MTT test, flow cytometry and direct cell counting. To study intracellular localization of hBD-2 immunocytofluorescent and immunocytochemical analyses were applied, and effect of hBD-2 on signal cascades involved in cell cycle regulation has been studied by Western blotting. Results: According to our data, rec-hBD-2 exerts a concentration-dependent effect on the viability of cultured A549 and A431 cells. It causes proproliferative effect at concentrations below 1 nM, significant suppression of cell proliferation at concentration range from 10 nM to 1 µM (p<0.05), and cell death at higher concentrations. Using flow cytometry we have demonstrated that hBD-2 dependent growth suppression is realized via cell cycle arrest at G1/S phase (p<0.05). Also, we have registered significant activation of pRB and decreased expression of Cyclin D1 in cells treated with the defensin compared to untreated control cells, while the expression of p53 remains unaffected. The study of intracellular localization of hBD-2 in these cells has revealed that exogeneously added defensin molecules enter the cells, are distributed throughout the cytoplasm and could be detected in cell nuclei. The model study using A549 cells treated with 1,25-(OH)2D3 has shown similar cell growth suppression effect of native endogenously produced hBD-2. Conclusion: The results of our study suggest that in malignant epithelial cells hBD-2 may control cell growth via arrest of G1/S transition and activation of pRB. We express our sincere gratitude to Dr. N.N. Khranovskaya and Dr.O.V. Skachkova (National Cancer Institute, Kyiv, Ukraine) for qualified help in flow cytometry analysis, Dr.V.M. Pushkarev (V.P.Komissarenko Institute of Endocrinology and Metabolism, Kyiv, Ukraine) for expertized guidance in analysis of cell cycle regulation, Dr. V. Kashuba (Karolinska Institute, Sweden) for kindly provided A549 cells, and Dr.L.Apukhovska,Palladin Institute of Biochemistry, Kyiv, Ukraine for kindly provided metabolites of vitamin D3. en Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України Experimental Oncology Original contributions Human beta-defensin-2 controlS cell cycle in malignant epithelial cells: in vitro study Article published earlier |
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Human beta-defensin-2 controlS cell cycle in malignant epithelial cells: in vitro study |
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Human beta-defensin-2 controlS cell cycle in malignant epithelial cells: in vitro study Zhuravel, E. Shestakova, T. Efanova, O. Yusefovich, Yu. Kytvin, D. Soldatkina, M. Pogrebnoy, P. Original contributions |
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Human beta-defensin-2 controlS cell cycle in malignant epithelial cells: in vitro study |
| title_full |
Human beta-defensin-2 controlS cell cycle in malignant epithelial cells: in vitro study |
| title_fullStr |
Human beta-defensin-2 controlS cell cycle in malignant epithelial cells: in vitro study |
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Human beta-defensin-2 controlS cell cycle in malignant epithelial cells: in vitro study |
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human beta-defensin-2 controls cell cycle in malignant epithelial cells: in vitro study |
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Zhuravel, E. Shestakova, T. Efanova, O. Yusefovich, Yu. Kytvin, D. Soldatkina, M. Pogrebnoy, P. |
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Zhuravel, E. Shestakova, T. Efanova, O. Yusefovich, Yu. Kytvin, D. Soldatkina, M. Pogrebnoy, P. |
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Original contributions |
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Original contributions |
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2011 |
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Experimental Oncology |
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Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
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Article |
| description |
Aim: In the present research we analyze the mechanism of human beta-defensin-2 (hBD-2) influence on cultured malignant epithelial cell growth. Materials and Methods: The analysis of a concentration-dependent effect of recombinant hBD-2 (rec-hBD-2) on cell growth patterns and cell cycle distribution has been performed in vitro with 2 cell lines (human lung adenocarcinoma A549 cells and human epidermoid carcinoma A431 cells) using MTT test, flow cytometry and direct cell counting. To study intracellular localization of hBD-2 immunocytofluorescent and immunocytochemical analyses were applied, and effect of hBD-2 on signal cascades involved in cell cycle regulation has been studied by Western blotting. Results: According to our data, rec-hBD-2 exerts a concentration-dependent effect on the viability of cultured A549 and A431 cells. It causes proproliferative effect at concentrations below 1 nM, significant suppression of cell proliferation at concentration range from 10 nM to 1 µM (p<0.05), and cell death at higher concentrations. Using flow cytometry we have demonstrated that hBD-2 dependent growth suppression is realized via cell cycle arrest at G1/S phase (p<0.05). Also, we have registered significant activation of pRB and decreased expression of Cyclin D1 in cells treated with the defensin compared to untreated control cells, while the expression of p53 remains unaffected. The study of intracellular localization of hBD-2 in these cells has revealed that exogeneously added defensin molecules enter the cells, are distributed throughout the cytoplasm and could be detected in cell nuclei. The model study using A549 cells treated with 1,25-(OH)2D3 has shown similar cell growth suppression effect of native endogenously produced hBD-2. Conclusion: The results of our study suggest that in malignant epithelial cells hBD-2 may control cell growth via arrest of G1/S transition and activation of pRB.
|
| issn |
1812-9269 |
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https://nasplib.isofts.kiev.ua/handle/123456789/138664 |
| citation_txt |
Human beta-defensin-2 controlS cell cycle in malignant epithelial cells: in vitro study / E. Zhuravel, T. Shestakova, O. Efanova, Yu. Yusefovich, D. Lytvin, M. Soldatkina, P. Pogrebnoy // Experimental Oncology. — 2011. — Т. 33, № 3. — С. 114-120. — Бібліогр.: 29 назв. — англ. |
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2025-11-24T23:49:18Z |
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| fulltext |
114 Experimental Oncology 33, 114–120, 2011 (September)
HUMAN BETA-DEFENSIN-2 CONTROLS CELL CYCLE
IN MALIGNANT EPITHELIAL CELLS: IN VITRO STUDY
E. Zhuravel1*, T. Shestakova1, O. Efanova1, Yu. Yusefovich1, D. Lytvin2, M. Soldatkina1, P. Pogrebnoy1
1R.E. Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology, NAS of Ukraine, Kyiv,Ukraine
2Institute of Food Biotechnology and Genomics,NAS of Ukraine, Kyiv, Ukraine
Aim: In the present research we analyze the mechanism of human beta-defensin-2 (hBD-2) influence on cultured malignant epithelial
cell growth. Materials and Methods: The analysis of a concentration-dependent effect of recombinant hBD-2 (rec-hBD-2) on cell
growth patterns and cell cycle distribution has been performed in vitro with 2 cell lines (human lung adenocarcinoma A549 cells and
human epidermoid carcinoma A431 cells) using MTT test, flow cytometry and direct cell counting. To study intracellular localization
of hBD-2 immunocytofluorescent and immunocytochemical analyses were applied, and effect of hBD-2 on signal cascades involved
in cell cycle regulation has been studied by Western blotting. Results: According to our data, rec-hBD-2 exerts a concentration-
dependent effect on the viability of cultured A549 and A431 cells. It causes proproliferative effect at concentrations below 1 nM,
significant suppression of cell proliferation at concentration range from 10 nM to 1 μM (p<0.05), and cell death at higher con-
centrations. Using flow cytometry we have demonstrated that hBD-2 dependent growth suppression is realized via cell cycle arrest
at G1/S phase (p<0.05). Also, we have registered significant activation of pRB and decreased expression of Cyclin D1 in cells
treated with the defensin compared to untreated control cells, while the expression of p53 remains unaffected. The study of intracel-
lular localization of hBD-2 in these cells has revealed that exogeneously added defensin molecules enter the cells, are distributed
throughout the cytoplasm and could be detected in cell nuclei. The model study using A549 cells treated with 1,25-(OH)2D3 has
shown similar cell growth suppression effect of native endogenously produced hBD-2. Conclusion: The results of our study suggest
that in malignant epithelial cells hBD-2 may control cell growth via arrest of G1/S transition and activation of pRB.
Key Words: human beta-defensin-2, cell cycle, signaling pathway, pRB, intracellular localization.
INTRODUCTION
Human beta-defensins (hBDs) belong to a family
of antimicrobial peptides that constitute an important
part of the innate immune defense system. hBDs are
expressed mostly in epithelial tissues. Up to date, four
hBDs, hBD-1–4 have been identified in human tissues.
These molecules are small, with a molecular weight rang-
ing from 3.5 to 4.5 kDa, possess high positive net charge
and hydrophobic properties that allow their interaction
with microbial membrane, its penetration or its destruc-
tion. They exert direct antimicrobial action and are active
against bacteria, fungi, viruses; in parallel, according
to the recent data, they possess multiple biologic activi-
ties, in particular, immunomodulatory ones, and are
implicated in anti-tumor response [reviewed in 1, 2].
Biophysical properties of defensin molecules allow
them to form dimers or octamers in water solutions
and to create pores in target membrane or disrupt/
penetrate the membranes causing lysis of bacterial
and tumor cells [3, 4]. Interestingly, it has been docu-
mented that at lower concentrations defensins (at par-
ticular, alpha-defensins) may cause a concentration-
dependent effect on cultured eucaryotic cells leading
to stimulation of cell proliferation [5–7]. Moreover,
in vivo effects of antimicrobial peptides could be also
of opposite character dependent on their concentra-
tions and patterns of tumor microenvironment [1].
In present research, we have studied the mechanism
of growth suppressing action of human beta-defen-
sin-2 (hBD-2) in vitro. hBD-2, a 41 residue peptide, was
originally isolated from extracts of lesional scales from
psoriatic skin in 1997 [8]; its expression is characteristic
for skin, respiratory tract and gastrointestinal epithe-
lium. hBD-2 expression is inducible upon treatment
with bacterial products and some cytokines [9]. Simi-
larly to neutrophylic defensins, HNPs, this antimicrobial
peptide has been shown to affect cell proliferation and
viability in a concentration-dependent manner [10, 11].
In this work, we have shown that recombinant
hBD-2 (rec-hBD-2) exogenously added into cell
cultivation medium at a wide concentration range
causes stimulation of cell proliferation in picomolar
concentrations and a killing effect at concentrations
higher than 100 nM; rec-hBD-2 at concentrations
of 10–100 nM is capable to enter cells, it accumulates
in cell nuclei and causes cell growth suppression via
cell cycle arrest in G1/S phase and activation of pRB.
MATERIALS AND METHODS
Cell lines and treatment with recombinant
hBD-2 peptide. Human non-small cell lung ad-
enocarcinoma A549 cells were kindly provided
by Dr. V. Kashuba (Karolinska Institute, Sweden)
and human epidermoid carcinoma A431 cells were
obtained from the Bank of Cell Lines from Human
and Animal Tissues, R.E. Kavetsky Institute of Experi-
mental Pathology, Oncology and Radiobiology of NAS
of Ukraine (Kyiv, Ukraine). The cells were cultured in vi-
tro in DMEM culture medium with high glucose content
supplemented with 10% fetal bovine serum (FBS),
100 units/ml penicillin G sodium, 100 μg/ml strepto-
mycin sulfate in 5% CO2 athmosphere at 37 ˚C.
Received: July 5, 2011.
*Correspondence: E-mail: pogrebnoy@onconet.kiev.ua
Abbreviations used: hBD-2 – human beta-defensin-2; pRB – retino-
blastoma protein; rec-hBD-2 — recombinant hBD-2; VD3 – vitamin D3.
Exp Oncol 2011
33, 3, 114–120
ORIGINAL CONTRIBUTIONS
Experimental Oncology 33, 114–120, 2011 (September) 115
To study the effect of exogenous defensin on cell
growth, we have used the preparation of rec-hBD-2 ex-
pressed in bacterial cells as GST-hBD-2 fusion protein
purified by standard two-step procedure as described
earlier [12]. Protein concentration was determined
by the method of Bradford [13] and by UV absor-
bance at 280 nm using spectrophotometer Nano-
drop-1000 (USA).
Cells were cultured for 24 h in serum-free me-
dium or (dependent on experimental conditions)
in culture medium supplemented with 2.5% FBS,
to nearly 50% confluence and then treated by ad-
dition of rec-hBD-2 at various concentrations (from
10 pM to 100 M) for 48 h. After the treatment, cells
were triply washed with PBS, detached with trypsin,
and counted in hemocytometer or were subjected
to flow cytometry analysis. The percent of dead cells
was analized using tripan blue staining. Each experi-
ment was repeated in triplicate.
For flow cytometry analysis, the attached cells
were harvested by trypsinization, pelleted at 4 ˚C
(500 g) for 5 min, washed twice in PBS, and resuspended
in 1 ml of hypotonic lysis buffer (0.1% sodium citrate, 0.1%
Triton X-100, 5 g/ml PI (Sigma, USA). The cells were in-
cubated at room temperaure for 30 min in the dark, and
analyzed by Becton Dickinson FACS Calibur. The data
were analyzed with the use of CellQuest software package
and ModFit LT2.0 program (BDIS, USA) for Mac comput-
ers. All analyses were performed three times.
To evaluate the effect of rec-hBD-2 on cell viability,
MTT-test has been applied [14]. Shortly, A549 and
A431 cells were seeded into 96-well plates and incu-
bated with rec-hBD-2 for 48 h in serum-free medium.
Then the cells were treated with МТТ (3-[4,5-dimeth-
ylthiazole-2-yl]-2,5-diphenyltetrazolium bromide),
and colorimetric reaction was evaluated with the use
of ELISA reader (Awareness Technology Inc, USA)
at the wave lengths min =545 and max=630.
In some experiments to study the effect of native
endogenously produced hBD-2, A549 cells were
treated with 40 ng/ml of 1,25-(OH)2D3 for 48 h. Then
the attached cells were triply washed with PBS and
counted in hemocytometer.
RT-PCR analysis of hBD-2 expression. Total
RNA was isolated from tissue samples by the method
of Chomzynski and Sacchi [15]. For detection of ex-
pression of hBD-2 mRNA in tissue samples, semiquan-
titative RT-PCR analysis was performed using specific
primers (hBD-2 — F: -5'-gaagctcccagccatcagcc; R: —
5'-gtcgcacgtctctgatgaggga; beta-actin — F: -5'-ctg-
gaacggtgaaggtgaca; R: 5'-aagggacttcttgtaacaatgca).
The expression level of beta-actin (the house-keeping
gene) served as a loading control. The products
of RT-PCR were routinely analyzed by electrophoresis
in agarose gel. The relative mRNA expression levels
were analyzed using TotalLab Program.
Western-blot analysis. To analyse the level of ex-
pression and/or phosphorylation of different proteins,
the cultured cells were treated with rec-hBD-2 for
48 h as described above, and then washed with PBS and
lyzed in PBS solution containing 50 mM Тris-НCl (pH 7.0),
50 mM EDTA, 2% Triton Х-100. The concentration of total
protein was determined by the method of Lowry.
The proteins were separated by electrophoresis
in 9–22% gradient polyacrylamide gel and transferred
to nitrocellulose membrane Hybond-ECL, RPN3032D
(Amersham Biosciences, USA). Nonspecific binding
sites were blocked with 1Х PBS-Т, 5% BSA solution for
1 h, and the blots were incubated with primary Abs,
and then with secondary polyclonal HRP-conjugated
anti-rabbit IgG or anti-mouse IgG Abs (DakoCytoma-
tion, Denmark). The ECL Western blotting detection
system (Amersham Pharmacia Biotech) was used
to reveal immunoreactivity. For Western blot analysis,
the following antibodies were used: anti-pRB-Abs,
anti-p53-Abs, anti-CyclinD1-Abs (Cell Signaling Tech,
USA) and MoAbs against beta-actine (Sigma, USA).
All antibodies were used at the working dilutions ac-
cording to manufacturer instructions.
Confocal microscopy and immunocytoche-
mistry. A431 and A549 cells were plated into 6 cm Petri
dishes on glass cover slides. Control cells were grown
in fresh growth media supplemented with 2.5% FBS,
while experimental cells were additionally treated for
48 h with 1 M rec-hBD-2. After the treatment, cells
were washed in PBS and fixed for 1 h at -20 ̊ C in meth-
anol-acetone (1:1) solution. Cells were then washed
three times with PBS and blocked with 5% normal goat
serum (VectorLaboratories, Inc., Burlingame, CA)
in PBS for 1 h at room temperature. Next, cells were
incubated for 1 h with rabbit anti-hBD-2 polyclonal
antibody (Santa-Cruz, USA) diluted 1:50 in blocking
solution. Following this, cells were washed and incu-
bated for 1 h with goat anti-rabbit IgG-FITC antibody
(Sigma F0382) at a dilution of 1:80. Finally, the cells
were incubated with 0.5 μg/ml DAPI (4–6-diamidino-
2-phenylindole). Confocal images were obtained using
a Zeiss LSM 510 META confocal microscope equipped
with lasers for 405 nm and 488 nm excitation for DAPI
and FITC, correspondingly. Images were collected
with Plan-Apochromate 63X/1,4 Oil DIC lens (Zeiss,
Germany) in multi-track mode using line switching with
averaging of 16 readings. Excitation of FITC and DAPI
was collected with LP 505 and LP 420 filters.
Also, to detect intracellular localization of recombi-
nant hBD-2, the cells were treated with 1 M hBD-2 for
48 h, washed with PBS, fixed in cold methanol/acetone
(1:1) mixture for 24 h at -20 °С, blocked with 5% nor-
mal goat serum for 1 h at RT. For immunocytochemi-
cal detection of hBD-2 staining we have used rabbit
polyclonal anti-hBD-2 Abs (Santa-Cruz, USA) at the
dilution 1:100. EnVision System and DAB (DAKO,
Denmark) were used for visualization. The slides were
analized using Axioplan microscope (Zeiss, Germany).
On average, 10 fields were examined for each sample,
Statistical analysis. The data are reported as the
mean±SD. The statistical significance of differences
between mean values was assessed by the Stu-
dent’s t-test. Values p<0.05 were considered as sta-
tistically significant.
116 Experimental Oncology 33, 114–120, 2011 (September)
RESULTS
Our research has been performed in vitro using
rec-hBD-2 exogenously added to the cell culture me-
dium. Control experiment was carried out on the model
of vitamin-D3-induced expression of native hBD-2. Re-
combinant hBD-2 was expressed in bacterial cells as GST-
hBD-2 fusion protein and purified by standard procedure
as described earlier [13]. The purified defensin preparation
was of 98% purity as justified by the data of electrophoretic
analysis, WB, HPLC, and active in antimicrobial test against
Pseudomonas aeruginosa (data not shown).
The analysis of rec-hBD-2 effect on cell growth
patterns in vitro. To study the effect of exogenous
rec-hBD-2 on growth patterns of cultured A431 and
A549 cells, the cells were treated with rec-hBD-2 that
was added into serum-free culture medium at different
concentrations (from 10 pM up to 100 M) for 48 h. Us-
ing MTT-analysis, we have revealed that hBD-2 caused
a concentration-dependent effect on cell growth rates,
in particular, it stimulated cell proliferation at concentra-
tion range from 10 pM to 1 nM, and significantly sup-
pressed cell growth at concentration from 10 nM to 1 M
(Fig. 1). Treatment of both cell lines with recombinant
defensin at concentrations higher than 1000 nM resulted
in cell death. The data of MTT-test were supported by the
results of direct cell counting (Fig. 2). These effects were
hBD-2-specific as far as pretreatment of rec-hBD-2 with
anti-hBD-2 Abs completely abolished the above men-
tioned effects (Fig. 2).
0
20
40
60
80
100
120
140
160
hBD2 concentration, μM
Ce
ll
nu
m
be
r,
%
A431
A549
0 0,0001 0,001 0,01 0,1 1 10 20 100
Fig. 1. Exogenous rec-hBD-2 affects the number of viable
cultured cells of A431 and A549 lines. Cells were cultured in 96-
well plates in serum free culture medium and treated with ex-
ogenously added recombinant hBD-2 at concentrations from
10 pM to 100 M for 48 h. The number of viable cells was evalu-
ated by MTT analysis. The data of three independent experiments
are presented as the mean ± SD
Flow cytometry analysis. To explore the mecha-
nism implicated in the growth-inhibition effect
of hBD-2 we have selected the hBD-2 concentration
range of 10–1000 nM, and using flow cytometry analy-
sis we have analyzed the influence of rec-hBD-2 ex-
ogenously added into the cell culture medium on cell
cycle distribution of cultured A549 and A431 cells after
48 h incubation. Flow cytometry analysis of A431 cells
treated with rec-hBD-2 has revealed (Fig. 3) a concen-
tration-dependent cell growth arrest at G1/S phase
(p<0.05). Similar results were registered for A549 cells
treated with rec-hBD-2 (data not shown). At the same
time treatment of cells with 10–1000 nM of hBD-2 had
0
20
40
60
80
100
120
A549 A431
Ce
ll
nu
m
be
r,
%
Control 10nM rec-hBD2 100nM rec-hBD2
1μM rec-hBD2 50ng rec-hBD2+antihBD2 ab
Fig. 2. A concentration-dependent effect of exogenous re-
combinant hBD-2 on the number of viable cultured A549 and
A431cells. The number of attached cells was evaluated by direct
cell counting. Cells were cultured in 96-well plates in serum free
medium and treated with exogenously added rec-hBD-2 concen-
trations of 10, 100 nM and 1 M for 48 h. To analyze the specificity
of growth-suppressing activity of rec-hBD-2, 50 ng of hBD-2 were
pre-incubated for 1 h with 0.8 μg of anti-hBD-2 Abs (Santa-Cruz,
USA), and then added to the cell incubation medium for 48 h. The
data of four independent experiments are presented as the mean ±
SD. *The difference is significant as compared to control (p < 0.05)
0
20
40
60
80
100
Control 10 100 1000
hBD-2 concentration, nМ
Ce
ll
nu
m
be
r,
%
G0/G1
G2/M
S
1600
1200
800
400
0
0 50 100 250150 200
Nu
m
be
r
Channels (FL2-A)
1200
1000
800
600
400
200
0
0 50 100 250150 200
Nu
m
be
r
Channels (FL2-A)
2000
1600
1200
800
400
0
0 50 100 250150 200
Nu
m
be
r
Channels (FL2-A)
2500
2000
1500
1000
500
0
0 50 100 250150 200
Nu
m
be
r
Channels (FL2-A)
b c
a
d e
Dip G1
Dip G2
Dip S
Fig. 3. Flow cytometry analysis of cell cycle distribution of A431 cells
treated or not treated with rec-hBD-2 for 48 h (a-e). Cells were
cultured in 10 cm Petri dishes in culture medium supplemented
with 10% FBS to 50% confluency, then the medium was replaced
by the one supplemented with 2.5% FBS, and the cells were treated
by addition of rec-hBD-2 for 48 h. After the treatment, the cells were
triply washed with PBS, detached with trypsin and subjected to flow
cytometry analysis as described in Materials and Methods section:
a — the data of three independent experiments are presented as the
mean ± SD; b — control A431 cells; c, d, e — the cells treated with
10, 100, and 1000 nM rec-hBD-2 respectively
Experimental Oncology 33, 114–120, 2011 (September) 117
no effect on cell mortality: the percent of apoptotic
cells in control and hBD-2 treated A431 cells didn’t dif-
fer significantly and did not exceed 4%. There data
were supported by the results of tripan blue staining.
The analysis of rec-hBD-2 effect on signal
pathways involved in cell cycle regulation in vitro.
To explore how rec-hBD-2 affects specific signal cas-
cades involved in cell cycle regulation in cultured cells,
we have studied the state of some proteins which ac-
tivity is required for G1/S cell cycle transition. We have
analyzed phosphorylation of pRB, expression levels
of cyclin D1 and p53 in A549 and A431 cells treated with
1 M rec-hBD-2 in comparison to control (untreated)
cells. We have shown, rec-hBD-2 treatment resulted
in the dramatic reduction of pRB phosphorylation, and
significant decrease of cyclin D1 expression, while
p53 expression remained unaffected (Fig. 4). So, one
may conclude that G1/S cell cycle arrest in hBD-2 treat-
ed cells could be possibly realized via activation of pRB.
0
20
40
60
80
100
120
p53 pRb Cyclin D1
Ex
pr
es
si
on
/p
ho
sp
ho
ril
at
io
n,
re
la
tiv
e
un
its
0
20
40
60
80
100
120
140
p53 pRb Cyclin D1
Ex
pr
es
si
on
/p
ho
sp
ho
ril
at
io
n,
re
la
tiv
e
un
itsA549 K
A549+hBD-2
A431 K
A431+hBD-2
β-actin
Cyclin D1
pRB
p53
1 2 3 4
cb
a
Fig. 4. Western blot analysis of pRB phosphorylation and expres-
sion levels of cyclin D1 and p53 in A549 and A431 cells treated
with 1 M rec-hBD-2 for 48 h. a: line 1 — intact A549 cells; line
2 — A549 cells treated with 1 M rec-hBD-2; line 3 — intact
A431 cells; line 4 — A431 cells treated with 1 M rec-hBD-2;
b, c — graphic representations of Western blot analysis data
counted with the use of GelPro 4.0 program
The study of intracellular localization of hBD-2.
To analyze whether exogenously added rec-hBD-2 en-
ters the cell, two methods were used — immunocyto-
chemical analysis and immunocytofluorimetry. In both
cases we have compared hBD-2-specific immunos-
taining in defensin-treated A431 and A549 cells versus
intact control cells, and have detected hBD-2 specific
immunostaining in the fist case. According to our re-
sults (Fig. 5), rec-hBD-2 enters the cells, is distrib-
uted in cytoplasm and is accumulated in cell nuclei
of A431 cells (Fig. 5) and A549 cells (Fig. 7).
250
200
150
100
50
0
0 5 10 2515 3520 30 40
a
b
c
In
te
ns
ity
Distance (µm)
Ch3-T1
Ch2-T2
Fig. 5. Rec-hBD-2 is detected in cytoplasm (a) and nuclei (b,
c) of hBD-2 treated A431 cells but not in control cells. Confocal
microscopy was applied for analysis of immunofluorescent stain-
ing of hBD-2 in A431 cells treated (1–3) or not treated (4–6) with
the recombinant defensin at the concentration of 1 M for 48 h.
1, 4- staining with FITC-anti-hBD-2 Abs; 2, 5 — DAPI staining, 3,
6 — overlay. c — evaluation of hBD-2 specific nuclear staining
of A431 cells treated with 1 M rec-hBD-2
Modeling of action of native hBD-2 in vitro. To find
out whether the data obtained with the use of recom-
binant defensin may reflect similar physiologic events
related to the action of native hBD-2, we have designed
an experiment on a model of A549 cells in which the
hBD-2 mRNA expression is induced by treatment with
metabolites of vitamin D3 (VD3). First of all, using RT-
PCR analysis we have determined that intact A549 cells
do not express hBD-2 mRNA, while its expression
in these cells could be induced if the cells are treated
with 4·10–7 M 1,25-(0H)2D3 or 25(0H)D3 for 12 h (Fig. 6).
Next, with the use of direct cell counting we have
revealed that A549 cells incubated with metabolites
of VD3 for 48 or 72 h demonstrated significantly
suppressed cell proliferation rate compared with the
control cells (by 1.3 and 1.6 fold respectively, p<0.05),
118 Experimental Oncology 33, 114–120, 2011 (September)
and that such growth suppression could be eliminated
by the addition of anti-hBD-2 Abs into the cell culture
medium. Next, using immunocytochemical and immu-
nofluorescent staining we have detected an appear-
ance of hBD-2-specific immunostaining in cytoplasm
and nuclei of A549 cells treated with VD3 metabolites
for 48–72 h (see Fig. 7).
DISCUSSION
In our research we have received new data that
allow to understand the possible mechanism of cell
growth-suppressing activity of hBD-2 toward human
cancer cells in vitro. As we have shown, rec-hBD-2 ex-
erts a concentration-dependent effect toward cultured
cells of two different lines — lung adenocarcinoma
A549 cells and vulval epidermoid carcinoma A431 cells.
This defensin stimulates cell proliferation at the con-
centrations below 1 nM, and suppress cell growth
at the concentrations higher than 10 nM. Such dual ef-
fect has been reported earlier for some defensins [16–
18], and beta-defensin-dependent stimulation of cell
proliferation has been studied in detail by few research
groups [19–22]. In particular, it has been shown that
hBD-2,-3,-4 stimulate keratinocyte proliferation, and
this process requires hBD-dependent phosphorylation
of EGFR, STAT1 and STAT3 [21]. Another study [23]
has shown that hBD-2-dependent proliferation of hu-
man conjunctival fibroblasts is mediated by activation
of p42/44 MAP kinase and Akt. Killing effects of hBDs
toward cultured human tumor cells have been studied
in many works, and they are thought to be realized via
membrane lysis [reviewed in 24].
At the same time the mechanism of action of defen-
sins at concentration range between pro-proliferative
and cytolytic doses was not analyzed yet, and our
research is the first one in this field. We have analyzed
the growth suppressing arm of hBD-2-concentration
curve, namely its concentrations between 10 and
1000 nM. These medium concentrations could be con-
sidered close enough to physiologic margins reported
in some related publications [25, 26].
First of all we have studied effect of nanomolar
concentrations of rec-hBD-2 on cell cycle distribution
and signal pathways that regulate G1/S cell cycle transi-
tion. The obtained results have shown that exogenously
added rec-hBD-2 caused G1/S cell cycle arrest in both
cell lines via significant dephosphorylation of pRB and
inhibition of cyclin D1 expression, while p53 expression
remained unaffected. So, the growth inhibitory action
of rec-hBD-2 could be in part realized through pRB
activation, and this fact is of significant importance.
It is well recognized that the pRB plays a tumor suppres-
sive function and is able to promote cell cycle exit via the
control of activity of E2F transcription factors regulating
a number of genes, involved into the G1 to S transi-
tion [27]. As far as we know, it is a first report on a new
biologic activity of hBD-2 as a native activator of pRB.
According to the data of immunocytochemical and
immunocytofluorescent analysis, rec-hBD-2 enters
the cells, may be found in cytoplasm and also — in cell
nuclei; the latest fact points on a possibility that this
defensin may play a role in gene transcription. Accord-
a b
1 2 3 4 1 2 3 4
Fig. 6. RT-PCR analysis of hBD-2 mRNA expresssion
in А-549 cells (a). The cells А549 (2,5х105 ) were grown
in 3 cm Petri dishes, cultured for 24 h in serum free medium, and
then treated with 1,25- (0H)2D3 or (25)(0H)D3 at the concentra-
tions of 40 ng/ml for 12 h. a — 1 — DNA ladder (Fermental,
Lithuania); 2 — control A549 cells; 3- the cells treated with 40 ng/
ml 1,25-(0H)2D3; 4 — the cells treated with 40 ng/ml 25(0H)D3.
β-actin is used as a house-keeping gene (b)
a
b
с
Fig. 7. Immunocytochemical staining of hBD-2 in intact A459 cells
(a) and the cells treated with the 1000 nM rec-hBD-2 for 48 h (b)
or 40 ng/ml 1,25-(0H)2D3 for 48 h (c). Magnification x400
Experimental Oncology 33, 114–120, 2011 (September) 119
ing to recent data, some antimicrobial peptides were
found in cancer cell nuclei, in particular, hBD-1 [28]
and cathelicidine LL-37 [29]. However, biologic role
of nuclear translocation of these peptides have got
no explanations so far.
To check if the observed effects of rec-hBD-2 may
reflect similar physiologic events related to the ac-
tion of native hBD-2 and are not related to artifacts,
we have analyzed cell growth patterns of A549 cells
in which hBD-2 mRNA expression is induced by treat-
ment with VD3 metabolites. We have demonstrated
that VD3-treated A549 cells express hBD-2 mRNA
and possess lower growth rates than control cells that
do not express the defensin. It is known that VD3 me-
tabolites regulate more than two hundred genes,
not only this one; so, we have analyzed if a depletion
of newly secreted defensin molecules from culture
medium of 1,25-(0H)2D3-treated cells will restore
growth rates of these cells. So, in a model experiment
with A549 cells we have induce hBD-2 expression
(treatment with 40 ng/ml 1,25-(0H)2D3; 24 h after such
induction we have added anti-hBD-2 Abs into cell
culture medium to bind defensin molecules that are
secreted from the cells; and 48 h after the beginning
of VD3 treatment we have revealed that anti-hBD-
2-Abs treated hBD-2 expressing A549 cells do not
differ by cell counts from control A549 cells. So,
we conclude that growth suppression of A549 cells
in which hBD-2 expression was induced by VD3 me-
tabolites, is related to action of native defensin. Also,
we have observed specific hBD-2 immunostaining
in (1,25)(0H)2D3 treated cells (Fig. 7) and its nuclear
localization (data not shown). So, one may conclude
that autocrine effect of native hBD-2 toward A549 cells
is similar to that of recombinant defensin.
All these data allowed us to propose a hypothesis
on a possible mechanism of hBD-2 dual concentra-
tion-dependent action on eucaryotic cells in vitro.
Let’s remember that this peptide is shown to be involved
in wound healing in vitro and in vivo stimulating kerati-
nocyte proliferation and migration [10, 21]; at the same
time this process couldn’t be endless. From other side,
biophysical studies evidence on an ability of the defensin
to form oligomers in water solutions [3] — hBD-2 may
exist in monomeric, dimeric or octameric forms, and the
latest ones, according to the authors conclusion, are
responsible for the disruption of bacterial membranes.
It’s tempting to speculate that the concentration-
dependent mode of influence of hBD-2 could be re-
lated to different mechanisms of action of monomeric
and oligomeric forms of these molecules — possibly,
receptor-mediated for monomeric forms (with pro-pro-
liferative mode of action), membrane-penetrating ones
for dimeric forms (with growth suppressing activity) and
cytolytic activity characteristic for octamers. However,
such hypothesis is a speculative one and requires further
experimental research.
In conclusion, the data of the present research al-
low to suggest that human beta-defensin-2 may act
as a native regulator of cell growth — either positive
at low concentrations or negative at higher ones, and
further research in this field may help to reveal new
activities of this peptide antibiotic in epithelial cells.
ACKNOWLEDGEMENTS
We express our sincere gratitude to Dr. N.N. Khranovs-
kaya and Dr.O.V. Skachkova (National Cancer Institute,
Kyiv, Ukraine) for qualified help in flow cytometry
analysis, Dr.V.M. Pushkarev (V.P.Komissarenko Institute
of Endocrinology and Metabolism, Kyiv, Ukraine) for
expertized guidance in analysis of cell cycle regulation
, Dr. V. Kashuba (Karolinska Institute, Sweden) for kindly
provided A549 cells, and Dr.L.Apukhovska,Palladin In-
stitute of Biochemistry, Kyiv, Ukraine for kindly provided
metabolites of vitamin D3.
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