Biologic activities of recombinant human-beta-defensin-4 toward cultured human cancer cells
Aim - the aim of the study was in vitro analysis of biological activity of recombinant human beta-defensin-4 (rec-hBD-4). Methods: hBD-4 cDNA was cloned into pGEX-2T vector, and recombinant plasmid was transformed into E. coli BL21(DE3) cells. To purify soluble fusion GST-hBD-4 protein, affinity chr...
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Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України
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| Цитувати: | Biologic activities of recombinant human-beta-defensin-4 toward cultured human cancer cells / O.L. Gerashchenko, E.V. Zhuravel, O.V. Skachkova, N.N. Khranovska, V.V. Filonenko, P.V. Pogrebnoy, A. Soldatkina // Experimental Oncology. — 2013. — Т. 35, № 2. — С. 76–82. — Бібліогр.: 37 назв. — англ. |
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irk-123456789-1452082019-01-21T01:23:18Z Biologic activities of recombinant human-beta-defensin-4 toward cultured human cancer cells Gerashchenko, O.L. Zhuravel, E.V. Skachkova, O.V. Khranovska, N.N. Filonenko, V.V. Pogrebnoy, P.V. Soldatkina, M.A. Original contributions Aim - the aim of the study was in vitro analysis of biological activity of recombinant human beta-defensin-4 (rec-hBD-4). Methods: hBD-4 cDNA was cloned into pGEX-2T vector, and recombinant plasmid was transformed into E. coli BL21(DE3) cells. To purify soluble fusion GST-hBD-4 protein, affinity chromatography was applied. Rec-hBD-4 was cleaved from the fusion protein with thrombin, and purified by reverse phase chromatography on Sep-Pack C18. Effects of rec-hBD-4 on proliferation, viability, cell cycle distribution, substrate-independent growth, and mobility of cultured human cancer cells of A431, A549, and TPC-1 lines were analyzed by direct cell counting technique, MTT assay, flow cytofluorometry, colony forming assay in semi-soft medium, and wound healing assay. Rech-BD-4 was expressed in bacterial cells as GST-hBD-4 fusion protein, and purified by routine 3-step procedure (affine chromatography on glutathione-agarose, cleavage of fusion protein by thrombin, and reverse phase chromatography). Analysis of in vitro activity of rec-hBD-4 toward three human cancer cell lines has demonstrated that the defensin is capable to affect cell behaviour in concentration-dependent manner. In 1 - 100 nM concentrations rec-hBD-4 significantly stimulates cancer cell proliferation and viability, and promotes cell cycle progression through G2/M checkpoint, greatly enhances colony-forming activity and mobility of the cells. Treatment of the cells with 500 nM of rec-hBD-4 resulted in opposite effects: significant suppression of cell proliferation and viability, blockage of cell cycle in G1/S checkpoint, significant inhibition of cell migration and colony forming activity. 2013 Article Biologic activities of recombinant human-beta-defensin-4 toward cultured human cancer cells / O.L. Gerashchenko, E.V. Zhuravel, O.V. Skachkova, N.N. Khranovska, V.V. Filonenko, P.V. Pogrebnoy, A. Soldatkina // Experimental Oncology. — 2013. — Т. 35, № 2. — С. 76–82. — Бібліогр.: 37 назв. — англ. 1812-9269 http://dspace.nbuv.gov.ua/handle/123456789/145208 en Experimental Oncology Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
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Original contributions Original contributions |
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Original contributions Original contributions Gerashchenko, O.L. Zhuravel, E.V. Skachkova, O.V. Khranovska, N.N. Filonenko, V.V. Pogrebnoy, P.V. Soldatkina, M.A. Biologic activities of recombinant human-beta-defensin-4 toward cultured human cancer cells Experimental Oncology |
| description |
Aim - the aim of the study was in vitro analysis of biological activity of recombinant human beta-defensin-4 (rec-hBD-4). Methods: hBD-4 cDNA was cloned into pGEX-2T vector, and recombinant plasmid was transformed into E. coli BL21(DE3) cells. To purify soluble fusion GST-hBD-4 protein, affinity chromatography was applied. Rec-hBD-4 was cleaved from the fusion protein with thrombin, and purified by reverse phase chromatography on Sep-Pack C18. Effects of rec-hBD-4 on proliferation, viability, cell cycle distribution, substrate-independent growth, and mobility of cultured human cancer cells of A431, A549, and TPC-1 lines were analyzed by direct cell counting technique, MTT assay, flow cytofluorometry, colony forming assay in semi-soft medium, and wound healing assay. Rech-BD-4 was expressed in bacterial cells as GST-hBD-4 fusion protein, and purified by routine 3-step procedure (affine chromatography on glutathione-agarose, cleavage of fusion protein by thrombin, and reverse phase chromatography). Analysis of in vitro activity of rec-hBD-4 toward three human cancer cell lines has demonstrated that the defensin is capable to affect cell behaviour in concentration-dependent manner. In 1 - 100 nM concentrations rec-hBD-4 significantly stimulates cancer cell proliferation and viability, and promotes cell cycle progression through G2/M checkpoint, greatly enhances colony-forming activity and mobility of the cells. Treatment of the cells with 500 nM of rec-hBD-4 resulted in opposite effects: significant suppression of cell proliferation and viability, blockage of cell cycle in G1/S checkpoint, significant inhibition of cell migration and colony forming activity. |
| format |
Article |
| author |
Gerashchenko, O.L. Zhuravel, E.V. Skachkova, O.V. Khranovska, N.N. Filonenko, V.V. Pogrebnoy, P.V. Soldatkina, M.A. |
| author_facet |
Gerashchenko, O.L. Zhuravel, E.V. Skachkova, O.V. Khranovska, N.N. Filonenko, V.V. Pogrebnoy, P.V. Soldatkina, M.A. |
| author_sort |
Gerashchenko, O.L. |
| title |
Biologic activities of recombinant human-beta-defensin-4 toward cultured human cancer cells |
| title_short |
Biologic activities of recombinant human-beta-defensin-4 toward cultured human cancer cells |
| title_full |
Biologic activities of recombinant human-beta-defensin-4 toward cultured human cancer cells |
| title_fullStr |
Biologic activities of recombinant human-beta-defensin-4 toward cultured human cancer cells |
| title_full_unstemmed |
Biologic activities of recombinant human-beta-defensin-4 toward cultured human cancer cells |
| title_sort |
biologic activities of recombinant human-beta-defensin-4 toward cultured human cancer cells |
| publisher |
Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
| publishDate |
2013 |
| topic_facet |
Original contributions |
| url |
http://dspace.nbuv.gov.ua/handle/123456789/145208 |
| citation_txt |
Biologic activities of recombinant human-beta-defensin-4 toward cultured human cancer cells / O.L. Gerashchenko, E.V. Zhuravel, O.V. Skachkova, N.N. Khranovska, V.V. Filonenko, P.V. Pogrebnoy, A. Soldatkina // Experimental Oncology. — 2013. — Т. 35, № 2. — С. 76–82. — Бібліогр.: 37 назв. — англ. |
| series |
Experimental Oncology |
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| fulltext |
76 Experimental Oncology 35, 76–82, 2013 (June)
BIOLOGIC ACTIVITIES OF RECOMBINANT HUMAN-BETA-
DEFENSIN-4 TOWARD CULTURED HUMAN CANCER CELLS
O.L. Gerashchenko1,*, E.V. Zhuravel1, O.V. Skachkova2,
N.N. Khranovska2, V.V. Filonenko3, P.V. Pogrebnoy1, M.A. Soldatkina1
1R.E. Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology, NAS of Ukraine, Kyiv 03022, Ukraine
2National Cancer Institute, Kyiv, Ukraine
3Institute of Molecular Biology and Genetics, NAS of Ukraine, Kyiv 03143, Ukraine
Aim: The aim of the study was in vitro analysis of biological activity of recombinant human beta-defensin-4 (rec-hBD-4). Meth-
ods: hBD-4 cDNA was cloned into pGEX-2T vector, and recombinant plasmid was transformed into E. coli BL21(DE3) cells.
To purify soluble fusion GST-hBD-4 protein, affinity chromatography was applied. Rec-hBD-4 was cleaved from the fusion
protein with thrombin, and purified by reverse phase chromatography on Sep-Pack C18. Effects of rec-hBD-4 on proliferation,
viability, cell cycle distribution, substrate-independent growth, and mobility of cultured human cancer cells of A431, A549, and
TPC-1 lines were analyzed by direct cell counting technique, MTT assay, flow cytofluorometry, colony forming assay in semi-
soft medium, and wound healing assay. Results: Rec-hBD-4 was expressed in bacterial cells as GST-hBD-4 fusion protein, and
purified by routine 3-step procedure (affine chromatography on glutathione-agarose, cleavage of fusion protein by thrombin,
and reverse phase chromatography). Analysis of in vitro activity of rec-hBD-4 toward three human cancer cell lines has dem-
onstrated that the defensin is capable to affect cell behaviour in concentration-dependent manner. In 1–100 nM concentrations
rec-hBD-4 significantly stimulates cancer cell proliferation and viability, and promotes cell cycle progression through G2/M check-
point, greatly enhances colony-forming activity and mobility of the cells. Treatment of the cells with 500 nM of rec-hBD-4 re-
sulted in opposite effects: significant suppression of cell proliferation and viability, blockage of cell cycle in G1/S checkpoint,
significant inhibition of cell migration and colony forming activity. Conclusion: Recombinant human beta-defensin-4 is bio-
logically active peptide capable to cause oppositely directed effects toward biologic features of cancer cells in vitro dependent
on its concentration.
Key Words: human beta-defensin-4, cancer cell, proliferation, migration, colony forming activity.
Human beta-defensins (hBDs), cationic antimi-
crobial peptides and important components of innate
immunity system in humans, are mainly expressed
in epithelial cells of different origin and provide first-
line defense of epithelial surfaces from microbial
challenge. According to modern knowledge, the family
of hBDs includes at least 30 members most of which
are indentified by computational analysis and functions
of which remain unknown [1, 2].
Up to date, the most studied members of beta-
defensin subfamily are hBD-1 and hBD-2, and
to the lesser extent — hBD-3 and hBD-4. hBD-1 was
originally isolated from hemofiltrate in 1995 [3],
and later was found in urogenital and respiratory
tracts [4]; this defensin is supposed to be consti-
tutively expressed. Three other hBDs are peptides
with inducible character of expression. hBD-2 was
isolated from psoriatic skin lesions [5] and later from
many epithelial tissue types; its expression is shown
to be regulated by different bacteria and inflamma-
tory factors due to the presence of binding site for
inflammatory mediator NF-κB in the promoter region
of hBD-2 gene [6]. hBD-3 and hBD-4 have been
firstly discovered by screening genomic sequences
of the Human Genome Project [7, 8]; hBD-3 expres-
sion is induced upon stimulation with interferon-γ via
STAT binding site in promoter region of hBD-3 gene
[7]. Also, in 2001 hBD-3 was isolated from psoriatic
lesions, cloned from keratinocytes and expressed
as His-Tag-fusion protein in E. coli [9]. hBD-4 gene
promoter contains no NF-kB or STAT binding sites,
and its expression is positively regulated with PMA
and bacteria and is thought to be mediated by protein
kinase C [8].
All these beta-defensins demonstrate potent anti-
microbial activity against a variety of bacterial patho-
gens, that’s why direct microbial killing is supposed
to be their main function. However, experimental data
suggest that defensins are multifunctional molecules
with a wide spectrum of important biological activities
and could be involved not only in local immediate anti-
microbial response but also in chemotaxis, modulation
of inflammatory response, angiogenesis, and wound
healing [10, 11]. The last process is of special inter-
est. It is shown in a number of studies that expression
of inducible hBDs is up-regulated in skin lesions after
injury and these peptides positively regulate kera-
tinocyte proliferation and migration thus promoting
wound healing [12, 13]. In a number of studies there
have been documented abilities of hBDs to influence
many vital cell processes — cell proliferation, viability,
differentiation, and apoptosis, and it has been shown
that such effects of hBDs are concentration dependent
and could be exerted against many cell types, not only
keratinocytes [14–17]. In a similar manner hBDs may
Received: December 12, 2012.
*Correspondence: E-mail: pogrebnoy@onconet.kiev.ua
Abbreviations used: GST — glutathione-S-transferase;
hBDs — human beta-defensins; hBD-4 — human beta-defensin-4;
rec-hBD-4 — recombinant hBD-4.
Exp Oncol 2013
35, 2, 76–82
ORIGINAL CONTRIBUTIONS
Experimental Oncology 35, 76–82, 2013 (June) 77
affect growth patterns of tumor cells and may play
a role in promotion or suppression of human cancer
cell growth.
In recent years, new insights on possible involve-
ment of hBDs in tumorigenesis have been gained
[17–27]. hBD-1 is considered a tumor suppressor:
its expression is absent in prostate cancer and renal
clear cell carcinoma; down-regulation of hBD-1 ex-
pression contributes to cancer cell survival while its
induction results in tumor cell death [17, 18]. The
role of hBD-2 in cancer cell biology is not so evident
because in different tumor types expression patterns
of this defensin could differ significantly. In salivary
gland tumors hBD-2 expression is down-regulated
[19]; the authors suppose that in salivary gland
hBD-2 may play antioncogenic functions [19]. Ac-
cording to our data [20, 21], in lung adenocarcinoma
expression level of hBD-2 correlates with tumor dif-
ferentiation grade, while in vitro this defensin causes
significant suppression of lung cancer cell growth via
cell cycle regulation. In oral squamous cell carcinoma
hBD-2 has been shown to be up-regulated and is sup-
posed to play a pro-oncogenic role [22]. So, functional
meaning of hBD-2 expression in cancer cells seems
to depend on histologic type of tumor and on its
microenvironment. This is true also for hBD-3 which
is considered as pro-oncogenic molecule in some
tumors (head and neck cancer, oral carcinoma [23,
24]) or tumor suppressor in salivary gland tumors [19].
Up to date, the role of hBD-4 in tumor cell biology has
not been analyzed.
In this work we report successful production
of biologically active recombinant hBD-4 (rec-hBD-4)
expressed in prokaryotic cells. Analysis of biologic
activity of this defensin against cultured human can-
cer cells has shown that hBD-4 is capable to affect
in vitro human tumor cell proliferation and viability,
migration potential and substrate-independent growth
in a concentration-dependent manner characteristic
to other representatives of beta-defensin family.
MATERIALS AND METHODS
Cell lines. Human epidermoid carcinoma A431 cells
and human lung adenocarcinoma A549 cells were ob-
tained from the Bank of Cell Lines from Human and
Animal Tissues, R.E. Kavetsky Institute of Experimental
Pathology, Oncology and Radiobiology, NAS of Ukraine
(Kyiv, Ukraine). Human papillary thyroid carcinoma
cell line TPC-1 was kindly provided by Dr. V.M. Push-
karev (V.P. Komissarenko Institute of Endocrinology
and Metabolism, AMS of Ukraine, Kyiv, Ukraine). The
cells were cultured in vitro in DMEM culture medium
supplemented with 10% fetal bovine serum (FBS),
100 units/mL penicillin G sodium, 100 μg/mL strepto-
mycin sulfate in 5% CO2 atmosphere at 37 °C.
The cloning of hBD-4 and its prokaryotic
expression. To clone the gene coding for mature
hBD-4 sequence, total RNA was isolated from
human lung squamous cell carcinoma sample
No 5 [27], and hBD-4 cDNA was obtained by re-
verse transcription using a pair of specific primers:
hBD-4-F: 5`-GTGTTGGATCCGAATTTGAATTG-
GACAGAAT-3`; hBD-4-R: 5`-TCTTGGAATTCT-
CAGGGTTTTGTACGATTCAGTA-3. Primer design
was based on analysis of NM_080389.2 sequence
from GenBank database.
HBD-4 cDNA was cloned into pGEX-2T vector
(GE Healthcare, Sweden). Restriction of plasmids,
ligation of fragments, bacterial cell transformation
were performed by standard protocols.
Production and purification of rec-hBD-4.
Rec-hBD-2 was purified by three-step proce-
dure similarly to rec-hBD-2 [28]. Shortly, E. coli
BL21(DE3) bacteria transformed with GST-hBD-
4-recombinant plasmid were induced by 1 mM IPTG
for 6 h, pelleted by centrifugation, resuspended in ly-
sis buffer (50 mМ Tris-HCl, pH 7,6; 250 mМ NaCl;
1% Triton Х-100 and cocktail of proteases inhibi-
tors), and sonicated using ultrasound disintegrator
(UD-11 Automatic, Poland). Then cell lysate was
applied to affine chromatography on glutathione-
agarose column (GE Healthcare, Sweden) with
following cleavage of the defensin from fusion
protein by thrombin digestion. hBD-4 peptide was
further purified by reverse phase chromatography
on Sep-Pack C18 cartridge (Waters, USA). Pro-
tein concentration was determined by UV absor-
bance at 280 nm using spectrophotometer Nano-
drop-1000 (USA), and purity of the peptide prepa-
ration was analyzed by 7–22% gradient SDS PAAG
electrophoresis.
Direct cell counting. To study the effect of rec-
hBD-4 on cell proliferation, A431, A549, and TPC-1 cells
were routinely cultured in 24-well plates (5x104 cells
per well) to nearly 50% confluence and then treated
by addition of rec-hBD-4 at various concentrations
(from 0.1 nM to 1 M) for 48 h in 2.5% FBS medium.
After the treatment, cells were triply washed with PBS,
detached with trypsin, and counted in hemocytometer.
The percentage of dead cells was analyzed using
trypan blue staining.
MT T assay. To evaluate the effect of rec-
hBD-4 on cell viability, MTT-test has been applied
[29]. A549, A431, and TPC-1 cells were seeded into
96-well plates (7x103 cells per well) and incubated with
rec-hBD-4 for 48 h in medium with 2.5% FBS. Then the
cells were routinely treated with МТТ (3-[4,5-dimeth-
ylthiazole-2-yl]-2,5-diphenyltetrazolium bromide)
by standard protocol, and colorimetric reaction was
evaluated with the use of ELISA reader (Awareness
Technology Inc, USA) at = 545.
Flow cytometry analysis. Cells were cultured
in 10 cm dishes in DMEM culture medium supple-
mented with 10% FBS to 50% confluency, then the me-
dium was replaced by the fresh DMEM supplemented
with 2.5% FBS, and the cells were treated by addition
of 1; 10; 100; 250; 500; 750, or 1000 nM rec-hBD-4 for
48 h. After the treatment, the attached cells were triply
washed with PBS, harvested by trypsinization, pelleted
at 4 °C (500 g) for 5 min, washed twice in PBS, and re-
78 Experimental Oncology 35, 76–82, 2013 (June)
suspended in 1 ml of hypotonic cell lysis buffer (0.1%
sodium citrate, 0.1% Triton X-100, 5 mg/ml PI (Sigma,
USA)). The cells were incubated at RT for 30 min in the
dark, and cell cycle distribution was analyzed using
cytometer Becton Dickinson FACS Calibur. The data
were analyzed with the use of CellQuest software
package and ModFit LT2.0 program (BDIS, USA) for
Mac computers.
Antimicrobial activity of rec-hBD-4 was analyzed
against P. aeruginosa by bacterial growth suppression
in radial diffusion assay [30]. P. aeruginosa ATCC
9027 strain was obtained from Ukrainian Collection
of Microorganisms (D.K. Zabolotny Institute of Micro-
biology and Virology, NAS of Ukraine, Kyiv, Ukraine).
Scratch wound healing assay. An effect of rec-
hBD-4 on mobility of cultured human cancer cells was
performed by scratch wound healing assay [31]. The
cells were seeded in 6-well plates (15x104 cells per
well) and routinely grown till 80% confluence, than
a scratch was performed using a 20 μl tip, and the
cells were grown for next 24 h in the presence of 100,
500 or 1000 nM rec-hBD-4 in serum free medium.
Then the cell migration into wound area was examined
using inverted microscope Axioplan (Zeiss, Germany).
Colony forming assay. Substrate independent
growth was studied by routine procedure [32]. The
cells were seeded in 3 cm Petri dishes (5х103 cells)
in the medium containing 0.8% methylcellulose, 2x
DMEM, 30% FBS and 100; 500, or 1000 nM rec-hBD-4,
and were cultured in 5% СО2 at 37 °С for 2 weeks.
Then the colonies were stained with 0.01% neutral red
in PBS for 30 min, and examined by light microscopy.
TotalLab program has been used for data calculation.
Statistical analysis. The data are reported as the
mean ± SD. The statistical significance of diffe rences
between mean values was assessed by the Stu-
dent’s t-test. Values p < 0.05 were considered statisti-
cally significant.
RESULTS AND DISCUSSION
hBD-4 cloning and prokaryotic expression
of hBD-4-GST fusion protein. To clone the gene
encoding mature hBD-4, a predicted 50 amino acid
protein of 5.9 kDa, hBD-4 cDNA was amplified from
total RNA isolated from human lung squamous cell
carcinoma sample No 5 [27]; in this particular tumor
sample we have registered hBD-4 up-regulation dur-
ing the screening of hBDs expression in human lung
tumors [27].
For expression of rec-hBD-4 we have used GST-
system for production of recombinant proteins that
allows protein expression in E. coli strains in a form
of chimeric proteins fused to GST tag. Both PCR
product and pGEX-2T vector were purified, digested
by BamHI and EcoRI restriction endonucleases, li-
gated and the hybrid plasmid was used for subsequent
transformation of E. coli BL21(DE3) cells. After selec-
tion of colonies, recombinant vectors were analyzed
by digestion with BamHI and EcoRI endonucleases;
the result evidenced correct 150 bp insertion (Fig. 1)
which sequence was shown to correspond to nucleo-
tide sequence of hBD-4 gene by direct DNA sequenc-
ing analysis.
150 bp.
1 2 3 4 5
Fig. 1. Recombinant hBD-4-pGEX-2T construct digested with
BamH1 and EcoRI endonucleases. Lines 1, 2 — undigested
plasmid; 3, 4 — recombinant plasmid digested with BamH1 and
EcoRI endonucleases; line 5 — DNA ladder (Fermentas, USA)
Purification of rec-hBD-4 has been performed
by routine three-step procedure including affine
chromatography, proteolysis of fusion protein with
thrombin and reverse phase chromatography. In brief,
E. coli BL21DE3 cells transformed with hBD-4-pGEX-
2T construct were induced with 1 mM IPTG for 6 h;
the cells were collected and disrupted by sonication
in lysis buffer containing 1% Triton Х-100 and cocktail
of protease inhibitors. Bacterial cell lysate was clari-
fied by centrifugation, and supernatant was applied
on glutathione-agarose column (GE Healthcare,
Sweden); affine chromatography was performed
by routine procedure according to instructons of the
manufacturer. Then the fusion protein was treated
with thrombin (1 U of thrombin per 1 mg of fusion
protein), and hBD-4 peptide was purified from pro-
teolytic mixture by reverse phase chromatography
on Sep-Pak С18 column (Water, USA) in acetonitrile
gradient. Pure hBD-4 fraction was eluted by 70–100%
acetonitrile, collected, dried in Speed-Vac, analyzed
by gradient 7–22% SDS-PAGE (Fig. 2) and by in-
hibitory zone test. Rec-hBD-4 was active against P.
aeruginosa in a dose of 10–15 μM (data not shown).
In summary, prokaryotic expression of hBD-4 and pu-
rification procedure allowed to purify up to 5 mg of bi-
ologically active rec-hBD-4 from 1 L of IPTG-induced
bacterial culture.
Effect of rec-hBD-4 on proliferation and viabi-
lity of human cancer cells in vitro. We have analyzed
an effect of rec-hBD-4 on proliferation and viability
of three human cancer cell lines — A431 (epidermoid
carcinoma cells), A549 (lung adenocarcinoma cells),
and TPC-1 (papillary thyroid cancer cells). As it has
been shown by direct cell counting technique (Fig. 3),
rec-hBD-4 exerts a concentration-dependent effect
on the cell proliferation and stimulates cell proliferation
in the concentrations from 1 to 100 nM and significantly
suppresses — in concentrations ≥ 500 nM. Effects
of hBD-4 were similar in A431 and A549 cells but
TPC-1 cells seems to be less sunsitive to low nano-
molar concentration of rec-hB44 (Fig. 3). According
to the data of trypan blue staining, the percentage
of dead cells in all samples didn’t exceed 4–5% for
any hBD-4 concentration, so one may conclude that
in nanomolar concentrations this defensin does not
cause cell death.
Experimental Oncology 35, 76–82, 2013 (June) 79
— rec-hBD-4 (5,9 kDa)
1 2 3 4
Fig. 2. SDS-PAGE analysis of rec-hBD-4 purified by reverse
phase chromatography on Sep-Pak С18 (Water, USA). 1 —
molecular weight marker (SM 0661, Fermentas, USA); fractions
of rec-hBD-4 eluted with 80% CH3CN (2), 70% CH3CN (3), 100%
CH3CN (4)
0
20
40
60
80
100
120
140
160
Control 0.1 1 100 500 1000
hBD-4 concentration, nM
Ce
ll
nu
m
be
r,
%
А431
А549
TPC-1 *
*
*
* *
* * *
*
Fig. 3. A concentrat ion-dependent ef fect of rec-
hBD-4 on the number of viable cultured cells of A431, A549,
and TPC-1 lines. The number of attached cells was evaluated
by direct cell counting. Cells were cultured in 24-well plates
(5x104 cells per well) to nearly 50% confluence, then culture
medium was replaced with fresh DMEM supplemented with
2.5% FBS and rec-hBD-4 was added into cell cultivation me-
dium in concentrations of 0.1; 1; 100; 500; 1000 nM, and the
cells were cultured with rec-hBD-4 for 48 h. The data of three
independent experiments are presented as the mean ± SD.
*The difference is significant as compared to appropriate
control (p < 0.05)
Next, we have analyzed the effect of rec-hBD-4 on vi-
ability of cultured A431, A549 and TPC-1 cells using MTT
assay. Our data have shown that in low concentrations
(0.1–100 nM) rec-hBD-2 caused insignificant increase
of viability of A431, TPC-1 and 549 cells (Fig. 4). In all
three cell lines treated with 500 nM of rec-hBD-4, a de-
crease of cell viability has been recorded while treatment
with 1 μM of rec-hBD-4 resulted in significant increase
of cell viability (Fig. 4). In concentrations more than
1 μM rec-hBD-4 caused significant suppression of vi-
ability of A431, A549 and TPC-1 cells.
Interestingly, such multimodal effects of rec-
hBD-4 on cell viability were found to be reflected in its
effects on cell cycle distribution. When A431 cells
treated with 1 nm — 1 μM rec-hBD-4 were subjected
to flow cytofluorometry analysis, it has been revealed
(Fig. 5) that treatment of the cells with 100 nM rec-
hBD-4 resulted in significant stimulation of cell cycle
in G2/M checkpoint, with 500 nM — in significant
blockage of cell cycle in G1/S checkpoint; higher
concentrations of hBD-4 insignificantly suppressed
cell cycle progression (Fig. 5). Similar results were
obtained with A549 and TPC-1 cells treated with rec-
hBD-4 (data not shown).
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
Control 0.1 1 10 100 500 1000 5000
HBD4 concentration, nM
Ab
so
rb
an
ce
(5
45
n
m
)
А431
А549
ТРС1
Fig. 4. A concentration-dependent effect of rec-hBD-4 on viabil-
ity of A431, A549, and TPC-1 cells. Cells were cultured in 96-well
plates (7x103 cells per well) and incubated with rec-hBD-4 in con-
centrations from 100 pM to 5 M for 48 h in medium supple-
mented with 2.5% FBS. The number of viable cells was evaluated
by MTT analysis. The data of three independent experiments are
presented as the mean ± SD
0
10
20
30
40
50
60
70
80
90
Control 1 10 100 250 500 750 1000
hBD-4 concentration, nM
Ce
ll
nu
m
be
r,
%
apoptosis
G0/G1
G2/M
S
*
*
*
*
*
* *
Fig. 5. Flow cytofluorometry analysis of cell cycle distribu-
tion of A431 cells treated with rec-hBD-4 for 48 h. Cells were
cultured in 10 cm dishes in culture medium supplemented with
10% FBS to 50% confluency, then the medium was replaced
by fresh DMEM supplemented with 2.5% FBS, and cells were
treated by addition of 1; 10; 100; 250; 500; 750, or 1000 nM rec-
hBD-4 for 48 h. After the treatment, the cells were triply washed
with PBS, detached with trypsin and subjected to flow cytofluo-
rometry analysis as described in Materials and Methods sec-
tion. The data of two independent experiments are presented
as the mean ± SD.
*The difference is significant as compared to appropriate
control (p < 0.05)
Rec-hBD-4 affects colony-forming activity
of epithelial cancer cells. We have performed
colony forming assay to evaluate possible effects
of rec-hBD-4 on important biological property
of cancer cells — their ability for substrate in-
dependent growth. In this experiment we have
used three concentrations of hBD-4 (100, 500,
1000 nM); addition of the defensin into cell in-
cubation medium significantly affected A431cell
ability to form colonies in semi-soft medium (Fig.
6): treatment with 100 nM rec-hBD-4 resulted
in significant increase of colony numbers com-
pared to control untreated cells (p < 0.05), with
500 nM — in significant decrease of colony counts
(p < 0.05), while in the presence of 1 M of the de-
fensin no visible colonies were developed. Similar
80 Experimental Oncology 35, 76–82, 2013 (June)
results were obtained with A549 cells treated with
rec-hBD-4 (data not shown).
0
20
40
60
80
100
120
140
160
180
A B C D
Co
lo
ny
n
um
be
rs
BA
DC
*
*
Fig. 6. Rec-hBD-4 significantly affects colony-forming activity
of A431 cells. The cells (5х103) were cultured in 3 cm Petri dishes
(5х103 cells) in the medium containing 0.8% methylcellulose, 2x
DMEM, 30% FBS in the absence (control cells, A) or presence
of rec-hBD-4 (100 nM (B), 500 nM (C) or 1 μM (D)). When visible
colonies developed the plates were stained with 0.01% neutral
red. The graph represents the numbers of colonies calculated
with the use of TotalLab program.
*The difference is significant as compared to appropriate
control (p < 0.05)
Effect of rec-hBD-4 on cancer cell migra-
tion ability. To analyze a possible effect of rec-
hBD-4 on cancer cell mobility, wound healing as-
say has been applied. A431 and TPC-1 cells were
seeded in 6-well plates (15x104 cells per well) and
routinely grown till 80% confluence, then a scratch
was performed using a 20 μL tip, and the cells
were grown for next 48 h in the presence of 100;
500 or 1000 nM of rec-hBD-4 in serum free me-
dium. Light microscopy examination has revealed
(Fig. 7) that 500 nM of rec-hBD-4 significantly sup-
pressed migration of TPC-1 cells while treatment with
100 or 1000 nM of rec-hBD-4 seems to have no notable
effect on TPC-1 cell mobility. In A431 cell line defen-
sin moderately affected cell migration: stimulation
of cell mobility has been detected in cells treated with
100 nM hBD-4 but 500 or 1000 nM of the defensin
had insignificant influence on migration of A431 cells
(Fig. 7).
DISCUSSION
Antimicrobial peptides in general and defensins
in particular are in spite of scientific interest in recent
years due to the search for new antibiotics which
could be used against highly resistant clinical isolates.
Really, defensins as natural compounds with potent
antimicrobial activity could be successfully used to kill
even antibiotic-resistant pathogens [33]. Apart from
this, defensins may be potentially used in other fields
of medicine, for example, as anticancer or wound-
healing agents [13, 34].
For experimental purposes defensins are produced
as synthetic peptides or recombinant molecules ex-
pressed in prokaryotes. In the latest case, to avoid host
cell autodestruction and to simplify purification proce-
dures, defensins are cloned and expressed in a form
of fusion proteins containing His-Tag, GST, TrxA,
or other tags. Such approach has allowed to produce
biologically active recombinant defensins — hBD-3 [9],
hBD-2 [29], hBD-26 and -27 [35], and some others.
Similarly, in present work we have produced soluble
biologically active recombinant hBD-4 expressed
in prokaryotic system as GST-hBD-4 fusion peptide
and for the first time characterized its in vitro activity
toward human cancer cells.
Up to date, just few articles have described the
biologic activity of hBD-4 [8, 36]. In these studies
synthetic peptide has been used; it has been demon-
strated that hBD-4 is active against P. aeruginosa and
Staphylococcus carnosus, and its antimicrobial activity
depends on salt concentration; hBD-4 is chemotac-
tic for human blood monocytes at the concentration
of 10 nM, and its chemotactic activity is independent
from intracellular calcium mobilization [8]. hBD-4 has
been supposed to play an important role in defense
of low respiratory tract against infections, especially
those caused by P. aeruginosa [36], and in H. pylori
associated gastritis [37].
b
a
Fig. 7. Wound healing assay in A431 (a) and TPC-1 (b) cells
treated with 100 nM (B), 500 nM (C) or 1000 nM (D) rec-hBD-4 for
48 h. A — control untreated cells. The results of typical experi-
ment are presented
Our research has demonstrated for the first time
that hBD-4 could exert a concentration dependent ef-
fect on biological properties of cultured human cancer
cells: in low nanomolar concentrations (1–100 nM) rec-
Experimental Oncology 35, 76–82, 2013 (June) 81
hBD-4 stimulates cancer cell proli feration and viability,
promotes cell cycle progression through G2/M check-
point, significantly enhances colony-forming activity
and mobility of A431, A549, and TPC-1 cells. Treatment
of cells with 500 nM of rec-hBD-2 resulted in opposite
effects: significant suppression of cell proliferation and
viability, blockage of cell cycle in G1/S checkpoint, sig-
nificant inhibition of cell migration and colony forming
activity. In concentrations more than 5 μM hBD-4 is cy-
totoxic to all studied cell lines. It’s necessary to note
that biologic effects of rec-hBD-4 were similar, but not
equal in all three studied cell lines: for example, thyroid
papillary carcinoma cell migration ability is more sensitive
to hBD-4 than that of A431 cells.
So, biologic effects of nanomolar concentrations
of rec-hBD-4 are of bimodal character, unlike to these
of recombinant hBD-2 described in our earlier publica-
tion [21]. So, hypothetically, at low nanomolar range
human beta-defensin-4 may play a role of pro-onco-
genic molecule via stimulation of cell proliferation and
cell malignancy potential, and in high nanomolar range
hBD-4 could play a role in tumor suppression. It’s of in-
terest to further explore the mechanisms of regulation
of hBD-4 expression in human epithelial cells, patterns
of its expression in human carcinoma versus normal
tissue, and its physiologic content in biologic fluids and
tissues to understand the possible role of this defensin
in tumorigenesis.
ACKNOWLEDGEMENT
This work was in part supported with NASU grant
0110U005758 “Fundamental basis of molecular and
cellular biotechnologies”.
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