Involvement of human beta-defensin-2 in regulation of malignant potential of cultured human melanoma cells
Background and Aim: Human beta-defensin-2 (hBD-2) is an antimicrobial cationic peptide capable to control human carcinoma cell growth via cell cycle regulation. The present study was aimed on determination of hBD-2 influence on the growth patterns and malignant potential of cultured human melanoma c...
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| Zitieren: | Involvement of human beta-defensin-2 in regulation of malignant potential of cultured human melanoma cells / O. Gerashchenko, E. Zhuravel, O. Skachkova, N. Khranovska, V. Pushkarev, P. Pogrebnoy, M. Soldatkina // Experimental Oncology. — 2014. — Т. 36, № 1. — С. 17-23. — Бібліогр.: 35 назв. — англ. |
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irk-123456789-1453132019-01-21T01:23:42Z Involvement of human beta-defensin-2 in regulation of malignant potential of cultured human melanoma cells Gerashchenko, O. Zhuravel, E. Skachkova, O. Khranovska, N. Pushkarev, V. Pogrebnoy, P. Soldatkina, M. Original contributions Background and Aim: Human beta-defensin-2 (hBD-2) is an antimicrobial cationic peptide capable to control human carcinoma cell growth via cell cycle regulation. The present study was aimed on determination of hBD-2 influence on the growth patterns and malignant potential of cultured human melanoma cells. Methods: The study was performed on cultured human melanoma cells of mel Z and mel Is lines treated with recombinant hBD-2 (rec-hBD-2); cell viability, proliferation, cell cycle distribution, and anchorage-independent growth were analyzed using MTT test, direct cell counting, flow cytometry, and colony forming assay respectively. Expression and/or phosphorylation levels of proteins involved in cell cycle control were evaluated by Western blotting. Results: The treatment of mel Z and mel Is cells with rec-hBD-2 in a concentration range of 100–1000 nM resulted in a concentration-dependent suppression of cell proliferation, viability, and colony forming activity. It has been shown that rec-hBD-2 exerts its growth suppression effects via significant downregulation of B-Raf expression, activation of pRB and upregulation of p21WAF1 expression, downregulation of cyclin D1 and cyclin E resulting in cell cycle arrest at G1/S checkpoint. Conclusion: According to obtained results, hBD-2 exerts its growth suppression effect toward human melanoma cells via downregulation of B-Raf, cyclin D1 and cyclin E expression, upregulation of p21WAF1 expression and activation of pRB. Key Words: human beta-defensin-2, melanoma, proliferation, viability, cell cycle, B-Raf, anchorage-independent growth. 2014 Article Involvement of human beta-defensin-2 in regulation of malignant potential of cultured human melanoma cells / O. Gerashchenko, E. Zhuravel, O. Skachkova, N. Khranovska, V. Pushkarev, P. Pogrebnoy, M. Soldatkina // Experimental Oncology. — 2014. — Т. 36, № 1. — С. 17-23. — Бібліогр.: 35 назв. — англ. 1812-9269 http://dspace.nbuv.gov.ua/handle/123456789/145313 en Experimental Oncology Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
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Original contributions Original contributions Gerashchenko, O. Zhuravel, E. Skachkova, O. Khranovska, N. Pushkarev, V. Pogrebnoy, P. Soldatkina, M. Involvement of human beta-defensin-2 in regulation of malignant potential of cultured human melanoma cells Experimental Oncology |
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Background and Aim: Human beta-defensin-2 (hBD-2) is an antimicrobial cationic peptide capable to control human carcinoma cell growth via cell cycle regulation. The present study was aimed on determination of hBD-2 influence on the growth patterns and malignant potential of cultured human melanoma cells. Methods: The study was performed on cultured human melanoma cells of mel Z and mel Is lines treated with recombinant hBD-2 (rec-hBD-2); cell viability, proliferation, cell cycle distribution, and anchorage-independent growth were analyzed using MTT test, direct cell counting, flow cytometry, and colony forming assay respectively. Expression and/or phosphorylation levels of proteins involved in cell cycle control were evaluated by Western blotting. Results: The treatment of mel Z and mel Is cells with rec-hBD-2 in a concentration range of 100–1000 nM resulted in a concentration-dependent suppression of cell proliferation, viability, and colony forming activity. It has been shown that rec-hBD-2 exerts its growth suppression effects via significant downregulation of B-Raf expression, activation of pRB and upregulation of p21WAF1 expression, downregulation of cyclin D1 and cyclin E resulting in cell cycle arrest at G1/S checkpoint. Conclusion: According to obtained results, hBD-2 exerts its growth suppression effect toward human melanoma cells via downregulation of B-Raf, cyclin D1 and cyclin E expression, upregulation of p21WAF1 expression and activation of pRB. Key Words: human beta-defensin-2, melanoma, proliferation, viability, cell cycle, B-Raf, anchorage-independent growth. |
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Article |
| author |
Gerashchenko, O. Zhuravel, E. Skachkova, O. Khranovska, N. Pushkarev, V. Pogrebnoy, P. Soldatkina, M. |
| author_facet |
Gerashchenko, O. Zhuravel, E. Skachkova, O. Khranovska, N. Pushkarev, V. Pogrebnoy, P. Soldatkina, M. |
| author_sort |
Gerashchenko, O. |
| title |
Involvement of human beta-defensin-2 in regulation of malignant potential of cultured human melanoma cells |
| title_short |
Involvement of human beta-defensin-2 in regulation of malignant potential of cultured human melanoma cells |
| title_full |
Involvement of human beta-defensin-2 in regulation of malignant potential of cultured human melanoma cells |
| title_fullStr |
Involvement of human beta-defensin-2 in regulation of malignant potential of cultured human melanoma cells |
| title_full_unstemmed |
Involvement of human beta-defensin-2 in regulation of malignant potential of cultured human melanoma cells |
| title_sort |
involvement of human beta-defensin-2 in regulation of malignant potential of cultured human melanoma cells |
| publisher |
Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
| publishDate |
2014 |
| topic_facet |
Original contributions |
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http://dspace.nbuv.gov.ua/handle/123456789/145313 |
| citation_txt |
Involvement of human beta-defensin-2 in regulation of malignant potential of cultured human melanoma cells / O. Gerashchenko, E. Zhuravel, O. Skachkova, N. Khranovska, V. Pushkarev, P. Pogrebnoy, M. Soldatkina // Experimental Oncology. — 2014. — Т. 36, № 1. — С. 17-23. — Бібліогр.: 35 назв. — англ. |
| series |
Experimental Oncology |
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2025-07-10T21:24:11Z |
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2025-07-10T21:24:11Z |
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| fulltext |
Experimental Oncology 36, 17–23, 2014 (March) 17
INVOLVEMENT OF HUMAN BETA-DEFENSIN-2 IN REGULATION
OF MALIGNANT POTENTIAL OF CULTURED HUMAN MELANOMA
CELLS
O. Gerashchenko1, E. Zhuravel1, O. Skachkova2, N. Khranovska2, V. Pushkarev3, P. Pogrebnoy1, M. Soldatkina1,*
1R.E. Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology, NAS of Ukraine, Kyiv 03022,
Ukraine
2National Cancer Institute MPH of Ukraine, Kyiv 03022, Ukraine
3State Institution “V.P. Komisarenko Institute of Endocrinology and Metabolism”, AMS of Ukraine, Kyiv 04114,
Ukraine
Background and Aim: Human beta-defensin-2 (hBD-2) is an antimicrobial cationic peptide capable to control human carcinoma
cell growth via cell cycle regulation. The present study was aimed on determination of hBD-2 influence on the growth patterns and
malignant potential of cultured human melanoma cells. Methods: The study was performed on cultured human melanoma cells
of mel Z and mel Is lines treated with recombinant hBD-2 (rec-hBD-2); cell viability, proliferation, cell cycle distribution, and
anchorage-independent growth were analyzed using MTT test, direct cell counting, flow cytometry, and colony forming assay
respectively. Expression and/or phosphorylation levels of proteins involved in cell cycle control were evaluated by Western blotting.
Results: The treatment of mel Z and mel Is cells with rec-hBD-2 in a concentration range of 100–1000 nM resulted in a concen-
tration-dependent suppression of cell proliferation, viability, and colony forming activity. It has been shown that rec-hBD-2 exerts
its growth suppression effects via significant downregulation of B-Raf expression, activation of pRB and upregulation of p21WAF1 ex-
pression, downregulation of cyclin D1 and cyclin E resulting in cell cycle arrest at G1/S checkpoint. Conclusion: According to ob-
tained results, hBD-2 exerts its growth suppression effect toward human melanoma cells via downregulation of B-Raf, cyclin D1 and
cyclin E expression, upregulation of p21WAF1 expression and activation of pRB.
Key Words: human beta-defensin-2, melanoma, proliferation, viability, cell cycle, B-Raf, anchorage-independent growth.
Human melanoma is the most lethal form of skin
cancer with constantly rising incidence worldwide.
While early stage melanoma is curable, melanoma
patients with advanced disease have poor prognosis
because this type of cancer is resistant to chemothe-
rapy [1]. That’s why there is a need for the develop-
ment of new strategies for melanoma treatment.
Cationic antimicrobial peptides (CAPs) of eukaryotic
origin are among many agents with potential anti-mel-
anoma activity. These peptide antibiotics are produced
by many living species including mammals. CAPs are
active against different bacterial pathogens, viruses,
and fungi. Therapeutic potential of peptide antibiotics
as anti-infective agents is considered pro mising despite
the difficulties related to their pharmacokinetics, toxicity
and immunogenicity [2]. Moreover, some of CAPs exert
cytotoxic activity toward cancer cells. This ability of CAPs
is thought to be related to the biophysical properties
of their molecules, in particular high cationic charge and
amphipathicity allowing interaction of CAP molecules
with negatively charged cancer cell membranes and cell
lysis [3]. The know ledge on cytotoxic activity of particular
CAPs against cancer cells in vitro and in vivo has been
gained. Moreover, a number of these peptides have
been studied in experimental models as anticancer
agents [4]. In particular, some antimicrobial peptides, like
cathelicidin-BF from the venom of the snake Bungarus
fasciatus [5], crotamine from the venom of Crotalus
durissus terrificus rattlesnake [6], or gomesin from
hemocytes of the spider Acanthoscurria gomesiana [7]
have been demonstrated to be effective for experimental
B16 melanoma treatment in vivo.
In humans, CAPs are represented by the host defense
peptides of defensin family (human α- and β-defensins,
hBDs) and cathelicidin LL-37, important components
of innate immunity protecting organism from pathogens
along with many other biologically active molecules [8, 9].
It has been assumed that hBDs and LL-37 play a critical
role in antimicrobial protection of human skin and in wound
healing processes, and dysregulation of hBDs expres-
sion (in particular, hBD-2) is implicated in pathogenesis
of many skin pathologies, in particular, psoriasis and atopic
dermatitis [10–12]. At the same time, despite the steady
progress in understanding hBDs role in inflammatory
skin disorders, little is known about possible implication
of these peptides in the development of skin cancer.
It has been shown recently that in cutaneous squa-
mous cell carcinoma (SCC) hBD-1 is downregulated,
while hBD-2 and hBD-3 are upregulated compared
to healthy skin. So, the authors supposed that in SCC
hBD-1 may play a role of tumor suppressor, while
hBD-2 and hBD-3 may promote skin cancer develop-
ment [13]. However, hypothetically, the resulting effect
of hBDs expression could depend on many factors, in-
cluding their local concentrations in particular tumor mi-
Received: February 2, 2014.
*Correspondence: E-mail: pogrebnoy@onconet.kiev.ua
Fax: (038) 044 2581656
Abbreviations used: CAP(s) — cationic antimicrobial peptide(s);
GST — glutathione-S-transferase; hBDs — human beta-defensins;
hBD-2 — human beta-defensin-2; MC1R — melanocortin-1 receptor;
rec-hBD-2 — recombinant hBD-2; SCC — squamous cell carcinoma.
Exp Oncol 2014
36, 1, 17–23
18 Experimental Oncology 36, 17–23, 2014 (March)
croenvironment as far as these antimicrobials are shown
to exert different effects in a concentration-dependent
manner [14–17]. For example, hBD-2 and hBD-4 in low
nanomolar concentration range stimulate carcinoma cell
proliferation, but at higher doses are capable to block cell
cycle and drastically suppress cancer cell growth in vitro
[15, 17]. In high concentrations some human defensins
exert potent cytotoxic activities against cancer cells in vi-
tro and in vivo, similarly to CAPs of other origin [18, 19].
Up-to-date, the effects of hBDs in physiological
(nanomolar) concentration range on melanocytes and
melanoma cells remains poorly studied. Melanoma arise
from melanocytes, pigment dendritic cells located in epi-
dermis and hair follicles. Theoretically, their behavior
could be regulated through paracrine way by defensins
produced by neighboring keratinocytes. Indeed, it has
been established that one of the inducible β-defensins,
namely hBD-3, expression of which could be induced
in keratinocytes by UVR [20], in low nanomolar concen-
trations exerts pigmentary effects and is a high-affinity
ligand of melanocortin-1 receptor (MC1R). It is well
recognized that MC1R is a receptor, which play a central
role in pigmentation and in melanoma susceptibility in hu-
mans. It is supposed that hBD-3 may play an important
role in melanocyte biology, response of human skin
to UVR and skin cancer susceptibility [21, 22]. The ef-
fects of other hBDs on melanoma cells remain unknown.
In the present study we addressed a question whe-
ther exogenous recombinant hBD-2 (rec-hBD-2) could
regulate the growth patterns and malignancy potential
of cultured human melanoma cells. We have revealed
that proliferation and viability of human melanoma cells
are regulated by hBD-2 in a concentration-dependent
manner. In particular, rec-hBD-2 in high nanomolar range
suppresses viability and proliferation of melanoma cells
via cell cycle arrest in G1/S checkpoint and markedly
reduces their colony forming activity. Significant down-
regulation of B-Raf, cyclin E and cyclin D1 expression
along with dephosphorylation of pRB in human mela-
noma cells treated with hBD-2 have been demonstrated.
These data may be useful for further development of new
approaches for melanoma treatment using CAPs.
MATERIALS AND METHODS
Cell lines and recombinant hBD-2 peptide.
The study was carried out on 2 human melanoma cell
lines — mel Z and mel Is, which were kindly provided
by prof. A.Y. Baryshnikov (N.N. Blokhin Russian Cancer
Research Center RAMS, Moscow, Russian Federa-
tion). Mel Z and mel Is cell lines were established from
surgically resected metastatic lesions of patients with
malignant disseminated melanoma by Dr. O.S. Bu-
rova (N.N. Blokhin Russian Cancer Research Center
RAMS, Moscow, Russian Federation) [23].
Cell lines were cultured in DMEM culture medium
supplemented with 10% fetal bovine serum (FBS),
100 units/mL penicillin G sodium, 100 μg/mL streptomy-
cin sulfate in humidified 5% CO2 atmosphere at 37 oC.
To study the effect of exogenous defensin on cell
growth, we used purified recombinant hBD-2 [24].
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 centrifugation,
resuspended in lysis buffer (50 mМ Tris-HCl, pH 7.6;
250 mМ NaCl; 1% Triton Х-100 and cocktail of prote-
ase and phosphatase inhibitors), and disrupted using
ultrasound disintegrator (UD-11 Automatic, Poland).
Then cell lysate was applied to affinity chromatography
on glutathione-agarose column (GE Healthcare, Swe-
den) with following cleavage of the defensin from fusion
protein by thrombin digestion. hBD-2 peptide was fur-
ther purified by reverse phase chromatography on Sep-
Pack C18 cartridge (Waters, USA), vacuum-dried, and
re-dissolved in acidified 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, mel Z and mel Is 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 concentrations of 100, 500, or 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.
MT T assay. To evaluate the effect of rec-
hBD-2 on cell viability, MTT-test has been applied
[25]. Mel Z and mel Is cells were seeded into 96-
well plates (7•103 cells per well) and incubated
with rec-hBD-2 at the concentration range from
1 pM to 2 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-diphenyltetrazoli-
um bromide) according to standard protocol, and colo-
rimetric reaction was evaluated with the use of ELISA
reader (Awareness Technology Inc, USA) at = 545.
Flow cytometry analysis. Mel Z and mel Is cells
were cultured in 10 cm Petri dishes in DMEM supple-
mented with 10% FBS to 50% confluence, then the me-
dium was replaced by the fresh DMEM supplemented
with 2.5% FBS, and cells were treated with 1, 10, 100,
500, or 1000 nM rec-hBD-2 for 48 h. After the treatment,
the attached cells were washed with PBS, harvested
by trypsinization, pelleted at 4 oC (500 g) for 5 min,
washed twice in PBS, and resuspended in 1 ml of hy-
potonic cell lysis buffer (0.1% sodium citrate, 0.1%
Triton X-100, 5 g/ml propidium iodide (Sigma, USA)).
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.
Western blot analysis. To analyze the level of ex-
pression and/or phosphorylation of some components
of signaling pathways involved in cell cycle regulation,
melanoma cells were cultured in 6-well plates and
treated with 1000 nM rec-hBD-2 for 48 h as described
above, washed with PBS and lyzed in RIPA buffer
Experimental Oncology 36, 17–23, 2014 (March) 19
with protease and phosphatase inhibitors. Proteins
were separated in 9–22% gradient SDS-PAGE and
transferred to nitrocellulose membrane Hybond-ECL,
RPN3032D (Amersham Biosciences, USA). Nonspecific
binding sites were blocked with 1Х PBS-Т, 5% milk solu-
tion for 3 h. Then the blots were incubated sequentially
with primary Abs, and then with secondary polyclonal
HRP-conjugated anti-rabbit IgG or anti-mouse IgG Abs
(DakoCytomation, Denmark). The ECL Western blot-
ting detection system (Amersham Pharmacia Biotech)
was used to reveal immunoreactivity. For Western blot-
ting, we have used antibodies against pRB, cyclin D1,
phospho-pRB (S780), and CDK4 from Cell Signaling
Tech (USA), p53 (IEPOR, Ukraine), p21WAF1 (Oncogene,
USA), cyclin E, B-Raf (Santa-Cruz, USA), and beta-actin
(Sigma, USA). All antibodies were used at the working
dilutions accor ding to manufacturer instructions.
Colony forming assay and melanin content anal-
ysis. Substrate independent growth was studied by rou-
tine procedure [26]. The cells were seeded in 3 cm Petri
dishes (5•103 cells) in the medium conta ining 0.5%
methylcellulose, DMEM, 15% FBS with or without addi-
tion of 100, 500, or 1000 nM rec-hBD-2. After 2-weeks
culturing in 5% СО2 at 37 оС, the developed colonies
were stained with 0.01% neutral red in PBS for 30 min,
and examined by light microscopy. TotalLab 2.0 pro-
gram (UK) has been used for data evaluation.
Melanin content in cultured human melanoma
cells was analyzed using standard Fontana — Masson
method [27].
Statistical analysis. The data are reported
as the mean ± SD. Data on direct cell counting, MTT,
flow cytometry analysis and colony forming assay were
analyzed by Student’s t-test to assess the statistical
significance of the difference between the groups.
A statistically significant difference was considered
to be present at p < 0.05.
RESULTS
The study has been performed in vitro using two hu-
man melanoma cell lines — mel Z cells originating from
moderately differentiated melanoma, and mel Is origi-
nating from low differentiated melanoma [23].
The analysis of rec-hBD-2 effect on proli-
feration and viability of cultured human melanoma
cells. To study the effect of rec-hBD-2 on growth rates
of cultured melanoma cell lines, direct cell counting
technique has been used. Мel Z and mel Is cells
were treated with rec-hBD-2 that was added into cell
culture medium at different concentrations (100 nM,
500 nM, 1 M) for 48 h. Rec-hBD-2 caused significant
concentration-dependent suppression of mel Z proli-
feration (p < 0.05), while in mel Is cells growth suppres-
sion has been observed only in the case of treatment
with 500 nM and 1 M rec-hBD-2 (p < 0.05) (Fig. 1).
Effect of the defensin on melanoma cell proliferation
was hBD-2-specific because preincubation of rec-
hBD-2 with anti-hBD-2 Abs (Santa-Cruz, USA) prior
to its addition into cell culture medium completely
abolished the above mentioned effects. According
to the data of trypan blue staining, the percentage
of dead cells in all samples didn’t exceed 3.5–4% for
all tested rec-hBD-2 concentrations.
0
20
40
60
80
100
120 Mel Z
1
**
*
*
*
*
2 3 4 5 1 2 3 4 5
Mel Is
Ce
ll
nu
m
be
r,%
Fig. 1. A concentration-dependent effect of rec-hBD-2 on the num-
ber of viable cultured human melanoma cells of mel Z and mel Is lines.
Mel Z and mel Is cells were treated with rec-hBD-2 in concentra-
tions of 100 nM (columns 2), 500 nM (column 3), 1000 nM (column
4); column 1 — control cells; column 5 — the cells were treated
with 500 nM rec-hBD-2 after its 30 min preincubation with anti-
hBD-2 antibodies (Santa-Cruz, USA). The number of attached cells
was evaluated by direct cell counting. The data of three independent
experiments are presented as the mean ± SD. *The diffe rence
is significant as compared to appropriate control (p < 0.05)
Next, we have analyzed the effect of rec-hBD-2 on vi-
ability of cultured mel Z and mel Is cells using MTT
assay. It has been shown that at low concentrations
(1 pM — 1 nM) rec-hBD-2 didn’t affect significantly
viability of both cell lines, while treatment of the cells
with higher concentrations of rec-hBD-2 (10 nM —
1000 nM) resulted in significant dose-dependent de-
crease of cell viability (p < 0.05) (Fig. 2).
0
20
40
60
80
100
120
Co
nt
ro
l
0.
00
1
0.
01
0.
05 0.
1 1 10 10
0
50
0
10
00
20
00
hBD-2 concentration, nM
Vi
ab
ili
ty
, %
fr
om
c
on
tro
l
Mel Z
Mel Is
*
*
*
* *
Fig. 2. A concentration-dependent effect of rec-hBD-2 on viabi-
lity of mel Z and mel Is cells. Cell viability was evaluated by MTT
analysis. The data of three independent experiments are present-
ed as the mean ± SD. *The difference is significant as compared
to appropriate control (p < 0.05)
So, the data of direct cell counting and MTT de-
monstrated that low-differentiated melanoma mel Is cells
are less sensitive to growth suppressing action of rec-
hBD-2 than moderately differentiated mel Z cells. An-
other important observation is an absence of apoptotic
effects of the defensin toward cultured melanoma cells:
the percentage of dead cells slightly differed between
the samples treated with different hBD-2 concentra-
tions. This fact was further confirmed by the data of flow
cytometry (see below) and analysis of PARP expression
in hBD-2 treated melanoma cells (data not shown).
20 Experimental Oncology 36, 17–23, 2014 (March)
Rec-hBD-2 affects cell cycle distribution and
signaling pathways involved in cell cycle regulation
in vitro. Flow cytometry analysis of cell cycle distribution
of mel Z cells treated with 10–1000 nM rec-hBD-2 for
48 h demonstrated a concentration-dependent cell
growth arrest at G1/S checkpoint (p < 0.05) (Fig. 3).
Similar results were registered for mel Is cells treated with
rec-hBD-2 (data not shown). Treatment of melanoma
cells with 10–1000 nM of hBD-2 did not affect the per-
centage of apoptotic cells (see Fig. 3).
0
10
20
30
40
50
60
70
80
90
100
Control 1 10 100 500 1000
hBD-2 concentration, nM
Ce
lls
,%
apoptosis
G0/G1
G2/M
S
*
*
*
*
*
*
* *
*
* *
Fig. 3. Flow cytometry analysis of cell cycle distribution of mel Z cells
treated with rec-hBD-2. Mel Z cells were treated with 1, 10, 100, 500,
or 1000 nM rec-hBD-2 for 48 h. The data of two independent experi-
ments are presented as the mean ± SD. *The difference is significant
compared to appropriate control (p < 0.05)
To explore the effect of rec-hBD-2 on the proteins
involved in cell cycle regulation and cell growth control,
the expression levels of cyclin D1, cyclin E, retino-
blastoma protein (pRB), CDK4, p21WAF1, p53, B-Raf
and phosphorylation of pRB in mel Z and mel Is cells
treated for 48 h with 1000 nM rec-hBD-2 in comparison
to control (untreated) cells were studied using Western
blotting. Treatment with rec-hBD-2 resulted in slight
downregulation of pRB expression and its complete
dephosphorylation. Moreover, we revealed suppres-
sion of cyclin D1 expression, significant downregula-
tion of cyclin E, CDK4 and B-Raf expression levels, and
significant upregulation of p21WAF1 in hBD-2-treated
melanoma cells. At the same time p53 expression level
remained unaffected (Fig. 4).
Effect of rec-hBD-2 on colony-forming acti-
vity of human melanoma cells. We have performed
colony forming assay to evaluate an effect of rec-
hBD-2 on anchorage-independent growth ability
of human melanoma cells. It was shown that treatment
with 100 nM of rec-hBD-2 resulted in insignificant de-
crease of colony numbers compared to control cells,
addition of 500 nM of rec-hBD-2 into cell incubation
medium significantly inhibited (p < 0.05) ability of mel
Is cells to form colonies in semi-soft medium (Fig. 5),
while in the pre sence of 1 M of the defensin no visible
colonies were developed. Also, rec-hBD-2 caused
a concentration-dependent effect on anchorage-
independent growth of mel Z cells (data not shown).
Mel Z
Control Control
Mel Is
Cyclin D1
ph-pRb
β-actin
CDK4
p53
pRb
β-actin
B-Raf
p21WAF1
Cyclin E
β-actin
a
b
0
1
2
3
4
5
6
B-Raf CyclinE P21
0
0,5
1
1,5
2
2,5
3
ph-pRb CyclinD1 pRb p53 CDK4
Mel Z control
Mel Z +1000 nM hBD-2
Mel Is control
Mel Is +1000 nM hBD-2
Fig. 4. Western blot analysis of expression levels of cyclin D1,
cyclin E, pRB, CDK4, p53, B-Raf, and p21WAF1, and phosphory-
lation of pRB (ph-pRB) in mel Z and mel Is cells treated with
1000 nM rec-hBD-2 for 48 h in comparison to control (untreated)
cells (a). Graphic representations of Western blotting data cal-
culated with the use of TotalLab program (b)
Experimental Oncology 36, 17–23, 2014 (March) 21
0
100
200
300
400
500
600
700
800
Control 1 nM hBD-2 500 nM hBD-2
Co
lo
ny
n
um
be
r
a
b
*
Fig. 5. Rec-hBD-2 significantly inhibits colony-forming activity
of mel Is cells. The graph represents the numbers of colonies
calculated with the use of TotalLab program
Melanin content in cultured human melanoma
cells of mel Z and mel Is cells treated with 1 nM,
100 nM or 500 nM rec-hBD-2 for 72 h was analyzed.
We did not found alterations in melanin content
in mel Z and mel Is cells treated with the defensin
compared to control cells (data not shown). Thus,
hBD-2 seems to be not involved in melanin synthesis
regulation in cultured human melanoma cells.
DISCUSSION
In the present study we have shown that re-
combinant hBD-2, cationic host defense peptide,
affects proliferation, viability, and cell cycle distribu-
tion of human melanoma cells in vitro. The effects
of rec-hBD-2 on melanoma cell proliferation and
viability were found to be similar to those in human
A431 and A549 carcinoma cells [15]. Indeed, treatment
of mel Z and mel Is cells with rec-hBD-2 at concentra-
tions higher than 100 nM resulted in significant inhibi-
tion of cell proliferation and viability evidently realized
via cell cycle arrest at G1/S checkpoint.
The mechanism of cell division suppression
by the defensin could be possibly based on inhibi-
tion of B-Raf expression and inactivation of signaling
pathways controlled by this kinase. It is known that de-
creased expression of B-Raf affects activity of down-
stream kinases such as МЕК1 and ERK, and induction
of transcription factors c-Fos, c-Jun and important
mediator of growth factors effects — c-Myc, which
in turn controls expression of cyclin D1 and CDK4 [28].
It’s necessary to note that treatment of melanoma cells
with rec-hBD-2 resulted in complete down-regulation
of cyclin D1 expression (see Fig. 4). In contrary to cy-
clins Е and В, the quantities of which after termination
of their functioning during cell cycle progression de-
crease to minimal values, cyclin D1 expression remains
at relatively high level — approximately equal to its
half-maximal value in G1 phase [29].
According to our data (see Fig. 4), the possible
mechanism of cell cycle arrest in G1/S checkpoint
in melanoma cells treated with rec-hBD-2 could
be based on activation (dephosphorylation) of pRB,
down-regulation of cyclins D1 and E, and increased
quantity of CDK inhibitor — р21WAF1. Up-regulation
of р21WAF1 is difficult to explain because expres-
sion of р53 in the melanoma cells treated with rec-
hBD-2 did not alter significantly. It can not be excluded
that an increase of р21WAF1 expression is realized via
alternative, p53-independent mechanisms [30, 31].
The mechanism of growth-suppressing acti-
vity of rec-hBD-2 toward human melanoma cells
in vitro was found to be similar to that in human
A431 and 549 carcinoma cells — via cell cycle ar-
rest at G1/S checkpoint [15]. Therefore, this activity
of the defensin is not cell-type specific.
In current study we found a significant influence
of rec-hBD-2 on expression of B-Raf — an important
serine/threonine kinase involved in regulation of cell
growth. Mutations in BRAF gene resulting in constitu-
tive activation of the kinase are often observed in many
human cancers, including melanoma [32]. Novel
drugs, specific B-Raf inhibitors, have been developed;
some of them (dabrafenib, vemurafenib) have been
evaluated in clinical trials for melanoma treatment
and the results of these trials are considered pro-
mising [33]. Our finding, which demonstrated a potent
down-regulation of B-Raf expression in human mela-
noma cells in vitro caused by innate defense molecule
hBD-2 can be potentially useful in the development
of new agents for combined treatment of melanoma.
Also, our data have shown that rec-hBD-2 is ca-
pable to suppress an important hallmark of malig-
nant cells — their ability for anchorage-independent
growth. Indeed, in the presence of 500 nM of rec-
hBD-2 the number of colonies formed by mel Z cells
was nearly 10 fold lower that in the control (p < 0.05)
(see Fig. 5). Similar ability to inhibit malignant potential
of human cancer cells in vitro has been described re-
cently for human beta-defensin-4 — another inducible
peptide antibiotic expressed by epithelial cells [17].
The results of our study raise new questions:
do human melanoma cells express β-defensins, and
could their behavior and growth patterns be affected
by paracrine way through hBDs expressed by neigh-
boring skin keratinocytes? Unfortunately, little is known
about expression of hBDs in normal melanocytes and
malignant melanoma cells. At the same time, experi-
mental studies have provided evidence on responsive-
ness of melanocytes to the action of β-defensins.
In particular, it has been demonstrated that some hu-
man β-defensins (hBD-1 and hBD-3) in low nanomolar
concentrations are high-affinity ligands for MC1R
in primary human melanocytes, and it has been sup-
posed that hBD-3 may activate MAPK and cAMP sig-
naling pathways via MC1R thus regulating melanocyte
responses [22, 34]. In a recent Spanish case-control
study [35] it has been shown that hBD-1 (but not
hBD-3) could be involved in malignant melanoma
22 Experimental Oncology 36, 17–23, 2014 (March)
susceptibility as far as polymorphisms in hBD-1 gene
may be associated with increased risk of melanoma.
According to the data of our research, treatment with
rec-hBD-2 had no influence on melanin production
levels in mel Z and mel Is cells. At the same time,
hBD-2 significantly affects melanoma cell growth pat-
terns and malignancy potential in vitro and hypotheti-
cally could be capable to exert such effects in vivo.
It will be reasonable to study possible associations
between hBD-2 expression patterns and/or genetic
polymorphisms of its gene in malignant melanoma
and the risk of the development of this type of cancer.
In conclusion, our data have shown that exo genous
hBD-2 can regulate the growth patterns of human
melanoma cells in a concentration-dependent manner.
In particular, in medium-to-high nanomolar concentra-
tion range this peptide is capable to suppress viability
and proliferation of melanoma cells via cell cycle arrest
in G1/S checkpoint and to reduce significantly their
colony forming activity. Importantly, growth-sup-
pressing activity of hBD-2 is mediated by significant
down-regulation of B-Raf, cyclin E and cyclin D1 ex-
pression along with dephosphorylation of pRB in hu-
man melanoma cells. Together, these results provide
new evidence on important role of hBD-2 in regulation
of malignant potential of human melanoma cells.
COMPETING INTERESTS
Authors declare that they have no competing in-
terests.
ACKNOWLEDGEMENTS
This work was in part supported with grant
0110U005758 of National Academy of Sciences
of Ukraine “Fundamental Basis of Molecular and Cel-
lular Biotechnologies”.
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