Down-regulation of murine beta-defensin-2 in Lewis lung carcinoma cells results in accelerated growth of tumor cells in vitro and in vivo
To evaluate the anti-tumor activity of murine beta-defensin-2 (mBD-2) expression in vitro and in vivo. Materials and Methods: Based on pcDNA3 vector, constructs containing mBD-2 cDNA coding mature defensin molecule (pcDNA3-mBD2), and Igk-mBD-2 insertion, coding secretory sequence plus mature defensi...
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| Published in: | Experimental Oncology |
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| Date: | 2008 |
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| Language: | English |
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Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України
2008
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| Cite this: | Down-regulation of murine beta-defensin-2 in Lewis lung carcinoma cells results in accelerated growth of tumor cells in vitro and in vivo / E. Zhuravel, T. Shestakova, N. Glushko, M. Soldatkina, P. Pogrebnoy // Experimental Oncology. — 2008. — Т. 30, № 3. — С. 206–211. — Бібліогр.: 21 назв. — англ. |
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Digital Library of Periodicals of National Academy of Sciences of Ukraine| _version_ | 1860037251859218432 |
|---|---|
| author | Zhuravel, E. Shestakova, T. Glushko, N. Soldatkina, M. Pogrebnoy, P. |
| author_facet | Zhuravel, E. Shestakova, T. Glushko, N. Soldatkina, M. Pogrebnoy, P. |
| citation_txt | Down-regulation of murine beta-defensin-2 in Lewis lung carcinoma cells results in accelerated growth of tumor cells in vitro and in vivo / E. Zhuravel, T. Shestakova, N. Glushko, M. Soldatkina, P. Pogrebnoy // Experimental Oncology. — 2008. — Т. 30, № 3. — С. 206–211. — Бібліогр.: 21 назв. — англ. |
| collection | DSpace DC |
| container_title | Experimental Oncology |
| description | To evaluate the anti-tumor activity of murine beta-defensin-2 (mBD-2) expression in vitro and in vivo. Materials and Methods: Based on pcDNA3 vector, constructs containing mBD-2 cDNA coding mature defensin molecule (pcDNA3-mBD2), and Igk-mBD-2 insertion, coding secretory sequence plus mature defensin molecule (pcDNA3-Igk-mBD-2) were generated. Lewis lung carcinoma (3LL) cells were transfected in vitro with these plasmids and with blank pcDNA3 vector, and the proliferative rate and clonogenic ability of obtained cell lines cultivated in vitro were analyzed using 3H-incorporation technique and colony formation in semi-soft medium, respectively. Expression of mBD-2 mRNA was studied by semiquantative RT-PCR analysis. Also, transfected cells were transplanted to C57B mice, and the patterns of tumor growth in vivo were analyzed by routine techniques. Results: We have found out that in the 3LL cells transfected with pcDNA3-mBD-2 and pcDNA3-Igk-mBD-2, the expression of mBD-2 mRNA is significantly down regulated compared to wild-type cells and 3LL cells transfected with blank vector. The cells with suppressed mBD-2 expression differed from parental cells and cells transfected with blank vector by higher proliferation rate (p < 0.001) and higher clonogenic ability. The 3LL-mBD-2 and 3LL-Igk-mBD-2 cells that are transplanted to C57B mice gave rise to more aggressive tumors that possessed significantly higher growth rate (p < 0.01) than those that arise from wild-type 3LL cells. Conclusion: The obtained results are evidencing on a possible tumor-suppressing role of mBD-2 expression.
Цель: настоящая работа посвящена анализу противоопухолевых свойств бета-дефенсина-2 мыши (mBD-2) in vitro and in
vivo. Материалы и методы: на основе pcDNA3.1+ вектора были созданы 2 плазмидных конструкта, кодирующих зрелую
форму mBD-2, содержащие или несодержащие сигнальную последовательность Igk (pcDNA3mBD-2 и pcDNA3Igk-mBD-2
соответственно). Путем трансфекции клеток 3LL полученными векторами, а также контрольным вектором pcDNA3.1+
были получены клеточные линии (3LL-mBD-2, 3LL-Igk-mBD-2 и 3LL-pcDNA3), для которых были проведены исследования
их пролиферативной активности, определенной по уровню включения 3
Н-тимидина в ДНК, и способности к
колониеобразованию в среде, содержащей метилцеллюлозу. Экспрессию гена mBD-2 исследовали с помощью полуколичественного
ОТ-ПЦР-анализа. Трансфецированные клетки были имплантированы мышам линии C57BL, после чего была
проанализирована динамика роста опухоли. Результаты: установлено, что в трансфектных клеточных линиях 3LL-mBD-2
и 3LL-Igk-mBD-2 уровень экспрессии mBD-2 снижен по сравнению с контрольными. Эти клетки характеризовались достоверным
повышением уровня пролиферации (р < 0,001) и способности к колониеобразованию. Клетки сублиний 3LLmBD-2
и 3LL-Igk-mBD-2, трансплантированные мышам линии C57BL, вызывали развитие более агрессивных опухолей,
обладающих значительно более высокой скоростью роста (p < 0,01), чем таковые, вызванные перевивкой клеток 3LL.
Выводы: полученные результаты свидетельствуют о возможной роли mBD-2 как опухолевого супрессора.
|
| first_indexed | 2025-12-07T16:54:40Z |
| format | Article |
| fulltext |
206 Experimental Oncology 30, 206–211, 2008 (September)
Defensins, mammalian antimicrobial peptides
with pluripotent biological activities, are a family of
cationic cysteine-rich peptides which spectrum of
action largely depends on their local concentration
and microenvironment [1]. By the differences in their
tertiary structure and disulfide bonding, defensins
are distributed in three families, namely, α-, β- and
θ-defensin; the places of their expression in the body
are different: while α-defensins are expressed by
netrophils/leucocytes, β-defensins are found mainly
in epithelial cells.
Recently these molecules are recognized also as
the possible members of antitumor defense arsenal
due to their ability to cause lyzis of tumor cells [2], and
immunomodulatory and chemokine activities [3–6], in
particular, ability to attract immature dendritic cells.
This important pattern of beta-defensins has been
tested in few experimental studies, where defensin
gene was introduced to tumor cells that lack its expres-
sion, and its in vivo expression resulted in suppression
of tumor growth in vivo [7, 8]. From the other hand, de-
fensins may have positive or negative impact on tumor
growth due to their proangiogenic properties (murine
beta-defensin-29 [9]) or antiangiogenic activities
(HNP-1 and HNP-3 [10]). Moreover, there is a number
of reports where pro-proliferative action of defensins
in vitro [11-13] has been demonstrated, or, in contrary,
no effect of hBD-2 on proliferation has been stated
[14]. So, the question on possible role of defensins in
tumorigenesis remains largely unanswered.
The present research continues our studies of the
functional role of beta-defensin expression in cancer
cells. For the study, Lewis lung carcinoma (3LL) model
was chosen. 3LL is an experimental murine tumor model
widely used in cancer research. 3LL cells are express-
ing murine beta-defensin-2 (mBD-2), the homolog of
human beta-defensin-1, at a constant moderate level.
We have introduced an additional copy of mBD-2 gene
in 3LL cells with the aim to receive its hyperexpression
to achieve possible suppression of 3LL growth in vivo.
However, after such manipulation we have received an
opposite effect: surprisingly, transfection of 3LL cells
with pcDNA-mBD-2 and pcDNA-Igk-mBD-2 vectors
resulted in down-regulation of mBD-2 mRNA expression
in these cells in parallel with acquired higher prolifera-
tive rate and colony forming ability in vitro, and higher
aggressiveness of experimental tumors that arised
from the cells with suppressed mBD-2 expression.
Taken together, such results are pointing on a possible
tumor-suppressor role of mBD-2.
MATERIALS AND METHODS
Cell lines and bacterial strains. In vitro culture
of Lewis lung carcinoma (3LL) cells was obtained from
the Bank of Cell Lines from Human and Animal Tissues
of R.E. Kavetsky Institute of Experimental Pathology,
Oncology and Radiobiology NAS of Ukraine (Kyiv,
Ukraine). The cells were cultivated in vitro in DMEM
culture medium with high glucose content supple-
mented with 10% fetal bovine serum, 100 units/mL
penicillin G sodium, 100 μg/mL streptomycin sulfate
and 0.25 μg/mL amphotericin B as fungizone in 5%
CO2 athmosphere at 37 °C.
For testing the antimicrobial activity of recombinant
mBD-2 in the test of Hultmark et al. [15], Pseudomonas
DOWN-REGULATION Of MURINE bETA-DEfENSIN-2 IN LEWIS
LUNG cARcINOMA cELLS RESULTS IN AccELERATED GROWTH
Of TUMOR cELLS IN VITRO AND IN VIVO
E. Zhuravel*, t. shestakova, N. Glushko, M. soldatkina, P. Pogrebnoy
R.E. Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology NAS of Ukraine, 03022
Kyiv, Ukraine
Aim: To evaluate the anti-tumor activity of murine beta-defensin-2 (mBD-2) expression in vitro and in vivo. Materials and Methods:
Based on pcDNA3 vector, constructs containing mBD-2 cDNA coding mature defensin molecule (pcDNA3-mBD2), and Igk-mBD-2
insertion, coding secretory sequence plus mature defensin molecule (pcDNA3-Igk-mBD-2) were generated. Lewis lung carcinoma
(3LL) cells were transfected in vitro with these plasmids and with blank pcDNA3 vector, and the proliferative rate and clonogenic
ability of obtained cell lines cultivated in vitro were analyzed using 3H-incorporation technique and colony formation in semi-soft
medium, respectively. Expression of mBD-2 mRNA was studied by semiquantative RT-PCR analysis. Also, transfected cells were
transplanted to C57B mice, and the patterns of tumor growth in vivo were analyzed by routine techniques. Results: We have found
out that in the 3LL cells transfected with pcDNA3-mBD-2 and pcDNA3-Igk-mBD-2, the expression of mBD-2 mRNA is signifi-
cantly down regulated compared to wild-type cells and 3LL cells transfected with blank vector. The cells with suppressed mBD-2
expression differed from parental cells and cells transfected with blank vector by higher proliferation rate (p < 0.001) and higher
clonogenic ability. The 3LL-mBD-2 and 3LL-Igk-mBD-2 cells that are transplanted to C57B mice gave rise to more aggressive
tumors that possessed significantly higher growth rate (p < 0.01) than those that arise from wild-type 3LL cells. Conclusion: The
obtained results are evidencing on a possible tumor-suppressing role of mBD-2 expression.
Key Words: murine beta-defensin, Lewis lung carcinoma, expression, proliferation, colony formation.
Received: August 7, 2008.
*Correspondence: E-mail: pogrebnoy@onconet.kiev.ua
Abbreviations used: 3LL — Lewis lung carcinoma; mBD-2 — murine
beta-defensin-2.
Exp Oncol 2008
30, 3, 206–211
Experimental Oncology 30, 206–211, 2008 (September)30, 206–211, 2008 (September) (September) 207
aeruginosa ATCC 9027 was used; the strain was kindly
gifted by Dr. E. Kiprianova from the D.K. Zabolotny
Institute of Microbiology and Virology NAS of Ukraine
(Kyiv, Ukraine).
Generation and purification of recombinant
mBD-2. To clone the gene coding mature mBD-2
molecule, mBD-2 cDNA was received by RT-PCR
with the use of specific primers (mBD-2-F: 5`-ACCG-
GATCCACCATGGAA CTTGACCACTGCCACACC-3`
mBD-2-R: 5`-GCCGAATTCTCATTTCAT GTACTTG-
CAACAGGG-3`) from total RNA isolated from murine
BALB/c macrophages incubated for 24 h with 10 ng/ml
lipopolysaccharides of P. aeruginosa. Primers were
designed on the base of AJ011800 sequence from
GenBank database.
For production of recombinant mBD-2, we have
chosen the GST-expression system, that allows the
production of recombinant proteins fused with gluta-
thione-S-transferase (GST) in bacterial cells upon in-
duction of isopropyl-β-D-thiogalactopiranoside (IPTG).
Respectively, mBD-2 cDNA fragment was cloned
in pGEX-2T vector by BamHI and EcoRI sites using
routine procedure. PCR product and pGEX-2T vector
were purified, treated with restriction endonucleases
BamHI and EcoRI, ligated by routine technique, and
correctness of insertion was verified by the DNA dide-
oxy sequencing method and restriction analyzis. Then
E. coli BL21DE3 cells were routinely transformed with
this vector, the selection of clones was performed
as described elsewhere. Recombinant mDD-2 was
isolated from bacterial cells by means of affine chro-
matography on Glutathione-Sepharose (Sigma, USA)
with the next proteolysis of GST-mBD-2 fusion protein
with thrombin and reverse phase chromatography on
Sep-Pack C18 (Waters, USA) carrier similarly to the
procedure described earlier for recombinant human
beta-defensin-2 [16].
Gene cloning, plasmid constructions and
transfection of 3LL cells. The gene coding mature
murine β-defensin-2 was cloned from total RNA
isolated from murine BALB/c macrophages incu-
bated for 24 h with 10 ng/ml lipopolysaccharides of
P. aeruginosa and cDNA was amplified by RT-PCR
using specific primers (mBD-2-F: 5`-ACCTAAGCT-
TCGAACTTGACCACTGCCACACC-3` and mBD-2-R:
5`-GGCGAATTCTCATTTCATGTACTTGCAACAGGG-3).
The PCR products were digested with HindIII and EcoRI
and cloned into the same sites of expression vector
pcDNA3.1+, which contained a selective marker — the
neomycin phosphotransferase gene. The constructs
were verified by the DNA dideoxy sequencing method
and purified using a standard protocol.
To receive a variant for secreted form of mBD-2,
leader sequence of Igk gene was recloned from pSec-
Tag2A vector (Invitrogene) and placed into pcDNA3.1+
vector before mBD-2 sequence.
The resulting constructs were transfected into
3LL cells using FuGENE 6 Reagent (Roche Molecular
Biochemicals) according to the instructions of the
manufacturer.
After transfection, the cells were selected in the
medium containing 600 μg/mL geneticin G418 (Gibco,
Grand Island, NY, USA) for 2 weeks, and stable cell sub-
lines 3LL-pcDNA3 (transfected with blank pcDNA3 vec-
tor), 3LL-mBD2 (expressing mBD2) and 3LL-Igk-mBD2
(carrying secreted form of mBD2) were obtained.
RT-PCR analysis of mBD2 expression. Total
RNA was isolated from tissue samples by the method
of Chromzynski and Sacchi [17]. Concentration of
RNA was evaluated at the wave length of 260 nm using
Beckman DU-8B spectrophotometer, its purity — by
OD relation at 280 nm and 260 nm, its quality — by
electrophoresis in 1% agarose gel containing 20%
formaldehyde. For detection of mBD-2 RNA expres-
sion, semiquantitive RT-PCR analysis was performed
with the use of specific primers. The expression level
of beta-actine served as the house-keeping gene.
The products of RT-PCR were routinely analyzed by
electrophoresis in agarose gel. The relative expression
level was analyzed with the use of TotalLab Program.
3H-thymidine incorporation. To evaluate cell
proliferation rate, radioisotope method was routinely
applied as described elsewhere [18]. Shortly, the
cells (1 x 105 cells/well) were grown on 24-well plate
in DMEM for 24 h. Then 0.1 μCi-3H-thymidine per well
was added, and the cells were incubated for 16 h. The
radioactivity of the cells was measured according
standard procedure using scintillation beta counter
Beckman 5801 LS.
Colony formation analysis. Analysis of colony
forming ability of the cells was carried out by routine
technique as described elsewhere [19] in DMEM me-
dium supplemented with 15% FBS and 0.4% methyl-
cellulose. The cells were plated in 6-well plates (1 х
103 cells per 1 ml of medium) and incubated in СО2-
incubator for 14–16 days. The colonies were visualized
using staining procedure with 0.2% p-iodonitrotetra-
zolium violet dye and counted.
In vivo study. For in vivo research, male C57BL
mice weighing 20–25 g bred in the vivarium of
R.E. Kavetsky Institute of Experimental Pathology,
Oncology and Radiobiology NAS of Ukraine (Kyiv,
Ukraine) were used. All animal procedures were carried
out according to the rules of local Ethic Committee and
were approved by the Ethic Board.
3LL cells and transfected cell sublines were trans-
planted i.m. ((3 x 105 cells/100 μl PBS per animal) to
C57BL mice mice. Animals were housed in 4 groups
(5 mice per group): animals with transplanted wild-
type 3LL cells (group 1), 3LL-pcDNA3 cells (group 2),
3LL-mBD2 cells (group 3), and 3LL-Igk-mBD2 cells
(group 4). Tumor growth dynamics was monitored
each week for 3 weeks by means of calipers. At the day
21 after tumor cell transplantation, the animals were
sacrificed by ether narcosis, tumors were removed
and weighted, and lung metastases were calculated.
By this scheme, 3 independent in vivo experiments
were performed.
Statistical analysis. The data are reported as the
mean ± SD. The statistical significance of differences
208 Experimental Oncology 30, 206–211, 2008 (September)
between mean values was assessed by the Student’s
t-test. Values p < 0.05 were considered statistically
significant.
RESULTS
Generation of recombinant mBD-2. For pro-
duction of recombinant mBD-2, the GST-expression
system was chosen. Respectively, recombinant
mBD-2 fused with GST was isolated from transformed
bacterial cells by means of affine chromatography on
Glutathione-agarose with the next proteolysis of GST-
mBD-2 fusion protein with thrombin and reverse phase
chromatography on Sep-Pack C18 carrier (Fig. 1, a)
similarly to the procedure described earlier for recom-
binant human beta-defensin-2. The obtained mBD-2
protein was demonstrated to be active in micromolar
concentrations against P. aeruginosa (Fig. 1, b).
fig. 1. a, Electrophoretic analysis of purity of the recombinant
mBD-2 preparation after reverse phase chromatography on
Sep-Pack C18. Line 1 — molecular weight markers (Fermentas,
Lietuva); lines 2 and 3 — elution with 30% CH3CN and 40%
CH3CN, respectively. b, Antimicrobial activity of recombinant
mBD-2 against P. aeruginosa: 1, 2, 3 — 1, 3 and 5 μM mBD-2
So, in this first part of our work, recombinant antimi-
crobially active mBD-2 was obtained for some special
tasks of the further research.
Generation of pcDNA3-Igk-pcDNA3 vectors,
transfection of 3LL cells and expression of mBD-2
mRNA in vitro. At the first stage of the research, us-
ing standard cloning technique, we have created two
vectors at the base of pcDNA3.1+: pcDNA3-mBD-2
and pcDNA3-Igk-mBD-2 that codes mature mBD-2
molecule and its secreted form, respectively. These
vectors and blank control pcDNA3.1+ vector were used
for the transfection of 3LL cells. After selection on G418
containing medium, the transfected cells were multi-
plied and analyzed for expression of mBD-2 mRNA.
3LL cells are expressing mBD-2 mRNA at a con-
stant moderate level. Performing such transfection,
we supposed that overexpression of mBD-2 mRNA
in 3LL cells will be achieved. Surprisingly, finally we
have observed quite the opposite effect: in the cells
transfected with pcDNA3-mBD-2 and pcDNA3-Igk-
mBD-2 (3LL-mBD2 and 3LL-Igk-mBD2 respectively),
a notable down-regulation of mBD-2 gene was regis-
tered (Fig. 2), while in the cells transfected with a blank
vector this parameter did not differ significantly from
that in parental 3LL cells (p < 0.01).
0
1
2
3
4
5
6
7
3LL 3LLmBD2 3LLIgkmBD2 3LLpcDNA3
m
BD
-2
e
xp
re
ss
io
n
le
ve
l (
re
la
tiv
e
un
its
)
a
b
139 bp
284 bp
1 2 3 4 5
fig. 2. a, RT-PCR analysis of mBD-2 gene expression in 3LL cells
(2), 3LL-mBD2 cells (3), 3LLIgk-mBD2 cells (4), 3LL-pcDNA3
cells (5); line 1 — DNA standard 50 bp ladder (Fermentas, Lie-
tuva) (a). Expression of beta-actine as house-keeping gene (b).
b, The data of mBD-2 gene expression in transfected cells are
normalized by beta-actine expression using TotalLab program
To find out whether down-regulation of mBD-2
gene may be caused by its own protein product, we
have performed in vitro culturing of wild-type 3LL cells
with exogeneously added 0.1 and 1.0 μg/ml recombi-
nant mBD-2 in serum-free medium for 24 h and 48 h.
Then the cells were lyzed, total RNA was isolated, and
semi-quantative RT-PCR analyzis was carried out.
Similarly to the above-mentioned data, in the cells cul-
tivated with recombinant mBD-2 significant decrease
of mBD-2 mRNA was detected, and mBD-2 mRNA was
nearly undetectable after 48 h incubation of 3LL cells
with 1.0 μg/ml recombinant mBD-2 (Fig. 3).
So, on the base of 3LL cells expressing mBD-2
mRNA we have generated the cell sublines 3LL-mBD2
and 3LL-Igk-mBD2 where expression of mBD-2 is
strongly down-regulated, possibly, by the feedback
mechanism. So, it looked reasonable to check the
patterns of 3LL cells with down-regulated mBD-2 gene
compared with parental strain. 3LL cells are widely
used in vivo for generation of aggressive experimental
murine tumors with high metastatic activity resulting
in multiple lung metastases. So, we have checked the
proliferative rate and colony forming capacity of sub-
lines 3LL-mBD2 and 3LL-Igk-mBD2 compared to wild
type 3LL cells as well as the growth dynamics of tumors
that arised from transplanted 3LL cell sublines.
Experimental Oncology 30, 206–211, 2008 (September)30, 206–211, 2008 (September) (September) 209
fig. 3. RT-PCR analysis of mBD-2 gene expression in 3LL cells
incubated in vitro with the recombinant mBD-2. Lines: 1 — DNA
standard 50 bp ladder (Fermentas, Lietuva); 2–6 — expression
of beta-actine as house-keeping gene; expression of mBD-2 in
control 3LL cells (7), 3LL cells induced by 0.01 μg/ml mBD-2 for
24 h (8), 0.01 μg/ml mBD-2 for 48 h (9); 1 μg/ml mBD-2 for 24 h
(10), 1 μg/ml mBD-2 for 48 h (11)
Colony formation and proliferation rate of
3LL cells transfected with pCDNA3- pcDNA3
and pCDNA3-Igk-pcDNA3plasmids. Ability to
form colonies in a semi-soft medium is an important
characteristic of cell culture indicating its tumotigenic
potential. The analysis of ability of 3LL, 3LL-pcDNA3,
3LL-mBD2 and 3LL-Igk-mBD2 cells to form colonies in
0.4% methylcellulose has demonstrated that the cells
where expression of mBD-2 gene is down-regulated,
formed significantly higher number of colonies than
those where expression of mBD-2 mRNA is not af-
fected, and that 3LL-mBD2 and 3LL-Igk-mBD2 cells
gave rise to larger colonies than 3LL and 3LL-pcDNA3
did (Table).
Table. Colony forming activity of 3LL cells and 3LL-mBD2 and 3LL-Igk-
mBD2 sublines
Cell line Number of colonies Colony size
3LL < 20 < 10 cells
3LL-pcDNA3 < 20 < 10 cells
3LL-mBD2 > 50 > 10 cells
3LL-Igk-mBD2 > 50 > 10 cells
Analysis of 3H-thymidine incorporation in DNA of
3LL, 3LL-pcDNA3, 3LL-mBD2 and 3LL-Igk-mBD2
cells has shown that 3LL-mBD2 and 3LL-Igk-mBD2
are characterized by significantly higher proliferation
rate (p < 0.001) than the control cells, while this index
did not differ significantly between wild-type 3LL cells
and the cells transfected with the blank vector (p >
0.05) (Fig. 4).
So, our data have indicated an existence of reverse
relation between colony-forming activity and prolifera-
tion potential of 3LL cells and the level of mBD-2 mRNA
expression. The next question to be answered was
whether such properties will influence the develop-
ment and growth dynamics of experimental 3LL tumors
that arise from trasfected 3LL cells.
0
5000
10000
15000
20000
25000
30000
35000
40000
45000
50000
3LL 3LLpcDNA3 3LLmBD-2 3LLIgkmBD-2
3 H-
ty
m
id
in
e
in
co
rp
or
at
io
n
(im
pu
ls
e/
m
in
)
*
*
fig. 4. Analysis of 3H-thymidine incorporation in the DNA of
3LL, 3LL-pcDNA3, 3LL-mBD2 and 3LL-Igk-mBD2 cells. The data
(Mean ± SD) of two independent experiments performed in four
parallels each are presented
*p < 0.001 compared to 3LL.
Influence of mBD-2 expression on the growth
dynamics of experimental murine tumors. To an-
swer this question, 3LL, 3LL-pcDNA3, 3LL-mBD2 and
3LL-Igk-mBD2 cells were routinely transplanted (3 x
105 cells per animal) in C57 Black mice, and tumor
growth dynamics was monitored each week during
21 days period. Our data have shown that the tumors
that arised from 3LL-mBD2 and especially from 3LL-
Igk-mBD2 cells possessed significantly higher growth
rate that was nearly 1.5-fold and 1.7-fold higher than
that of 3LL- and 3LL-pcDNA3-derived tumors (p <
0.05) (Fig. 5), while 3LL- and 3LL-pcDNA3 tumors
did not differ significantly in the growth dynamics (p >
0.05). At the day 21 after tumor cell transplantation,
the metastatic rate was high in all 4 studied groups,
and it was impossible to make correct comparisons
between the groups; however, there was observed a
tendency for more aggressive metastasis in the case
of 3LL-Igk-mBD2-derived tumors (data not shown).
0
20
40
60
80
100
120
140
160
180
200
3LL 3LLpcDNA3 3LLmBD-2 3LLIgkmBD-2
Re
la
tiv
e
tu
m
or
w
ei
gh
t (
%
)
*
*
fig. 5. Relative weight of the tumors derived from transplanted
3LL, 3LL-pcDNA3, 3LL-mBD2 and 3LL-Igk-mBD2 cells at the
day 21 after tumor cell transplantation. The data (Mean ± SD)
of three independent experiments are presented
*p < 0.01 compared to 3LL.
So, these results have demonstrated that down-
regulation of mBD-2 gene expression resulted in
acceleration of the development of experimental
murine tumors; which, taking into account the above-
mentioned data on in vitro consequences of mBD-2
down-regulation, may suggest a tumor-suppressing
role of murine beta-defensin-2 expression.
210 Experimental Oncology 30, 206–211, 2008 (September)
DIScUSSION
During the last few years, the defensins became
a subject of intensive research due to their recently
revealed multiple biological activities that allow to con-
sider them not only as an important component of host
antibacterial defense, but as an agents influencing
tumorigenesis as well. First of all, in vitro studies have
shown mitogenic action of defensins, for example,
pro-proliferative effects of alpha- and beta-defensins
(HNP-1 and hBD-2) at micromolar concentration range
toward conjunctival fibroblasts [20] and airway epithe-
lial cells [11]; similar data were reported in our earlier
publications where we have shown a dose-dependent
mitogenic effect of hBD-2 on human cancer A431 and
HeLa cells [13]. However, there are also reports where
0.1–10 μg hBD-2 has no influence of proliferation rate
of intestinal epithelial cells, but strongly promotes
epithelial cell survival [14].
In the present study, we have introduced an ad-
ditional copy of mBD-2 gene in 3LL cells with an aim
to receive its hyperexpression to achieve possible
suppression of 3LL growth in vivo, but received quite
the opposite effect and revealed an interesting phe-
nomenon: if expression of mBD-2 gene was down-
regulated, 3LL cancer cells acquire significantly higher
proliferative rate (p < 0.001). Moreover, such aggres-
sive growth pattern was analogous in vivo, when these
cells with down-regulated mBD-2 gene gave rise to
experimental tumors. We propose a hypothesis that in
fact mBD-2 may play a role of anti-mitogenic molecule
that, being expressed at physiologically normal low
level, protects cells from excessive proliferation. Some
evidence presented in this report point to the existence
of some kind of a feedback mechanism: excessive
defensin production may result in the down-regulation
of its own gene, allowing in turn the cell proliferation.
In our earlier studies we did not check expression
levels of hBD-2 in the A431 and HeLa cells treated with
extracellular defensin; it could not be excluded that
in the case of hBD-2 the mechanism of action could
be similar. In part, such hypothesis is supported by
our research of human embryonal kidney (HEK293)
cells transfected with Igk-hBD-2-containing vector
(not published data): if wild-type cells do not express
hBD-2 and possess high proliferative rate and high
colony forming activity, transfected cells are express-
ing picomolar concentrations of hBD-2 and largely lost
colony formation ability along with decreased prolifera-
tion rate. Of course, such hypothesis requires further
more intense studies for its support. At the same time,
these unexpected data received in in vitro and in vivo
models are in agreement with an opinion about pos-
sible tumor-suppression functions of defensins [21]
delineated from a number of recent studies including
those carried on human prostate and renal tumors [7].
Fundamental studies of defensin signaling machinery
will strongly facilitate our knowledge on exact func-
tional role of these antimicrobials in cancer cells.
AckNOWLEDGEMENTS
The work was supported by NASU grants
UI0107U005545, the Program “Newest Medico-Bio-
logical Problems and Environment”, Part 2. “Biologi-
cally Active Compounds for Human Health” (Ukraine)
and “Fundamental Problems of Genomix and Proteo-
mix” UI0107U002243.
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Copyright © Experimental Oncology, 2008
СНИЖЕНИЕ УРОВНЯ ЭКСПРЕССИИ БЕТА-ДЕФЕНСИНА-2
МЫШИ В КЛЕТКАХ КАРЦИНОМЫ ЛЕГКИХ ЛЬЮИС ПРИВОДИТ
К УСКОРЕНИЮ РОСТА ОПУХОЛЕВЫХ КЛЕТОК IN VITRO И IN VIVO
Цель: настоящая работа посвящена анализу противоопухолевых свойств бета-дефенсина-2 мыши (mBD-2) in vitro and in
vivo. Материалы и методы: на основе pcDNA3.1+ вектора были созданы 2 плазмидных конструкта, кодирующих зрелую
форму mBD-2, содержащие или несодержащие сигнальную последовательность Igk (pcDNA3mBD-2 и pcDNA3Igk-mBD-2
соответственно). Путем трансфекции клеток 3LL полученными векторами, а также контрольным вектором pcDNA3.1+
были получены клеточные линии (3LL-mBD-2, 3LL-Igk-mBD-2 и 3LL-pcDNA3), для которых были проведены иссле-
дования их пролиферативной активности, определенной по уровню включения 3Н-тимидина в ДНК, и способности к
колониеобразованию в среде, содержащей метилцеллюлозу. Экспрессию гена mBD-2 исследовали с помощью полуколи-
чественного ОТ-ПЦР-анализа. Трансфецированные клетки были имплантированы мышам линии C57BL, после чего была
проанализирована динамика роста опухоли. Результаты: установлено, что в трансфектных клеточных линиях 3LL-mBD-2
и 3LL-Igk-mBD-2 уровень экспрессии mBD-2 снижен по сравнению с контрольными. Эти клетки характеризовались до-
стоверным повышением уровня пролиферации (р < 0,001) и способности к колониеобразованию. Клетки сублиний 3LL-
mBD-2 и 3LL-Igk-mBD-2, трансплантированные мышам линии C57BL, вызывали развитие более агрессивных опухолей,
обладающих значительно более высокой скоростью роста (p < 0,01), чем таковые, вызванные перевивкой клеток 3LL.
Выводы: полученные результаты свидетельствуют о возможной роли mBD-2 как опухолевого супрессора.
Ключевые слова: бета-дефенсин-2 мыши, карцинома легких Льюис (3LL), экспрессия, пролиферация, колониеобразование.
|
| id | nasplib_isofts_kiev_ua-123456789-139913 |
| institution | Digital Library of Periodicals of National Academy of Sciences of Ukraine |
| issn | 1812-9269 |
| language | English |
| last_indexed | 2025-12-07T16:54:40Z |
| publishDate | 2008 |
| publisher | Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
| record_format | dspace |
| spelling | Zhuravel, E. Shestakova, T. Glushko, N. Soldatkina, M. Pogrebnoy, P. 2018-06-21T13:40:19Z 2018-06-21T13:40:19Z 2008 Down-regulation of murine beta-defensin-2 in Lewis lung carcinoma cells results in accelerated growth of tumor cells in vitro and in vivo / E. Zhuravel, T. Shestakova, N. Glushko, M. Soldatkina, P. Pogrebnoy // Experimental Oncology. — 2008. — Т. 30, № 3. — С. 206–211. — Бібліогр.: 21 назв. — англ. 1812-9269 https://nasplib.isofts.kiev.ua/handle/123456789/139913 To evaluate the anti-tumor activity of murine beta-defensin-2 (mBD-2) expression in vitro and in vivo. Materials and Methods: Based on pcDNA3 vector, constructs containing mBD-2 cDNA coding mature defensin molecule (pcDNA3-mBD2), and Igk-mBD-2 insertion, coding secretory sequence plus mature defensin molecule (pcDNA3-Igk-mBD-2) were generated. Lewis lung carcinoma (3LL) cells were transfected in vitro with these plasmids and with blank pcDNA3 vector, and the proliferative rate and clonogenic ability of obtained cell lines cultivated in vitro were analyzed using 3H-incorporation technique and colony formation in semi-soft medium, respectively. Expression of mBD-2 mRNA was studied by semiquantative RT-PCR analysis. Also, transfected cells were transplanted to C57B mice, and the patterns of tumor growth in vivo were analyzed by routine techniques. Results: We have found out that in the 3LL cells transfected with pcDNA3-mBD-2 and pcDNA3-Igk-mBD-2, the expression of mBD-2 mRNA is significantly down regulated compared to wild-type cells and 3LL cells transfected with blank vector. The cells with suppressed mBD-2 expression differed from parental cells and cells transfected with blank vector by higher proliferation rate (p < 0.001) and higher clonogenic ability. The 3LL-mBD-2 and 3LL-Igk-mBD-2 cells that are transplanted to C57B mice gave rise to more aggressive tumors that possessed significantly higher growth rate (p < 0.01) than those that arise from wild-type 3LL cells. Conclusion: The obtained results are evidencing on a possible tumor-suppressing role of mBD-2 expression. Цель: настоящая работа посвящена анализу противоопухолевых свойств бета-дефенсина-2 мыши (mBD-2) in vitro and in
 vivo. Материалы и методы: на основе pcDNA3.1+ вектора были созданы 2 плазмидных конструкта, кодирующих зрелую
 форму mBD-2, содержащие или несодержащие сигнальную последовательность Igk (pcDNA3mBD-2 и pcDNA3Igk-mBD-2
 соответственно). Путем трансфекции клеток 3LL полученными векторами, а также контрольным вектором pcDNA3.1+
 были получены клеточные линии (3LL-mBD-2, 3LL-Igk-mBD-2 и 3LL-pcDNA3), для которых были проведены исследования
 их пролиферативной активности, определенной по уровню включения 3
 Н-тимидина в ДНК, и способности к
 колониеобразованию в среде, содержащей метилцеллюлозу. Экспрессию гена mBD-2 исследовали с помощью полуколичественного
 ОТ-ПЦР-анализа. Трансфецированные клетки были имплантированы мышам линии C57BL, после чего была
 проанализирована динамика роста опухоли. Результаты: установлено, что в трансфектных клеточных линиях 3LL-mBD-2
 и 3LL-Igk-mBD-2 уровень экспрессии mBD-2 снижен по сравнению с контрольными. Эти клетки характеризовались достоверным
 повышением уровня пролиферации (р < 0,001) и способности к колониеобразованию. Клетки сублиний 3LLmBD-2
 и 3LL-Igk-mBD-2, трансплантированные мышам линии C57BL, вызывали развитие более агрессивных опухолей,
 обладающих значительно более высокой скоростью роста (p < 0,01), чем таковые, вызванные перевивкой клеток 3LL.
 Выводы: полученные результаты свидетельствуют о возможной роли mBD-2 как опухолевого супрессора. The work was supported by NASU grants
 UI0107U005545, the Program “Newest Medico-Biological
 Problems and Environment”, Part 2. “Biologically
 Active Compounds for Human Health” (Ukraine)
 and “Fundamental Problems of Genomix and Proteomix”
 UI0107U002243. en Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України Experimental Oncology Uncategorized Down-regulation of murine beta-defensin-2 in Lewis lung carcinoma cells results in accelerated growth of tumor cells in vitro and in vivo Снижение уровня экспрессии бета-дефенсина-2 мыши в клетках карциномы легких Льюис приводит к ускорению роста опухолевых клеток in vitro и in vivo Article published earlier |
| spellingShingle | Down-regulation of murine beta-defensin-2 in Lewis lung carcinoma cells results in accelerated growth of tumor cells in vitro and in vivo Zhuravel, E. Shestakova, T. Glushko, N. Soldatkina, M. Pogrebnoy, P. Uncategorized |
| title | Down-regulation of murine beta-defensin-2 in Lewis lung carcinoma cells results in accelerated growth of tumor cells in vitro and in vivo |
| title_alt | Снижение уровня экспрессии бета-дефенсина-2 мыши в клетках карциномы легких Льюис приводит к ускорению роста опухолевых клеток in vitro и in vivo |
| title_full | Down-regulation of murine beta-defensin-2 in Lewis lung carcinoma cells results in accelerated growth of tumor cells in vitro and in vivo |
| title_fullStr | Down-regulation of murine beta-defensin-2 in Lewis lung carcinoma cells results in accelerated growth of tumor cells in vitro and in vivo |
| title_full_unstemmed | Down-regulation of murine beta-defensin-2 in Lewis lung carcinoma cells results in accelerated growth of tumor cells in vitro and in vivo |
| title_short | Down-regulation of murine beta-defensin-2 in Lewis lung carcinoma cells results in accelerated growth of tumor cells in vitro and in vivo |
| title_sort | down-regulation of murine beta-defensin-2 in lewis lung carcinoma cells results in accelerated growth of tumor cells in vitro and in vivo |
| topic | Uncategorized |
| topic_facet | Uncategorized |
| url | https://nasplib.isofts.kiev.ua/handle/123456789/139913 |
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