Administration of vitamin D3 improves antimetastatic efficacy of cancer vaccine therapy of Lewis lung carcinoma
Aim: To analyze antitumor efficacy of experimental cancer vaccine therapy combined with introduction of vitamin D3 (VD3) for treatment of Lewis lung carcinoma (3LL). Materials and Methods: Cancer vaccines composed from recombinant murine beta-defensin-2 (mBD-2) and 3LL cell lysate, or DNA, coding fo...
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| Cite this: | Administration of vitamin D3 improves antimetastatic efficacy of cancer vaccine therapy of Lewis lung carcinoma / E. Zhuravel, O. Efanova, T. Shestakova, N. Glushko, O. Mezhuev, M. Soldatkina, P.V. Pogrebnoy // Experimental Oncology. — 2010. — Т. 32, № 1. — С. 33-39. — Бібліогр.: 37 назв. — англ. |
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Zhuravel, E. Efanova, O. Shestakova, T. Glushko, N. Mezhuev, O. Soldatkina, M. Pogrebnoy, P.V. 2018-06-19T10:17:39Z 2018-06-19T10:17:39Z 2010 Administration of vitamin D3 improves antimetastatic efficacy of cancer vaccine therapy of Lewis lung carcinoma / E. Zhuravel, O. Efanova, T. Shestakova, N. Glushko, O. Mezhuev, M. Soldatkina, P.V. Pogrebnoy // Experimental Oncology. — 2010. — Т. 32, № 1. — С. 33-39. — Бібліогр.: 37 назв. — англ. 1812-9269 https://nasplib.isofts.kiev.ua/handle/123456789/138597 Aim: To analyze antitumor efficacy of experimental cancer vaccine therapy combined with introduction of vitamin D3 (VD3) for treatment of Lewis lung carcinoma (3LL). Materials and Methods: Cancer vaccines composed from recombinant murine beta-defensin-2 (mBD-2) and 3LL cell lysate, or DNA, coding for mBD-2-Muc1 fusion construct cloned in pcDNA3+ vector, were prepared and used for intradermal vaccination. Experimental cancer vaccines introduced i. d. at therapeutic and prophylactic regimens to 3LLbearing C57Bl mice, were applied alone or in combination with VD3 (administered per os) and/or low-dose cyclophosphamide (CP, administered intraperitoneal). Efficacy of treatments was analyzed by primary tumor growth dynamics indexes and by metastasis rate in vaccinated animals. Results: As it has been shown, administration of the protein-based vaccine composed from mBD-2 and 3LL cell lysate in combination with VD3 and CP, but not in VD3 free setting, led to significant suppression of primary tumor growth (p < 0.005) and had significant antimetastatic effect. Introduction of VD3 with or without CP in the scheme of treatment with mBD-2-Muc1-DNA vaccine at therapeutic regimen has led to significant suppression of primary tumor (p < 0.05) and metastasis volumes (p < 0.005), while in the groups of animals treated with DNA-vaccine + VD3 with or without CP at prophylactic regimen, significant antimetastatic effect (p < 0.05) and elevation of average life-span (p < 0.05) have been registered. Conclusion: The results of this pilot study have shown promising clinical effects of VD3 administration in combination with cancer vaccinotherapy in vivo. The work was supported by NASU grants 0107U005545, the Program “Newest Medico- Biological Problems and Environment”, Part 2. “Biologically Active Compounds for Human Health” (Ukraine), and U002243 “Fundamental Problems of Genomics and Proteomics”. en Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України Experimental Oncology Original contributions Administration of vitamin D3 improves antimetastatic efficacy of cancer vaccine therapy of Lewis lung carcinoma Article published earlier |
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Administration of vitamin D3 improves antimetastatic efficacy of cancer vaccine therapy of Lewis lung carcinoma |
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Administration of vitamin D3 improves antimetastatic efficacy of cancer vaccine therapy of Lewis lung carcinoma Zhuravel, E. Efanova, O. Shestakova, T. Glushko, N. Mezhuev, O. Soldatkina, M. Pogrebnoy, P.V. Original contributions |
| title_short |
Administration of vitamin D3 improves antimetastatic efficacy of cancer vaccine therapy of Lewis lung carcinoma |
| title_full |
Administration of vitamin D3 improves antimetastatic efficacy of cancer vaccine therapy of Lewis lung carcinoma |
| title_fullStr |
Administration of vitamin D3 improves antimetastatic efficacy of cancer vaccine therapy of Lewis lung carcinoma |
| title_full_unstemmed |
Administration of vitamin D3 improves antimetastatic efficacy of cancer vaccine therapy of Lewis lung carcinoma |
| title_sort |
administration of vitamin d3 improves antimetastatic efficacy of cancer vaccine therapy of lewis lung carcinoma |
| author |
Zhuravel, E. Efanova, O. Shestakova, T. Glushko, N. Mezhuev, O. Soldatkina, M. Pogrebnoy, P.V. |
| author_facet |
Zhuravel, E. Efanova, O. Shestakova, T. Glushko, N. Mezhuev, O. Soldatkina, M. Pogrebnoy, P.V. |
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Original contributions |
| topic_facet |
Original contributions |
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2010 |
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English |
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Experimental Oncology |
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Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
| format |
Article |
| description |
Aim: To analyze antitumor efficacy of experimental cancer vaccine therapy combined with introduction of vitamin D3 (VD3) for treatment of Lewis lung carcinoma (3LL). Materials and Methods: Cancer vaccines composed from recombinant murine beta-defensin-2 (mBD-2) and 3LL cell lysate, or DNA, coding for mBD-2-Muc1 fusion construct cloned in pcDNA3+ vector, were prepared and used for intradermal vaccination. Experimental cancer vaccines introduced i. d. at therapeutic and prophylactic regimens to 3LLbearing C57Bl mice, were applied alone or in combination with VD3 (administered per os) and/or low-dose cyclophosphamide (CP, administered intraperitoneal). Efficacy of treatments was analyzed by primary tumor growth dynamics indexes and by metastasis rate in vaccinated animals. Results: As it has been shown, administration of the protein-based vaccine composed from mBD-2 and 3LL cell lysate in combination with VD3 and CP, but not in VD3 free setting, led to significant suppression of primary tumor growth (p < 0.005) and had significant antimetastatic effect. Introduction of VD3 with or without CP in the scheme of treatment with mBD-2-Muc1-DNA vaccine at therapeutic regimen has led to significant suppression of primary tumor (p < 0.05) and metastasis volumes (p < 0.005), while in the groups of animals treated with DNA-vaccine + VD3 with or without CP at prophylactic regimen, significant antimetastatic effect (p < 0.05) and elevation of average life-span (p < 0.05) have been registered. Conclusion: The results of this pilot study have shown promising clinical effects of VD3 administration in combination with cancer vaccinotherapy in vivo.
|
| issn |
1812-9269 |
| url |
https://nasplib.isofts.kiev.ua/handle/123456789/138597 |
| citation_txt |
Administration of vitamin D3 improves antimetastatic efficacy of cancer vaccine therapy of Lewis lung carcinoma / E. Zhuravel, O. Efanova, T. Shestakova, N. Glushko, O. Mezhuev, M. Soldatkina, P.V. Pogrebnoy // Experimental Oncology. — 2010. — Т. 32, № 1. — С. 33-39. — Бібліогр.: 37 назв. — англ. |
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| fulltext |
Experimental Oncology 32, 33–39, 2010 (March) 33
Immunotherapy with the use of rationally designed
cancer vaccines is presently considered as a promising
approach for cancer treatment directed on enhancement
of immune response against specific antigens expressed
in tumor cells. As a rule, tumor antigens are poorly immu-
nogenic, that’s why vaccination with tumor-associated
antigens (TAAs) only doesn’t lead to induction of effective
antitumor immunity. For generation of effective antitumor
response it is considered reasonable to enhance it via
introduction of specific chemokine molecules used in
vaccine formulation as TAA carriers to antigen-presenting
cells [1–3]. In the last years, along with a number of che-
mokines (CCL7, CCL20, CXCL10 etc.), an antimicrobial
peptide murine beta-defensin-2 (mBD-2), a molecule
which ability to bind CCR6 on iDCs [4], has been used
in experimental cancer immunotherapy as the carrier of
genetically fused TAA, what improved significantly the
clinical efficacy of the vaccinations [5–7]. In the pres-
ent research devoted to experimental cancer vaccine
therapy, taking into account the data of immunologic
studies [5–7], we have chosen mBD-2 as a chemokine
in the content of cancer vaccines; from the other side we
wish to explore whether activation of in vivo expression of
this peptide antibiotic may be beneficial for clinical effect
of cancer immunotherapy.
mBD-2 belongs to the family of small (2–6 kD)
cationic microbicidal peptides that are produced by
epithelial cells in response to bacterial products and
proinflammatory cytokines, possess multiple biologic
activities, in particular immunomodulatory ones, and
compose an important chain of innate immunity sys-
tem [8, 9]. Moreover, similarly to other mammalian
defensins [10–12], mBD-2 may be involved in tumori-
genesis playing a complex and poorly understood yet
role in cancer cells and tumor microenvironment. Ac-
cording to our recent results [12], mBD-2 expressed
in murine Lewis lung carcinoma (3LL) cells, may pos-
sibly be involved in regulation of 3LL cell proliferation
in vitro and in vivo playing antiproliferative role in this
experimental tumor. Down-regulation of mBD-2 mRNA
expression in 3LL cells in vivo led to accelerated tumor
growth and more aggressive metastasis [12]. There-
fore, we hypothesized that up-regulation of mBD-
2 expression in epithelial tissues of 3LL-bearing mice
in addition to immunotherapy could improve total an-
ticancer effect of the treatment. In this regard we have
decided to analyze whether it is possible to activate
mBD-2 mRNA expression in vivo by introduction of vi-
tamin D3 (VD3) to experimental animals. According to
the literature data, some human antimicrobial peptides
(cathelicidin [13] and human beta-defensin-2 [14]) are
positively regulated by 1.25(OH)2D3 — metabolic form
of VD3. The study [15] performed on mice has shown
that mBD-2, mBD3 and cathelin-related antimicrobial
peptide are up-regulated in mouse skin exposed to
low-dose UV irradiation, and this process is mediated
by cutaneous VD3 activation.
To our knowledge, despite wide interest to VD3 as
potent pleiotropic immunomodulating agent that may
be used alone or in combination with chemothera py
[16] significantly improving anticancer effect of treat-
ment, there are just few publications in the field on its
combined use in a setting of cancer immunotherapy.
For example, in the work [17], the research has been
ADMINISTRATION OF VITAMIN D3 IMPROVES ANTIMETASTATIC
EFFICACY OF CANCER VACCINE THERAPY OF LEWIS LUNG
CARCINOMA
E. Zhuravel, O. Efanova, T. Shestakova, N. Glushko, O. Mezhuev, M. Soldatkina, P. Pogrebnoy*
R.E. Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology, NAS of Ukraine, Kyiv, Ukraine
Aim: To analyze antitumor efficacy of experimental cancer vaccine therapy combined with introduction of vitamin D3 (VD3) for treat-
ment of Lewis lung carcinoma (3LL). Materials and Methods: Cancer vaccines composed from recombinant murine beta-defensin-2
(mBD-2) and 3LL cell lysate, or DNA, coding for mBD-2-Muc1 fusion construct cloned in pcDNA3+ vector, were prepared and
used for intradermal vaccination. Experimental cancer vaccines introduced i. d. at therapeutic and prophylactic regimens to 3LL-
bearing C57Bl mice, were applied alone or in combination with VD3 (administered per os) and/or low-dose cyclophosphamide (CP,
administered intraperitoneal). Efficacy of treatments was analyzed by primary tumor growth dynamics indexes and by metastasis
rate in vaccinated animals. Results: As it has been shown, administration of the protein-based vaccine composed from mBD-2 and
3LL cell lysate in combination with VD3 and CP, but not in VD3 free setting, led to significant suppression of primary tumor growth
(p < 0.005) and had significant antimetastatic effect. Introduction of VD3 with or without CP in the scheme of treatment with mBD-
2-Muc1-DNA vaccine at therapeutic regimen has led to significant suppression of primary tumor (p < 0.05) and metastasis volumes
(p < 0.005), while in the groups of animals treated with DNA-vaccine + VD3 with or without CP at prophylactic regimen, significant
antimetastatic effect (p < 0.05) and elevation of average life-span (p < 0.05) have been registered. Conclusion: The results of this pilot
study have shown promising clinical effects of VD3 administration in combination with cancer vaccinotherapy in vivo.
Key Words: experimental cancer vaccine, murine beta-defensin-2, mucin-1, Lewis lung carcinoma, vitamin D3, cyclophosphamide.
Received: February 4, 2010.
*Correspondence: E-mail: pogrebnoy@onconet.kiev.ua
Abbreviations used: 3LL — Lewis lung carcinoma; СР — cyclophos-
phamide; DC — dendritic cell; mBD-2 — murine beta-defensin-2;
Muc1 — murine mucin-1; TAA — tumor-associated antigen; VD3 —
vitamin D3.
Exp Oncol 2010
32, 1, 33–39
34 Experimental Oncology 32, 33–39, 2010 (March)
performed on 3LL model, where the efficacy of applied
immunotherapy (adoptively transferred tumor-reactive
lymph node cells) has been shown to be significantly
elevated by introduction of VD3; the authors have
registered the significant antimetastatic effect of such
treatment. In a series of works [18–20] it has been
demonstrated that VD3/calcitriol may be considered
an effective mucosal adjuvant agent potently promot-
ing immune responses to cutaneously administered
vaccines.
So, the first task of our work was to study the ex-
pression patterns of mBD-2 mRNA in lung tissue of
healthy mice treated with VD3, and the second one
was to prepare anticancer protein-based and DNA-
based vaccines and analyze whether their efficacy
in vivo in 3LL tumor model may be increased by an
introduction of VD3.
MATERIALS AND METHODS
Cell lines and bacterial strains. In vitro culture of
transplantable 3LL cells was obtained from the Bank of
Cell Lines from Human and Animal Tissues of R.E. Ka-
vetsky 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 supplemented with 10%
fetal bovine serum, 100 units/mL penicillin, 100 μg/mL
streptomycin sulfate, and 0.25 μg/mL amphotericin B
as fungizone in 5% CO2 athmosphere at 37 °C.
Cultivation of bacterial cells (strain E. coli DH5α)
was carried out on LB medium at 37 °C. Selection and
storage of recombinant colonies was performed on
agarized LB medium supplemented with 50 μg/mL
ampicilline or kanamycine.
Gene cloning and plasmid construction. The
pcDNA3-Igk-mBD-2 expression plasmid contai-
ning secretable mBD-2 was created by the following
way. Mouse Igk signal sequence was cloned from
plasmid pSecTag2a to pcDNA3.1.+. Gene for ma-
ture mBD2 was obtained from lipopolysaccharide
(10 ng/ml)-treated murine BALB/c macrophages
by RT-PCR using the following primers: mBD-2-F:
5’-ACCTAAGCTTCGAACTTGACCACTGCCACACC-3’
and mBD-2-R: 5’-GCCGAATTCTCATTTCATGTACTT-
GCAACAGGG-3. The mature mBD2 cDNA was cloned
in frame with Igk signal sequence.
For construction of pcDNA3-Igk-mBD-2-Muc1, ex-
tracellular domain of Muc1 fragment containing 11 tan-
dem repeats was cloned by RT-PCR from total RNA
of 3LL cells with the use of the following PCR pri mers:
Muc1-F: 5’-GCCGAATTCACCAGCAGTTCCTTAG-
CATC-3’; Muc1-R: 5’-CTACTCGAGTCATGCAGAGCT-
GGTAGTTGTGAC-3’; and for mBD-2 fragment:
mBD-2-F and CmBD-R: 5’-GGCGAATTCAAGATCG-
GCTTTCATGTACTTGCAACAGGG-3’. The N-terminus
of Muc1 was fused in frame with mBD-2 through the
linker sequence. The Muc1 fragment for the control
vector pcDNA3-Igk-Muc1 was amplified using the
following primers pair: CMuc1-F: 5’-AACAAGCTTCAC-
CAGCAGTTCCTTAGCATC -3’; Muc1-R.
All constructs were verified by DNA dideoxyse-
quencing method using T7 and BGH primers and puri-
fied using plasmid purification kit Quiagen EndoFree
(USA). Content of LPS in plasmid preparation and pro-
tein-based vaccine was evaluated by standard LAL-test.
Control transfection of human embryonal kidney
(HEK293) cells was performed with the use of FuGene
6 reagent (Roche Molecular Biochemicals, USA) ac-
cording to the instructions of the manufacturer.
RT-PCR analysis. Total RNA was isolated from tis-
sue samples by the method of Chromzynski and Sacchi
[21]. For detection of mBD-2 or Muc1 RNA expression,
semiquantitative RT-PCR analysis was performed
with the use of specific primers. The expression level
of beta-actin as the house-keeping gene served as
a control. The relative expression level was analyzed
with the use of TotalLab Program.
Preparation of protein-based vaccine. For the
preparation of protein-based vaccine, active recombi-
nant mBD-2 produced in bacterial cells as GST-fusion
protein [12], that was additionally purified from endo-
toxin contaminants has been used. 3LL cell lysate was
prepared from in vitro cultured 3LL cells by standard
lysis of the cells with modified SDS-free RIPA buffer,
and stored at –70 °C until use.
In vivo study. For in vivo research, male C57Bl
mice 2 months old bred in the animal facility of R.E. Ka-
vetsky Institute of Experimental Pathology, Oncology
and Radiobiology, NAS of Ukraine (Kyiv, Ukraine) were
used. All animal procedures were carried out accor-
ding to the rules of local Ethic Committee and were
approved by the Ethic Board of IEPOR NASU.
3LL cells (5 x 104 cells/100 μl PBS per animal) were
transplanted i.m. in right hind leg of C57Bl mice. Tu-
mor growth dynamics was monitored each 5 days by
means of calipers starting from day 17 after tumor cell
transplantation when tumors became palpable, and till
the end of experiment. At 34-th day after tumor cell
transplantation, the animals were sacrificed by ether
narcosis, primary tumors and lungs were removed and
weighted, lung metastases were calculated, and blood
serum was collected.
Immunotherapy of experimental murine tumors
with the use of cancer DNA vaccine or protein-based
vaccine was performed at therapeutic and prophy-
lactic regimens by similar schedules. In therapeutic
setting, vaccine was introduced i.d. at days 2, 6, 10,
and 20 after tumor cell transplantation [5]; in prophy-
lactic one — at days 0, 4, 8, 18; in 2 weeks after the
last immunization, 3LL cells were transplanted. For
i.d. administration of DNA vaccine, the first three im-
munizations (20 μg DNA/10 μL PBS) were performed
with the use of tattoo device in the skin of left hind leg
by the method described in [22], while the fourth vac-
cination (20 μg DNA/10 μL PBS) was done i. d. in the
ear of animals. In the case of protein-based cancer
vaccine, it was injected i. d. only in the ear of animals
(recombinant mBD-2 peptide (2 g/10 l PBS) and
3LL cell lysate (20 g/20 l PBS)).
Experimental Oncology 32, 33–39, 2010 (March) 35
VD3 (10 IU/day/animal) was administered with
drinking water. Cyclophosphamide (СР, 2.5 mg/100 μL
PBS/animal) was administered i. p. at the days 1, 9,
19 after tumor cell transplantation as described in [23].
Experimental animals were housed in 10 groups
per each protein-based vaccination setting (А — thera-
peutic regimen, B — prophylactic regimen; n = 5 per
group) and received the following treatment: А1 (B1) —
control; А2 — mBD-2; А3 — 3LL cell lysate; А4 —
3LL + VD3; A5 — 3LL + D3 + CP; A6 — mBD-2 + 3LL;
А7 — VD3; А8 — СР; А9 — mBD-2 + 3LL + VD3; А10 —
mBD-2 + 3LL + VD3 + СР.
Similarly, for DNA-vaccination there were 10 groups
for therapy (C) and 10 groups for prophylaxis (D)
(n = 5 per group) that received complete or incom-
plete plasmid constructs: C1 (D1) — control (blank
vector); C2 (D2) — pcDNA3-Igk-mBD-2; C3 (D3)
–pcDNA3-Igk-Muc1; C4 (D4) — pcDNA3-Igk-Muc1
+ VD3; C5 (D5) — pcDNA3-Igk-Muc1 + VD3 +CP; C6
(D6) — pcDNA3-Igk-mBD-2-Muc1; C7 (D7) — VD3;
C8 (D8) — CP; C9 (D9) — pcDNA3-Igk-Muc1 + VD3;
C10 (D10) — pcDNA3-Igk-mBD-2-Muc1 + VD3 + СР.
Antitumor and antimetastatic effects of vaccina-
tion were evaluated by suppression of primary tumor
growth and the number of lung metastases. Each
experiment was repeated twice.
Statistical analysis. The data were reported as the
mean SD. The statistical significance of the differen ces
between mean values was assessed by the Student’s
t-test. Values p < 0.05 were considered statistically
significant. Differences in survival between the groups
were determined by nonparametric log-rank test.
RESULTS AND DISCUSSION
VD3 treatment up-regulates mBD-2 mRNA
expression in murine lung tissue. Prior to vaccina-
tions, we have performed an in vivo study in order to
analyze whether it is possible to activate mBD-2 mRNA
expression in vivo in epithelial lung cells of healthy mice
by introduction of VD3. Experiment has been carried
out on healthy C57Bl male mice (n = 3 per group)
that received VD3 (10 IU per animal per day) per os
for 10 days, or did not receive the vitamin. Then the
animals were sacrificed, and mBD-2 mRNA expression
level in healthy lung tissues of the animals was ana-
lyzed with the use of semiquantitative RT-PCR analysis.
Our data have demonstrated (Fig. 1) that consumption
of VD3 resulted in up-regulation of mBD-2 expression
in healthy lungs of C57Bl mice compared to the con-
trol. So, we have expected that introduction of VD3 in
immunotherapy schedule may lead to up-regulation
of mBD-2 expression in lung tissues of experimental
animals and, respectively, to elevation of its local an-
tiproliferative activity [12] what may possibly improve
the results of experimental immunotherapy.
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
K VD3
Re
la
tiv
e
le
ve
l o
f m
BD
-2
e
xp
re
ss
io
n
1 2 3 4
β-actin 284 bp mBD-2 120 bp
a
b
Fig. 1. RT-PCR analysis of mBD-2 mRNA expression in lung
tissue of healthy mice (a): mBD-2 mRNA expression in lungs
of healthy animals that were treated with VD3 (line 4) vs that in
control animals (line 3). Graphical representation of mBD2 mRNA
expression level normalized by beta-actin expression (b)
Design of experiment and selection of tumor
model. For performance of vaccinations, we have
used 3LL, an experimental model of murine solid tumor
with high level metastasis in lungs. Transplantation of
just 5 x 104 3LL cells/animal results in 100% lethality
in 5 weeks period.
In a setting of protein-based vaccine, we have used
3LL cell lysate as total tumor antigen.
In a setting of DNA-based vaccine, to select
a protein target in 3LL cells, we have performed an
analysis of literature data in the field, and decided to
introduce murine mucin-1 (Muc1) coding sequence in
DNA vaccine. Muc1 is a highly glycosylated 200 kDa
membrane protein [24], a non-polymorphic homo-
log of human polymorphic epithelial mucin MUC1.
The human MUC1 is expressed by a wide variety of
epithelial tissues where the protein plays the important
physiologic and antiinflammatory roles at normal state
[25, 26]. In human epithelial cancers, MUC1 is often
overexpressed, underglycosylated and lost polarized
type of expression, that’s why it is recognized as uni-
versal tumor antigen of human carcinoma. There are
numerous experimental researches of cancer vaccines
directed against MUC1 performed on MUC1-transgenic
animals, and clinical trials of cancer vaccines directed
against MUC1 [27, 28]; and their results are promising.
In contrary, little is known about expression patterns of
Muc1 in murine tumors, and experimental vaccinations
with the use of wtMuc1 have been done rarely. Accor-
ding to the data of literature [29], earlier with the use
of experimental breast cancer model there were per-
formed immunizations in C3H/HeOuj mice with mouse
mucin-1 fusion protein, and in a case of combination
of such immunotherapy with CP treatment antitumor
effect has been registered. As far as we did not found
the data on Muc1 expression patterns in murine tumor
cells and in particular, in 3LL cells, we have performed
semiquantitative RT-PCR analysis of Muc1 mRNA
expression in 3LL-derived tumors, healthy lungs and
36 Experimental Oncology 32, 33–39, 2010 (March)
heavily metastasized lungs of 3LL-bearing mice. We
have registered high expression levels of Muc1 mRNA
both in 3LL derived tumors, metastatic lesions, and
in healthy lung cells of C57Bl mice (Fig. 2); so, being
certain that Muc1 is expressed in 3LL cells, we have
supposed to direct our vaccine against Muc1 in this
particular tumor model despite the understandable risk
of its low specificity and autoimmunity.
0
0.05
0.1
0.15
0.2
0.25
0.3
1 2 3 4
Re
la
tiv
e
le
ve
l o
f M
uc
1
ex
pr
es
si
on
1 2 3 4
β-actin
284 bp
Muc1
750 bp
a
b
Fig. 2. RT-PCR analysis of Muc1 mRNA expression in largely
metastasized murine lung tissue (line 4), healthy lung tissue
(lines 1, 2) and primary 3LL tumor sample (line 3). Expression
of beta-actin served as a control (a). Graphical representation
of mBD2 mRNA expression level normalized by beta-actin ex-
pression (b)
At last, according to literature data [23, 30], cancer
immunotherapy efficacy could be elevated by its com-
bination with low-dose chemotherapy for inhibition of
immunosuppressive cancer network. That’s why we
have supplemented the vaccination schedule with
low-dose CP therapy (100 mg/kg).
Vaccination of 3LL-bearing mice with cancer
vaccine composed from recombinant mBD-2 pep-
tide and 3LL cell lysate. To analyze antitumor effect
of vaccination with the use of cancer vaccine com-
posed from recombinant mBD-2 peptide (2 g/10 l
PBS) and 3LL cell lysate (20 g/20 l PBS), we have
applied both therapeutic and prophylactic administra-
tion of the vaccine and its separate components with or
without VD3 and CP therapy by the schemes described
in Materials and Methods section.
The results of therapeutic vaccination have shown
(Fig. 3, 4) that administration of mBD-2 peptide to-
gether with 3LL cell lysate led to statistically insignifi-
cant suppression of primary tumor growth and meta-
static levels in 3LL-bearing mice compared with control
groups. However, introduction of VD3 into treatment
schedule, especially in combination with CP therapy at
metronomic regimen, has notably affected an efficacy
of such vaccination and resulted in significant decrease
of primary tumor volumes (group A10, p < 0.005, see
Fig. 3) and the number of lung metastases (see Fig. 4).
Interestingly, similar antimetastatic effect of VD3 ad-
ministration has been observed in a control group of
animals treated with 3LL cell lysate + VD3 (see Fig. 4)
but not in the case of separate VD3 or 3LL cell lysate
administration, and there were insignificant differences
in lung metastasis numbers bet ween 3LL + VD3 and
mBD-2 + 3LL + VD3 treated groups. Administration of
CP only didn’t influence significantly primary tumor
volumes, but had significant antimetastatic effect
(group A8). Upon prophylactic administration of the
vaccine, also the highest antimetastatic effect has been
observed in the case of administration of the vaccine si-
multaneously with VD3 consumption (mean lung weight
in vaccine + VD3 treated animals was 190 ± 15 mg vs
295 ± 145 mg in control groups). Both prophylactic and
therapeutic vaccinations had statistically insignificant
influence on average life span of tumor-bearing mice
(Fig. 5).
0
1
2
3
4
5
6
7
8
A1 A2 A3 A4 A5 A6 A7 A8 A9 A10
Tu
m
or
w
ei
gh
t,
r.u
.
*
Fig. 3. Comparative analysis of primary tumor weight (rel. units)
in experimental animals from groups A1–A10 treated with pro-
tein-based vaccine at therapeutic regimen at the day 34 after 3LL
transplantation: А1 — control; А2 — mBD-2; А3 — 3LL cell lysate;
А4 — 3LL + VD3; A5 — 3LL + D3 + CP; A6 — mBD-2 + 3LL; А7 —
VD3; А8 — СР; А9 — mBD-2 + 3LL + VD3; А10 — mBD-2 + 3LL +
VD3 + СР. Each group was composed from 5 animals, experi-
ment was twice repeated. *Difference is significant (p < 0.005)
compared to group A1.
0
0.1
0.2
0.3
0.4
0.5
0.6
A1 A2 A3 A4 A5 A6 A7 A8 A9 A10
Lu
ng
w
ei
gh
t
Fig. 4. Lung weight of experimental animals from groups A1–
A10 treated with protein-based vaccine at therapeutic regimen
at the day 34 after 3LL transplantation. Treatment groups: А1 —
control; А2 — mBD-2; А3 — 3LL cell lysate; А4 — 3LL + VD3;
A5 — 3LL + D3 + CP; A6 — mBD-2 + 3LL; А7 — VD3; А8 — СР;
А9 — mBD-2 + 3LL + VD3; А10 — mBD-2 + 3LL + VD3 + СР.
Each group was composed from 5 animals, experiment was
twice repeated
So, from the data of mentioned above experiments
we have figure out two main conclusions. Firstly, an
anticancer efficacy of protein [mBD-2 + 3LL lysate]
vaccination is moderate enough and did not generate
significant therapeutic and protective immunity in 3LL
model. Such results could be explained by the data of
investigation [4] where the authors have shown that
Experimental Oncology 32, 33–39, 2010 (March) 37
effective vaccinations using beta-defensin-2 require
an existence of physical link between peptide and TAA
to elicit antitumor immunity. Possibly, an absence of
chemical coupling between recombinant mBD-2 pep-
tide and TAAs in 3LL cell lysate may explain mode rately
good results of vaccination of 3LL bearing mice. Sec-
ondly, our results have demonstrated that introduction
of VD3 into the scheme of cancer immunotherapy may
be considered reasonable and, without being toxic,
notably elevates antimetastatic effect of vaccination,
even in its defensin-free setting (see Fig. 4). Besides,
the use of VD3 alone also led to some decrease of
secondary tumors volumes, however such rates were
statistically insignificant; it couldn’t be excluded that
mentioned effect may be in part realized via up-regu-
lation of mBD-2 gene in murine epithelial tissues. Both
mentioned conclusions we have taken into account in
the second part of our research directed on generation
of mBD-2-containing cancer DNA vaccine.
0 10 20 30 40 50 60
0
10
20
30
40
50
60
70
80
90
100
A1
A2
A3
A4
A5
A6
A7
A8
A9
A10
Days
Su
rv
iva
l,
%
0 10 20 30 40 50
0
10
20
30
40
50
60
70
80
90
100
B1
B2
B3
B4
B5
B6
B7
B8
B9
B10
Days
Su
rv
iva
l,
%
a
b
Fig. 5. Survival of 3LL-bearing mice treated with protein-based
vaccine at therapeutic (a) and prophylactic (b) regimens. Each
group of animals was composed from 10 animals. Log-rank
test. Treatment groups: А1 (B1) — control; А2 (B2) — mBD-2;
А3 (B3) — 3LL cell lysate; А4 (B4) — 3LL + VD3; A5 (B5) — 3LL +
D3 + CP; A6 (B6) — mBD-2 + 3LL; А7 (B7) — VD3; А8 (B8) —
СР; А9 (B9) — mBD-2 + 3LL + VD3; А10 (B10) — mBD-2 + 3LL +
VD3 + СР. *Difference is significant (p < 0.05) compared to the
control groups treated with blank vector.
Construction of cancer DNA vaccine composed
from mBD-2 and murine Muc1. To generate cancer
DNA vaccine composed from mBD-2 and murine
Muc1, we have constructed DNA vector — chimeric
molecule containing the coding sequences of mature
mBD-2 and murine Muc1 genes by the procedure
described in Materials and Methods section.
The constructs were tested by control transfec-
tion of human embryonal kidney HEK293 cells, with
the next RT-PCR analysis of Muc1 mRNA expres-
sion (Fig. 6). RT-PCR analysis has demonstrated
that transfection was effective, and the transfected
HEK293 cells express Muc1 mRNA (see Fig. 6).
1 2 3 4 5 6
β-actin 284 bp Muc1 750 bp
Fig. 6. RT-PCR analysis of Muc1 mRNA expression in control HEK
293 cells (4) and transfected НЕК-pcDNA3-Igk-Muc1 (5) and
НЕК-pcDNA3-Igk-mBD-2-Muc1 cells (6). Lines 1–3 — expres-
sion of beta-actin gene in HEK 293, НЕК-pcDNA3Igk-Muc1 and
НЕК-pcDNA3Igk-mBD-2-Muc1 cells served as a control
So, in following research we have used the next panel
of vector constructs: pcDNA3.1+, pcDNA3-Igk-mBD-2,
pcDNA3-Igk-Muc1, pcDNA3-Igk-mBD-2-Muc1.
Evaluation of anticancer efficacy of mBD-2-
Muc1-cancer DNA vaccine. To evaluate anticancer
efficacy of the developed mBD-2-Muc1-DNA vaccine,
we have apply vaccination protocol consisting from
four sequential intradermal immunizations (20 μg
DNA/10 μl PBS per animal) — first three with the use
of tattoo device in skin of animal’s leg, and the forth
one — i. d. in ears of mice, at therapeutic (series C)
and prophylactic (series D) regimens, as described in
Materials & Methods section.
As it has been mentioned above, transplantation
of 5 x 104 3LL cells/animal results in 100% mortality
in 5 weeks period; as we have found out, vaccination
with the use of DNA vaccine + VD3 + CP led to genera-
tion of tumor protection in 20% of mice that received
immunotherapy at prophylactic and therapeutic
regimens. In the rest of animals that developed the
tumors, therapeutic vaccination suppressed signifi-
cantly primary tumors growth (p < 0.05) (Fig. 7) and
led to significant reduction of metastasis (p < 0.005)
in animals treated with vaccine + VD3 and with/without
CP (Fig. 8). Prophylactic vaccination led to statistically
insignificant suppression of primary tumor growth in
animals treated with vaccine + VD3 + CP and significant
decrease of metastasis rate in groups D4, D9, D10
(see Fig. 8); in groups C9, C10, D9 and D10 (pcDNA3-
Igk-mBD-2-Muc1 + VD3 with or without CP) we have
registered also the significant increase of average
life-span of tumor-bearing animals (p < 0.05, Fig. 9).
In conclusion, the data of in vivo experiments evi-
dence on positive clinical effect of VD3 introduction
into scheme of mBD-2-Muc1-DNA-based vaccination
combined with low-dose CP treatment, reflected in sig-
nificant antimetastatic effect and increase of average
life-span of tumor-bearing animals. To be explained,
the aforementioned results, without a doubt, require
immunological studies, and we’ll perform them in our
further research.
According to recent knowledge, VD3 is a pleiotropic
potent regulator of mammalian immune system [30].
Its ability to suppress autoimmune reacti vity is dem-
onstrated in experimental models of type 1 diabetes,
systemic lupus erythematosus, inflammatory bowel
disease, autoimmune thyroiditis etc. [31, 32]. Along with
immunosuppressive activity, paradoxically, VD3 has a
38 Experimental Oncology 32, 33–39, 2010 (March)
positive effect as well on the system of innate immunity
enhancing its response against inva ding microorgan-
isms; VD3 deficiency is believed to be associated with
susceptibility to some infectious diseases, in particular,
tuberculosis [33]. In the last case, some possible mecha-
nisms of innate defense against Mycobacterium tuber-
culosis are supposed to be realized via VD3-dependent
up-regulation of cathelicidin in host macrophages [34].
We believe that in present research the favoring effect of
VD3 on antimetastatic efficacy of experimental cancer
vaccines possibly may be in some part mediated by
VD3-dependent up-regulation of mBD-2 in lung epithelial
cells. Meanwhile, recent publications have shown that
antitumor activity of VD3 and 1.25(OH)2D3 in vivo, due
to multiple target genes of the vitamin, could be reali-
zed by many ways including induction of cancer cell
apoptosis, regulation of the cell cycle, up-regulation
of VD3-receptor (VDR) playing a role in p53-mediated
suppression of tumor growth, or regulation of a balance
between proangiogenic and antiangiogenic factors thus
influencing tumor angiogenesis [35–37]. Further studies
in this field will help us understand the mechanisms of
immunomodulatory effects of VD3 as potentially effec-
tive adjuvant in cancer immunotherapy, and of its direct
antitumor action.
0
1
2
3
4
5
6
7
C1 C2 C3 C4 C5 C6 C7 C8 C9 C10
Tu
m
or
w
ei
gh
t,
r.u
.
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0.5
C1 C2 C3 C4 C5 C6 C7 C8 C9 C10
Lu
ng
w
ei
gh
t,
r.u
.
a
b
* *
*
* *
Fig. 7. Primary tumors volume (a) and lung weight (b) in
3LL-bearing mice, treated with DNA vaccine at therapeutic regi-
men at the day 34 after 3LL transplantation: C1 — control (blank
vector); C2 — pcDNA3-Igk-mBD-2; C3 — pcDNA3-Igk-Muc1;
C4 — pcDNA3-Igk-Muc1 + VD3; C5 — pcDNA3-Igk-Muc1 +
VD3 +CP; C6 — pcDNA3-Igk-mBD-2-Muc1; C7 — VD3; C8 —
CP; C9 — pcDNA3-Igk-mBD-2-Muc1 + VD3; C10 — pcDNA3-
Igk-mBD-2-Muc1 + VD3 + СР. a — *Difference is significant
(p < 0.05) compared to group C1. b — *Difference is significant
(p < 0.005) compared to group C1. Each group of animals was
composed from 5 animals, experiment was twice repeated.
0
1
2
3
4
5
6
D1 D2 D3 D4 D5 D6 D7 D8 D9 D10
Tu
m
or
w
ei
gh
t,
r.u
.
0
0.1
0.2
0.3
0.4
0.5
0.6
D1 D2 D3 D4 D5 D6 D7 D8 D9 D10
Lu
ng
w
ei
gh
t,
r.u
.
a
b
***
Fig. 8. Primary tumor volume (a) and lung weight (b) in 3LL-
bearing mice, treated at prophylactic regimen with DNA vaccine
at the day 34 after 3LL transplantation: D1 — control (blank vec-
tor); D2 — pcDNA3-Igk-mBD-2; D3 — pcDNA3-Igk-Muc1; D4 —
pcDNA3-Igk-Muc1 + VD3; D5 — pcDNA3-Igk-Muc1 + VD3 +
CP; D6 — pcDNA3-Igk-mBD-2-Muc1; D7 — VD3; D8 — CP;
D9 — pcDNA3-Igk-mBD-2-Muc1 + VD3; D10 — pcDNA3-Igk-
mBD-2-Muc1 + VD3 + СР. *Difference is significant (p < 0.05)
compared to group D1. Each group of animals was composed
from 5 animals, experiment was twice repeated.
0 10 20 30 40 50 60 70 80 90 100 110
0
10
20
30
40
50
60
70
80
90
100 D1
D2
D3
D4
D5
D6
D7
D8
D9
D10
Days
Days
Su
rv
iva
l,
%
0 10 20 30 40 50 60 70 80 90 100 110
0
10
20
30
40
50
60
70
80
90
100
C1
C2
C3
C4
C5
C6
C7
C8
C9
C10
Su
rv
iva
l,
%
a
b
Fig. 9. Survival of 3LL-bearing mice treated with DNA vaccine
at therapeutic (a) and prophylactic (b) regimens. Each group
of animals was composed from 10 animals. Log-rank test. C1
(D1) — control (blank vector); C2 (D2) — pcDNA3-Igk-mBD-2;
C3 (D3) — pcDNA3-Igk-Muc1; C4 (D4) — pcDNA3-Igk-Muc1 +
VD3; C5 (D5) — pcDNA3-Igk-Muc1 + VD3 + CP; C6 (D6) —
pcDNA3-Igk-mBD-2-Muc1; C7 (D7) — VD3; C8 (D8) — CP; C9
(D9) — pcDNA3-Igk-mBD-2-Muc1 + VD3; C10 (D10) — pcDNA3-
Igk-mBD-2-Muc1 + VD3 + СР. *Difference between groups C9 vs
C1, and D9 vs D1 are significant (p < 0.05).
Experimental Oncology 32, 33–39, 2010 (March) 39
ACKNOWLEDGEMENTS
The work was supported by NASU grants
0107U005545, the Program “Newest Medico-Biological
Problems and Environment”, Part 2. “Biologically Active
Compounds for Human Health” (Ukraine), and U002243
“Fundamental Problems of Genomics and Proteomics”.
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