The effect of NSC-631570 (Ukrain) alone and in combination with pathogen-associated molecules on cell cycle distribution and apoptosis induction of mouse melanoma cells with different biological properties
Monotherapy and combined application of antitumor drug NSC-631570 (Ukrain) are s uccessfully used for treatment of malignant melanoma since 1996. Melanoma cells of different origin have distinct susceptibility to components of Ukrain. Aim: To carry out comparative investigation of the effect of Ukra...
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Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України
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| Cite this: | The effect of NSC-631570 (Ukrain) alone and in combination with pathogen-associated molecules on cell cycle distribution and apoptosis induction of mouse melanoma cells with different biological properties / L.M. Skivka, O.O. Trompak, Yu.I. Kudryavets, N.A. Bezdenezhnykh, Ya.M. Susak // Experimental Oncology. — 2010. — Т. 32, № 2. — С. 92-96. — Бібліогр.: 26 назв. — англ. |
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Skivka, L.M. Trompak, O.O. Kudryavets, Yu.I. Bezdenezhnykh, N.A. Susak, Ya.M. 2018-06-19T10:21:08Z 2018-06-19T10:21:08Z 2010 The effect of NSC-631570 (Ukrain) alone and in combination with pathogen-associated molecules on cell cycle distribution and apoptosis induction of mouse melanoma cells with different biological properties / L.M. Skivka, O.O. Trompak, Yu.I. Kudryavets, N.A. Bezdenezhnykh, Ya.M. Susak // Experimental Oncology. — 2010. — Т. 32, № 2. — С. 92-96. — Бібліогр.: 26 назв. — англ. 1812-9269 https://nasplib.isofts.kiev.ua/handle/123456789/138600 Monotherapy and combined application of antitumor drug NSC-631570 (Ukrain) are s uccessfully used for treatment of malignant melanoma since 1996. Melanoma cells of different origin have distinct susceptibility to components of Ukrain. Aim: To carry out comparative investigation of the effect of Ukrain used alone and in combination with pathogen associated molecules (PAM) on mitotic cycle and apoptosis induction in mouse melanoma cell lines with different biological properties. Methods: Two cell lines with different biological properties (rate of cell division, level of hematogenous metastasis, sensitivity to tumor necrosis factor (TNF)-induced apoptosis) established from B16 mouse melanoma cell line, were used. Apoptosis induction and cell viability were analyzed using trypan blue exclusion test, morphological criteria, DNA gel electrophoresis and flow cytometry. Cell cycle distribution of tumor cells was determined by flow cytometry. Transporters associated with antigen processing (TAP) genes expression was analyzed using reverse transcriptase-polymerase chain reaction (RT-PCR) method. Results: The melanoma cells with different metastatic capabilities differed ma rkedly by the growth rate, sensitivity to apoptosis inducers, and the character of TAP gene expression. Treatment of melanoma cells with Ukrain resulted in apoptosis induction in a dose dependent manner. Melanoma cells with high-metastatic properties were more sensitive to Ukrain than their low metastatic variants. However combined use of drug with PAM induced apoptosis more effectively in melanoma cells with low-metasta tic potential. Conclusion: Sensitivity to Ukrain in vitro may depend on biological properties of melanoma cells and may be modified by combined treatment of cells with TLR ligands. The results can be useful to optimize the regimen of mono and combined treatment of melanoma with Ukrain. en Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України Experimental Oncology Original contributions The effect of NSC-631570 (Ukrain) alone and in combination with pathogen-associated molecules on cell cycle distribution and apoptosis induction of mouse melanoma cells with different biological properties Article published earlier |
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The effect of NSC-631570 (Ukrain) alone and in combination with pathogen-associated molecules on cell cycle distribution and apoptosis induction of mouse melanoma cells with different biological properties |
| spellingShingle |
The effect of NSC-631570 (Ukrain) alone and in combination with pathogen-associated molecules on cell cycle distribution and apoptosis induction of mouse melanoma cells with different biological properties Skivka, L.M. Trompak, O.O. Kudryavets, Yu.I. Bezdenezhnykh, N.A. Susak, Ya.M. Original contributions |
| title_short |
The effect of NSC-631570 (Ukrain) alone and in combination with pathogen-associated molecules on cell cycle distribution and apoptosis induction of mouse melanoma cells with different biological properties |
| title_full |
The effect of NSC-631570 (Ukrain) alone and in combination with pathogen-associated molecules on cell cycle distribution and apoptosis induction of mouse melanoma cells with different biological properties |
| title_fullStr |
The effect of NSC-631570 (Ukrain) alone and in combination with pathogen-associated molecules on cell cycle distribution and apoptosis induction of mouse melanoma cells with different biological properties |
| title_full_unstemmed |
The effect of NSC-631570 (Ukrain) alone and in combination with pathogen-associated molecules on cell cycle distribution and apoptosis induction of mouse melanoma cells with different biological properties |
| title_sort |
effect of nsc-631570 (ukrain) alone and in combination with pathogen-associated molecules on cell cycle distribution and apoptosis induction of mouse melanoma cells with different biological properties |
| author |
Skivka, L.M. Trompak, O.O. Kudryavets, Yu.I. Bezdenezhnykh, N.A. Susak, Ya.M. |
| author_facet |
Skivka, L.M. Trompak, O.O. Kudryavets, Yu.I. Bezdenezhnykh, N.A. Susak, Ya.M. |
| topic |
Original contributions |
| topic_facet |
Original contributions |
| publishDate |
2010 |
| language |
English |
| container_title |
Experimental Oncology |
| publisher |
Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
| format |
Article |
| description |
Monotherapy and combined application of antitumor drug NSC-631570 (Ukrain) are s uccessfully used for treatment of malignant melanoma since 1996. Melanoma cells of different origin have distinct susceptibility to components of Ukrain. Aim: To carry out comparative investigation of the effect of Ukrain used alone and in combination with pathogen associated molecules (PAM) on mitotic cycle and apoptosis induction in mouse melanoma cell lines with different biological properties. Methods: Two cell lines with different biological properties (rate of cell division, level of hematogenous metastasis, sensitivity to tumor necrosis factor (TNF)-induced apoptosis) established from B16 mouse melanoma cell line, were used. Apoptosis induction and cell viability were analyzed using trypan blue exclusion test, morphological criteria, DNA gel electrophoresis and flow cytometry. Cell cycle distribution of tumor cells was determined by flow cytometry. Transporters associated with antigen processing (TAP) genes expression was analyzed using reverse transcriptase-polymerase chain reaction (RT-PCR) method. Results: The melanoma cells with different metastatic capabilities differed ma rkedly by the growth rate, sensitivity to apoptosis inducers, and the character of TAP gene expression. Treatment of melanoma cells with Ukrain resulted in apoptosis induction in a dose dependent manner. Melanoma cells with high-metastatic properties were more sensitive to Ukrain than their low metastatic variants. However combined use of drug with PAM induced apoptosis more effectively in melanoma cells with low-metasta tic potential. Conclusion: Sensitivity to Ukrain in vitro may depend on biological properties of melanoma cells and may be modified by combined treatment of cells with TLR ligands. The results can be useful to optimize the regimen of mono and combined treatment of melanoma with Ukrain.
|
| issn |
1812-9269 |
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https://nasplib.isofts.kiev.ua/handle/123456789/138600 |
| citation_txt |
The effect of NSC-631570 (Ukrain) alone and in combination with pathogen-associated molecules on cell cycle distribution and apoptosis induction of mouse melanoma cells with different biological properties / L.M. Skivka, O.O. Trompak, Yu.I. Kudryavets, N.A. Bezdenezhnykh, Ya.M. Susak // Experimental Oncology. — 2010. — Т. 32, № 2. — С. 92-96. — Бібліогр.: 26 назв. — англ. |
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2025-11-24T02:34:50Z |
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2025-11-24T02:34:50Z |
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| fulltext |
92 Experimental Oncology 32, 92–96, 2010 (June)
Malignant melanoma incidence increases steadily
all over the world. The number of patients with this pa-
thology had been doubled in the last 20 years. At the
early stages of melanoma development the patients
are treated surgically, but the advanced disease is in-
curable [1, 2]. Melanoma research experts consider in-
stability of melanoma cell biological properties among
the primary reasons of aggressiveness of the tumor
and its resistance to antitumor drugs. Melanoma cells
with different metastatic ability can differ significantly
in their sensitivity to anticancer agents [3–5].
NSC-631570 (Ukrain) is a semisynthetic product
of thiosulfuric acid and celandine alkaloids (Chelido-
nium majus L.). The drug has cytotoxic and cytostatic
action on tumor cells in vitro and in vivo due to its ability
to accumulate in tumor tissue selectively and cause
cancer cell death but not normal cells death [6, 7].
For more than 20 years NSC-631570 had been used
for benign and malignant tumor treatment [8]. Ukrain-
based monotherapy, and also its usage in combina-
tion with vaccine based on self-restricted autologic
dendritic cells demonstrated significant therapeutic
effect in the treatment of experimental В-16 mouse
melanoma [9, 10].
NSC-631570 monotherapy was successfully intro-
duced into human melanoma therapy in 1996, and has
been shown to be effective in combination with adjuvants
[11, 12]. However, melanoma cells of different origin
possess different sensitivity to Ukrain components [13].
The Toll-like receptor (TLR) agonists are among
the most promising adjuvants used for more than
10 years in combined therapy of malignant melanoma.
Pathogen associated molecules (PAM), such as pep-
tidoglycane, lipopolysaccharide, flagellin, CpG-DNA
etc. are the natural TLR agonists. It is known that TLR
are expressed in a broad spectrum of cells and tis-
sues including the effector cells of immune system.
Stimulation of TLR-dependent immune cell mediates
activation of inflammatory reactions and increases
antitumor immunity [14]. TLR are also expressed on
many tumor cells including melanoma cells [15, 16].
The use of natural (PAM) and synthetic TLR1, 2, 4,
7 and 9 agonists are shown to be effective for mela-
noma treatment [17]. In the present study, the extract
of S. aureus Wood 46 CPM was used as PAM. TLR6 in a
complex with TLR1 and/or TLR2 is considered as a
potential receptor for CPM [18].
The goal of the study was comparative assessment
of the effect of NSC-631570 (Ukrain) used alone and
in combination with PAM, on the mitotic cycle and
THE EFFECT OF NSC-631570 (UKRAIN) ALONE AND IN COMBINATION
WITH PATHOGEN-ASSOCIATED MOLECULES ON CELL CYCLE
DISTRIBUTION AND APOPTOSIS INDUCTION OF MOUSE MELANOMA
CELLS WITH DIFFERENT BIOLOGICAL PROPERTIES
L.M. Skivka1, *, O.O. Trompak2, Yu.I. Kudryavets3, N.A. Bezdenezhnykh3, Ya.M. Susak4
1T. Shevchenko Kyiv National University, Vladymyrska 64, Kyiv 01033, Ukraine
2National University of “Kyiv-Mohyla Academy”, Skovorody 2, Kyiv 04070, Ukraine
3R.E. Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology of NAS of Ukraine,
Vasylkivska 45, Kyiv 03022, Ukraine
4O.O. Bogomolets National Medical University, Shevchenko Ave 13, Kyiv 1601, Ukraine
Monotherapy and combined application of antitumor drug NSC-631570 (Ukrain) are s uccessfully used for treatment of malignant
melanoma since 1996. Melanoma cells of different origin have distinct susceptibility to components of Ukrain. Aim: To carry out
comparative investigation of the effect of Ukrain used alone and in combination with pathogen associated molecules (PAM) on mitotic
cycle and apoptosis induction in mouse melanoma cell lines with different biological properties. Methods: Two cell lines with different
biological properties (rate of cell division, level of hematogenous metastasis, sensitivity to tumor necrosis factor (TNF)-induced
apoptosis) established from B16 mouse melanoma cell line, were used. Apoptosis induction and cell viability were analyzed using
trypan blue exclusion test, morphological criteria, DNA gel electrophoresis and flow cytometry. Cell cycle distribution of tumor
cells was determined by flow cytometry. Transporters associated with antigen processing (TAP) genes expression was analyzed using
reverse transcriptase-polymerase chain reaction (RT-PCR) method. Results: The melanoma cells with different metastatic capabili-
ties differed ma rkedly by the growth rate, sensitivity to apoptosis inducers, and the character of TAP gene expression. Treatment
of melanoma cells with Ukrain resulted in apoptosis induction in a dose dependent manner. Melanoma cells with high-metastatic
properties were more sensitive to Ukrain than their low metastatic variants. However combined use of drug with PAM induced
apoptosis more effectively in melanoma cells with low-metasta tic potential. Conclusion: Sensitivity to Ukrain in vitro may depend
on biological properties of melanoma cells and may be modified by combined treatment of cells with TLR ligands. The results can
be useful to optimize the regimen of mono and combined treatment of melanoma with Ukrain.
Key Words: antitumor drug NSC-631570 (Ukrain), melanoma cell lines, metastatic capabilities, TAP genes.
Received: April 10, 2010.
*Correspondence: E-mail: realmed@i.com.ua
Abbreviations used: CPM — cytoplasmic membrane extract; MHC —
major histocompatibility complex; PAM — pathogen associated
mole cules; PCR — polymerase chain reaction; RT — reverse trans-
criptase; TLR — toll-like receptor; TNF — tumor necrosis factor.
Exp Oncol 2010
32, 2, 92–96
Experimental Oncology 32, 92–96, 2010 (June) 93
apoptosis of melanoma cells which possess various
biological properties.
MATERIALS AND METHODS
Cell cultivation, treatment and cell analysis.
Mouse melanoma MM-4 and ММ-4М2 cell lines were
obtained from Bank of Cell Lines of R.E. Kavetsky Institute
of Experimental Pathology, Oncology and Radiobiology,
National Academy of Sciences of Ukraine. The cells were
cultured in vitro in Dulbecco modified Eagle medium
(DMEM, Sigma, USA), supplemented by 10% fetal bo-
vine serum (FBS), penicillin (100 U/mL) and streptomycin
(100 μl/mL) at 37 °C in 5% CO2 atmosphere.
To study cell growth kinetics, melanoma cells were
seeded on 24-well plates at a density of 1 x 104 cells
per well in 1.5 ml of medium containing 10% FBS and
incubated at standard conditions for 6 days. The num-
ber of viable cells was determined daily with Trypan
Blue 0.4% staining as described in [19–21].
To test the cytotoxicity of drugs, cells were treated
at logarithmic phase of growth with Ukrain (Nowicky
Pharma, Austria) at the concentrations of 20 μg/ml
and 250 μg/ml for 24–48 h. Staphylococcus aureus
cytoplasmic membrane extract (CPM), which was kindly
granted by prof V.K. Pozur (T. Shevchenko Kyiv National
University), was used as PAM and was added in culture
medium at the concentrations of 2 μg/ml and 25 μg/ml
for 24–48 h. Ukrain (20 μg/ml) + PAM (2 μg/ml) and
Ukrain (250 μg/ml) + PAM (25 μg/ml) were used for
combined treatment. Solvent was added to the controls.
For a morphologic investigation, cytospin speci-
mens of cells were dried, Pappenheim-stained and
analyzed using Axiostar Plus microscope (Carl Zeiss,
Germany) at x 400–1000 magnification.
DNA extraction, gel electrophoresis and flow
cytofluorimetry were performed as it was described
earlier [19–21].
RNA isolation and RT-PCR analysis of TAP1 and
TAP2 mRNA expression. Total RNA was isolated from
homogenized cells using the “RiboSorb” KIT (AmpliSens,
Russia). Extracted RNA was transcribed into cDNA
using M-MuLV Reverse Transc riptase (Fermentas,
Lithuania) following recommendations of the manu-
facturer. The cDNA concentration was measured and
2 μl of each cDNA samples were taken for semiquan-
titative analysis. To circumvent false negative results,
primers detecting the GAPDH gene were used in the
same reaction tube. Each reaction mixture for PCR
contained: Taq-buffer with (NH4)2SO4, 2.5 μМ Mg2+,
0.2 μМ each dNTP, 1U Taq-polimerase (Fermentas,
Lithuania), 50 μМ of each primer and 2 μl of cDNA.
Following primers were used: TAP1 primers — forward
5’GGACTGTCAGCAGCGGCAACC-3’ and reverse
5’CAAGGCCTTTCATGTTTGAGGG-3’; TAP2 primers —
forward 5’CAGGATGCAGTGGCCAGGGCG-3’ and reverse
5’TAGATACACGTCTTTTTCCAGG-3’; GAPDH primers —
forward 5’GCCAAGGTCATCCATGACAACTTTGG-3’ and
reverse 5’GCCTGCTTCACCACCTTCTTGATGTC-3’. PCR
program: initial denaturation step at 94 °C, 4 min, and
30 cycles (93 °C 35 s, 60 °C 35 s, 72 °C 35 s). PCR pro-
ducts were visualized by electrophoresis in 2% agarose
gels with ethidium bromide using UV-transilluminator.
Semiquantitative analysis of the expression level was
performed in TotalLab TL120 program (Great Britain).
The statistical significance of the differences between
mean values was assessed by the Student’s t-test.
RESULTS AND DISCUSSION
ММ-4 cell line and ММ-4М2 subline, which were
used as the models in the experiments, were estab-
lished earlier from transplantable В-16 melanoma
of C57BL/6 mice [1]. ММ-4 cells have relatively low
metastatic ability: the cells rarely metastasize spon-
taneously when transplanted intramuscularly or
subcutaneously, and upon intravenous application
of 2 х 105 cells at average 10–20 pigmented metasta-
ses will be formed in the lungs of 70–100% of animals.
ММ-4М2 highly metastatic subline was obtained by
double in vivo passage of ММ-4 cells in C57BL/6 mice:
the cells were administered intravenously with further
in vitro recultivation of the largest lung metastases
that developed after 21 days. After the preparative
cultivation of metastatic cells, they were intravenously
administered to mice, and then the cells from large me-
tastases were again recultivated. The cells of newly es-
tablished line possessed much higher metastatic po-
tential than the initial cells: the metastases are formed
in 100% of animals, their average number reached
206.6 ± 56 (ranging from 68 to 360). ММ-4М2 cells
don’t differ significantly from the initial cell line by mor-
phology and pigmentation, although they have a clear
epithelioid morphology and higher adhesion (data not
presented). MM-4 and MM-4M2 cell lines dramatically
differ by growth rate: doubling time at logarithmic
phase of growth for MM-4 cells is 19.3 h, and for
MM-4M2 line — 22.7 h; maximal density of MM-4 cell
growth reaches 5.7 x 105/cm2, and of MM-4M2 cells —
only 2.78 x 105/cm2 (Fig. 1).
0
2
4
6
8
10
12
14
1 2 3 4 5 6
Days
Ce
ll
nu
m
be
r x
1
0-5
/w
el
l
MM-4
MM4-M2
Fig. 1. Cell growth kinetics of MM-4 and MM-4M2 melanoma
cell lines
The ММ-4 and ММ-4М2 cell lines are sensitive
to various apoptosis inducers, particularly to tumor
necrosis factor (TNF) (Fig. 2, a, b).
Interestingly, the sensitivity to growth inhibition
and apoptosis induction caused by TNF is much
higher in ММ-4М2 highly metastatic subline than
in ММ-4 cells. The difference between cell lines
is especially expressed at TNF concentrations
< 8–10 000 IU/ml, but at higher TNF concentrations
94 Experimental Oncology 32, 92–96, 2010 (June)
the cells of both lines become equally sensitive to TNF
(Fig. 2, b; Fig. 3).
1 2 M
a b
Fig. 2. Apoptosis in ММ-4 (a) and ММ-4М2 (a, b) cells treated
with TNF: a, morphology of apoptotic MM4 cells treated with TNF;
b, DNA laddering of MM4-M2 and MM4 cells treated with TNF
0
10
20
30
40
50
60
70
80
128 32 8 2
TNF un/ml x 10-3
In
hi
bi
tio
n
of
c
el
l g
ro
wt
h,
%
MM-4
MM-4M2
Fig. 3. Inhibition of cell growth of MM-4 and MM-4M2 mela-
noma cells
Expression of TAP1 and TAP2 in low- and high-
metastatic melanoma B16 cells. Level of metastasis
is dependent on antigenic characteristics of tumor cells,
and an enhanced or decreased antigenicity of dissemi-
nated tumor cells will lead to their immune recognition
and rejection or, conversely, enhancement of metasta-
sis. TAP proteins (transporters associated with antigen
processing) take part in the transport of oligopeptides
from proteasomes into endoplasmic reticulum. In the
endoplasmic reticulum these oligopeptides are bound
to MHC class I molecules and transported to the cell
surface. TAP proteins consist of two subunits: TAP1 and
TAP2. It has been previously shown that TAP protein
expression can be decreased in malignant cells, fol-
lowed by reduced protein expression or complete lack
of MHC class I antigens on the cell surface [11, 12].
Thus, character of TAP proteins expression may be
closely linked to metastatic ability of melanoma cells.
RT-PCR analysis revealed a total inhibition of mRNA
transcription of the peptide transporter TAP1 in high-
metastatic MM-4M2 cells compared to low-metastatic
MM-4 cells. mRNA levels of TAP2 had no significant
differences in these melanoma sublines (Fig. 4).
TAP1 and TAP2 down-regulation in primary mela-
noma lesions is associated with the decrease of tumor
immunogenicity and significantly correlated with tumor
progression and development of metastases [13].
Thus, ММ-4М2 cell subline is a useful model which
allows to reproduce post-invasive stage of hematoge-
nous metastasis, and also to examine the mechanisms
of melanoma cell sensitivity to various programmed
cell death inducers.
MM4 MM-4M2
TAP1
TAP2
GAPDH
Fig. 4. RT-PCR analysis of TAP mRNA expression in mouse
melanoma cells with different metastatic potential
It should be noted that some characteristics of high
metastatic MM-4M2 cells, such as a reduced rate of cell
proliferation and increased sensitivity to TNF-induced
apoptosis contradict, in part, to known characteristics of
some metastatic models. It is considered that resistance
to TNF is accompanied by an increase of the metastatic
ability. However, in this case the sensitivity to TNF did
not affect the selection of in vivo highly metastatic cells.
In the first part of the study we have examined the
effect of Ukrain alone or in combination with CPM on
the growth of ММ-4 and ММ-4М2 cell sublines [18].
The results have shown that the effect of Ukrain used
alone or in combination with PAM differs for ММ-4 and
ММ-4М2 cells (at least for 48 h treatment period).
Ukrain used alone at low concentration (20 μg/ml)
caused significant (treatment time independent) growth
inhibition of ММ-4 cells at average by 65% (p < 0.001)
(Fig. 5, a). In ММ-4М2 cells, the progressive inhibitory
action of Ukrain has been observed: the number of
viable cells decreases by 64.6% in 24 h and by 79.4%
in 48 h (p < 0.001) (Fig. 5, b). In this case, the sensitiv-
ity of MM-4M2 cells to Ukrain was significantly higher
(p < 0.01) than that of MM-4 cells. At high concentration
(250 μg/ml) the drug caused death of nearly 100% cells
of both sublines after 24–48 h of incubation.
PAM treatment of MM-4 and MM-4M2 cells influ-
enced their growth only during first 24 h of incubation,
but not later. PAM at the low concentration (2 μg/ml)
significantly (p < 0.01) inhibited cell growth in MM-4 cell
line. However, at the high concentration (25 μg/ml) led to
the increase of the number of viable cells (see Fig. 5a).
In MM-4M2 cells, by contrast, increased concentrations
of PAM (from 2 to 25 ug/ml) was accompanied by a sig-
nificant decrease in the number of living cells (p < 0.01).
The PAM, regardless of concentration after short-time
exposure, did not change the sensitivity of both cell lines
to the cytotoxic action of Ukrain (Fig. 5, a, b). Apoptosis
inducing effect after prolonged treatment of MM-4 cells
with Ukrain (48 h) in combination with PAM was more
expressed than that after treatment with Ukraine alone.
It is known that Ukrain attenuates tumor cell growth
by inducing arrest of the cells which are at G2/M phase
(or G0/G1 phase in vitro) with further apoptosis induction
[22, 23]. Analysis of cell cycle of MM-4 and MM-4M2 cells
Experimental Oncology 32, 92–96, 2010 (June) 95
treated by Ukrain in vitro has distinctly shown the ability of
the drug to modulate the cell cycle in a dose-dependent
way differently for the cells with various biological pro-
perties (Table). NSC-631570 did not affect apoptosis
rate of MM-4 cells at concentration of 20 μg/ml after
24 h of incubation. However, the number of G0/G1 cells
increased significantly (by 56% compared to control
cells), the number of cells in G2/M phase was 1.4 times
lower, and S-phase cells — two times lower, what can
point on cell cycle arrest in G0/G1. 24-hour incubation
of MM-4 cells with Ukrain at 250 μg/ml concentration
resulted in apoptosis of 98.4% cells.
0
20
40
60
80
100
120
140
1 2 3 4 5 6
Drugs
Vi
ab
le
c
el
ls
, %
o
f c
on
tro
l
MM-4
MM-4M2
0
20
40
60
80
100
120
140
1 2 3 4 5 6
Drugs
Vi
ab
le
c
el
ls
, %
o
f c
on
tro
l
a
b
Fig. 5. The effect of Ukrain and PAM on the growth of mouse
melanoma cells with different biological properties: 24 h (a) and
48 h (b): 1 — Ukrain 20 μg/ml; 2 — 250 μg/ml; 3 — PAM 2 μg/ml;
4 — PAM 25 μg/ml; 5 — Ukrain 20 μg/ml + PAM 2 μg/ml; 6 —
Ukrain 250 μg/ml + PAM 25 μg/ml
The MM-4M2 cells appear to be more sensitive
to NSC-631570 at the concentration of 20 μg/ml:
the number of apoptotic cells was 26.67% (4 times
higher than in MM-4 cells). Moreover, the number of
cells in G0/G1 phase has increased while the number
of G2/M and S-phase cells has dropped. Incubation
of MM-4M2 cells with Ukrain for 24 h at 250 μg/ml
concentration resulted in apoptosis of 97.2% of cells.
Prolonged (for 48 h) MM-4 cells incubation with
Ukrain at the concentration of 20 μg/ml has resulted
in the increase of apoptotic cell number (from 1% of
control to 25%). Apoptosis rate in MM-4M2 after 48-hour
incubation with NSC-631570 at concentration of 20 μg/ml
was lower than that after short-term (24-hour) exposure,
though 15 times higher than that in the control cells.
Ukrain caused stable apoptotic effect in both cell lines at
the concentration of 250 μg/ml after 48 h of incubation.
Thus, melanoma cells with different metastatic po-
tential are characterized by different sensitivity to NSC-
631570. Short-term drug treatment at low concentra-
tions had caused the apoptosis of significant number
of MM-4M2 cells only. The response of MM-4 cells
to Ukrain had developed more slowly (after 48-hour
of incubation). The results suggest that sensitivity of
melanoma cells to apoptosis induced by TNF may
coincide with sensitivity to Ukrain and possibly may
have some common mechanisms. These biological
patterns of the cells may be useful for the development
of therapeutic schedule of NSC-631570 drug usage.
Table. Cell cycle distribution in low- (MM-4) and high-metastatic
(MM-4M2) cell line after their treatment with Ukrain and PAM in vitro
Cells and
treatment
Cell number (%) in cell cycle phases
Apoptosis G0/G1 S G2/M
Incubation for 24 h
Control ММ-4 4.73 ± 0.14 44.06 ± 2.12 30.68 ± 0.99 25.26 ± 1.94
Ukrain 20 μg/ml 5.66 ± 0.18 68.74 ± 1.60 13.39 ± 1.07* 17.87 ± 0.98*
Ukrain 250 μg/ml 98.4 + 0.06 * Apoptosis
PAM 2 μg/ml 1.13 ±0.02* 43.55 ± 1.83 32.68 ± 0.88 23.76 ± 2.00
PAM 25 μg/ml 1.66 ±0.02* 49.10 ± 1.99 32.49 ± 0.76 18.41 ± 1.34
Ukrain 20 μg/ml +
PAM 2 μg/ml
29.2 ± 1.94* 70.15 ± 2.09* 17.16 ± 0.99* 12.69 ± 1.10*
Ukrain
250 μg/ml + PAM
25 μg/ml
94.46 ± 0.09* Apoptosis
Control ММ-4М2 5.61 ±0.11 44.64 ± 2.17 33.64 ± 2.01 21.72 ± 0.21
Ukrain 20 μg/ml 26.67 ± 0.79* 67.7 ± 1.60* 16.20 ± 0.98* 16.10 ± 1.04*
Ukrain 250 μg/ml 97.18 ± 0.09 Apoptosis
PAM 2 μg/ml 1.96 ± 0.07* 48.06 ± 2.98 18.49 ± 1.00 33.44 ± 1.01
PAM 25 μg/ml 6.45 ± 0.09 44.45 ± 1.06 21.20±0.86 34.34 ± 1.07
Ukrain 20 μg/ml +
PAM 2 μg/ml
9.22 ± 0.09* 74.62 ± 1.35* 13.02 + 1.00 12.36 ± 1.01
Ukrain
250 μg/ml + PAM
25 μg/ml
96.82 ± 1.00* Apoptosis
Incubation for 48 h
Control ММ-4 1.06 ± 0.08 72.43 ± 3.08 15.54 ± 1.16 12.03 ± 1.10
Ukrain 20 μg/ml 24.98 ± 1.94* 66.38 ± 2.96 16.76 ± 1.12 16.86 ± 1.10*
Ukrain 250 μg/ml 97.74 ± 1.04* Apoptosis
PAM 2 μg/ml 0.61 ± 0.02* 77.20 ± 1.98 8.01 ± 1.31* 14.79 ± 1.20
PAM 25 μg/ml 0.61 ± 0.05* 78.32 ± 3.11 7.11 ± 1.12* 14.57 ± 1.22
Ukrain 20 μg/ml +
PAM 2 μg/ml
18.73 ± 1.00* 63.95 ± 2.99 24.97 ±
0.85 *
11.08 ± 1.02
Ukrain
250 μg/ml + PAM
25 μg/ml
92.51 ± 1.00* Apoptosis
Control ММ-4М2 0.58 ± 0.03 64.29 + 2.08 22.72 ± 1.55 13.00 ± 1.01
Ukrain 20 μg/ml 8.83 ± 0.99* 68.81 ±2.08 15.85 ± 1.48 15.34 ± 1.11
Ukrain 250μg/ml 83.80 ± 3.06* 63.88 + 1.77 23.41 ± 2.00 12.71 ± 3.16
PAM 2 μg/ml 0.57 ± 0.08 69.10 ± 2.68 16.06 ± 1.90 14.85 ± 1.05
PAM 25 μg/ml 0.64 ± 0.02 69.65 ± 2.41 17.53 ± 1.10 12.92 ± 1.10
Ukrain 20 μg/ml +
PAM 2 μg/ml
14.28 ± 1.07* 72.59 ± 1.36* 14.70 ± 0.96* 12.64 ± 2.13
Ukrain
250 μg/ml + PAM
25 μg/ml
78.95 ± 3.03* 68.03 ± 1.42 17.54 ± 1.29 14.43 ± 1.16
*Difference is significant compared to the control (p < 0.05).
Nowadays information concerning TLR agonists effect
on cell cycle distribution is quite scanty and controversial.
It is known that TLR activation by natural ligands causes
increase of malignant cell proliferation under certain con-
ditions [24, 25]. It is recently shown that TLR agonists can
activate apoptosis of tumor cells [26]. Our experiments
have shown that in almost all cases and regardless of the
dose, PAM causes suppression of basal level of apop-
tosis in MM-4 cells. The PAM response pattern of MM-
4M2 subline differed from that of MM-4 cells. Exposure of
MM-4 cells to 2 μg/ml PAM for 24 h had led to decrease
of the apoptosis level by 65%. Usage of high PAM con-
centration (25 μg/ml) did not cause any reliable influence
on mitotic cycle of MM-4 cells. Reliable influence on cell
cycle was also absent when the exposure was prolonged.
96 Experimental Oncology 32, 92–96, 2010 (June)
Thus, В16 melanoma cell sublines which possess
different biological properties, had different response
to TLR agonists. We suggest that it could be considered
upon its usage in antitumor adjuvant immunotherapy,
especially in the case of low metastatic tumor variant.
However, PAM treatment causes different effect on
cell cycle in melanoma cell sublines if it used in combina-
tion with Ukrain.
Ukrain at the concentration of 20 μg/ml combined
with PAM at the concentration of 2 μg/ml caused
increased MM-4 tumor cell death rate. The number of
apoptotic cells in some samples was 6 times higher in
comparison to the control, 5 times higher compared to
Ukrain-treated samples and 22 times higher than that
in PAM-treated samples (see Table).
Effect of Ukrain and PAM combination (20 μg/ml
and 2 μg/ml correspondingly) on highly metastatic MM-
4M2 cells was weaker than that of Ukrain used alone
after shot-term exposure. However, when the exposure
was prolonged, apoptosis inducing effect of combined
treatment was more expressed than that after treatment
of MM-4M2 cells with Ukrain alone.
Total apoptosis was observed in the cells of both cell
sublines treated by combination of Ukrain and PAM at high
concentrations (250 μg/ml and 25 μg/ml correspond-
ingly), as well as in the samples treated with Ukrain at the
same concentration.These data have demonstrated that
the PAM does not reduce the apoptotic effect of Ukrain.
Thus, melanoma cells with different metastatic po-
tential had different response pattern to NSC-631570
(Ukrain) used alone or in combination with PAM. 24-hour
treatment with drug at the low concentration resulted
in considerable death rate of MM-4M2 cells only.
MM-4 cells were more sensitive to combined treatment
at low concentrations. It is possible that such effect could
be caused by different TLR expression level in these cells.
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