Anti-tumor activity of murine peritoneal macrophages induced by porcine skin gelatin
Aim: To study the induction of anti-tumor activity of murine peritoneal macrophages in vitro by porcine skin gelatin. Methods: Anti-tumor activity of the macrophages was evaluated with tritium thymidine uptake by target tumor cells. ELISA was used to measure amounts of cytokines secreted in culture...
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Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України
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| Цитувати: | Anti-tumor activity of murine peritoneal macrophages induced by porcine skin gelatin / T. Koide, T. Kojima, Y. Inamura, H. Nagata, Y. Hashimoto, Y. Sugita, H. Maeda // Experimental Oncology. — 2008. — Т. 30, № 4. — С. 300–305. — Бібліогр.: 15 назв. — англ. |
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Koide, T. Kojima, T. Inamura, Y. Nagata, H. Hashimoto, Y. Sugita, Y. Maeda, H. 2018-06-21T13:55:53Z 2018-06-21T13:55:53Z 2008 Anti-tumor activity of murine peritoneal macrophages induced by porcine skin gelatin / T. Koide, T. Kojima, Y. Inamura, H. Nagata, Y. Hashimoto, Y. Sugita, H. Maeda // Experimental Oncology. — 2008. — Т. 30, № 4. — С. 300–305. — Бібліогр.: 15 назв. — англ. 1812-9269 https://nasplib.isofts.kiev.ua/handle/123456789/139943 Aim: To study the induction of anti-tumor activity of murine peritoneal macrophages in vitro by porcine skin gelatin. Methods: Anti-tumor activity of the macrophages was evaluated with tritium thymidine uptake by target tumor cells. ELISA was used to measure amounts of cytokines secreted in culture medium. Results: The ability of the gelatin to induce anti-tumor activity of the macrophages was stronger than that of lipopolysaccharide of E. coli. Combination of the lipopolysaccharide and interferon-g synergistically stimulated the macrophages but that of the gelatin and interferon-g additionally did. The culture supernatant of the macrophages incubated with the gelatin also showed higher anti-tumor activity than that with the lipopolysaccharide though the lipopolysaccharide was more excellent than the gelatin in stimulating secretion of anti-tumor cytokines (IL-1, IL-6, TNF-a, IFN-g) by the macrophages. Anti-TNF-a antibody partially suppressed the anti-tumor activity of the culture supernatant of the macrophages incubated with the lipopolysaccharide but not with the gelatin. The gelatin induced anti-tumor activity of the macrophages of C3H/HeJ as well as C3H/HeN mice whereas the lipopolysaccharide did only in C3H/HeN mice. The macrophages stimulated in vitro by the gelatin exerted anti-tumor activity in vivo. Moreover, the gelatin stimulated peritoneal exudates cells in vivo when subcutaneously administered with them. Conclusions: Porcine skin gelatin induces anti-tumor activity of macrophages in mice and its magnitude is greater than that of lipopolysaccharide of E. coli. Its mechanism is different from that of the lipopolysaccharide but not fully clarified. Цель: изучить in vitro противоопухолевую активность мышиных перитонеальных макрофагов, индуцированную желатином кожи свиньи. Методы: противоопухолевую активность макрофагов оценивали по включению меченного тимидина опухолевыми клетками-мишенями. Уровень цитокинов, секретируемых в культуральную среду, определяли с помощью ELISA. Результаты: cпособность желатина индуцировать противоопухолевую активность макрофагов была сильнее, чем у липополисахарида E. coli. Комбинация липополисахарида и интерферона-γ (IFN-γ) синергично стимулировала макрофаги, что показано и для комбинации желатина с IFN-γ. Противоопухолевая активность культурального супернатанта макрофагов, инкубированных с желатином, была выше, чем в случае применения липополисахарида, хотя липополисахарид индуцировал более сильную секрецию противоопухолевых цитокинов (IL-1, IL-6, TNF-α, IFN-γ) макрофагами. Антитела против TNF-α частично угнетали противоопухолевую активность культурального супернатанта макрофагов, инкубированных с липополисахаридом, но не с желатином. Желатин индуцировал противоопухолевую активность макрофагов как C3H/HeJ мышей, так и мышей C3H/ HeN, в то время как липополисахарид влиял только на макрофаги C3H/HeN мышей. Макрофаги, стимулированные in vitro, показывали противоопухолевую активность in vivo. Более того, желатин стимулировал клетки перитонеального экссудата in vivo при одновременном подкожном введении. Выводы: желатин свиной кожи индуцирует противоопухолевую активность макрофагов у мышей, причем более эффективно, чем липополисахарид E. coli. Механизм действия желатина отличается от механизма действия липополисахарида и остается пока невыясненным до конца en Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України Experimental Oncology Original contributions Anti-tumor activity of murine peritoneal macrophages induced by porcine skin gelatin Противоопухолевая активность мышиных перитонеальных макрофагов, индуцированная желатином кожи свиньи Article published earlier |
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Digital Library of Periodicals of National Academy of Sciences of Ukraine |
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DSpace DC |
| title |
Anti-tumor activity of murine peritoneal macrophages induced by porcine skin gelatin |
| spellingShingle |
Anti-tumor activity of murine peritoneal macrophages induced by porcine skin gelatin Koide, T. Kojima, T. Inamura, Y. Nagata, H. Hashimoto, Y. Sugita, Y. Maeda, H. Original contributions |
| title_short |
Anti-tumor activity of murine peritoneal macrophages induced by porcine skin gelatin |
| title_full |
Anti-tumor activity of murine peritoneal macrophages induced by porcine skin gelatin |
| title_fullStr |
Anti-tumor activity of murine peritoneal macrophages induced by porcine skin gelatin |
| title_full_unstemmed |
Anti-tumor activity of murine peritoneal macrophages induced by porcine skin gelatin |
| title_sort |
anti-tumor activity of murine peritoneal macrophages induced by porcine skin gelatin |
| author |
Koide, T. Kojima, T. Inamura, Y. Nagata, H. Hashimoto, Y. Sugita, Y. Maeda, H. |
| author_facet |
Koide, T. Kojima, T. Inamura, Y. Nagata, H. Hashimoto, Y. Sugita, Y. Maeda, H. |
| topic |
Original contributions |
| topic_facet |
Original contributions |
| publishDate |
2008 |
| language |
English |
| container_title |
Experimental Oncology |
| publisher |
Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
| format |
Article |
| title_alt |
Противоопухолевая активность мышиных перитонеальных макрофагов, индуцированная желатином кожи свиньи |
| description |
Aim: To study the induction of anti-tumor activity of murine peritoneal macrophages in vitro by porcine skin gelatin. Methods: Anti-tumor activity of the macrophages was evaluated with tritium thymidine uptake by target tumor cells. ELISA was used to measure amounts of cytokines secreted in culture medium. Results: The ability of the gelatin to induce anti-tumor activity of the macrophages was stronger than that of lipopolysaccharide of E. coli. Combination of the lipopolysaccharide and interferon-g synergistically stimulated the macrophages but that of the gelatin and interferon-g additionally did. The culture supernatant of the macrophages incubated with the gelatin also showed higher anti-tumor activity than that with the lipopolysaccharide though the lipopolysaccharide was more excellent than the gelatin in stimulating secretion of anti-tumor cytokines (IL-1, IL-6, TNF-a, IFN-g) by the macrophages. Anti-TNF-a antibody partially suppressed the anti-tumor activity of the culture supernatant of the macrophages incubated with the lipopolysaccharide but not with the gelatin. The gelatin induced anti-tumor activity of the macrophages of C3H/HeJ as well as C3H/HeN mice whereas the lipopolysaccharide did only in C3H/HeN mice. The macrophages stimulated in vitro by the gelatin exerted anti-tumor activity in vivo. Moreover, the gelatin stimulated peritoneal exudates cells in vivo when subcutaneously administered with them. Conclusions: Porcine skin gelatin induces anti-tumor activity of macrophages in mice and its magnitude is greater than that of lipopolysaccharide of E. coli. Its mechanism is different from that of the lipopolysaccharide but not fully clarified.
Цель: изучить in vitro противоопухолевую активность мышиных перитонеальных макрофагов, индуцированную желатином
кожи свиньи. Методы: противоопухолевую активность макрофагов оценивали по включению меченного тимидина опухолевыми
клетками-мишенями. Уровень цитокинов, секретируемых в культуральную среду, определяли с помощью ELISA. Результаты:
cпособность желатина индуцировать противоопухолевую активность макрофагов была сильнее, чем у липополисахарида
E. coli. Комбинация липополисахарида и интерферона-γ (IFN-γ) синергично стимулировала макрофаги, что показано и для
комбинации желатина с IFN-γ. Противоопухолевая активность культурального супернатанта макрофагов, инкубированных
с желатином, была выше, чем в случае применения липополисахарида, хотя липополисахарид индуцировал более сильную
секрецию противоопухолевых цитокинов (IL-1, IL-6, TNF-α, IFN-γ) макрофагами. Антитела против TNF-α частично угнетали
противоопухолевую активность культурального супернатанта макрофагов, инкубированных с липополисахаридом, но не с
желатином. Желатин индуцировал противоопухолевую активность макрофагов как C3H/HeJ мышей, так и мышей C3H/
HeN, в то время как липополисахарид влиял только на макрофаги C3H/HeN мышей. Макрофаги, стимулированные in vitro,
показывали противоопухолевую активность in vivo. Более того, желатин стимулировал клетки перитонеального экссудата
in vivo при одновременном подкожном введении. Выводы: желатин свиной кожи индуцирует противоопухолевую активность
макрофагов у мышей, причем более эффективно, чем липополисахарид E. coli. Механизм действия желатина отличается от
механизма действия липополисахарида и остается пока невыясненным до конца
|
| issn |
1812-9269 |
| url |
https://nasplib.isofts.kiev.ua/handle/123456789/139943 |
| citation_txt |
Anti-tumor activity of murine peritoneal macrophages induced by porcine skin gelatin / T. Koide, T. Kojima, Y. Inamura, H. Nagata, Y. Hashimoto, Y. Sugita, H. Maeda // Experimental Oncology. — 2008. — Т. 30, № 4. — С. 300–305. — Бібліогр.: 15 назв. — англ. |
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300 Experimental Oncology 30, 300–305, 2008 (December)
We have observed that different gelatins exhibit diffe
rent biological activities. Bovine bone (BB) gelatin stimu
lated proliferation of murine spleen cells in vitro [1]. BB and
porcine skin (PS) gelatins stimulated murine peritoneal
macrophages (mPMs) to secrete several cyokines, e.g.,
interleukin (IL)6, 12, TNFα and MCP1 [2]. Alternatively
PS gelatin suppressed proliferation of cells of human as
well as murine cell lines [1, 3, 4]. In this study, we investi
gated whether or not PS gelatin induced antitumor activity
of mPMs. There are many reports about stimulants that
induce antitumor activity of macrophages. Mantovani [5]
described that activated mononuclear phagocytes killed
transformed target cells via different mediators, e. g.,
cyto kines (IL1 [6], TNF [7], IL6 [8]), reactive intermedi
ates of oxygen [9], or nitrogen [10]. It was documented
that neutral serine protease [11] secreted by activated
macrophages was involved in the effector function. The
present study demonstrated that PS gelatin induced anti
tumor activity of mPMs and that its mechanism was mostly
different from all described above.
MATERIALS AND METHODS
Mice. Five to eight weekold female C3H/HeN and
C3H/HeJ mice were purchased from Chubu Kagaku
Shizai Co. Ltd. (Nagoya, Japan). All the experiments
were performed accoding to the approved animalcare
protocols of the Ethical Committee of institution.
Cell Lines. A murine hepatic cell carcinoma cell line,
MH134, a murine fibrosarcoma cell line, Meth A and
a murine T cell lymphoma cell line, BW5147 have been
maintained in suspension culture in our laboratory.
Reagents. PS gelatin and Interferonγ(IFNγ)
were purchased from Sigma Chemical Co. (St. Louis,
MO, USA). Lipopolysaccharide (LPS) derived from
E. coli 026: B6 was purchased from Difco Laboratories
(Detroit, MI, USA). Antimouse TNFα antibody was
purchased from upstate biotechnology (Lake Placid,
NY 12946, USA).
Peritoneal exudate cells. A mouse was injected
with 3 ml of 3% thioglycollate medium (Sigma Chemical
Co.) intraperitoneally (i.p.). Three days after, about 8 ml of
physiological buffered saline (PBS) was injected into the
peritoneal cavity and the PBS containing peritoneal exu
date cells (PECs) was aspirated. PECs were washed with
modified MEM twice. Modified MEM was composed of
2 mM Lglutamine, 5 x 105 M 2mercaptoethanol, 25 mM
HEPES and Eagle’s minimum essential medium(Nissui
Pharmaceutical Co. Ltd., Tokyo, Japan).
In vitro anti-tumor activity assay using tritium
thymidine. PECs (1 x 105/well) were incubated in wells
of a 96well roundbottomed microtiter plate, each
containing 0.1 ml of 10% FCS modified MEM at 37 °C
for 90 min. The PECs equally adhered to the round
bottoms, which was confirmed microscopically. The
adherent PECs were used as mPMs. In the preliminary
experiment using a kit, CytoTox 96® Nonradioactive
Cytotoxicity Assay (Promega, Woods, Hollow Road
ANTI-TUMOR ACTIVITY OF MURINE PERITONEAL MACROPHAGES
INDUCED BY PORCINE SKIN GELATIN
T. Koide1, T. Kojima2, *, Y. Inamura2, H. Nagata3, Y. Hashimoto4, Y. Sugita5, H. Maeda5
1Health Research Center, Aichi-Gakuin University, Aichi 470-0195, Japan
2Departments of Surgery, Aichi-Gakuin University School of Dentistry, Nagoya 464-8651, Japan
3Department of Digestive Surgery, Aichi Medical University, Aichi 480-1195, Japan
4Department of Biochemistry, Aichi-Gakuin University School of Dentistry, Nagoya 464-8650, Japan
5Department of Oral Pathology, Aichi-Gakuin University School of Dentistry, Nagoya 464-8650, Japan
Aim: To study the induction of anti-tumor activity of murine peritoneal macrophages in vitro by porcine skin gelatin. Methods: Anti-
tumor activity of the macrophages was evaluated with tritium thymidine uptake by target tumor cells. ELISA was used to measure
amounts of cytokines secreted in culture medium. Results: The ability of the gelatin to induce anti-tumor activity of the macrophages
was stronger than that of lipopolysaccharide of E. coli. Combination of the lipopolysaccharide and interferon-γ synergistically stimu-
lated the macrophages but that of the gelatin and interferon-γ additionally did. The culture supernatant of the macrophages incubated
with the gelatin also showed higher anti-tumor activity than that with the lipopolysaccharide though the lipopolysaccharide was
more excellent than the gelatin in stimulating secretion of anti-tumor cytokines (IL-1, IL-6, TNF-α, IFN-γ) by the macrophages.
Anti-TNF-α antibody partially suppressed the anti-tumor activity of the culture supernatant of the macrophages incubated with the
lipopolysaccharide but not with the gelatin. The gelatin induced anti-tumor activity of the macrophages of C3H/HeJ as well as C3H/
HeN mice whereas the lipopolysaccharide did only in C3H/HeN mice. The macrophages stimulated in vitro by the gelatin exerted
anti-tumor activity in vivo. Moreover, the gelatin stimulated peritoneal exudates cells in vivo when subcutaneously administered with
them. Conclusions: Porcine skin gelatin induces anti-tumor activity of macrophages in mice and its magnitude is greater than that of
lipopolysaccharide of E. coli. Its mechanism is different from that of the lipopolysaccharide but not fully clarified.
Key Words: porcine skin gelatin, macrophages, cytotoxicity.
Received: October 11, 2008.
*Correspondence: E-mail: taku@dpc.aichi-gakuin.ac.jp
Abbreviations used: 3H-TdR — tritium thymidine; BB — bovine bone;
ET ratio — effector : target ratio; IFN-γ — interferon-gamma; i. p. —
intraperitoneally; LPS — lipopolysaccharide; MCP-1 — monocyte
chemoattractant protein-1; mPMs — murine peritoneal mac-
rophages; PBS — physiological buffered saline; PECs — peritoneal
exudate cells; PS — porcine skin; TLR4 — Toll-like receptor 4.
Exp Oncol 2008
30, 4, 300–305
Experimental Oncology 30, 300–305, 2008 (December) 301
Madison, WI, USA), the amount of LDH released from
1 x 105 of lysed PECs was similar to that of the lysed
adherent population of 1 x 105 of PECs. It showed
that the number of adherent PECs approximated that
of PECs. In order to easily wash nonadherent target
cells by centrifugation, roundbottomed microtiter
plates were used. Nonadherent target cells equally
settled on the round bottoms when the plate was not
centrifuged. mPMs (approximately 1 x 105/well) were
incubated with reagents in the wells, each containing
0.2 ml of 5% FCS modified MEM at 37 °C for 24 h.
After washing the wells with PBS twice, target cells
(5 x 103/well) were added to the wells, each contai
ning 0.2 ml of 5% FCS modified MEM followed by an
additional 48 h incubation. Tritium thymidine (3HTdR)
was added to all wells (final concentration: 1 μCi/ml)
approximately 7 h before the termination of the incu
bation. Thereafter they were washed with 0.2 ml of
PBS twice and mixed with 0.2 ml of 1% sodium lauryl
sulfate, and 0.1 ml of the 0.2 ml was mixed with 3 ml
of a scintillator (Ready Flow III, Beckman). Scintillation
of the mixture was counted with a scintillation counter
(Beckman LS3801). When target cells were incubated
with effector cells, 3HTdR uptake by the target cells
was calculated by subtracting 3HTdR uptake by effec
tor cells from that by both effector and target cells.
Culture supernatants of mPMs incubated with
a stimulant. mPMs (1 x 106/well) were incubated
with or without 15 mg/ml of PS gelatin or 500 ng/ml
of LPS in wells of a 24well culture plate, each contai
ning 2 ml of 5% FCS modified MEM at 37 °C for 24 h.
After washing the cells, they were incubated in 2 ml
of 5% FCS modified MEM at 37 °C for 24 h. Then the
supernatants were collected and stored at –20 °C until
used for antitumor assay or cytokine detection as
say. Target cells (5 x 103/well) were incubated in wells
of a 96well roundbottomed microtiter plate, each
containing 50 μl of 5% FCS modified MEM and the
supernatant (150 μl) at 37 °C for 48 h. The manner of
pulse by 3HTdR was described above.
Ultrafiltration. The supernatant was filtered with
centrifugal filter devices, models YM10 at 14 000 g
for 30 min (Millipore Corporation, Bedford, MA, USA)
and filtrates and retentates were reconstituted with the
original medium. The membrane nominal molecular
weight limit of YM10 is 10 000 in Daltons.
Fluorescent immunohistochemistry. The cell
samples were fixed with 4% formaldehyde solution in
phosphate buffered saline (PBS) for 15 min at room
temperature. Then, they were washed three times with
PBS, after which incubated with 0.1% Triton X100 in
PBS for 5 min at room temperature. Then they were in
cubated 10% normal goat serum (NGS) in PBS at room
temperature for 30 min to block nonspecific binding.
Further they were incubated with optimum concentra
tions of primary antibody diluted with 10% NGS in PBS
for 60 min at room temperature. As primary antibody,
rabbit polyclonal antibody against asialo GM1 (natural
killer cell marker) and rat antimouse F4/80 (IgG2b)
(macrophage marker) were obtained from Wako Pure
Chemical Industries, Ltd. (Osaka, Japan) and AbD
Serotec (Oxford, UK), respectively. All antibodies were
labeled using Zenon Labeling Kits (Molecular Probes,
OR, USA). The cells and polyclonal antibodies were
observed using an immunofluorescence microscope
(Axio Observer. Z1; Zeiss, Oberkochen, Germany) with
a magnification of x100 and a halogen lamp of 100 W.
Images were photographed using a digital camera
(AioCam MRm, HRc; Zeiss) and AxioVision 4.6 soft
ware (Zeiss). The photographs made with 4 different
filters were later merged with the AxioVision 4.6 soft
ware to get the quadruplestained pictures.
Statistics. Difference between 2 groups was
evaluated using Student’s ttest. Difference between
2 groups in tumor sizes was evaluated using Mann
Whitney test because each population of tumor size
did not show statistic normal distribution. Difference
between 2 groups in survival was evaluated using
generalized Wilcoxon test. p values of 5% or less were
considered statistically significant.
RESULTS
Comparison of PS gelatin with LPS in inducing
anti-tumor activities of mPMs. Fig. 1 shows that
mPMs incubated with PS gelatin exerted antitumor
activity at effector : target ratio (ET ratio) 2, which
was statistically significant (p < 0.05). On the other
hand, mPMs intact or incubated with LPS enhanced
proliferation of target cells at ET ratio 1~2. Intact
mPMs exerted antitumor activity at ET ratio 16. mPMs
incubated with LPS exerted antitumor activity at ET
ratio 8. LPS of 500 ng/ml was optimal in stimulating
cytokine secretion and 15 mg/ml of PS gelatin also
optimal in suppressing tumor cell proliferation (data
not shown).
0
5000
10 000
15 000
20 000
25 000
30 000
35 000
40 000
45 000
0 1 2 4 8 16
ET ratio
3H
-T
dR
u
pt
ak
e
by
B
W
51
47
c
el
ls
(d
pm
/w
el
l)
Fig. 1. Comparison between LPS and PS gelatin in induction
of antitumor activities in mPMs. mPMs were incubated with
500 ng/ml of LPS ( ) or 15 mg/ml of PS gelatin ( ) or without
both ( ) in 5% FCS modified MEM at 37 °C for 24 h. Those mPMs
were collected with a scraper. Then they or no mPMs ( ) were
incubated with BW5147 cells at the indicated ET ratio at 37 °C
for additional 24 h
Induction of anti-tumor activity of mPMs by
synergistic action of LPS and IFN-γ. Fig. 2 shows
that mPMs stimulated with 1 ng/ml of IFNγ to exert
antitumor activity, that as low as 0.1 ng/ml of IFNγ
synergistically enhanced antitumor activity of mPMs
with 500 ng/ml of LPS and that 1 ng/ml of IFNγ ad
ditionally augmented antitumor activity of mPMs with
10 mg/ml of PS gelatin.
302 Experimental Oncology 30, 300–305, 2008 (December)
0
2000
4000
6000
8000
10 000
12 000
14 000
16 000
0 0,1 1
IFN-γ (ng/ml)
3H
-T
dR
u
pt
ak
e
by
B
W
51
47
c
el
ls
(d
pm
/w
el
l)
*
Fig. 2. Synergistic action of LPS and IFNγ inducing antitumor activi
ty in mPMs. mPMs were incubated with the indicated concentration of
IFNγ in the presence of 500 ng/ml of LPS ( ), 10 mg/ml of PS gelatin
( ) or absence of both ( ) at 37 °C for 24 h, and those reagents were
washed out. Then they were incubated with BW5147 cells at 37 °C for
additional 24 h. *Statistically different from control ( , 0) at p < 0.05.
Anti-Tumor Activity of the Culture Superna-
tant of mPMs Incubated with LPS or PS Gelatin.
Fig. 3 shows that both supernatants of mPMs incu
bated with LPS and PS gelatin suppressed 3HTdR
uptake by MH134 cells and that the activity of the
supernatant of mPMs incubated with PS gelatin was
higher than that with LPS. The supernatant of mPMs
alone enhanced 3HTdR uptake by MH134 cells.
0
5000
10 000
15 000
20 000
25 000
30 000
35 000
40 000
45 000
None PS LPS
Stimulator
3H
-T
dR
u
pt
ak
e
by
M
H
13
4
ce
lls
(d
pm
/w
el
l)
*
Fig. 3. Antitumor activity of culture supernatant of mPMs stimu
lated by LPS or PS gelatin. mPMs were incubated with or without
(None) 15 mg/ml of PS gelatin (PS) or 500 ng/ml of LPS (LPS) in
5% FCS modified MEM at 37 °C for 24 h. After discarding stimula
tors, they were incubated in the new medium at 37 °C for additional
24 h. The supernatants were collected. MH134 cells were incu
bated in 150 μl of the supernatant and 50 μl of 5% FCS modified
MEM (closed) or in 200 μl of the medium alone (opened) at 37 °C
for 48 h. *Statistically different from LPS at p < 0.01.
Molecular size of the factor having the anti-
tumor activity in the culture supernatant. The filter
which does not pass molecules of more than 10 kDa
suggested that the molecular weight of the antitumor
activity was over 10 kDa (Fig. 4).
0
2000
4000
6000
8000
10 000
12 000
14 000
16 000
18 000
20 000
<10 000 >10 000
Stimulant
3H
-T
dR
u
pt
ak
e
by
M
H
13
4
ce
lls
(d
pm
/w
el
l)
*
*
Fig. 4. Molecular size of the factor having the antitumor activi
ty in the culture supernatant. The culture supernatant of mPMs
with or without (black) 15 g/ml of PS gelatin (white) or 500 ng/ml
of LPS (grey) was ultrafiltered with microcon YM10 (10 kDa).
*Statistically different from control (black) at p < 0.01.
IL-1β, IL-6, TNF-α and IFN-γ in the culture
supernatant of mPMs incubated with LPS or PS
gelatin. In the experiment shown in Fig. 5, the anti
tumor activity of the supernatant of mPMs incubated
with PS gelatin or LPS was examined. In this experi
ment, concentrations of antitumor cytokines, i. e.,
IL1β, IL6, TNFα and IFNγ in those supernatants
were measured. Fig. 5 shows that LPS was more ex
cellent than PS gelatin in stimulating mPMs to secrete
cytokines (IL1β, IL6, TNFα and IFNγ).
Effect of anti-TNF-α antibody on the anti-tumor
activity of the supernatant. Fig. 6 shows that the
antitumor activity of the supernatant of mPMs incu
bated with PS gelatin was not affected by antiTNFα
antibody, but that with LPS was partially inhibited by
the antibody.
Involvement of Toll-like receptor 4 in induction
of anti-tumor activity of mPMs by PS gelatin. Mac
rophages of C3H/HeJ mice lack Tolllike receptor 4
(TLR4). Fig. 7 shows that PS gelatin stimulated mPMs of
both C3H/HeN and /HeJ mice to exert antitumor acti
vity whereas LPS did mPMs of C3H/HeN mice only.
0
10
20
30
40
50
60
70
80
90
100
None PS LPS
Stimulator
IL
-1
β
(p
g/
m
l)
0
500
1000
1500
2000
2500
None PS LPS
Stimulator
IL
-6
(p
g/
m
l)
0
500
1000
1500
2000
2500
3000
3500
4000
None PS LPS
Stimulator
TN
F-
α
(p
g/
m
l)
0
10
20
30
40
50
60
70
80
None PS LPS
Stimulator
IF
N
- γ
(p
g/
m
l)
a b c d
Fig. 5. IL1β, IL6, TNFα and IFNγ in culture supernatants of mPMs incubated with LPS or PS gelatin. mPMs were incubated with
or without (None) 15 mg/ml of PS gelatin (PS) or 500 ng/ml of LPS (LPS) in 5% FCS modified MEM at 37 °C for 24 h. After discarding
stimulators, they were incubated in the new medium at 37 °C for additional 24 h. Supernatants were collected and the amounts of
IL1β (a), IL6 (b), TNFα (c) and IFNγ (d) were measured
Experimental Oncology 30, 300–305, 2008 (December) 303
0
5000
10 000
15 000
20 000
25 000
30 000
0 120
Anti-TNF-α antibody (ng/ml)
3H
-T
dR
u
pt
ak
e
by
M
et
h
A
ce
lls
(d
pm
/w
el
l) *
**
Fig. 6. Effect of antiTNFα antibody on the antitumor activi
ty of the culture supernatant. The supernatant (150 μl/well) of
mPMs incubated with or without (black) 15 mg/ml of PS gelatin
(white) or 500 ng/ml of LPS (grey) was incubated with or without
120 ng/ml of antimouse TNFα antibody (25 μl/well) at 37 °C for
1 h, and Meth A cells (5 x 103/25 μl/well) were added to the wells
followed by an additional 24 h incubation. *Statistically different
from control (black) at p < 0.01, **not different.
0
2000
4000
6000
8000
10 000
12 000
14 000
C3H/HeN C3H/HeJ
3H
-T
dR
u
pt
ak
e
by
M
et
h
A
ce
llo
s
(d
pm
/w
el
l)
*
**
Fig. 7. Involvement of TLR 4 in inducing antitumor mPMs by PS
gelatin. mPMs of C3H/HeN or /HeJ were incubated with or without
(black) 10 mg/ml of PS gelatin (white) or 500 ng/ml of LPS (grey)
at 37 °C for 24 h. After removing those reagents, MH134 cells were
incubated with the mPMs for additional 24 h. *Statistically different
from control (black) at p < 0.05, **not different.
Winn’s test to prove generation of anti-tumor
mPMs by PS gelatin in vitro. When mPMs incubated
with PS gelatin in vitro were injected with MH134 cells
i.p. or subcutaneously (s. c.), tumorbearers of each
group survived for longer periods than those injected
with mPMs incubated in the absence of PS gelatin
(Fig. 8, a, c) and the subcutaneous tumors grew more
slowly than those of the control mice (Fig. 8, b).
Effects of PECs, PS gelatin and both on tumor
growth in vivo. When PECs, PS gelatin or both were
injecetd with MH134 cells i. p, the survival of each
group did not differ statistically (Fig. 9, a). On the
other hand, PS gelatin plus PECs or PS gelatin alone
suppressed the tumor growth as compared with PECs
alone when they were injected with MH134 cells s. c.
(Fig. 9, b) and moreover PS gelatin plus PECs but not
PS gelatin alone had the tumorbearers survive for
a longer period than PECs alone (Fig. 9, c).
Identification of Adherent PECs. Fluorescent
immunohistochemistry showed that more than 90%
of adherent PECs was F4/80positive and at most 10%
of them was asialo GM1positive (data not shown).
DISCUSSION
PS gelatin was observed to suppress prolifera
tion of tumor cells in vitro [2, 3]. However, when PS
gelatin administered in vivo, it was expected to be
easily degraded by various proteases, e. g., lysosomal
enzymes in mPMs. Therefore, the effect of mPMs on
degradation of PS gelatin was investigated by means of
in vitro antitumor activity of PS gelatin. In this experi
ment, adherent PECs were regarded as mPMs since
more than 90% of them had F4/80 antigen. mPMs
were added to the incubation of tumor cells with PS
gelatin. If mPMs degrade PS gelatin, the antitumor
activity must be attenuated. Unexpectedly mPMs
0
10
20
30
40
50
60
70
80
90
100
11 13 15 17 19
Survival (day)
%
s
ur
vi
va
l
0
50
100
150
200
250
300
350
400
450
18 33 49 62
Day
Tu
m
or
s
iz
e
(m
m
x
m
m
)
0
10
20
30
40
50
60
70
80
90
100
18 33 49 62
Survival (day)
%
s
ur
vi
va
l
Fig. 8. Winn’s test to prove the stimu
lation of mPMs by PS gelatin in vitro.
mPMs (4 × 106/dish) were incubated
with ( ) or without ( ) 15 mg/ml of
PS gelatin in dishes at 37 °C for 48 h.
Then they were collected with a
scraper, and mPMs/dish were mixed
with 1 × 105 of MH134 cells and the
mixture was inoculated i. p. (a) or
s. c. (b, c). Both are statistically dif
ferent at p < 0.05 (a), at p < 0.05 on
day 18, 26, 33, 38 and 64 (b) and at
p < 0.01 (c)
0
10
20
30
40
50
60
70
80
90
100
12 14 16 18 22
Survival (day)
%
s
ur
vi
va
l
0
50
100
150
200
250
300
350
400
12 16 19 22 26 29
Day
Tu
m
or
s
iz
e
(m
m
x
m
m
)
0
10
20
30
40
50
60
70
80
90
100
26 33 36 38 41 48
Survival (day)
%
s
ur
vi
va
l
Fig. 9. Effects of PECs, PS gelatin
and both on tumor growth in vivo.
PECs (1 × 107) ( ), 25 mg of PS gela
tin ( ) or both ( ) were inoculated into
the peritoneal cavity with 5 × 105 of
MH134 cells (a). PECs (2 × 106) ( ),
1.75 mg of PS gelatin ( ) or both ( )
were inoculated s. c. with 1 × 105 of
MH134 cells (b, c). Both ( , ) are
not different from control ( ) statisti
cally (a) and statistically different from
control ( ) at p < 0.05 on day 16, 19,
22 and 26 (b). Both ( , ) are statisti
cally different at p < 0.05, but both ( ,
) are not different statistically (c)
304 Experimental Oncology 30, 300–305, 2008 (December)
augmented the antitumor activity of PS gelatin (data
not shown), and so it was examined whether or not PS
gelatin stimulated mPMs to be cytostatic or cytocidal in
the present study. In addition, BB gelatin also became
to obtain antitumor activity in the same experimental
system whereas it did not exert antitumor activity by
itself (data not shown). This may be related with its ac
tivity to stimulate mPMs to secrete various cytokines,
which was greater than that of PS gelatin [2].
mPMs incubated with PS gelatin exerted antitumor
activity in vitro. The experiment using double chamber
culture indicated that the antitumor activity of mPMs
incubated with PS gelatin did not require the contact
with target cells (data not shown). However, a possibili
ty that the contact of target cells with activated mPMs
exerts an additional antitumor effect is not deniable.
The culture supernatant of mPMs incubated with PS
gelatin exerted higher antitumor activity than that
with LPS (Fig. 3).
LPS stimulated macrophages [2] or cells of a mac
rophage cell line [12] to secrete a variety of cytokines
or an antitumor factor other than IL1β, IL6 and
TNFα, respectively. Previously we reported that PS
gelatin stimulated mPMs to secrete IL6, IL12 and
TNFα and other cytokines but its activity was lower
than that of BB gelatin and by far lower than that of
LPS [2]. Clarke et al. [13] described that inhibition of
macrophagemediated killing was associated with
decreases in superoxide, nitric oxide and TNFα pro
duction in response to provocative stimulus. In this
study, LPS induced larger amounts of cytokines, i. e.,
IL1β, IL6, TNFα and IFNγ in mPMs than PS gela
tin. However, mPMs stimulated by LPS exerted lower
antitumor activity than those stimulated by PS gelatin.
The antitumor activity of the supernatant of mPMs
stimulated by LPS but not PS gelatin partly depends on
TNFα. The ultrafiltration study showed that the active
molecule did not pass through 10 kDa filter. Therefore
superoxide or nitric oxide is suggested not to be the
candidate for the antitumor activity of the culture
supernatants generated by PS gelatin and LPS.
Lorsbach et al. [10] reported that synthesis of NO
was increased when cells of a mouse macrophage
cell line, RAW 264.7 were costimulated with LPS and
IFNγ. This increase paralleled increase in cytotoxicity.
In our experiment, the synergism between LPS and
IFNγ also was shown in exerting antitumor activity
of mPMs. However, the synergism between IFNγ and
PS gelatin was not shown (Fig. 2).
It is well known that LPS stimulates macrophages
via TLR4 [13, 14]. Previously we reported that PS gela
tin as well as LPS stimulated mPMs to secrete TNFα
via TLR4 [2]. In the present study, however, mPMs
of C3H/HeJ mice (TLR4 deficient) incubated with
PS gelatin exerted antitumor activity in vitro whereas
those incubated with LPS did not. This finding sug
gests that induction of antitumor activity in mPMs by
PS gelatin depends on another pathway than cytokine
signaling pathway via TLR 4.
The antitumor activity of mPMs incubated with PS
gelatin was exerted in vivo as well as in vitro regardless
of inoculation site. Miyasaka et al. [15] reported that
PECs elicited by i.p. inoculation of 3% thioglycollate
medium exerted antitumor activity in vitro but not
in vivo. The PECs rather increased the number of
lung metastases when injected with tumor cells intra
venously. When the mixture of PS gelatin, PECs and
tumor cells or that of PS gelatin and tumor cells was
inoculated s. c., the tumor growth was suppressed to
the same extent in each group as compared with the
mixture of PECs and tumor cells as control, and the
survival of the former but not the latter was significantly
prolonged as compared with the control. These fin
dings suggest that some immunological mechanism in
which PECs, especially PECs activated by PS gelatin,
participated induced the prolonged survival. When the
mixture of PS gelatin, PECs and tumor cells or that of
PS gelatin and tumor cells was inoculated i.p., the sur
vival of tumor bearers was not prolonged. These in vivo
experiments suggest that the contact of PECs with PS
gelatin for an appropriate duration in vivo is required for
their activation and that once they are activated, e.g.,
incubation in vitro, they exert antitumor activity also
in such a large space as peritoneal cavity. PS gelatin
might disappear rapidly from peritoneal cavity when
injected i. p., and might remain in situ when injected
s. c. However, results of this experiment using PECs
but not adherent PECs cannot exclude involvement of
natural killer cells since PECs are expected to contain
more natural killer cells than adherent PECs.
This study using female mice cannot discuss the
effect of sex.
In conclusion, PS gelatin is a new macrophage
stimulant and its ability to induce antitumor mac
rophages is higher than that of LPS or IFNγ. The
mechanism remains to be clarified. One study is under
investigation whether or not PS gelatin administered
repeatedly i. p. would prolong the survival of mice
bearing MH134 tumor, and another is identifying the
antitumor molecule in the culture supernatant of
mPMs stimulated by PS gelatin.
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ПРОТИВООПУХОЛЕВАЯ АКТИВНОСТЬ МЫШИНЫХ
ПЕРИТОНЕАЛЬНЫХ МАКРОФАГОВ, ИНДУЦИРОВАННАЯ
ЖЕЛАТИНОМ КОЖИ СВИНЬИ
Цель: изучить in vitro противоопухолевую активность мышиных перитонеальных макрофагов, индуцированную желатином
кожи свиньи. Методы: противоопухолевую активность макрофагов оценивали по включению меченного тимидина опухолевы-
ми клетками-мишенями. Уровень цитокинов, секретируемых в культуральную среду, определяли с помощью ELISA. Результа-
ты: cпособность желатина индуцировать противоопухолевую активность макрофагов была сильнее, чем у липополисахарида
E. coli. Комбинация липополисахарида и интерферона-γ (IFN-γ) синергично стимулировала макрофаги, что показано и для
комбинации желатина с IFN-γ. Противоопухолевая активность культурального супернатанта макрофагов, инкубированных
с желатином, была выше, чем в случае применения липополисахарида, хотя липополисахарид индуцировал более сильную
секрецию противоопухолевых цитокинов (IL-1, IL-6, TNF-α, IFN-γ) макрофагами. Антитела против TNF-α частично угнетали
противоопухолевую активность культурального супернатанта макрофагов, инкубированных с липополисахаридом, но не с
желатином. Желатин индуцировал противоопухолевую активность макрофагов как C3H/HeJ мышей, так и мышей C3H/
HeN, в то время как липополисахарид влиял только на макрофаги C3H/HeN мышей. Макрофаги, стимулированные in vitro,
показывали противоопухолевую активность in vivo. Более того, желатин стимулировал клетки перитонеального экссудата
in vivo при одновременном подкожном введении. Выводы: желатин свиной кожи индуцирует противоопухолевую активность
макрофагов у мышей, причем более эффективно, чем липополисахарид E. coli. Механизм действия желатина отличается от
механизма действия липополисахарида и остается пока невыясненным до конца.
Ключевые слова: желатин свиной кожи, макрофаги, цитотоксичность.
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