Gastric cancer cell supernatant causes apoptosis and fibrosis in the peritoneal tissues and results in an environment favorable to peritoneal metastases, in vitro and in vivo

Article (PDF Available)inBMC Gastroenterology 12(1):34 · April 2012with40 Reads
DOI: 10.1186/1471-230X-12-34 · Source: PubMed
Abstract
In this study, we examined effects of soluble factors released by gastric cancer cells on peritoneal mesothelial cells in vitro and in vivo. HMrSV5, a human peritoneal mesothelial cell line, was incubated with supernatants from gastric cancer cells. Morphological changes of HMrSV5 cells were observed. Apoptosis of HMrSV5 cells was observed under a transmission electron microscope and quantitatively determined by MTT assay and flow cytometry. Expressions of apoptosis-related proteins (caspase-3, caspase-8, Bax, bcl-2) were immunochemically evaluated. Conspicuous morphological changes indicating apoptosis were observed in HMrSV5 cells 24 h after treatment with the supernatants of gastric cancer cells. In vivo, peritoneal tissues treated with gastric cancer cell supernatant were substantially thickened and contained extensive fibrosis. These findings demonstrate that supernatants of gastric cancer cells can induce apoptosis and fibrosis in HMrSV5 human peritoneal mesothelial cells through supernatants in the early peritoneal metastasis, in a time-dependent manner, and indicate that soluble factors in the peritoneal cavity affect the morphology and function of mesothelial cells so that the resulting environment can become favorable to peritoneal metastases.
RE S E A R C H A R T I C L E Open Access
Gastric cancer cell supernatant causes apoptosis
and fibrosis in the peritoneal tissues and results
in an environment favorable to peritoneal
metastases, in vitro and in vivo
Di Na
1
, Zhi-Dong Lv
1
, Fu-Nan Liu
1
, Yan Xu
1
, Cheng-Gang Jiang
1
, Zhe Sun
1
, Zhi-Feng Miao
1
, Feng Li
2
and
Hui-Mian Xu
1*
Abstract
Background: In this study, we examined effects of soluble factors released by gastric cancer cells on peritoneal
mesothelial cells in vitro and in vivo.
Methods: HMrSV5, a human peritoneal mesothelial cell line, was incubated with supernatants from gastric cancer
cells. Morphological changes of HMrSV5 cells were observed. Apoptosis of HMrSV5 cells was observ ed under a
transmission electron microscope and quantitatively determined by MTT assay and flow cytometry. Expressions of
apoptosis-related proteins (caspase-3, caspase-8, Bax, bcl-2) were immunochemically evaluated.
Results: Conspicuous morphological changes indicating apoptosis were observed in HMrSV5 cells 24 h after
treatment with the supernatants of gastric cancer cells. In vivo, peritoneal tissues treated with gastric cancer cell
supernatant were substantially thickened and contained exten sive fibrosis.
Conclusions: These findings demonstrate that supernatants of gastric cancer cells can induce apoptosis and
fibrosis in HMrSV5 human peritoneal mesothelial cells through supernatants in the early peritoneal metastasis, in a
time-dependent manner, and indicate that soluble factors in the peritoneal cavity affect the m orphology and
function of mesothelial cells so that the resulting environment can become favorable to peritoneal metastases.
Keywords: Peritoneal carcinomatosis, Stomach neoplasms, M esothelial cell, Apoptosis, Fibrosis
Background
Peritoneal carcinomatosis severely limits the improve-
ment of gastric cancer patients prognoses after surgery
[1]. Peritoneal metastasis results in a metastatic cascade,
usually occurring at late-stage tumor development, which
significantly contributes to gastric cancer-related mortal-
ity. The mechanisms of peritoneal metastasis of diffusely
infiltrating carcinoma are not clearly understood.
The perito neal s trom a environment favors prolifera-
tion of tumor cells by serving as a rich source of growth
factors and chemokin es known to be involved in tumor
metastasis. Molecules mediating this c a scade have not
been extensively investigated [2]. Reportedly, mesothe-
lial cells could preven t cancer invasion and undergo
morpho logical changes in response to soluble factors
released by cancer cells [3]. The specific molecules
involved in this process are dictated by intrinsic charac-
teristics of the metastatic tumor cells. Tumor cells need
to attach firmly onto the submesothelial monolayer and
penetrate the mesothelial monolayer for inva sion. Buck
et al. [4] explored the protective effect of the mesothelial
layer, using a rat model in which the mesothelial lining
of parietal peritoneum wa s stripped off in the experi-
mental rats, leaving the basal membrane intact. We pre-
viously showed that the layer of confluen t, intact
mesothelial cells hinders cancer cell invasion of the ab-
dominal cavity. However, once the integrity of this b ar-
rier is disrupted, metastasis may occur, because the
* Correspondence: xuhuimian@yahoo.cn; dawangmo@yahoo.cn
1
Department of Oncology, The First Affiliated Hospital, China Medical
University, Shenyang 110001, Liaoning Province, China
Full list of author information is available at the end of the article
© 2012 Na et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.
Na et al. BMC Gastroenterology 2012, 12:34
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peritoneum provides a favorable environment for gastric
cancer cells to grow [5-9]. Additional studies have
shown that, prior to the attachment of gastric cancer
cells onto peritoneum, mesothelial cells acquire hemi-
spherical shape and start to shed. As a result, naked
areas of the submesothelial connective tissue are
exposed to the peritoneal cavity [10-12]. It is likely that
penetration of the mesothelial monolayer b y tumor cells
initiates with tumor-induced mesothelial cell apoptosis.
In this study, we examined the effects of soluble factors
released by gastric cancer cells on mo rphology and bio-
logical activity of peritoneal mesothelial cells in vitro
and in vivo.
Methods
Reagents
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazoliumbro-
mide (MTT) was obtained from Fluka, USA. Propidium
iodide (PI) was obtained from Biosharp, USA. Bcl-2, Bax,
caspase-3, caspase-8 and actin primary antibodies, and the
secondary antibody, goat anti-mouse IgG were obtained
from Santa Cruz Biotechnology Inc., CA, USA. Dulbeccos
modified Eagles medium (DMEM) and fetal calf serum
(FCS) were obtained from GIBCO BRL (Grand Island, NY,
USA). Other laboratory reagents were obtained from
Sigma, USA. A phase-contrast microscope (Japan Nikon),
transmission electron microscope (Hitachi H-6001, Japan)
was used.
Cell lines and cell culture
The human peritoneal mesothelial cell line HMrSV5 was
obtained from the Department of Cell Biology, China
Medical University, China. HMrSV5 was originally iso-
lated from human omentum. Briefly, omentum collected
from consenting non-uremic patients who were under-
going elective abdominal surgery, and was incubated in
0.05 % (w/v) trypsin and 0.01 % (w/v) EDTA for 20 min
at 37°C. The harvested mesothelial cells were centrifuged
at 150 g for 5 min and then transferred into 75 cm
2
tis-
sue culture flasks and cultured in a humidified 5 % CO
2
incubator, in DMEM supplemented with 10 % fetal calf
serum (FCS), 100 U/mL penicillin, 100 μg/mL strepto-
mycin, 2 mmol/L L-glutamine and 20 mmol/L hydro-
xyethyl piperazine ethanesulfonic acid (HEPES, GIBCo
BRL, USA). Medium was changed every 23 days.
Human gastric carcinoma cell lines, MKN-45, MKN-1,
SGC-7901, BGC-823 and MGC-803, were obtained from
the Department of Cell Biology, China Medical Univer-
sity, China. These cells were cultured in DMEM supple-
mented with 10 % FCS, 100 U/mL penicillin, 100 μg/mL
streptomycin, and 2 mmol/L L-glutamine in a humidified
5%CO
2
incubator at 37°C.
Preparation of serum-free conditioned media
Serum-free conditioned media (SF-CM) was prepared
from gastric cancer cells or normal gastric epithelial cells
as previously reported [1]. Briefly, 5.0 × 10
5
cells were
seeded into 100-mm tissue culture dishes with 10-mL
DMEM, supplemented with 10 % FCS and incubated at
37°C for 3 days. To obtain SF-CM, the cells were washed
twice with phosphate-buffered solution (PBS) and then
incubated for 2 days with 3 mL of serum-free DMEM.
The SF-CM was eluted and centrifuged at 1000 g for
5 min, passed through filters (pore size: 0.45 μm) and
stored at
20°C until used.
Animals
Male C57BL/6 mice (eight weeks old, weighing 20 ± 2 g),
were obtained from China Medical University animal fa-
cility and fed with purified water and a commercial stock
diet in an air-conditioned room at 2022°C.Animals used
in this study were maintained in accordance with the
Guide for Care and Use of Laboratory Animals published
by the US National Institutes of Health (NIH publication
No. 8523, revised 1996) and the Policy of Animal Care
and Use Committee of China Medical University.
Morphological Evaluation under a Phase-contrast
Microscope
Human peritoneal mesothelial cells (HPMCs) were cul-
tured to subconfluence in a 50-cm
2
dish with DMEM
containing 10 % FCS. The medium then was changed to
(1) serum-free DMEM or (2) SF-CM from gastric cancer
cell lines. The HPMCs in 24-well chambers were
exposed to test solutions for 24 h, and gently washed
with PBS. They were then examined under a phase-con-
trast microscope for size, shape, and integrity of the cell
membrane, cytoplasm, and nucleus.
Transmission electron microscopy
The HPMCs were cultured to subconfluence in a 50-cm
2
dish with DMEM containing 10 % FCS. The medium then
was changed to (1) serum-free DMEM or (2) SF-CM from
gastric cancer cell lines. After incubation for 24 h, the cells
were trypsinized and then fixed in ice-cold 2.5 % electron
microscopy grade glutaraldehyde in PBS (pH 7.3). The spe-
cimens were rinsed with PBS, post-fixed in 1 % osmium
tetroxide with 0.1 % potassium ferricyanide, dehydrated
through a graded ethanol series (30 %90 %), and embed-
ded in Epon. Semi-thin (300 nm) sections were cut using a
Reichart Ultracut (Reichart Ultracut (Leica, Germany),
stained with 0.5 % toluidine blue, and examined under a
light microscope. Ultrathin sections (65 nm) were stained
with 2 % uranyl acetate and Reynoldsleadcitrate,and
examined on a transmission electron microscope at × 5000
or × 8000 magnification.
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Quantitative determination of cell damage by MTT assay
The MTT assay was performed to assess viability of the
human peritoneal mesothelial cells after treatment with
SF-CM from gastric cancer cell lines. Cells in 96-well
plate cultures, after exposure to control or test solutions
for a specific time period (observed at 12 h, 24 h and
48 h), were incubated with 50 μg/mL MTT at a dilution
of 1:10, based on the volume of culture medium for 3 h
at 37°C. At the end of the incubation time, the MTT so-
lution was removed and 150 μL DMSO was added to
each well, and stirred to dissolve the dark-blue formazon
crystals which had formed. The proportion of viable cells
was determined by measuring the optical density of each
sample at 480 nm with a spectrophotometer. Three cul-
tures were exposed to each solution at each time period.
The means of each group of cultures were compared.
Flow cytometry
Following incubation in the test solutions for 24 h, 48 h
and 72 h, cells were harvested using trypsinization. Cells
were resuspended in PBS at a concentration of 1 × 10
6
/
mL and fixed in 2 mL methanol for 30 min at 4°C. After
the CNE2 cells were fixed, the mixture was incubated in
0.5 mL of PI solution (0.05 mg/mL in 3.8 mol/L Na cit-
rate) and 0.5 mL of RNAse A (0.5 mg/mL) at room
temperature for 30 min. Finally, the cells were resus-
pended in 1 mL PBS and analyzed by flow cytometry
according to the manufacturers instructions. The cells in
the subdiploid peak were considered apoptotic.
Histological appearance of peritoneum
Male C57BL/6 mice (eight weeks old, weighing 20 ± 2 g)
were used in the present study. The experiment followed
the guidelines for the use of Laboratory Animals in Re-
search and Teaching. The mice were fed a standard pel-
let laboratory chow and were provided with water ad
libitum. Mice were randomly assigned to one of three
groups (n = 5 or 6 in each group). Mice in the DMEM
group were treated with DMEM (100 ml/Kg) by intra-
peritoneal injections on days 1, 3, 5 and 7. Mice in the
SF-CM group were treated with 100 ml/Kg of SF-CM
from gastric cancer cell lines by intraperitoneal injections
on days 1, 3, 5 and 7. Nothing was added to injections
for the control mice. After 29 days, mice were anesthe-
tized with ethyl ether and sacrificed. Parietal periton-
eums were stained with hematoxylin and eosin (H&E)
and Massons trichrome staining. Morphologic changes
of the parietal peritoneum were observed by light micro-
scope. Thickness of the submesothelial extracellular
matrix was determined after the tissue sections had H&E
and Masson staining. The average for 10 independent
measurements was calculated for each section; data were
then summarized.
Western blotting
Human peritoneal mesothelial cells were cultured to sub-
confluence in a 50-cm
2
dish with DMEM containing
10 % FCS. The media were then changed to (1) SF-CM
from gastric cancer cell MKN-45, MKN-1, SGC-7901,
BGC-823 and MGC-803; and (2) serum-free DMEM
serving as control. Protein was extracted in a standard
lysis buffer with proteinase inhibitors (sodium orthovana-
date, phenylmethylsulfonyl fluoride, leupeptin, and apro-
tinin obtained from BioShop, Burlington, ON, Canada).
Aliquots of 20 μg of protein lysate was electrophoresed
with a 12 % SDS-PAGE gel, transferred to a nylon mem-
brane, and separately probed with antibodies for Bcl-2,
Bax, caspase-3 and caspase-8. Following incubation with
the secondary antibody, blots were developed using an
ECL Western Blot Substrate Kit (Abcam, USA).
Statistical analysis
All data are expressed as x ± sd. Comparisons of drug
effects were made using Students t-test. A P value < 0.05
was considered to be significant.
Results
Morphological evaluation under a phase-contrast
microscope
While HPMCs treated only in serum-free DMEM showed
a typical polygonal and cobblestone pattern (Figure 1A.),
cells treated with SF-CM from gastric cancer cell MKN45
for 24 h began to have morphological changes, the most
obvious of which were exfoliation and the appearance of
naked areas (Figure 1B).
Transmission electron microscopy
After 24 h of SF-CM from gastric cancer cell treatment,
apoptotic features (such as condensation of the nuclear
chromatin, wrinkling of nuclear membranes, dilation of
endoplasmic reticulum, and relatively normal structure
of the mitochondria) were observed under transmission
electron microscope ( TEM; Figures 2A, B). Under TEM,
the nuclear membrane was seen to be intact, the chro-
matin condensed into masses, and on the boundary of
the membrane or forming arch (Figure 2C). The budding
and the formation of the apoptosis bodies were also
observed (Figure 2C).
MTT assay
To evaluate potential suppressive effects of gastric cancer
cell SF-CM on HPMCs, we examined its growth curve
on the HPMC line HMrSV5. Gastric cancer cell SF-CM
induced growth suppression in HPMC cells, and did so
in a time-dependent manner (Figure 3A). This effect was
observed at 0 h, 12 h, 24 h and 48 h. These results indi-
cate that tumor supernatant induces mesothelial cell
damage or apoptosis.
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Figure 2 Human peritoneal mesothelial cells (HPMC) 24 h after incubation with and without SF-CM from gastric cancer cells. (A) Control
cells display normal nuclei and endoplasmic reticula. (B) Cells treated with SF-CM from MKN1 gastric cancer cells show chromatin condensed into
masses, and on the boundary of the membrane or forming arch. Budding and the formation of the apoptosis bodies were observed (arrows in B).
(C) .Cells treated with SF-CM from MKN45 gastric cancer cells show condensation of nuclear chromatin (arrows in C). (D) Cells treated with gastric
cancer cell line MGC-803 were similar to B. Budding and the formation of the apoptosis bodies were also observed.
Figure 1 Morphological changes of human peritoneal mesothelial cells under phase-contrast microscope. (A) Monolayer of polygonal
and cobblestone-like HPMCs cultured in serum-free DMEM. (BD) Exfoliated appearance of naked areas after treatment with SF-CM from gastric
cancer cell lines MKN45, SGC7901, and BGC823. (Magnification: ×40).
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Flow cytometry
To quantify the percentage of apoptotic cells after treat-
ment at various time periods, mesothelial cells were
stained with PI. Gastric cancer cell SF-CM effectively
induced apoptosis in mesothelial cells and did so in a
dose-dependent manner after 48 h (Figure 3.B). These
results were the same as those for the MTT assay.
Histology and morphometric analysis
Morphologic changes of the parietal p eritoneum were
analyzed using H&E and Massons trichrome staining.
Among normal mice, a mesothelial cell monolayer
covered the peritoneal surface without any thickening
(Figure 4 a,d). Due to apparent incompatibility, mice
receiving intraperitoneal injections of DMEM had
slight thickening in the peritoneal submesothelial col-
lagenous zone (Figure 4 b, e); those injected intraperi-
toneally with gastric cancer cell SF-CM had marked
thickening of the submesothelial compact zone and
increased cellularity (Figure 4 c, f).
Western blotting
We then sought to further delineate the mechanisms
which underlie the combined effects of gastric cancer
cell SF-CM on apoptosis-related proteins (caspase-3, cas-
pase-8, Bax, bcl-2). Levels of these proteins were evalu-
ated using western blot analysis. Caspase-3, caspase-8,
and Bax protein levels increased after 48 h of treatment
with SF-CM from most gastric cancer cells, while bcl-2
protein levels decreased (Figure 5). Beta-actin was used
as the loading control.
Discussion
Most studies of post-operative tumor recurrence show
that traumatized mesothelial surfaces are preferred sites
for tumor cell adhesion. Recently, disassociated cancer
cells inside peritoneal cavities, and proteins specifically
expressed in peritoneal metastasis of gastric carcinoma
were found to be linked to cancer prognoses. While
immunogenetic approaches show great promise in the
treatment of peritoneal metastasis of gastric carcinoma
[13-15], the effects of gastric cancer cells on mesothelial
cells are poorly understood.
Study showed that mesothelial cells provided protec-
tion against peritoneal metastasis of tumor in intact
mesothelia [9,16,17]. Paget et al proposed a seed and
soil theory: metastasis only occurs when tumor cells live
and grow in a favorable environment [18]. The periton-
eum might be such a favorable environment for scir-
rhous gastric cancer cells; possibly mesothelial cells
prevent cancer cells from infiltrating into submesothelial
connective tissue. Ma sakazu et al. [1] showed that adja-
cent confluent mesothelial cells hindered invasion by
cancer cells. In addition, Kiyasu et al. [3] reported that,
prior to peritoneal implantation of cancer cells, mesothe-
lial cells become hemispherical and exfoliate from the
peritoneum. Our hypothesis is that after serosa are
exposed, free cancer cells shed from primary gastric can-
cer sites into the abdominal cavity induce apoptosis in
peritoneal mesothelial cells [19-21]. As a result, mesothe-
lial cells become hemispherical and exfoliation takes
place. Naked areas of submesothelial connective tissue
are thus exposed to the peritoneal cavity; this injured
peritoneal site becomes a favorable environment for peri-
toneal metastasis [22-24].
We had previously shown gastric cancer cell super-
natant to significantly reduce the viability of mesothelial
cells, but normal gastric epithelial cell line GES-1 exerts
no effect on mesothelial cells [5]. Our present study also
demonstrates that cultured mesothelial cells become
hemispherical, and exfoliation occur when serum-free
medium conditioned by gastric cancer cells was added, as
shown by contrast phase microscopy. Furthermore, cyto-
plasmic reduction, nuclear condensation, and formation
Figure 3 Apoptosis was quantified by two methods: MTT and flow cytometry. (A) Viability of mesothelial cell HMrSV5 after treatment with
SF-CM from gastric cancer cells. (B) Apoptosis was quantified by flow cytometry after treatment with SF-CM from gastric cancer cells.
Na et al. BMC Gastroenterology 2012, 12:34 Page 5 of 8
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of extracellular and/or intracellular apoptotic bodies were
observed under transmission electron microscope. Apop-
tosis was quantified by two methods: MTT and flow cyto-
metry. We speculate that free gastric cancer cells in the
abdominal cavity induce apoptosis of mesothelial cells
and cause exfoliation, eventually leading to metastasis.
This may be the mechanism by which cancer cells adhere
to submesothelial connective tissue, although the meso-
thelial cells are still well-organized and confluent. Further
studies are needed to characterize SF-CM released from
gastric cancer cells.
Gastric cancer cells may induce apoptosis through
mitochondria- and death receptor-dependent apoptotic
pathways. Gastric cancer cells suppress mesothelial cell
Figure 4 Hematoxylin/eosin (H&E) and Masson staining of peritoneum tissues. Peritoneum tissues from different groups were obtained
surgically and subjected to H&E and Masson staining. (A) All photos were obtained at 40 × magnification. Normal peritoneum consists of only a
monolayer of mesothelium with little fibrosis (a, d). Peritoneum treated with DMEM also showed small amounts of connective tissue under the
mesothelial cells(arrows in b,e). In contrast, peritoneum treated by SF-CM from gastric cancer cells was substantially thickened and contained
extensive fibrosis (arrows in c,f). (B) Morphometric parameters of peritoneal tissues. Data are expressed as the mean ± standard error of the mean
of at least 3 separate experiments. *P < 0.05.
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growth by inhibiting proliferation through the promotion
of caspase-dependent apoptosis. Caspases are cytopla s-
mic aspartate-specific cysteine proteases, and play im-
portant roles in apoptosis [25]. The death receptor-
dependent apoptotic pathway is triggered at the cell sur-
face and requires activation of caspase-8, whereas the
mitochondrion-dependent pathway is initiated by the re-
lease of mitochondrial cytochrome c into the cytoplasm
and requires activation of caspase-9. Subsequently, cas-
pase-8 or 9 can activate caspas e-3, which in turn tar-
gets and degrades specific and vital cellular proteins,
ultimately resulting in nuclear DNA degradation and
apoptotic cell death [26]. B cl-2, an inhibitor of the mito-
chondrial apoptosis pathway, exerts its action by block-
ing proapoptotic counterparts, which in turn prevents
the release of cytochrome c and the activation of cas-
pases [27]. Bax is a death promoter, which is neutralized
by heterodimerization with Bcl-2. Bax translocates into
the outer mitochondrial membrane followed by leakage
of cytochrome c from the mitochondria into the cytosol
[28]. Caspase-9 and caspase-3 are activated sequentially,
and these events lead to the breakdown of chromosomal
DNA. As there is a significant possibility that gastric
cancer cell-mediated apoptosis of mesothelial cells is the
result of regulation of Bcl-2 and Bax, identification of
their target compounds is necessary.
In this study, we utilized a mouse experimental model
of peritoneal sclerosis induced by repeated injections of
gastric cancer cell SF-CM. Experimental peritoneal fibro-
sis induced by repeated intraperitoneal injections of gas-
tric cancer cell SF-CM might not completely mimic
peritoneal sclerosis observed in patients (diffusely infil-
trating carcinoma or Bormanns Type VI carcinoma). In
fact, pathologic findings of peritoneal carcinomatosis and
peritoneal sclerosis are not uniform and various factors
are involved. In addition, certain common features are
observed during the development of peritoneal sclerosis
between gastric cancer cell SF-CM-induced experimental
animal models and human patients undergoing periton-
eal carcinomatosis. These common histological findings
include increased accumulation of interstitial collagens
such as type I and III collagen, infiltration of monocytes/
macrophages, increase in a-SMA
+
myofibroblasts, and
vascular density in the peritoneum [7,8]. These similar-
ities in alterations of the peritoneal membranes between
experimental models and human peritoneal carcinoma-
tosis patients suggest that this is an appropriate model
for examining the efficacy of various potential thera-
peutic reagents for regulating peritoneal carcinomatosis.
Conclusions
These findings demonstrate that gastric cancer cells can
induce apoptosis and fibrosis of human peritoneal meso-
thelial cells through supernatants in the early peritoneal
metastasis, and indicate that soluble factors in the peri-
toneal cavity can affe ct the morphology and function of
mesothelial cells so that the resulting environment
becomes favorable to peritoneal metasta ses.
Abbreviations
HPMCs: Human peritoneal mesothelial cells; SF-CM: Serum-free conditional
media.
Competing interests
The authors declare that they have no competing interests.
Authors contributions
DN, Z-DL and F-NL carried out the studies on morphology and biological
activity of peritoneal mesothelial cells in vitro and in vivo. ZS, Z-FM and FL
participated in the in vivo studies. YX and C-GJ participated in the
morphology studies. DN and HX participated in the design of the study and
performed the statistical analysis. HX and DN conceived of the study, and
participated in its design and coordination and helped to draft the
manuscript. All authors read and approved the final manuscript.
Acknowledgements
The authors wish to express their sincere thanks to Dr. Yan Song for his
technical assistance. This study was supported by a grant from the National
Natural Sciences Foundation of China (NO. 81071956 and 81101884).
Author details
1
Department of Oncology, The First Affiliated Hospital, China Medical
University, Shenyang, 110001, Liaoning Province, China.
2
Department of Cell
Biology, China Medical University, Shenyang 110001, Liaoning Province,
China.
Received: 4 July 2011 Accepted: 20 April 2012
Published: 20 April 2012
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doi:10.1186/1471-230X-12-34
Cite this article as: Na et al.: Gastric cancer cell supernatant causes
apoptosis and fibrosis in the peritoneal tissues and results in an
environment favorable to peritoneal metastases, in vitro and in vivo.
BMC Gastroenterology 2012 12:34.
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    • "Although mesothelial cells can prevent against tumor invasion , they may become damaged in response to soluble factors released by tumor cells during metastasis [23, 24]. Damaged PMCs become hemispherical in shape and undergo exfoliation , which results in exposure of sub-mesothelial connective tissue and collagen fiber [25, 26]. "
    [Show abstract] [Hide abstract] ABSTRACT: Pleural dissemination is commonly associated with metastatic advanced lung cancer. The injury of pleural mesothelial cells (PMCs) by soluble factors, such as transforming growth factor-beta1 (TGF-β1), is a major driver of lung cancer pleural dissemination (LCPD). In this study, we examine the effects of TGF-β1 on PMC injury and the ability of TGF-β1 inhibition to alleviate this effect both in vitro and in vivo. PMCs were co-cultured with the high TGF-β1-expressing lung cancer cell line A549 and with various TGF-β1 signaling inhibitors. Expression of cleaved-caspase 3, cleaved-caspase 9, p21, and p16 were evaluated by Western blot and immunofluorescent confocal imaging. Apoptosis was measured by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltrazoliumbromide assay and AnnexinV-propidium iodide (PI) staining. PMC senescence was assessed by staining for senescence-associated β-galactosidase (SA-β-Gal). The ability of lung cancer cells (LCCs) to adhere to injured PMCs was investigated using an LCC-PMC adhesion assay. In our mouse model, PMC injury status was monitored by hematoxylin-eosin (H&E) and Masson's trichrome staining. LCCs expressing high levels of TGF-β1 induce apoptosis and senescence of PMCs in a co-culture system. Injured PMCs adhere to LCCs, which may further promote LCPD. Importantly, PMC monolayer injury could be reversed with TGF-β1 inhibitors. This was consistent with our in vivo data showing that the TGF-β1 inhibitor SB-431542 attenuated PMC barrier injury induced by A549 culture medium in our mouse model. Our study highlights the importance of TGF-β1 signaling in LCPD and establishes this signaling pathway as a potential therapeutic target in the disease.
    Full-text · Article · Nov 2014
  • [Show abstract] [Hide abstract] ABSTRACT: Peritoneal dissemination (PD) is the most frequent metastatic pattern of advanced gastric cancer (GC) and the main cause of death in GC patients. Human peritoneal mesothelial cell (HPMC) injury induced by gastric cancer cells (GCCs) and GCC outgrowths supported by peritoneal milky spot macrophages (PMSMs) are the key events during gastric cancer peritoneal dissemination (GCPD). In this study, we investigated whether PMSMs remodeled by GCC can induce HPMC injury and create a favorable microenvironment for GCPD. We established a tumor-associated macrophage (TAM) model using in vitro cell coculture. Normal macrophages cocultured with GCCs down-regulated expression of antigen-presenting surface molecules CD80, CD86, and MHC-II, but, notably, they up-regulated expression of phagocytic scavenger receptor CD206, which is similar to the M2 macrophage phenotype. In further experiments, various experimental methods were applied to detect the injurious effect of TAMs on HPMCs in another TAM-HPMC coculture. Our results showed that GCCs can induce HPMC apoptosis by unregulated apoptosis associated with cleaved caspase3, cleaved caspase9, and p21 proteins. HPMC growth ceased, and both early- and late-stage apoptosis were observed. Additionally, GCCs can induce HPMC fibrosis via increased expression of epithelial cell marker E-cadherin and decreased expression of mesenchymal cell marker α-SMA. Our results demonstrate that, in the GCPD process, PMSMs were remodeled by GCCs, resulting in phenotypic and functional transformation. In turn, this transformation induced HPMC injury and provided a favorable microenvironment for GCC anchorage and growth. These results may provide new insight into the mechanisms of GCPD.
    Full-text · Article · Aug 2013
  • [Show abstract] [Hide abstract] ABSTRACT: Peritoneal dissemination is the most frequent metastatic pattern of advanced gastric cancer and the main cause of death in gastric cancer patients. Transforming growth factor-beta1 (TGF- ß1), one of the most potent fibrotic stimuli for human peritoneal mesothelial cells, has been shown to play an important role in this process. In this study, we investigated the effect of TGF- ß1 signaling blockade in gastric cancer cell (GCC)-induced human peritoneal mesothelial cell (HPMC) fibrosis. HPMCs were cocultured with the high TGF- ß1 expressing GCC line SGC-7901 and various TGF- ß1 signaling inhibitors or SGC-7901 transfected with TGF-ß1-specific siRNA. HPMC fibrosis was monitored on the basis of morphology. Expression of the epithelial cell marker, E-cadherin, and the mesenchymal marker, α-smooth muscle actin (α-SMA), was evaluated by Western blotting and immunofluorescence confocal imaging. GCC adhesion to HPMC was also assayed. In nude mouse tumor model, the peritoneal fibrotic status was monitored by immunofluorescent confocal imaging and Masson's trichrome staining; formation of metastatic nodular and ascites fluid was also evaluated. Our study demonstrated that GCC expressing high levels of TGF-ß1 induced HMPC fibrosis, which is characterized by both upregulation of E-cadherin and downregulation of α-SMA. Furthermore, HPMC monolayers fibrosis was reversed by TGF- ß1 signaling blockade. In vivo, the TGF- ß1 receptor inhibitor SB-431542 partially attenuated early-stage gastric cancer peritoneal dissemination (GCPD). In conclusion, our study confirms the significance of TGFß1 signaling blockade in attenuating GCPD and may provide a therapeutic target for clinical therapy.
    Full-text · Article · Dec 2013
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