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Ersilia Dolfini, Leda Roncoroni,
Department of Biology and Genetics for Health Sciences, University
of Milan, Milan, Italy
Luca Elli, Maria Teresa Bardella,
Department of Gastroenterology, University of Milan, Ospedale
Maggiore Policlinico, Mangiagalli e Regina Elena IRCCS, Milan, Italy
Barbara Costa,
Department of Biotechnology and Bioscience, University of Milan-Bicocca,
Milan, Italy
Maria Pia Colleoni,
Department of Pharmacology, Chemotherapy and Toxicology, University
of Milan, Milan, Italy
Vito
Lorusso, Simona Ramponi,
Bracco Imaging SpA, Milan Research Centre, Milan, Italy
Paola Braidotti, Stefano Ferrero,
Department of Medicine, Surgery and Dentistry, S.
Paolo Hospital and Ospedale Maggiore Policlinico, Mangiagalli e
Regina Elena IRCCS, Milan, Italy
Maria Letizia Falini,
Department of Agrifood Molecular Science, University
of Milan, Milan, Italy
Supported by
the San Paolo Foundation grant to
“Centro
per lo Studio della Celiachia”
Correspondence to:
Maria Teresa Bardella, MD, Department of Gastroenterology, Ospedale
Maggiore Policlinico, Mangiagalli e Regina Elena IRCCS, Via F.
Sforza 35, Milan 20122,
Italy.
mariateresa.bardella@unimi.it
Telephone:
+39-2-55033384
Fax:
+39-2-50320403
Received:
2004-11-18
Accepted:
2005-01-26
Abstract
AIM:
To evaluate the effects of gliadin on the oxidative environment in
the “ in
vivo-like
”model of a three-dimensional cell culture system.
METHODS:
LoVo cell line (intestinal adenocarcinoma) multicellular spheroids
were treated with digested gliadin (with albumin used as a control).
Spheroid volumes, cell viability and morphology, lactate
dehydrogenase (LDH) release, content of reduced glutathione (GSH)
and activity of GSH-related enzymes were examined. The data were
statistically analyzed using the Student抯
t-test
(P<0.05). was considered statistically significant.
RESULTS:
Gliadin reduced cell viability (from 20% to 60%) and led to
morphological alterations characterized by apoptotic findings and
cytoskeletal injuries. LDH activity increased. The content of GSH
reduced (-20% vs controls), and activity of GSH-related
enzymes was significantly inhibited.
CONCLUSION:
Gliadin treatment induces an imbalance in the antioxidative
mechanism of cells cultured by the three-dimensional technique. This
alteration may explain the cell damage directly caused by gliadin
and the subsequent morphological abnormalities.
©2005
The WJG Press and Elsevier Inc. All rights reserved.
Key words:
Gliadin; Celiac disease; Cytotoxicity; Multicellular spheroids
Dolfini E, Elli L, Roncoroni L, Costa B, Colleoni MP, Lorusso V,
Ramponi S, Braidotti P, Ferrero S, Falini ML, Bardella MT. Damaging
effects of gliadin on three-dimensional cell culture model. World
J Gastroenterol 2005; 11(38): 5973-5977
http://www.wjgnet.com/1007-9327/11/5973.asp
INTRODUCTION
Since Hudson et al.[1],
first observed the inhibited growth and morphological modifications
in different human cell lines induced by gliadin exposure, a number
of in vitro studies have confirmed the cytotoxicity of the
peptide. Gliadin has agglutinating activity on K562 cells (human
chronic myeloid leukemia), reduces F-actin content in intestine 407
cells, inhibits cell growth, induces apoptosis and alters redox
equilibrium in Caco-2 cells (human intestinal adenocarcinoma), and
causes a rearrangement of the cytoskeleton through the zonulin
pathway and the loss of tight junction permeability in IEC-6 cells[2].
Three-dimensional cell cultures (multicellular tumor
spheroids, MCTSs) were first introduced by Sutherland et al.[3],
in the early 1970s, and are now considered as an interesting model
in biomedical research[4].
Unlike conventional monolayer cell culture systems, MCTSs maintain
the specific morphological and biochemical properties of the
corresponding in vivo tissue, and remain in a differentiated
and functionally active state for many weeks, thus making it
possible to study the long-term effects of various xenobiotics[5].
We have recently used MCTSs from the LoVo cell line to test gliadin,
and have shown that it has a direct cytotoxic effect on cell growth
and morphology[6].
Redox equilibrium plays a pivotal role in cell homeostasis,
and can affect biological functions[7,8].
Glutathione (GSH), which consists of L-glutamine, L-cysteine and
glycine, is the most important low-molecular-weight peptide involved
in redox equilibrium. It has a reductive action on the cell
environment and neutralizes the reactive oxygen compounds and free
radicals formed during metabolism. GSH depletion induces low thiol
protein stores, a determinant of cell homeostasis, and triggers
cytotoxicity[9,10].
GSH depletion has also been shown in a number of human diseases
(alcoholic liver disease, HIV infection, acute respiratory distress
syndrome, and inflammatory bowel disease), and it has been shown
that depressed intracellular GSH levels in liver and mammary tissues
promote carcinogenesis[11].
The aim of this study was to evaluate the content
of reduced GSH and the activity of GSH-related enzymes in LoVo MCTSs
treated with gliadin.
MATERIALS AND METHODS
Protein digestion
Gliadin was purified from Triticum aestivum
flour (Hereward cultivar) according to Capelli et al.[12].
Bovine serum albumin (BSA) used as a control was purchased from
Sigma (Milan, Italy). Pepsin was supplied by Sigma (Milan, Italy)
and pancreatin by Merck (Milan, Italy). All the chemicals were of
analytical grade. Digestion was performed as previously described[6]:
briefly, gliadin was first incubated with pepsin at 37
℃
for 24 h, and then with pancreatin at 37
℃
for 3 h, adjusting to pH 8.0.
The digested proteins were analytically
controlled by reverse-phase HPLC, size-exclusion HPLC and SDS-PAGE
before being freeze-dried and stored.
Cell culture, treatment, volumetric analysis and viability test
LoVo human colon adenocarcinoma cell line (ATCC,
Rockville, USA) was maintained in exponential monolayer growth and
routinely checked for mycoplasma contamination as previously
described[6].
MCTSs were initiated according to Dolfini et al.[6],
by seeding 2105 cells/mL
in 25 mL of complete medium in polycarbonate Erlenmeyer flasks
(Corning, Milan, Italy), and incubated in a gyratory rotation
incubator. MCTS volumes were evaluated according to Chignola et
al.[13].
On the 7th d,
MCTSs (mean±SD:
22070 mm)
were exposed to digested gliadin (PT-gliadin, 500
mg/mL)
or BSA (PT-BSA, 500
mg/mL)
in completely renewed medium for further 5 d, and subsequently taken
for the evaluation of lactate dehydrogenase (LDH) release, GSH
content and GSH-related enzyme activity, and morphological analysis.
MCTS viability was tested (colony-forming assay) by plating a
cell suspension obtained after trypsin disaggregation of MCTSs (on
the 5th d
of treatment at PT-gliadin concentrations of 125, 500, 750, and 1
000
mg/mL,
and a PT-BSA concentration of 1 000
mg/mL)
in triplicate in six-well plates (500 cells/well). The surviving
cell fraction was calculated after 10 d and compared with the
plating efficiency of the controls.
Lactate dehydrogenase activity
LDH released from damaged cells was measured in
free aliquots of medium from the cell cultures, and cell-free
complete medium was included as a negative control.
Briefly, 50
mL
of the aliquots of cell supernatants was mixed with 25
mL
of LDH reagent (Sigma, Milan, Italy) and incubated at room
temperature for 30 min. LDH activity was calculated by measuring the
increase in absorbance at 490 nm according to Legrand et al.[14],
and related to the protein content of MTCSs[15].
LDH activity was reported as percentages of control values.
GSH content and GSH-related enzyme activity
MTCSs were washed with PBS (Sigma, Milan, Italy),
and then sonicated and centrifuged. The cytosolic supernatant was
used to measure GSH content and activity of the GSH-related enzymes,
namely reductase (GSR), peroxidase (GPOX) and GSH-S-transferase
(GST).
GSH content and enzyme activity were analyzed
according to previously described methods[16],
and expressed as percentages of control values.
Light and electron microscopy
LoVo cell line MCTS samples were prepared as
previously described[6],
fixed in 2.5% glutaraldehyde in a phosphate buffer, and then washed
in the same buffer. In order to avoid injury or loss of spheroids,
they were encapsulated in a solidifying agar solution and small
spheroid-containing cubes were routinely processed for transmission
electron microscopy (TEM)[17].
The semi-thin (0.5
mm)
sections used for light microscopy analysis were stained with
toluidine blue, the ultra-thin sections (50-70 nm) used for the
ultrastructural study were counterstained with uranyl acetate and
lead citrate. The MCTSs were studied for the presence of microvilli
and intercellular junctions, and the appearance of nuclei, cytoplasm
and intracytoplasmic organelles.
Scanning electron microscopy (SEM) was performed using a
Philips mod. XL20 scanning electron microscope. MCTSs were washed
twice with PBS, and then fixed in 2.5% glutaraldehyde in a phosphate
buffer at 4
℃
for a minimum of 24 h. At the time of analysis, a representative
spheroid sample was recovered, immediately placed on a filter paper,
and observed in low vacuum modality at a high voltage of 10 kV.
Statistical analysis
Each experiment was repeated four times. All the data
were expressed as mean±SD
and analyzed using the two-tailed Student's
test. P<0.05 was
considered statistically significant.
RESULTS
Volume, viability, and LDH activity in medium
The untreated and control MCTSs (treated with PT-BSA)
did not show any statistical difference in terms of diameter (27710-3 mm3 vs
261010-3 mm3),
viability (98% of the colonies formed by untreated MCTSs), LDH
release (102% of the activity of untreated MCTSs), or microscopic
appearance (data not shown). The volumes of MCTSs treated with
PT-gliadin were similar to those of the untreated or control MCTSs
(Figure 1), but their viability was 20-50% less than that of the
controls and inversely related to PT-gliadin concentrations (Figure
2A). LDH activity in the medium of treated MCTSs was significantly
increased by about 30% (Figure 2B).
GSH content and GSH-related enzyme activity
As shown in Figure 3A, the GSH content of treated MCTSs was about
20% less than that observed in the controls (P<0.05). The
activity of GSH-related enzymes (GSR, GST, and GPOX) was also
significantly decreased (by respectively 56%, 34%, and 27%, Figure
3B).
Figure 1
Volumes of MCTSs treated with PT-gliadin.
Figure 2
Viability test of MCTSs after treatment with different doses of
digested gliadin (A) and lactate dehydrogenase (LDH) activity after
gliadin treatment (B).
Figure 3
Content of reduced GSH (A) and GSH-related enzyme activity (B) in
MCTSs treated with PT-gliadin.
Morphological analysis
At light microscopy, both the treated and control
MCTSs were spherical or oval in shape, but the control cells grew in
a solid manner (with pseudoglandular differentiation, and without
necrosis or apoptosis), whereas those in PT-gliadin-treated MCTSs
showed nucleic displacement, a foamy cytoplasm and loss of cell
adhesion: 20-50% had pycnotic nuclei and condensed chromatin
(Figures 4A and B).
TEM showed that the control MCTSs had microvilli on their
external surface and normal nuclei, cytoplasmatic organelles (rough
endoplasmic reticulum, mitochondria, Golgi complex, and lysosomes),
and cytokeratin tonofilaments. The cells were joined by complexes of
tight, intermediate and desmosome junctions. In the treated MCTSs,
microvilli disappeared from the cell surface and there were
cytoskeletal and tight junction injuries. The cytoplasm contained
electron-dense material in numerous phagosomes and frequent vacuoles
of various sizes. Some cells showed cytoplasmatic lipid-like
droplets and cannibalism (Figures 4C and D).
SEM of the control MCTSs revealed an ovoid or spherical shape
with compact cells, which were densely organized and tightly packed
together, but clearly distinguishable from each other. The treated
MCTSs were irregular in shape with loosely packed cells, and the
external surface was focally interrupted by irregularly distributed
holes and blebs (Figures 4E, F, and 5).
Figure 4
Microscopic analysis of MCTSs treated with digested gliadin or
untreated. (A and B) Untreated and gliadin-treated
MCTSs with cytoplasmatic vacuoles, (C and D) images of
untreated and treated MCTSs, and (E) no holes and blebs on
untreated controls, (F) holes and blebs on the surface of
treated MCTSs.
Figure 5
Smooth surface of untreated MCTS (A)
and bleb on surface of MCTS treated with digested gliadin (B).
DISCUSSION
Our findings show that gliadin had significant functional and
morphological cytotoxic effects on intestinal adenocarcinoma cell
line (LoVo) cultured in a three-dimensional model.
Gliadin is a protein characterized by a particularly
high proline and glutamine content (respectively 15% and 35% of the
residues) that forms a kink in the polypeptide structure that
prevents peptidase attack[18].
Over the last few years, a number of studies based on the
in vitro two-dimensional cell culture system have investigated
the cellular effects of gliadin in order to clarify its role in the
pathogenetic "Puzzle
"of celiac disease (CD)[19].
Gliadin alters the cytoskeleton through the network of actin
filaments, and damages tight junctions (TJ) by creating leaks in
intercellular spaces, and these effects may be responsible for the
loss of epithelial permeability and the changes in cell-cell
signaling associated with CD[20].
Furthermore, gliadin also has a pro-apoptotic and agglutinating
effect, inhibits cell growth and viability[2],
and modifies the redox status of Caco-2 cells, thus causing a loss
in reductive potential and an increase in the levels of reactive
oxygen species[21].
Oxidative balance plays a pivotal role in cell homeostasis,
and it has been suggested that its imbalance may be involved in
various human diseases (liver diseases, HIV infection, pulmonary
diseases, tumors, Parkinson'S
disease, myocardial ischemia, and inflammatory bowel disease)[9,11].
GSH and its enzymatic machinery represent one of the most important
cellular defenses against oxidative agents and harmful xenobiotics.
Three enzymes are mainly involved in the GSH cycle: GPOX, which
converts peroxides into less dangerous fatty acids, water, and GSH
disulfide (GSSG); GSR, which reduces GSSG to GSH in NADPH-dependent
reaction; and GST, which is involved in detoxification from
xenobiotic compounds[22].
GSH has also been implicated in other cell functions: apoptosis,
cell differentiation, prostaglandin synthesis, DNA repair, amino
acid transport, enhanced immune response, and enzymatic activation[23].
The reduced GSH content and GSH-related enzyme activity
observed in our gliadin-treated MCTSs confirm the dangerous effect
of this peptide in an experimental model that maintains some of the
biochemical and morphological features of the corresponding in
vivo tissue[5],
and support the hypothesis that relates a deficiency in an oxidable
substrate[24].
The disturbed redox equilibrium is associated with the reduced cell
viability in treated MCTSs and the alteration in the integrity of
plasma membrane, as demonstrated by the leakage of large molecules
such as LDH into the medium[25].
These functional alterations affect the morphology of MCTSs
and their cells, whose clearly foamy cytoplasm and peripherally
displaced nuclei, together with the fact that many are pycnotic with
marginally condensed chromatin, support the presence of apoptotic
processes[26].
TEM confirmed cell injury by revealing the presence of numerous
phagosomes and frequent vacuoles of different sizes in the
cytoplasm, as well as intra-cytoplasmic lipid-like droplets. The
external ring normally consisting of columnar cells with serrated
intercellular TJs and surface microvilli resembling normal
enterocyte epithelia was altered in our treated MCTSs, which showed
disrupted TJs and microvilli (Figure 4). SEM also showed that the
treatment causes a loss of organization in cells contained in the
external layer of spheroid, with the formation of hole-like
structures and an abundant presence of apoptotic blebs[27].
Although our data cannot explain whether the redox imbalance
is due to a resource-consuming mechanism, decreased nuclear
expression or protein synthesis (an effect previously associated
with gliadin exposure[28]),
they clearly demonstrate that gliadin has a direct damaging effect
on human intestinal cells. Furthermore, we have confirmed that the
three-dimensional cell culture system is a good experimental model
for investigating the effects of different peptides, and can
therefore be added to the techniques used to study CD.
ACKNOWLEDGMENTS
The authors would like to thank the "Centro
per lo Studio della Celiachia, University of Milan, for its logistic
support and Kevin Smart (LINK Srl, Milan) for his help in preparing
the manuscript.
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