1 Clinica di Gastroenterologia ed Epatologia and 3 Sezione di Medicina Interna e Scienze Oncologiche, Dipartimento di Medicina Clinica e Sperimentale, Università degli Studi di Perugia, 06100 Perugia, Italy; 2 Mucosal Inflammation Research Group, University of Calgary, Calgary T2N 4N1, Alberta, Canada; and 4 Nicox, Sophia Antipolis 06906, France
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ABSTRACT |
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The activation of
a self-amplifying cascade of caspases, of which caspase-8 is the apical
protease, mediates Fas-, tumor necrosis factor (TNF)-related
apoptosis-inducing ligand (TRAIL)-, and TNF--induced
apoptosis in colon cell lines. Nitric oxide (NO) protects from
apoptosis induced by Fas and TNF-
. We examined whether
NCX-456, an NO-releasing derivative of mesalamine, protects colon
epithelial cells from cytokine-induced apoptosis. Caco-2 and
HT-29 cell lines express death factor receptors and are driven to
apoptosis in response to incubation with Fas-agonistic
antibody, TNF-
/interferon-
, and TRAIL. The two novel observations
reported here are that 1) cotreatment of cells with NCX-456,
but not mesalamine, resulted in concentration-dependent protection
against death factor-induced apoptosis and inhibition of
caspase activity, and 2) exposure to dithiothreitol, an
agent that effectively removes NO from thiol groups, resulted in a 70%
recovery of caspase activity, which is consistent with
S-nitrosation as a major mechanism for caspase inactivation.
These data suggest that caspase S-nitrosation represents a
mechanism for protection of colonic mucosal epithelial cells from death
factor-induced death.
colon cancer cells; apoptosis; death factors; nitromesalamine
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INTRODUCTION |
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CROHN'S DISEASE and
ulcerative colitis, the two main forms of inflammatory bowel disease
(IBD), are chronic, spontaneously relapsing disorders that appear to be
immunologically mediated (13). Although the underlying
genetic and environmental causes remain to be elucidated,
CD4+ T helper 1 (Th1) lymphocytes play a pivotal role in
the pathogenesis of these diseases. A Th1-like phenotype, with its
signature cytokines interferon (IFN)- and tumor necrosis factor
(TNF)-
, is shared among many colitis models and is found in patients
with active Crohn's disease (6, 8, 13). The role of T
cell-derived cytokines seems to be critical because IFN-
and TNF-
drive colonic epithelial cells to apoptosis and injection of
anti-TNF-
antibodies in patients with active Crohn's disease leads
to a transient improvement of the disease and attenuates the
development of colitis in some experimental mouse models
(13). Moreover, IFN-
not only cooperates with TNF-
to cause colonic cell death, it also modulates epithelial Fas
expression and sensitizes colonic epithelial cells to Fas-induced apoptosis (13).
Apoptosis, or programmed cell death, is regulated by tightly
controlled intracellular signaling events in response to
pathological cytotoxic stimuli including TNF-, TNF-related
apoptosis-inducing ligand (TRAIL), and Fas (3, 4,
37). Fas, a transmembrane receptor that belongs to the TNF-
receptor family, is constitutively expressed by the basolateral
membrane of normal colon and small intestinal epithelium (6, 8,
16, 22, 36, 38). Fas ligation induces apoptosis in
colonic epithelial cells and is implicated in the epithelial damage
seen in ulcerative colitis (6, 8, 16, 22, 36, 38). The
ligand for Fas (FasL) is expressed by intraepithelial and lamina
propria lymphocytes, and its expression is increased in the lamina
propria of ulcerative colitis patients, suggesting that
Fas-FasL-induced apoptosis participates in the mucosal damage
of ulcerative colitis (6, 8). Inhibition of
cytokine-regulated apoptosis may therefore be useful in
preventing or treating intestinal lesions in patients with IBD.
One way of interfering with the progression of disease is to reduce
intracellular events that lead to apoptotic cell death. The
intracellular domains of Fas, TRAIL receptors 1 and 2 (TRAIL-R1 and
-R2), and TNF- receptor 1 (TNF-R1) contain a region termed the death
domain (DD), which is required for induction of apoptosis (4, 37). Binding of Fas by FasL, TRAIL-R1 or -R2 by TRAIL, and TNF-R1 by TNF-
leads to the association of Fas-associated DD (FADD) and TNF-R1-associated DD (TRADD), respectively, with Fas, TRAIL-R1 and R2, or TNF-R1 via their homologous DD (4, 37). In their turn, FADD and TRADD recruit the zymogen form of
the apoptosis-initiating protease, caspase-8, a member of the interleukin 1 (IL)-
-converting enzyme (ICE) family (3,
37), through homophilic interaction of "death effector
domains" leading to the assembly of a death-inducing signaling
complex (DISC) at the cytoplasmic DD of Fas, TRAIL-R1, TRAIL-R2, or
TNF-R1 (4). The proximity of caspase-8 zymogens
facilitates activation through self-processing, leading to cleavage of
downstream caspases including caspase-3, which is largely responsible,
directly and indirectly, for dismantling the apoptotic cells from
within (37). Inhibition of proapoptotic caspases is
therefore a new pharmacological means of protection from death
factor-induced apoptosis (11).
Mesalamine (5-aminosalicylic acid) is one of the most commonly used
drugs for the treatment of active IBD and for maintenance of remission
(34). However, the mechanism by which mesalamine reduces
mucosal injury in colitis is not clear. Mesalamine exerts many effects
that could contribute to its anti-inflammatory activity. These include
inhibition of leukotriene synthesis, scavenging of oxygen-derived free
radicals, scavenging of peroxynitrite, and inhibition of IL-1
synthesis (34). Mesalamine has also been demonstrated to
reduce peroxynitrite-induced apoptosis in Caco-2 cells, but
whether it is also active in protecting from cytokine-induced
apoptosis is unknown (33).
In recent years, we (14, 15) and others (12,
39) have demonstrated that the addition of a nitric oxide
(NO)-releasing moiety to conventional drugs, such as aspirin and other
anti-inflammatory analgesic drugs, and more recently to acetaminophen,
results in new chemical entities that share the property of releasing
small amounts of NO at target tissues. An increasing body of evidence demonstrates that the addition of the NO-releasing moiety to these compounds markedly enhances their anti-inflammatory, antipyretic, and/or analgesic effects (14, 15, 39, 40). Although the molecular mechanisms underlying the enhanced anti-inflammatory properties of NO-releasing compounds are poorly defined, we
demonstrated previously (14, 15) that, similarly to NO,
they cause the nitrosation/inhibition of cysteine proteases that
mediate inflammation (ICE/caspase-1) and apoptosis (caspases-9
and -3). On the basis of these findings we hypothesized that adding an
NO-releasing moiety to mesalamine would increase its effectiveness in
reducing colonic inflammation. Indeed, we reported (40)
that an NO-mesalamine derivative (hereafter referred to as NCX-456)
exhibited improved efficacy over mesalamine in a rat model of
chemically induced colitis. Compared with mesalamine, NCX-456 was more
effective in reducing inflammation and chemotaxin-induced leukocyte
adherence to the mesenteric endothelium and released NO when incubated
with colonic mucosa fragments (40). However, NCX-456
failed to inhibit IFN- release in the inflamed colon, suggesting
that it may protect colon cells by acting downstream to this cytokine.
Because protection against cytokine-induced apoptosis/necrosis
contributes to increased drug efficacy, the present study was designed
to investigate whether NCX-456 protects colonic epithelial cells
against apoptosis induced by Fas, IFN-
, TRAIL, and TNF-
and to define the intracellular mechanism involved in this effect.
Moreover, because it is still unknown whether colon cell lines express
functionally active TRAIL receptors, we have evaluated the presence and
functional activity of receptors for this death factor.
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MATERIALS AND METHODS |
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Materials.
RPMI-1640 medium, L-glutamine, EGTA, FCS, concanavalin A,
aprotinin, leupeptin, dithiothreitol (DTT), HEPES, propidium iodide, BSA fraction V, phenylmethylsulfonyl fluoride (PMSF), and mesalamine were from Sigma Chemical (St. Louis, MO). Recombinant human IFN- and
TNF-
were from ICN Flow (Milan, Italy), whereas other media and
serum were purchased from GIBCO (Paisley, UK). The anti-human Fas
antibody (APO-1-3) and
2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO) were from
Alexis (San Diego, CA). The anti-human caspase-8 antibody (p20 subunit)
was from New England Biolab (Beverly, MA), and anti-human polyclonal
cytochrome c (Cyt c) antibody was from Santa Cruz
Biotechnology (Santa Cruz, CA). TRAIL peptide was from Biomol
(Plymouth Meeting, PA), and 4,5-diaminofluorescein diacetate (DAF-DA)
was from Calbiochem (Darmstadt, Germany). NO-mesalamine [NCX-456;
5-amino-2-hydroxybenzoic acid 4-(nitroxybutylester)] was
synthesized by Nicox (Nice, France).
Cell culture. HT-29 and Caco-2, two human colon adenocarcinoma cell lines, were obtained from American Type Culture Collection (Rockville, MD). HT-29 cells were cultured in RPMI-1640 medium containing 10% FCS, glutamine (2 mM), and antibiotics (50 U/ml penicillin, 50 µg/ml streptomycin) (1, 2). Caco-2 cells were cultured in MEM containing 20% FCS, glutamine, and antibiotics and supplemented with nonessential amino acids and 1 mM sodium pyruvate. Both types of cells were grown in 75-cm2 culture flasks, and the medium was changed every other day. The cells were passaged weekly and, for experiments, were aliquoted into 96-well plates (104 cells/well) and allowed to adhere overnight before cytokine stimulation in medium without serum.
Induction and detection of apoptosis.
To test whether cytokines and death factors drive colon cells to
apoptosis, HT-29 and Caco-2 cells were incubated with medium alone or IFN- (1,000 U/ml) and TNF-
(100 ng/ml) or 10 µg/ml of
APO-1-3 monoclonal anti-human Fas antibody or human recombinant TRAIL (100 ng/ml) alone or in combination for 8 h. In experiments in which IFN-
and Fas monoclonal antibody were used to induce apoptosis, cells were preincubated for 3 h with IFN-
and then Fas monoclonal antibody was added. To test whether NCX-456 and mesalamine modulate cytokine-induced apoptosis, cells were
incubated with the cytokine mixture (i.e., IFN-
+ Fas agonistic
monoclonal antibody or IFN-
+ TNF-
at the concentration
described) with or without 1-100 µM NCX-456 or mesalamine for
8 h. In some experiments, cells were incubated with the NO
scavenger PTIO (100 µM) to assess whether NO scavenging reversed the
protective effect of NCX-456 on cytokine-induced cell death (3,
4). At the end of the culture period, adherent cells were
detached and mixed with floating cells by gentle centrifugation.
Apoptosis was detected by staining the cells with propidium
iodide (PI). Briefly, cell pellets were washed twice in PBS,
resuspended in hypotonic fluorochrome solution (50 µg/ml PI in 0.1%
sodium citrate + 0.1% Triton X-100), kept 4-8 h at 4°C in
the dark, and analyzed using a Epics XL flow cytometer (Beckman-Coulter, Miami, FL). The percentage of apoptotic cells was
determined by evaluating hypodiploid nuclei after proper gating on DNA
content. Alternatively, cells were recovered from cultures (both floating and adherent), fixed with 3.7% paraformaldehyde-PBS, and stained with 0.1 µg/ml 4,6-diamino-2-phenylindole (DAPI). The
percentage of cells with condensed chromatin and fragmented nuclei was
determined by ultraviolet (UV) microscopy (14). Each experiment was performed in triplicate on separate days.
Detection of apoptosis by ELISA assay. ELISA is based on the photometric sandwich-enzyme-immunoassay principle and uses mouse monoclonal antibodies directed against cytosolic DNA fragments and histones. Briefly, three incubation steps were performed. First, the antihistone antibody was fixed on the wall of a microtiter plate, Second, the nucleosomes contained in the sample were bound via their histone to the antihistone antibody. Third, anti-DNA-peroxidase was added to react with the DNA part of the nucleosome. The amount of peroxidase retained in the sample was determined photometrically (absorbance at 405/490 nm) with 2,2-azino-di-(3-ethylbenzthiazoline sulfonate) as a substrate (14, 15). Assays were performed using a cell death detection ELISA kit following the recommendations of the manufacturer (Boehringer Mannheim, Indianapolis, IN).
DNA fragmentation assay. In some experiments apoptosis was also assessed by DNA laddering assay, as previously described (14). Briefly, the floating cells and detached cells were collected and sedimented by centrifugation. Washed cell pellets were resuspended in cell lysis buffer [10 mM Tris · HCl (pH 7.4), 10 mM EDTA (pH 8.0), 0.5% Triton X-100] and incubated. RNase A (0.5 mg/ml) and proteinase K (0.5 mg/ml) were added, respectively, and incubated for 2 h. DNA was precipitated by ethanol, and 3 µg (or 1 × 106 cells) of water-diluted sample was run on a 2% agarose gel. Gels were stained with ethidium bromide, and DNA was visualized by UV transilluminator.
RT-PCR. Total RNA was isolated from human biopsies using TRIzol reagent (Life Technologies, Milan, Italy). First-strand cDNA was synthesized from total cellular RNA (1 µg) with 200 U of SuperScript II (Life Technologies), 500 ng of oligo(dT)15 primer (Sigma Genosys, Gallarate, Italy), and dNTPs (200 µM; Promega). Ten microliters of sample and two microliters of oligo(dT) primer were heated to 94°C for 2 min and cooled on ice for 5 min. To this mixture was added 4 µl of 5× first-strand buffer [250 mM Tris · HCl (pH 8.3), 375 mM KCl, 15 mM MgCl2], 2 µl of DTT (0.1 M), and 1 µl of mixed dNTPs (10 mM), and the mixture was incubated at 42°C for 2 min. One microliter of SuperScript II then added, and the reaction was incubated at 42°C for 50 min, heated at 70°C for 10 min to inactivate the enzyme, and cooled at 4°C.
Multiplex PCR was performed with a Human Apoptosis Genes Set-3 kit (Maxim Biotech, San Francisco, CA) using human Fas gene, human Fas ligand gene, human TRAIL gene, human FLICE gene (caspase-8), and human GAPDH gene as a control. PCR was performed using specific primers (Sigma Genosys). For human TRAIL R-1 the sense primer was 5'-CGCACGAACTCAGCCAACGATT-3' and antisense was 5'-TTTCCACAGTGGCATTGGCACC-3'; for human TRAIL R-2 the sense primer was 5'-CAAAATACACCGACGATGCCCG-3' and antisense was 5'-GTGCAGCGCAAGCAGAAAAGGA-3'; for human TRAIL R-3 the sense primer was 5'-CGCACGAACTCAGCCAACGAT T-3' and antisense was 5'-CGACGATGACGACGACGAACTT-3'; for human TRAIL R-4 the sense primer was 5'-TGGGACTTTGGGGCAAAAGCG T-3' and antisense was 5'-AGCAGAACCGCGACGATGAAGA-3'. The cDNA was amplified with a "hot start" reaction in a 20-µl reaction containing 5 µl of cDNA product, 2 µl of PCR buffer [200 mM Tris · HCl (pH 8.4), 500 mM KCl], 200 µM dNTPs, 1 µM sense and antisense primers, 1.5 mM MgCl2, 1 U of Platinum Taq polymerase (Life Technologies), and water in a Hybaid PCR Sprint thermocycler (Celbio, Milan, Italy). PCR was carried out for 35 cycles (30 min for amplification ofAssessment of caspase activity. After incubation with appropriate agents (see Fig. 2) HT-29 and Caco-2 cells were recovered into lysis buffer [10 mM Tris · HCl (pH 7.3), 25 mM NaCl, 0.25% Triton X-100, 1 mM EDTA]. After centrifugation at 16,000 g for 30 min, the resulting supernatants were adjusted to 1 mg/ml with lysis buffer and 25 µg total protein was incubated in 100 µl of caspase buffer [50 mM HEPES (pH 7.2), 100 mM NaCl, 1 mM EDTA (pH 8.0), 10% sucrose, 0.1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), and 1 mM DTT] with various fluorogenic substrate peptides (100 µM) including acetyl-Asp-Glu-Val-Asp-(7-amino-4-trifluoromethyl-coumarin) (Ac-DEVD-AFC) for caspase-3, Ac-Ile-Glu-Thr-Asp-AFC (Ac-IETD-AFC) for caspase-8, and Ac-Leu-Glu-His-Asp-AFC (Ac-LEHD-AFC) for caspase-9. Caspase-8, -3, and -9 activity was assayed using a fluorimeter plate reader in kinetic mode with excitation and emission wavelengths of 405 and 519 nm, respectively, continuously measuring release of AFC from substrate peptides as previously described (14, 15)
Western blot analysis of caspase-8 cleavage. Cells (1 × 107) were lysed in 100 µl of lysis buffer [20 mM Tris · HCl (pH 7.5), 150 mM NaCl, 10% glycerol, 1% Triton X-100, 2 mM EDTA, 1 mM PMSF, 40 µg/ml aprotinin, 20 µg/ml leupeptin] at 4°C. Ten microliters of sample were then run on 8-16% linear gradient polyacrylamide gels (Bio-Rad Laboratories, Hercules, CA). Gels were transferred to nitrocellulose membrane (Hybond-C Extra, Amersham). Membranes were blocked in 5% milk powder in PBS and probed with either anti-FLICE antibody (M2, Sigma Chemical) or a rabbit polyclonal anti-caspase-8 antibody, both at dilutions of 1:1000. The secondary antibody was goat anti-mouse IgG horseradish peroxidase-conjugated antibody (PharMingen, San Diego, CA) used at a dilution of 1:1,000.
Analysis of Cyt c release. Caco-2 cells were scraped off in isotonic isolation buffer (in mM: 1 EDTA, 10 HEPES, 250 sucrose, pH 7.6), collected by centrifugation at 2,500 g for 5 min at 4°C, and resuspended in hypotonic isolation buffer (in mM: 1 EDTA, 10 HEPES, 50 sucrose, pH 7.6). Cells were then incubated at 37°C for 5 min and homogenized under a Teflon pestle. Hypertonic isolation buffer (in mM: 1 EDTA, 10 HEPES, 450 sucrose, pH 7.6) was added to balance the buffer's tonicity. Samples were centrifuged at 2,000 g for 5 min at 4°C. Supernatants were recovered and centrifuged again at 10,000 g for 10 min. The pellet contained the mitochondrial fraction, which was resuspended in isotonic isolation buffer, whereas the supernatant contained cytosolic proteins. Protein concentrations in pellets and supernatants were determined using the Bio-Rad protein assay kit according to the manufacturer's specifications. After electrophoresis separation of 50 µg of protein in sodium dodecyl sulfate-12% polyacrylamide, gels were transferred by semidry transfer (Bio-Rad) to nitrocellulose membranes. Immunoblots were blocked in TTBS [10 mM Tris · HCl, 150 mM NaCl (pH 7.5), 0.05% Tween 20] containing 5% nonfat dried milk and incubated overnight with the anti-Cyt c antibody (diluted 1:1,000 in TTBS with 0.5% nonfat dried milk). After being washed, membranes were incubated with peroxide-conjugated secondary antibody (1:5,000 in TTBS with 0.5% nonfat dried milk) for 2 h, and the blot was developed with the ECL system (ECL Western blotting kit, Amersham International).
Detection of intracellular NO formation. To assess whether colon cells generate intracellular NO or NO-derived compounds from NCX-456, adherent Caco-2 cells were incubated with 100 µM NCX-456, mesalamine, or S-nitroso-DL-penicillamine (SNAP) and intracellular NO formation was assessed according to the method of Nakatsubo et al. (30) with DAF-DA. Briefly, adherent cells (1 × 106/ml) were preincubated with 1 mM L-N6-(1-iminoethyl)lysine (L-NIL) for 30 min to suppress endogenous NO generation and then loaded by incubating them in PBS in the presence of 10 µM DAF-DA at 37°C for 30 min. Cells were then washed three times with PBS and placed on a confocal microscope (Bio-Rad 1024, Bio-Rad, Milan, Italy), and images were taken every 5 s as previously described (30). Image analysis was carried out using the LaserSharp software (Bio-Rad). Intracellular NO formation was also assessed by fluorimetry in detached Caco-2 cells. Briefly, after detachment, cells (1 × 106) were preincubated with 1 mM L-NIL for 30 min, loaded with 10 µM DAF-DA, and then added to a quartz cuvette with continuous stirring and temperature thermostatically maintained at 37°C with a Hitachi 2000 (Hitachi, Milan, Italy) fluorescence spectrophotometer. SNAP, NCX-4016, or mesalamine (100 µM) was then added to the cell suspension. Cells were excited at 395-nm wavelengths with a 10-nm slit, and the intensity of fluorescence emitted at 515 nm was recorded. NO generation was expressed in arbitrary units of absorbance (16).
Nitrite/nitrate assay. Nitrite/nitrate concentrations in cell supernatants were measured by a fluorimetric detection kit (Cayman Chemical, Ann Arbor, MI). The lower detection limit, as reported by the manufacturer, was ~4 pM/well (16).
Assessment of caspase S-nitrosation.
To investigate whether the inhibition of caspaselike proteases exerted
by NCX-456 was caused by protein S-nitrosation, cell lysates
obtained from Caco-2 cells were incubated alone or with a cytokine
mixture in combination with 100 µM NCX-456, mesalamine, or SNAP and
were exposed to DTT (20 mM) to remove thiol-bound NO. The DTT and
excess of NO were then removed by passing the sample through a Sephadex
G-25 column preequilibrated with the lysis buffer and enzyme activity
was assessed (14-16, 24-28). In another set of
experiments, lysates obtained from Caco-2 cells incubated with Fas or
IFN-/TNF-
alone or in combination with NCX-456 were exposed to
DTT (20 mM) and/or HgCl2 (5 mM) for 40 min on ice and
caspase-8-like activity was measured (14-16,
24-28).
Data analysis. All values are expressed as means ± SE of n observations. Groups of data were compared using a one-way analysis of variance followed by a Student-Newman-Keuls test. With all analyses, an associated P value of <5% was considered significant (5).
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RESULTS |
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Death receptor expression on colon cancer lines.
We first investigated whether HT-29 and Caco-2 cells express death
receptors. As illustrated in Fig. 1,
RT-PCR analysis demonstrated that Fas and TNF-R1 are expressed in both
cell lines, although to a different extent, whereas TRAIL was not
expressed. Both cell lines also expressed FLICE (caspase-8). As
illustrated in Fig. 1B, both colon cancer cell lines
expressed TRAIL receptors (R1-R4). Incubation of the cells with
IFN- markedly upregulated Fas mRNA expression in both cell lines,
although it had no effect on TNF and TRAIL receptors (data not shown).
Thus colon cell lines express death receptors.
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Cytokines and Fas drive colon epithelial cells to apoptosis
and require caspase activation.
Incubation of growth-arrested HT-29 and Caco-2 monolayers with a
combination of IFN- (1000 U/ml) and TNF-
(100 ng/ml) or Fas-agonistic monoclonal antibody for 8 h resulted in a
30-35% increase in the expression of apoptotic markers, as
assessed by PI incorporation, nuclei staining with DAPI, DNA
fragmentation, and DNA-histone association (Figs.
2 and 3). In contrast, treatment with
individual cytokines or a Fas-agonistic monoclonal antibody alone did
not increase cell death above basal levels (Fig. 2). However, there
were differences in the sensitivity of the two cell lines to
apoptosis induced by the cytokine mixture, because HT-29 cells
were sensitive to Fas- and TNF-
-induced apoptosis and Caco-2
cells were resistant to apoptosis induced by the Fas-agonistic monoclonal antibody. In contrast, both cell lines were sensitive to
TRAIL-induced apoptosis (Fig. 2, B and
C). In both cell lines morphological changes suggestive of
apoptosis were associated with an increased activity of
caspase-8 like proteases (Fig. 2C) as well as caspase-3- and
-9-like proteases (data not shown). As shown in Fig. 2D,
pretreatment of the cells with 100 µM Z-VAD-FMK, a pancaspase
inhibitor, rescued both cell lines from apoptosis induced
by IFN-
/TNF-
, IFN-
/Fas-agonistic antibody, and TRAIL and caused an ~80% reduction of caspase-8 activation caused by proapoptotic agents. Thus caspase activation is required for
apoptotic signal transduction in colon cell lines
challenged with TNF-
or Fas in the presence of IFN-
and TRAIL.
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NCX-456 protects colon epithelial cells against cytokine-induced
apoptosis.
Exposure of HT-29 and Caco-2 cells to NCX-456, but not mesalamine,
protected against cytokine-induced cell death (Figs.
3 and 4). Indeed, at a concentration of
100 µM, the NO-mesalamine derivative caused ~80% reduction of
death factor-induced lethality (P < 0.001 vs. cytokine
alone) as measured by PI incorporation. Mesalamine (100 µM) also
caused ~30% reduction of apoptosis caused by a combination
of IFN- with Fas or TNF-
, although it was significantly less
effective than NCX-456 (P < 0.001). As shown in Fig.
4, the protective effect exerted by
NCX-456 was concentration dependent and lasted for at least 24 h.
Protection exerted by NCX-456 was detectable at a concentration of 1 µM, half-maximal at 6 µM, and maximal at 100 µM. Coincubation of
Caco-2 cells with NCX-456 (100 µM) was also effective in preventing
changes of nuclear morphology as evaluated by DAPI staining and DNA
fragmentation as assessed by DNA ladder assay and DNA-histone formation
(Fig. 3). Moreover, as shown in Fig. 3E, NCX-456 inhibited
cytokine-induced Cyt c release from mitochondria. Mesalamine
was significantly less effective than NCX-456 in reducing these markers
of apoptosis (Fig. 3), and at a concentration of 100 µM it
had no effect on DNA fragmentation or Cyt c release.
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Inhibition of apoptosis caused by NCX-456 is mediated by an
NO-dependent mechanism.
To gain insight on the mechanism responsible for apoptosis
inhibition, we then evaluated whether incubating the cells with an NO
scavenger reversed the protection exerted by NCX-456. As shown in Fig.
5, the ability of NCX-456 to rescue cells
from death induced by IFN-/TNF-
, IFN-
/FasL, and TRAIL was
significantly reduced when the NO scavenger PTIO (100 µM) was added
to the incubation medium. Thus NCX-456 acts through an NO-dependent
pathway.
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NCX-456 inhibits caspase activity.
Because activation of the caspase cascade is pivotal to the death
execution phase of apoptosis, we tested the effect of NCX-456 on proapoptotic caspases. As illustrated in Fig.
6, incubation of Caco-2 (Fig. 6,
A-C) and HT-29 (data not shown) cells with 100 µM
NCX-456 resulted in a 70-80% reduction of cytokine-induced caspase-8, -9, and -3 activity. To confirm that NCX-456 directly inhibits caspase-8 activation, we examined the cleavage of the procaspase-8 zymogen by Western blotting with an antibody that specifically detects the p20 subunit of this caspase. After treatment with IFN-/TNF-
, procaspase-8 was cleaved in Caco-2 and HT-29 cells. Cotreatment of the cells with NCX-456, but not with mesalamine, almost completely abolished procaspase-8 cleavage (Fig.
6D). Thus NCX-456 protects colon epithelial cell
against cytokine-induced apoptosis by inhibiting
proapoptotic caspases.
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NCX-456 inhibits caspase activity by nitrosation.
The association between the suppression of cytokine-induced
apoptosis and caspase inhibition suggests that NCX-456 inhibits apoptosis by blocking caspase activation. Because there is
evidence that NO-releasing compounds inhibit caspase activities by
causing enzyme nitrosation, we next investigated biochemically whether exposure to NCX-456 results in caspase-8 nitrosation/inhibition. To
ascertain this point, lysates obtained from cytokine-treated cells were
incubated with 20 mM DTT, an agent that effectively displaces
thiol-bound group from proteins. As shown in Fig.
7, exposure to DTT resulted in ~70%
recovery of protease activity in lysates obtained from cells incubated
with cytokine plus NCX-456. Confirming the role of NO in the inhibition
of caspase-8, SNAP (100 µM) caused 80% reduction of caspase-8-like
activity induced by TNF- and IFN-
(Fig. 7A), an effect
that was significantly reduced by DTT (P < 0.01).
Caspase-8-like activity was also reduced by incubating lysates obtained
from cytokine-treated cells with HgCl2, an agent that binds
thiol groups and removes NO. Indeed, at a concentration of 5 mM,
HgCl2 caused a 90% loss of proteolytic activity (Fig.
7B). Inhibition induced by HgCl2 was partially reversed by DTT. Thus NCX-456 causes caspase nitrosation.
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NCX-456 causes intracellular NO formation.
Because these data indicate that NCX-456-derived NO was responsible for
the antiapoptotic effect exerted by NCX-456, we next investigated
whether incubation of Caco-2 cells with this compound results in
intracellular NO formation. As shown in Fig.
8, exposure of DAF-DA-loaded cells to
NCX-456 (100 µM) resulted in a time-dependent increase in
intracellular fluorescence, suggesting that NCX-456 penetrates colon
cell membranes and is metabolized to release NO. The cell metabolism,
however, is slow, because maximal fluorescence was evident after 3 h of incubation. In contrast to NCX-456, the addition of SNAP caused a
rapid increase in intracellular fluorescence (Fig. 8D). No
changes in intracellular fluorescence were documented in cells
incubated with mesalamine alone (data not shown). To confirm these
findings by another means, we measured intracellular NO fluorescence in
nonadherent Caco-2 cells incubated with 100 µM NCX-456, SNAP, or
mesalamine. As illustrated in Fig. 8E, although exposure of
the DAF-DA-loaded cells to SNAP resulted in a rapid rise of
intracellular fluorescence, NCX-456 caused a slow but sustained
increase of intracellular fluorescence. Again, no changes were measured
in cells loaded with mesalamine. In confirmation of these findings,
incubation of the cells with SNAP or NCX-456 resulted in a significant
increase in nitrite/nitrate release (Fig. 8F). Exposure of
Caco-2 cells to 100 µM NCX-456 resulted in a high output of
nitrite/nitrate in cell supernatants (~150 µM/107 cells
at 3 h of incubation). No changes in nitrite/nitrate production were observed in cells incubated with mesalamine alone
(P < 0.001 compared with NCX-456). Thus NCX-456 is
metabolized by colon epithelial cells to release intracellular NO.
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DISCUSSION |
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T lymphocyte-derived cytokines not only recruit inflammatory cells
at the site of inflammation but directly kill colonic epithelial cells
or sensitize them to death induced by death factors such as TNF- and
FasL. TNF-
and FasL are expressed by intraepithelial and lamina
propria lymphocytes, and their expression is increased in the lamina
propria of ulcerative colitis patients (6, 8, 13). The
effects of T cell-derived cytokines on cultured intestinal epithelial
cells have also been well documented because human lamina propria T
lymphocytes produce IFN-
and TNF-
, which act in concert to kill
colon epithelial cells. The effects of IFN-
on colon epithelial
cells may have clinical relevance, as this cytokine has been implicated
in a variety of inflammatory conditions including ulcerative colitis,
Crohn's disease, and human immunodeficiency virus-related
enterocolitis (13). Indeed, biological agents that
inhibit TNF-
and/or IFN-
production (IL-10 and thalidomide) or
TNF-
activity (infliximab) effectively reduce inflammation in IBD
patients (31, 34). In the present study we demonstrated that exposure of two cell lines, which express different stages of
epithelial cell maturation, to IFN-
sensitizes them to the proapoptotic/necrotic effect of a Fas-agonistic monoclonal antibody and TNF-
and that this effect is mediated by activation of the caspase cascade. However, our results demonstrated that Caco-2 and
HT-29 cells have different sensitivities to the apoptosis induced by death factors, because Caco-2 cells, in contrast to HT-29
cells, are resistant to Fas-mediated apoptosis (1,
2). In line with this finding, it was demonstrated previously
(1, 2) that Caco-2 cells not only express very low amounts
of Fas but lack one or more presently unidentified factor(s) necessary to activate the chain of reactions that leads to caspase-8 recruitment. In the present study we also demonstrated for the first time that colon
cancer lines express TRAIL receptors and are sensitive to TRAIL-induced
apoptosis (4). These cells are therefore a useful model for the study of the effect of drugs on cytokine-regulated apoptosis in colonic mucosa.
NCX-456 is a derivative of mesalamine that consists of the parent drug
linked to an NO-releasing moiety through an ester linkage. In a
previous study (40) we demonstrated that this compound was
significantly more effective than mesalamine in reducing colonic inflammation in a rat model of colitis. In the present study we have
extended these observations by demonstrating that NCX-456 directly
protects IFN--sensitized colon epithelial cells from apoptotic/necrotic death induced by TNF-
or Fas-agonistic
monoclonal antibody by causing the S-nitrosation/inhibition
of proapoptotic caspases. Activation of the caspase cascade appears to
be essential to cytokine-induced apoptosis of HT-29 and Caco-2
cells, in light of our observation that pretreatment with the
pancaspase inhibitor Z-VAD-FMK completely prevents apoptosis
induced by TNF-
and Fas (Fig. 2). Our current understanding of
caspase involvement in Fas/TNF-
-induced apoptosis indicates
that Fas and/or TNF-R1 cross-linking leads to the activation of a
self-amplifying cascade of caspases, of which caspase-8 is the apical
protease (37). This particular caspase is recruited and
probably auto-proteolytically activated through protein-protein
interaction with FADD among a complex of proteins (DISC) that dock onto
the cytoplasmic DD of oligomerized Fas. Activated caspase-8
releases active caspase-8 subunits that can then cleave other caspases
(e.g., caspase-3), which can in turn cleave a large number of
intracellular death substrates. In the present study, we demonstrated
that, in contrast to mesalamine, NCX-456 prevents caspase activation in
Fas- and TNF-
-challenged cells and that this effect is mainly
mediated by protein nitrosation/inhibition. Supporting this view, we
demonstrated that 1) caspases-8, -3, and -9 are inhibited in
NCX-456-treated cells but not in cells treated with mesalamine, which
suggests that the NO group of NCX-456 is responsible for this effect;
2) exposure to PTIO, an NO scavenger, reversed protection
exerted by NCX-456 on cytokine-induced apoptosis (Fig. 5);
3) exposure of NCX-456-treated cells to DTT, an agent that
effectively removes NO from thiol groups, resulted in a 70% recovery
of caspase activity, which is consistent with nitrosation as a major
mechanism for caspase inhibition; 4) HgCl2,
which binds thiol groups, caused a DTT-reversible inhibition of caspase
activity, again suggesting a role for protein nitrosation in caspase
inhibition (14-16, 24-26); 5)
similarly to NCX-456, the NO donor SNAP inhibited caspase activity and
rescued cells from death induced by Fas and TNF-
; and 6)
incubation of nontreated Caco-2 cells with NCX-456 resulted in a time-
and concentration-dependent increase in intracellular NO as detected by
intracellular fluorescence in adherent and nonadherent cells and
nitrite/nitrate in cell supernatants. In contrast to NCX-456,
mesalamine did not cause NO formation and was significantly less
effective than NCX-456 in protecting from apoptosis caused by
Fas and TNF-
(33). Together these data suggest that NO
released from the nitroxy moiety of NCX-456 is responsible for caspase inhibition in this experimental setting (7, 29).
NO has emerged as a potent inhibitor of apoptosis in many cell
types, and we and others (9, 15, 24-26) have
previously demonstrated that a dominant mechanism for apoptosis
prevention is the inhibition of caspase activity by
nitrosation. NO-dependent caspase inhibition was demonstrated
previously to prevent Bcl-2 cleavage and Cyt c release as
well as activation of proteases that become activated after the release
of Cyt c in hepatocytes and other cell types
(24-27). Because of the capacity of NO to rapidly
diffuse intracellularly and from cell to cell, this would represent an
efficient mechanism to guard against the consequences of the activation
of caspases, which might result from cell injury or exposure to other
activators such as IFN-, TNF-
, and FasL in the local environment
(6, 7, 10, 13, 18, 21). Caspases are likely targets for
nitrosation because their catalytic mechanisms require a
redox-sensitive cysteine residue in the catalytic core
(9). Previous studies carried out with purified subunits of caspases-1 and -3 demonstrated that the p17 subunit of caspase-3 and
the p20 subunit of caspase-1 are selective targets for NO compounds and
that the nitrosation of these subunits leads to a
concentration-dependent inhibition of enzyme activity (9, 27,
28). More generally, there is now evidence that nitrosation is a
mechanism that is extensively involved in caspase regulation (10,
19, 24-28). A recent report from Mannick et al.
(27) indicates that in resting human cell lines caspase-3
zymogens are nitrosated and denitrosated on Fas/FasL cross-linking,
indicating that caspase activation requires both denitrosation
and zymogen cleavage. Although the reversal of NO-mediated inhibition
by DTT is consistent with nitrosation being the main mechanism for
caspase inhibition by NCX-456 and our results demonstrated NO formation in colon cell lines incubated with this compound, NO radical does not
effectively nitrosate thiol groups, suggesting that a NO reaction product is implicated in caspase nitrosation (10, 17).
NO+ equivalent nitrosates thiols and mimics the effect of
NO donors when added to cells (10). Reactive nitrogen
oxide species, including NO+ and its equivalents, can be
generated by the reaction of NO with O2 or iron-sulfur
clusters (17). These complexes have been shown to carry
out transnitrosative reactions (10). In our in vitro experiments, the cells were grown under aerobic conditions and the
interaction of NO with O2 could form
N2O3, which has been shown to cause nitrosation
via the formation of NO+ equivalents.
The failure of DTT treatment to induce a full recovery of all caspase-8 activity, however, raises the possibility that NO may also suppress caspase-8 activation (27). Supporting this concept, NCX-456 markedly reduced the amount of the caspase-8 p17 subunit released under Fas cross-linking (Fig. 6). Indeed, because caspase-8 activation is partially due to the autocatalytic cleavage of the inactive proenzyme, it cannot be excluded that S-nitrosation/inhibition of caspase-8 reduces the amount of active enzyme that is further generated through this pathway (19, 37). However, because caspase-8 may be activated via caspase-6 and NCX-456 behaves as a pancaspase inhibitor, it cannot be excluded that the NO-mesalamine derivative acts on multiple points of the caspase pathway (26).
In summary, we demonstrated that in contrast to mesalamine, NCX-456, an
NO-mesalamine derivative, protects IFN--sensitized colon epithelial
cells from death induced by TNF-
and Fas. The ability of NCX-456 to
interfere with TNF-
-mediated signals may be of clinical relevance,
considering that anti-TNF-
therapies effectively reduce inflammation
in IBD patients. The ability of NCX-456 to prevent caspase activation
may contribute to further downregulation of TNF-
signals in the
inflamed mucosa and may hold some promise for biological therapies to
attenuate T cell-dependent inflammation in IBD patients
(11).
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ACKNOWLEDGEMENTS |
---|
We thank Barbara Federici for technical assistance.
![]() |
FOOTNOTES |
---|
This work was supported in part by a grant from Ministero dell' Universita' e della Ricerca Scientifica (MURST) to S. Fiorucci.
Address for reprint requests and other correspondence: S. Fiorucci, Clinica di Gastroenterologia ed Endoscopia Digestiva, Policlinico Monteluce, 06100 Perugia, Italy (E-mail: fiorucci{at}unipg.it).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 10 November 2000; accepted in final form 30 April 2001.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Abreu-Martin, MT,
Vidrich A,
Lynch DH,
and
Targan SR.
Divergent induction of apoptosis and IL-8 secretion in HT-29 cells in response to TNF-alpha and ligation of Fas antigen.
J Immunol
155:
4147-4154,
1995[Abstract].
2.
Abreu-Martin, MT,
Palladino AA,
Faris M,
Carramanzana NM,
Nel AE,
and
Targan SR.
Fas activates the JNK pathway in human colonic epithelial cells: lack of a direct role in apoptosis.
Am J Physiol Gastrointest Liver Physiol
276:
G599-G605,
1999
3.
Alnemri, ES,
Livingston DJ,
Nicholson DW,
Salvesen G,
Thornberry NA,
Wong WW,
and
Yuan J.
Human ICE/CED-3 protease nomenclature.
Cell
87:
171,
1996[ISI][Medline].
4.
Ashkenazi, A,
and
Dixit VM.
Death receptors: signaling and modulation.
Science
281:
1305-1308,
1998
5.
Boirivant, M,
Pica R,
DeMaria R,
Testi R,
Pallone F,
and
Strober W.
Stimulated human lamina propria T cells manifest enhanced Fas-mediated apoptosis.
J Clin Invest
98:
2616-2622,
1996
6.
Boughton-Smith, NK,
Evans SM,
Hawkey CJ,
Cole AT,
Balsitis M,
Whittle BJR,
and
Moncada S.
Nitric oxide synthase activity in ulcerative colitis and Crohn's disease.
Lancet
342:
338-340,
1993[ISI][Medline].
7.
Colton, T.
Statistics in Medicine. Boston: Little, Brown, 1978.
8.
De Maria, R,
Boirivant M,
Cifone MG,
Roncaioli P,
Hahne M,
Tschopp J,
Pallone F,
Santoni A,
and
Testi R.
Functional expression of Fas and Fas ligand on human gut lamina propria T lymphocytes. A potential role for the acidic sphingomyelinase pathway in normal immunoregulation.
J Clin Invest
97:
316-322,
1996
9.
Dimmeler, S,
Haendeler J,
Nehls M,
and
Andreas M.
Suppression of apoptosis by nitric oxide via inhibition of interleukin-1 converting enzyme (ICE)-like and cysteine protease protein (CPP)-32-like proteases.
J Exp Med
185:
601-607,
1997
10.
Dimmeler, S,
and
Zeiher AM.
Nitric oxide and apoptosis: another paradigm for the double-edged role of nitric oxide.
Nitric Oxide
1:
275-281,
1997[ISI][Medline].
11.
Dinarello, CA,
and
Margolis NH.
Stopping the cuts: the recently discovered enzymes that process the precursors of inflammatory cytokines are good targets for the design of new anti-inflammatory therapeutic agents.
Curr Biol
5:
587-592,
1995[ISI][Medline].
12.
Elliott, SN,
McKnight W,
Cirino G,
and
Wallace JL.
A nitric oxide-releasing nonsteroidal anti-inflammatory drug accelerates gastric ulcer healing in rats.
Gastroenterology
109:
524-530,
1995[ISI][Medline].
13.
Fiocchi, C.
Inflammatory bowel disease: etiology and pathogenesis.
Gastroenterology
115:
182-205,
1998[ISI][Medline].
14.
Fiorucci, S,
Antonelli E,
Santucci L,
Morelli O,
Miglietti M,
Federici B,
Mannucci R,
Del Soldato P,
and
Morelli A.
Gastrointestinal safety of nitric oxide-derived aspirin is related to inhibition of ICE-like cysteine proteases in rats.
Gastroenterology
116:
1089-1106,
1999[ISI][Medline].
15.
Fiorucci, S,
Santucci L,
Antonelli E,
Distrutti E,
del Sero G,
Morelli O,
Romani L,
Federici B,
del Soldato P,
and
Morelli A.
NO-aspirin protects from T-cell mediated liver injury by inhibiting caspase-dependent processing of Th1-like cytokines.
Gastroenterology
118:
404-422,
2000[ISI][Medline].
16.
Fiorucci, S,
Santucci L,
Cirino G,
Mencarelli A,
Familiari L,
del Soldato P,
and
Morelli A.
ICE is a target for NO-releasing aspirin. New insights in the anti-inflammatory mechanism of NO-NSAIDs.
J Immunol
165:
5245-5254,
2000
17.
Grisham, MB,
Jourd'Heuil D,
and
Wink DA.
Physiological chemistry of nitric oxide and its metabolites: implications in inflammation.
Am J Physiol Gastrointest Liver Physiol
276:
G315-G321,
1999
18.
Grossmann, J,
Mohr S,
Lapetina EG,
Fiocchi C,
and
Levine AD.
Sequential and rapid activation of select caspases during apoptosis of normal intestinal epithelial cells.
Am J Physiol Gastrointest Liver Physiol
274:
G1117-G1124,
1998
19.
Hebestreit, H,
Dibbert B,
Balatti I,
Braun D,
Schapowal A,
Blaser K,
and
Simon HU.
Disruption of Fas receptor signaling by nitric oxide in eosinophils.
J Exp Med
187:
415-425,
1998
20.
Hooper, DH,
Bagasra O,
Marini JC,
Zborek A,
Ohnishi ST,
Kean R,
Champion JM,
Sarker AB,
Bobroski L,
Farber JL,
Akaike T,
Maeda H,
and
Koprowski H.
Prevention of experimental allergic encephalomyelitis by targeting nitric oxide and peroxynitrite: implications for the treatment of multiple sclerosis.
Proc Natl Acad Sci USA
94:
2528-2533,
1997
21.
Hsu, H,
Shu HB,
Pan MG,
and
Goedell DV.
TRADD-TRAF2 and TRADD-FADD interactions define two distinct TNF receptor 1 signal transduction pathways.
Cell
84:
299-308,
1996[ISI][Medline].
22.
Iwamoto, M,
Koji T,
Makiyama K,
Kobayashi N,
and
Nakane PK.
Apoptosis of crypt epithelial cells in ulcerative colitis.
J Pathol
180:
152-159,
1996[ISI][Medline].
23.
Jones, BA,
and
Gores GJ.
Physiology and pathophysiology of apoptosis in epithelial cells of the liver, pancreas, and intestine.
Am J Physiol Gastrointest Liver Physiol
273:
G1174-G1188,
1997
24.
Kim, Y-M,
de Vera ME,
Watkins SC,
and
Billiar TR.
Nitric oxide protects cultured rat hepatocytes from tumor necrosis factor--induced apoptosis by inducing heat shock protein 70 expression.
J Biol Chem
272:
1402-1411,
1997
25.
Kim, YM,
Kim TH,
Seol DW,
Talanian RV,
and
Billiar TR.
Nitric oxide suppression of apoptosis occurs in association with an inhibition of Bcl-2 cleavage and cytochrome c release.
J Biol Chem
273:
31437-31441,
1998
26.
Li, J,
Billiar TR,
Talanian RV,
and
Kim YM.
Nitric oxide inhibits seven members of the caspase family via S-nitrosylation.
Biochem Biophys Res Commun
240:
419-424,
1997[ISI][Medline].
27.
Mannick, JB,
Hausladen A,
Liu L,
Hess DT,
Zeng M,
Miao QX,
Kane LS,
Gow AJ,
and
Stamler JS.
Fas-induced caspase denitrosylation.
Science
294:
651-657,
1999.
28.
Mannick, JB,
Miao XQ,
and
Stamler JS.
Nitric oxide inhibits Fas-induced apoptosis.
J Biol Chem
272:
24125-24128,
1997
29.
Miller, MJS,
Thompson JH,
Zhang XJ,
Sadowska-Krowicka H,
Kakkis JL,
Munshi UK,
Sandoval M,
Rossi JL,
Eloby-Childress S,
Beckman JS,
Ye YZ,
Rodi CP,
Manning PT,
Currie MG,
and
Clark DA.
Role of inducible nitric oxide synthase expression and peroxynitrite formation in guinea pig ileitis.
Gastroenterology
109:
1475-1483,
1995[ISI][Medline].
30.
Nakatsubo, N,
Kojima H,
Kikuchi K,
Nagoshi H,
Hirata Y,
Maeda D,
Imai Y,
Imura T,
and
Nagamo T.
Direct evidence of nitric oxide production from bovine aortic endothelial cells using new fluorescence indicators: diaminofluoresceins.
FEBS Lett
427:
263-266,
1998[ISI][Medline].
31.
Papadakis, KA,
and
Targan SR.
Tumor necrosis factor: biology and therapeutic inhibitors.
Gastroenterology
119:
1148-1157,
2000[ISI][Medline].
32.
Pinkoski, MJ,
Brunner T,
Green DR,
and
Lin T.
Fas and Fas ligand in gut and liver.
Am J Physiol Gastrointest Liver Physiol
278:
G354-G366,
2000
33.
Sandoval, M,
Liu X,
Mannick EE,
Clark DA,
and
Miller MJ.
Peroxynitrite-induced apoptosis in human intestinal epithelial cells is attenuated by mesalamine.
Gastroenterology
113:
1480-1488,
1997[ISI][Medline].
34.
Sands, BE.
Therapy of inflammatory bowel disease.
Gastroenterology
118:
68S-82S,
2000.
35.
Southey, A,
Tanaka S,
Murakami T,
Miyoshi H,
Ishizuka T,
Sugiura M,
Kawashima K,
and
Sugita T.
Pathophysiological role of nitric oxide in experimental colitis.
Int J Immunopharmacol
19:
669-676,
1997[ISI][Medline].
36.
Strater, J,
Wellisch I,
Riedl S,
Walczak H,
Koretz K,
Tandara A,
Krammer PH,
and
Moller P.
CD95 (APO-1/Fas)-mediated apoptosis in colon epithelial cells: a possible role in ulcerative colitis.
Gastroenterology
113:
160-167,
1997[ISI][Medline].
37.
Thornberry, NA,
and
Lazebnik Y.
Caspases: enemies within.
Science
281:
1312-1316,
1998
38.
Ueyama, H,
Kiyohara T,
Sawada N,
Isozaki K,
Kitamura S,
Kondo S,
Miyagawa J,
Kanayama S,
Shinomura Y,
Ishikawa H,
Ohtani T,
Nezu R,
Nagata S,
and
Matsuzawa Y.
High Fas ligand expression on lymphocytes in lesions of ulcerative colitis.
Gut
43:
48-55,
1998
39.
Wallace, JL,
Reuter B,
Cicala C,
McKnight W,
Grisham MB,
and
Cirino G.
Novel nonsteroidal anti-inflammatory drug derivatives with markedly reduced ulcerogenic properties in the rat.
Gastroenterology
107:
173-179,
1994[ISI][Medline].
40.
Wallace, JL,
Vergnolle N,
Muscara MN,
Asfaha S,
Chapman K,
McKnight W,
del Soldato P,
Morelli A,
and
Fiorucci S.
Enhanced anti-inflammatory effects of a nitric oxide-releasing derivative of mesalamine in rats.
Gastroenterology
1117:
557-566,
1999.