1 Faculty of Pharmaceutical Sciences, Okayama University and 3 Precursory Research for Embryonic Science and Technology, Japan Science and Technology Corporation, Okayama 700-8530; and 2 Department of Nutrition, School of Medicine, University of Tokushima, Tokushima 770-8503, Japan
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ABSTRACT |
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A major clinical problem encountered with the use of nonsteroidal anti-inflammatory drugs (NSAIDs) such as indomethacin is gastropathy. In this study, we examined, using guinea pig gastric mucosal cells in primary culture, how NSAIDs damage gastric mucosal cells. The short-term treatment of cells with high concentrations of indomethacin decreased cell viability in the absence of apoptotic DNA fragmentation, chromatin condensation, or caspase activation. Cells lost membrane integrity with this short-term indomethacin treatment, suggesting that indomethacin induced necrosis under these conditions. In contrast, the long-term treatment of cells with low concentrations of indomethacin decreased cell viability and was accompanied by apoptotic DNA fragmentation, chromatin condensation, and caspase activation. Pretreatment of cells with inhibitors of caspases or protein synthesis suppressed cell death caused by long-term indomethacin treatment, suggesting that apoptosis was induced when the inhibitors were not present. These results imply that NSAIDs cause gastric mucosal damage through both necrosis and apoptosis of gastric mucosal cells.
gastric mucosal injury; indomethacin; aspirin; caspases; cell death
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INTRODUCTION |
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GASTROPATHY ASSOCIATED WITH the use of nonsteroidal anti-inflammatory drugs (NSAIDs) is one of the most frequent types of gastric mucosal injury (10, 22, 25). To establish a clinical protocol for overcoming this side effect, the molecular mechanism governing NSAID-induced gastric mucosal injury needs to be elucidated. Inhibition of the synthesis of PGs, which have cytoprotective effects on gastric mucosa, has been thought to be the major mechanism of the gastrotoxic effects of NSAIDs. However, evidence has suggested that the gastrotoxic effects of NSAIDs cannot be explained only by their inhibitory effects on PG synthesis. For example, administration of aspirin (25 mg/kg) inhibited ~90% of PG synthesis in rats, but it did not cause gastric mucosal lesion (19). It has also been proposed that NSAID-dependent gastric hypermotility, disturbances in mucosal blood flow, and activation of neutrophils are involved in the gastrotoxic effects of NSAIDs (10, 22, 25). In addition to these indirect effects of NSAIDs, direct cytotoxic effects of NSAIDs on gastric mucosal cells are also important in relation to gastropathy associated with the use of NSAIDs in vivo (10, 22, 25).
Various stresses cause cell death through either apoptosis or necrosis. Several in vivo experiments (1, 7, 28) have suggested that NSAIDs induce apoptosis in gastric mucosal cells. Although there are no reports demonstrating that NSAIDs induce necrosis in gastric mucosal cells in vivo and in vitro, the activation by NSAIDs of neutrophils and macrophages accompanying gastropathy suggests that NSAID-induced necrosis may be also involved in the gastrotoxic effects of NSAIDs in vivo.
Studies (5, 26) on the mechanism of how NSAIDs prevent colon cancer showed that NSAIDs could induce apoptosis in colon cancer cells. Several experiments (9, 17) using cell lines derived from gastric mucosa suggested that NSAIDs could induce apoptosis in gastric mucosal cells. The use of cells in primary culture is thought to mimic cells in vivo better than the use of cell lines, in addition to which the primary culture of guinea pig gastric mucosal cells has been established and well characterized (13, 23). Thus an examination of the cytotoxicity of NSAIDs using primary culture cells may provide useful information about the mechanism of NSAID-induced gastropathy. We (32) recently reported, using primary cultures of guinea pig gastric mucosal cells, that the short-term exposure of cells to indomethacin rapidly decreased cell viability in vitro in a dose-dependent manner. However, the mechanism of this cell death is yet to be elucidated. In this study, we have examined the mechanism of indomethacin-dependent cell death in primary cultures of guinea pig gastric mucosal cells. The results clearly show that NSAIDs induce both necrosis and apoptosis depending on the treatment conditions; short-term treatment of cells with high concentrations of NSAIDs and long-term treatment of cells with low concentrations of NSAIDs induce necrosis and apoptosis, respectively.
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MATERIALS AND METHODS |
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Chemicals and media. RPMI 1640 was obtained from Nissui Pharmaceutical (Tokyo, Japan). FCS, trypsin solution, and trypan blue were from GIBCO-BRL (Grand Island, NY). Pronase E and type I collagenase were obtained from Kaken Pharmaceutical (Kyoto, Japan) and Nitta Gelatin (Osaka, Japan), respectively. Indomethacin, aspirin, sodium-N-lauroylsarcosinate, and cycloheximide were from Wako (Tokyo, Japan). Proteinase K, RNase A, Hoechst 33342, and propidium iodide (PI) were from Sigma (Tokyo, Japan). Peptides for assays of caspases and an inhibitor of caspase activity [N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-fmk)] were from Peptide Institute (Osaka, Japan). [3H]glucosamine (18.7 Ci/mmol) was from Amersham Pharmacia Biotech (Tokyo, Japan).
Preparation and culture of gastric mucosal cells. Male guinea pigs (4 wk of age) were obtained from Shimizu (Kyoto, Japan). Gastric mucosal cells were isolated from guinea pig fundic glands, as described previously (13, 21, 23). Isolated gastric mucosal cells were cultured for 12 h in RPMI 1640, containing 0.3% vol/vol FCS, 100 U/ml penicillin, and 100 µg/ml streptomycin, in type I collagen-coated plastic culture plates (Iwaki) under the conditions of 5% CO2-95% air and 37°C. After nonadherent cells were removed by washing with RPMI 1640, cells attached to the plate at ~50% confluence were used. We (13, 21) previously characterized guinea pig gastric mucosal cell preparations under these conditions, with the majority (~90%) of cells being identified as pit cells.
Treatment of cells with indomethacin, aspirin, cycloheximide, or Z-VAD-fmk and trypan blue exclusion test. Cells were exposed to drugs by changing the entire bathing medium. Because indomethacin and aspirin were dissolved in 0.2 M Na2CO3 and DMSO, respectively, control experiments were carried out with final concentrations of 2 mM Na2CO3 or 0.3% vol/vol DMSO, which corresponded to the concentrations of each of the compounds in NSAID-treatment experiments. We confirmed that the concentrations of these compounds did not affect cell growth or viability or the pH value of the medium. For some experiments, cells were preincubated with 30 µM Z-VAD-fmk or 10 µg/ml cycloheximide for 1 h. After treatment with NSAIDs, cells were treated with 1% trypsin and collected by centrifugation. Cells were resuspended in PBS containing 0.2% trypan blue dye and observed under a microscope.
DNA fragmentation assay. Apoptotic DNA fragmentation was monitored, as described previously (20). Cells were collected with a rubber policeman and suspended with 70 µl of lysis buffer, consisting of 50 mM Tris · HCl (pH7.8), 10 mM EDTA, and 0.5% sodium-N-lauroylsarcosinate. Proteinase K was added to a final concentration of 1 mg/ml, and the lysate was incubated at 50°C for 2 h. RNaseA was then added to a final concentration of 0.5 mg/ml and incubated at 50°C for 30 min. These samples were analyzed by 2% agarose gel electrophoresis in the presence of 0.5 µg/ml ethidium bromide.
Nuclear staining assay for necrosis and apoptosis. As previously described (12) for the determination of the extent of necrosis after NSAID treatment, cells were washed with PBS and incubated with 0.17 mM Hoechst 33342 and 100 µg/ml PI for 20 min. Cells were then analyzed using fluorescence microscopy.
To determine the level of apoptosis after NSAID treatment, cells were washed with PBS and fixed with 10% vol/vol formaldehyde for 4 h (12). Cells were then washed with PBS, stained with 0.17 mM Hoechst 33342, and observed under a fluorescence microscope as previously described (12).Caspase activity assay. The activities of caspase-3, caspase-8, and caspase-9 were determined as described previously (11, 31). Briefly, cells were collected by centrifugation and suspended in extraction buffer (50 mM PIPES, pH 7.0, 50 mM KCl, 5 mM EGTA, 2 mM MgCl2, and 1 mM dithiothreitol). Suspensions were sonicated and centrifuged, after which the supernatants were incubated with fluorogenic peptide substrates [N-acetyl-DEVD-MCA (caspase-3), N-acetyl-IETD-MCA (caspase-8), and N-acetyl-LEHD-MCA (caspase-9)] in reaction buffer (100 mM HEPES-KOH, pH 7.5, 10% sucrose, 0.1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, and 1 mg/ml BSA) for 15 min at 37°C. The release of 7-amino-4-methylcoumarin (AMC) was determined using a fluorescence spectrophotometer. One unit of protease activity was defined as the amount of enzyme required to release 1 pmol AMC/min.
Measurement of incorporation of [3H]glucosamine into cells. Measurement of incorporation of [3H]glucosamine into cells was done as described previously (23). Gastric mucosal cells were incubated with [3H]glucosamine (18.7 Ci/mmol) in the presence or absence of indomethacin for 16 h. Cells were washed with PBS three times and lysed by the solution containing Tris · HCl (pH 7.2) and 2% Triton X-100. Samples were incubated for 15 min on ice in the presence of 50% ethanol and washed with 70% ethanol three times. The precipitates were suspended with 1 N NaOH and neutralized by acetic acid. Radioactivity was calculated in a liquid scintillation counter.
Statistical analysis. All values are expressed as means ± SE. Student's t-test for paired results was performed for the evaluation of differences between the groups. Differences were considered to be significant for values of P < 0.05.
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RESULTS |
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Effect of short- and long-term treatments with NSAIDs on cell
viability and chromosomal DNA.
We (32) previously reported that treatment of gastric
mucosal cells in primary culture with indomethacin for 1 h
decreased cell viability in a dose-dependent manner. To understand the
mechanism of this cell death, we examined the state of chromosomal DNA
under conditions of indomethacin treatment. Chromosomal DNA was
extracted after the treatment of cells for 1 h with various
concentrations of indomethacin, and the change in size of chromosomal
DNA was analyzed by agarose gel electrophoresis in the presence of
ethidium bromide. As shown in Fig.
1A, treatment of cells with
indomethacin for 1 h decreased cell viability in a dose-dependent
manner. The IC50 of indomethacin for cell viability was
estimated to be ~2 mM, which is much the same as that reported
previously (32). Treatment of cells with indomethacin for
1 h did not affect the size of chromosomal DNA, even at high
concentrations (2.5 mM) (Fig. 1B). These results suggest
that the short-term treatment of gastric mucosal cells with
indomethacin does not induce apoptosis, which would be
accompanied with apoptotic DNA fragmentation. These indomethacin
treatments may, however, induce necrosis in gastric mucosal cells.
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Necrosis by short-term treatment with indomethacin.
The results described above suggest that short-term treatments of
gastric mucosal cells with indomethacin induce necrosis. To confirm
this finding, we carried out double-staining experiments with PI and
Hoechst 33342. Because necrotic cells lose their membrane integrity, PI
staining causes pink nuclear staining in necrotic cells, whereas living
cells and apoptotic cells are not stained with PI
(12). For these experiments, we used concentrations of
indomethacin that caused an ~70% decrease in cell viability (1-h
treatment, 2.5 mM; 16-h treatment, 1 mM). Control cells (for both 1- and 16-h incubations) and cells treated with indomethacin for 16 h
did not stain with PI (Fig. 2). In
contrast, cells treated with 2.5 mM indomethacin for 1 h showed
pink nuclear staining (Fig. 2). These results strongly suggest that the
short-term treatment of cells with indomethacin causes cell death
through necrosis. Because cells were not fixed in these experiments,
the staining of cells with Hoechst 33342 was weak.
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Apoptosis by long-term treatment with indomethacin.
One of characteristic features of apoptosis in cells is
chromatin condensation. We therefore examined the state of chromatin in
cells treated with indomethacin or aspirin by employing Hoechst 33342 staining. Cells were fixed with 10% formaldehyde and stained with 0.17 mM Hoechst 33342. We used concentrations of NSAIDs that caused an
~50-70% decrease in cell viability (1-h treatment, 2.5 mM
indomethacin and 30 mM aspirin; 16-h treatment, 1 mM indomethacin and
10 mM aspirin). Observations using fluorescence microscopy showed that
long-term treatment of cells with indomethacin or aspirin clearly
induced chromatin condensation (Fig. 3).
On the other hand, chromatin condensation was not apparent after the short-term treatment of cells with indomethacin or aspirin (Fig. 3).
These results support the idea that long- and short-term treatments of
gastric mucosal cells with NSAIDs induce apoptosis and
necrosis, respectively.
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Activation of caspases by indomethacin.
Most apoptotic events are mediated by the sequential activation of
caspases (4, 16). Among them, caspase-3 is located downstream of the pathway and directly activates proteins responsible for DNA fragmentation and chromatin condensation (4).
Because caspase-8 and caspase-9 activate caspase-3 (4), we
subsequently examined whether or not NSAIDs were able to activate these
caspases in gastric mucosal cells in primary culture. After incubation with indomethacin for 1 or 16 h, cells were extracted, and
their caspase activities were examined by the use of specific
fluorogenic peptide substrates [N-acetyl-DEVD-MCA
(caspase-3), N-acetyl-IETD-MCA (caspase-8), and
N-acetyl-LEHD-MCA (caspase-9)]. We used concentrations of
indomethacin that caused an ~70% decrease in cell viability (1-h
treatment, 2.5 mM; 16-h treatment, 1 mM). As shown in Fig. 4, the long-term treatment of cells with
indomethacin induced significant activation of all caspases tested.
Treatment of cells with 1 mM indomethacin for 16 h caused 17-, 8-, and 11-fold increases in the activation of caspase-3, caspase-8, and
caspase-9, respectively, suggesting that apoptosis by long-term
treatment with indomethacin is mediated by the activation of caspases.
We also measured the activity of each caspase in the culture medium
after indomethacin treatment of cells. Treatment of cells with 1 mM
indomethacin for 16 h increased the activities of all these
caspases. The activity of caspase-3, caspase-8, or caspase-9 in the
culture medium was 0.528, 0.078, or 0.033 U/ml in the presence of
indomethacin and 0.167, 0.033, or 0 U/ml in the absence of
indomethacin. We calculated the total units of each caspase activity in
both cells and culture medium and found that the majority of caspase
activity was recovered in cells, suggesting that the apoptotic
cells were not disrupted under the present conditions. On the other
hand, 1-h treatment of cells with 2.5 mM indomethacin did not affect
the activities of any of the caspases tested (Fig. 4). The results
confirm that the short-term treatment of gastric mucosal cells in
primary culture with indomethacin was mediated by necrosis. It should
be noted that the background activities of caspases in control
experiments were relatively high compared with published reports
(11, 31) for other cells. This may be due to the fact that
gastric mucosal cells in primary culture undergo maturation-dependent
spontaneous apoptosis, which mimics the turnover of gastric mucosal
cells in vivo (34).
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Effect of inhibitors of caspases and protein synthesis on cell
death induced by indomethacin.
For further confirmation that long-term treatment of gastric mucosal
cells in primary culture with indomethacin caused apoptosis, we
examined whether or not the pretreatment of cells with an inhibitor of
caspase activity was able to inhibit the cell death induced by
indomethacin. We used a broad-spectrum inhibitor of caspases, Z-VAD-fmk, which inhibits the activities of most caspases, including caspase-3, caspase-8, and caspase-9 (35). Z-VAD-fmk (30 µM) did not affect the cell viability or the state of chromosomal DNA
in the absence of indomethacin (Fig. 5,
A and C). As shown in Fig. 5A,
pretreatment of gastric mucosal cells with 30 µM Z-VAD-fmk clearly
inhibited the dose-dependent cell death seen after treatment with
indomethacin (1 mM) for 16 h. We subsequently confirmed that 30 µM Z-VAD-fmk almost completely inhibited the activation of caspase-3,
caspase-8, and caspase-9 by NSAIDs (data not shown). We also found that
pretreatment of gastric mucosal cells with 30 µM Z-VAD-fmk clearly
inhibited the dose-dependent apoptotic DNA fragmentation seen after
treatment with 1 mM indomethacin for 16 h (Fig. 5C).
These results support the proposal that the long-term treatment of
cells with indomethacin caused cell death through apoptosis,
which is mediated by the activation of caspases. In contrast, the
pretreatment of cells with 30 µM Z-VAD-fmk did not prevent the cell
death seen in response to treatment with 2.5 mM indomethacin for 1 h, which again suggests that the short-term treatment of cells with
high concentrations of indomethacin caused cell death by necrosis.
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Effect of indomethacin on glucosamine uptake.
Gastric mucosal cells are protected from various gastric irritants by
secreted mucin in vivo. The guinea pig gastric mucosal cells in primary
culture were shown to produce and secrete mucin (13, 21).
We examined the effect of indomethacin on the incorporation of
[3H]glucosamine into cells. As shown in Fig.
7, indomethacin inhibited [3H]glucosamine uptake in a dose-dependent manner. We
also examined the amounts of mucin in cells after indomethacin
treatment by the periodic acid-Schiff (PAS) staining method.
Treatment of gastric mucosal cells with 1 or 2.5 mM indomethacin did
not affect the pattern of PAS staining until 4 h (data not shown),
suggesting that indomethacin did not affect the retention of mucin in
cells.
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DISCUSSION |
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In this study, we have clearly shown that long- and short-term treatment with NSAIDs of gastric mucosal cells in primary culture caused cell death by apoptosis and necrosis, respectively. These findings are based on results obtained from experiments in which we examined apoptotic DNA fragmentation, PI staining, chromatin condensation, caspase activation, and the effects of inhibitors of caspase activity and protein synthesis. This study is the first to demonstrate that NSAIDs cause cell necrosis in vitro. Because necrotic cells activate neutrophils and macrophages and cause inflammation, we believe that much more attention should be paid not only to apoptosis but also to necrosis to understand NSAID-induced gastritis. As for apoptosis, our results were basically consistent with those previously reported (9, 17) using cell lines derived from gastric mucosa. Because primary culture of gastric mucosal cells is thought to effectively mimic gastric mucosal cells in vivo, our results suggest that NSAIDs may induce both necrosis and apoptosis in gastric mucosal cells in vivo.
The concentrations of indomethacin required for necrosis and apoptosis in vitro were 2.5 and 1 mM, respectively, whereas those for aspirin were 30 and 10 mM, respectively. Studies (7, 8, 18) on the oral administration of indomethacin and aspirin in rats showed that the concentrations of indomethacin and aspirin in the stomach were ~1-8 and ~10-100 mM, respectively, when these drugs caused gastric injury. Thus the in vitro concentrations of NSAIDs used in our study are physiologically significant in vivo. In other words, necrosis and apoptosis in vitro may mimic gastric mucosal injury after the single and repeated oral administration of NSAIDs in vivo. In fact, there are data (1, 7, 28) supporting the concept that indomethacin induces apoptosis in gastric mucosa in vivo. Serum concentration of NSAIDs after oral administration in rats is relatively low (7, 8, 18) compared with that required for apoptosis in vitro. Thus NSAIDs in serum may not be involved in apoptosis by NSAIDs in vivo. However, PGs, whose synthesis is inhibited by NSAIDs, have been reported (15) to be involved in cytoprotection, because PGs affect various physiological parameters such as blood flow. In other words, NSAIDs have a negative effect on the balance between aggressive and defensive factors for gastric mucosa by inhibiting the synthesis of PGs . It is therefore possible that reduction of PGs by NSAIDs increases the sensitivity of cells to NSAIDs and consequently low serum concentrations of NSAIDs induce apoptosis in vivo.
The identification of a pathway of NSAID-dependent apoptosis is important to understand the gastropathy associated with the use of NSAIDs and to establish a clinical protocol to protect gastric mucosal cells against the deleterious effects of NSAIDs. Recently (36), a novel pathway for stress-induced apoptosis was proposed, in which caspase-8 stimulates the release of cytochrome c from mitochondria through the degradation of Bid, resulting in the activation of caspase-9. Thus it is possible that apoptosis by NSAIDs is mediated by this pathway, because NSAIDs activated caspase-8 activity (Fig. 4). Because caspase-8 can be activated by caspase-3 (27), it is also possible that the activation of caspase-8 by NSAIDs is the result of caspase-3 activation by caspase-9.
Adaptation of gastric mucosal cells to NSAIDs has been reported (14, 29); after long-term NSAID exposure, gastric mucosal cells become resistant to further NSAID treatment in vivo. Recently (1), this adaptation was shown to be due to the increased resistance of gastric mucosal cells to apoptosis caused by NSAIDs. Combined with our current results, it is possible that treatment of gastric mucosal cells with NSAIDs induces proteins that inhibit the pathways of apoptosis discussed above. Heat shock proteins (HSPs) are apparent candidates for these proteins. Not only did some HSPs directly interact with apoptotic protease-activating factor-1 and cytochrome c and inhibit apoptosis (2, 3, 6, 24) but also high concentrations of NSAIDs induced HSPs in gastric mucosal cells (32). However, HSPs were not induced in gastric mucosa when the adaptation to NSAIDs occurred in vivo (1). The identification of the proteins responsible for the adaptation upon chronic NSAID treatment may be important in establishing a clinical protocol for the prevention of the gastric mucosal injury induced by NSAIDs.
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ACKNOWLEDGEMENTS |
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This work was supported by grants-in-aid for Scientific Research from the Japanese Ministry of Education, Science, Sports, and Culture and the Mochida Memorial Foundation for Medical and Pharmaceutical Research.
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FOOTNOTES |
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Address for reprint requests and other correspondence: T. Mizushima, Faculty of Pharmaceutical Sciences, Okayama Univ., Okayama 700-8530, Japan (E-mail: mizushima{at}pharm.okayama-u.ac.jp).
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 4 December 2000; accepted in final form 14 May 2001.
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