Non-steroidal anti-inflammatory drugs induce apoptosis in gastric cancer cells through up-regulation of bax and bak
Xin Min Zhou1,3,*,
Benjamin Chun Yu Wong1,4,*,
Xiao Ming Fan1,
Hong Bo Zhang3,
Marie Chia Mi Lin2,
Hsiang Fu Kung2,
Dai Ming Fan3 and
Shiu Kum Lam1
1 Department of Medicine and
2 Institute of Molecular Biology, University of Hong Kong, Hong Kong and
3 Institute of Digestive Disease, Xijing Hospital, Fourth Military Medical University, Xian, People's Republic of China
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Abstract
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Aspirin- and non-steroidal anti-inflammatory drug (NSAID)-induced apoptosis is one of the important mechanisms for their anti-tumour effect in gastric cancer. We aimed at determining the role of bcl-2 family proteins and caspases in the apoptotic process. Gastric cancer cell lines AGS (wild-type p53) and MKN-28 (mutant p53) were used. Cell proliferation was measured by MTT assay. Apoptosis was determined by acridine orange staining. Protein expressions were determined by western blotting. Aspirin and indomethacin inhibited cell proliferation and induced apoptosis in both cells. AGS cells were more sensitive compared with MKN-28 cells. The pro-apoptotic proteins bax and bak were overexpressed after treatment, while the protein level of bcl-2 remained unchanged. Apoptosis was accompanied by an increase in caspase-3 activity and cleavage of caspase-3 and poly(ADP-ribose) polymerase. Inhibition of caspase-3 rescued aspirin-induced apoptosis. Our results suggest that one of the major pathways which mediates the anti-tumour response of aspirin and indomethacin in gastric cancer cells is through up-regulation of bax and bak and activation of caspase-3. Bax and bak are important in the chemoprevention of gastric cancer.
Abbreviations: NSAIDs, non-steroidal anti-inflammatory drugs; PARP, poly(ADP-ribose) polymerase; TBS-T, 10 mM TrisHCl buffered saline; pH 7.6, plus 0.05% Tween-20.
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Introduction
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Gastric cancer is the most common malignant tumour of the gastrointestinal tract in the world. In the past 10 years aspirin and other non-steroidal anti-inflammatory drugs (NSAIDs) have received wide attention for their potential role in reducing the risk of several cancer types, including colon and stomach (15). NSAIDs, including aspirin, inhibit cell proliferation and induce apoptosis in a number of cancer cell lines in vitro, which is considered to be an important mechanism for their anti-tumour activity and prevention of carcinogenesis (68). However, the molecular pathways involved are not completely elucidated.
We have previously reported that aspirin and indomethacin induce apoptosis in gastric cancer cell lines (9). This process is regulated by differential expression of apoptosis-related genes and their proteins, such as p53, p21 and c-myc (10,11). Downstream effectors to these proteins include the bcl-2 family, cytochrome c release from the mitochondria and activation of caspases. Bcl-2 and bax proteins influence cell survival by regulating activation of key caspases (12). Activation of caspases eventually leads to the execution of apoptosis in cancer cells. One key step in this cascade is activation of caspase-3 (CPP32/YAMA/apopain), which cleaves several substrates, such as the DNA repair enzyme poly(ADP-ribose) polymerase (PARP) and DNA fragmentation factor 45, leading to the typical 180 bp DNA strand breaks observed in the course of apoptosis (13).
In this study we have investigated the role of caspases in NSAID-induced apoptosis in gastric cancer cells. We have further examined the role of bcl-2 family proteins in the regulation of caspase-3-dependent apoptosis.
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Materials and methods
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Cell line and culture
Human gastric cancer cell line AGS with wild-type p53 was purchased from the American Type Culture Collection. MKN-28 cells, a gastric adenocarcinoma cell line with mutant p53, were purchased from the RIKEN Cell Bank (The Institute of Physical and Chemical Research, Japan). The cells were cultured at 37°C in an atmosphere of 5% CO2/95% air in RPMI 1640 medium containing 10% fetal bovine serum, 100 U/ml penicillin, 100 µg/ml streptomycin (Gibco BRL, Life Technologies, Grand Island, NY) in 25 cm2 culture flasks (Corning, Corning, NY).
Reagents
Both aspirin and indomethacin (Sigma, St Louis, MO) were freshly prepared in DMSO before use. Stock preparations of the reagents were stored at 20°C. The caspase general inhibitor Z-VAD-fmk [Z-Val-Ala-Asp(Ome)-CH2F] and specific caspase-3 inhibitor Z-DEVD-fmk [Z-Asp(OCH3)-Glu (OCH3)-Val-Asp(OCH3)-FMK] (Calbiochem, San Diego, CA) were prepared in DMSO at fixed concentrations before use.
MTT assay
The effects on cell proliferation were measured by a modified MTT assay, based on the ability of live cells to utilize thiazolyl blue and convert it into dark blue formazan. In brief, 6x103 cells/well (96-well microtitre plates) were incubated with 200 µl of culture medium overnight. Then the cells were treated with 1.0 mM aspirin and 400 µM indomethacin for fixed periods of time. Twenty microliters of stock MTT solution (2.5 mg/ml) were added to each well and the cells were further incubated at 37°C for 4 h. The supernatant was removed and 100 µl of 40 mM hydrochloric acid in isopropanol was added to each well at 37°C for 2 h. The absorbency at a wavelength of 595 nm was measured with a micro-ELISA reader (Bio-Rad, Hercules, CA). The negative control wells had no cells, with culture medium only. Each assay was performed three times in triplicate.
Apoptosis assay
DNA fragmentation was analyzed as described previously (9). DNA fragments in the form of a laddering pattern were visualized by UV transillumination (data not shown). Morphological changes were determined by acridine orange staining. Single cell suspensions were fixed in 1% formalin/phosphate-buffered saline and stained with 10 µg/ml acridine orange (Sigma). A drop of the stained cell suspension was placed on a microscope slide. Cells were visualized under a fluorescence microscope with a blue-green filter. Apoptotic cells were defined as cells showing cytoplasmic and nuclear shrinkage and chromatin condensation or fragmentation. At least 300 cells were counted and the percentage of apoptotic cells was determined.
Western blotting analysis
After aspirin and indomethacin treatment the cells were extracted with lysis buffer containing protease inhibitors (20 mM TrisHCl, pH 7.4, 50 mM sodium cholride, 1% Triton X-100, 1 mM EDTA, 1 mM EGTA, 1 mM sodium vanadate, 0.2 mM phenylmethylsulfonyl fluoride, 0.5% NP-40). The protein concentration was determined by bicinchoninic acid assay with bovine serum albumin (Sigma) as the standard. Western blotting was carried out as described previously (11,14). Briefly, an equal amount of total cell lysate (40 µg) was solubilized in sample buffer and boiled for 5 min. Twenty-five microliters of this lysate were electrophoresed on a 10% SDSPAGE gel and then the proteins were transferred to polyvinylidene difluoride membranes (Millipore) using transfer buffer at 400 mA for 1 h. Non-specific binding was blocked with 10 mM TrisHCl buffered saline, pH 7.6, plus 0.05% Tween-20 (TBS-T) containing 5% skimmed milk powder for 1 h at room temperature. Membranes were incubated with primary antibody overnight at 4°C. The primary antibodies used included: a polyclonal rabbit anti-caspase-3 antibody (1:1000) (PharMingen, San Diego, CA); a purified mouse anti-human PARP monoclonal antibody (1:1000) (PharMingen); a monoclonal mouse anti-human bcl-2 antibody (1:100) (Santa Cruz Biotechnology, Santa Cruz, CA); a polyclonal rabbit anti-bcl-xL antibody (1:100) (Santa Cruz); a monoclonal mouse anti-bax antibody (1:500) (Santa Cruz); a polyclonal rabbit anti-bak antibody (1:200) (Santa Cruz). After washing three times with TBS-T solution and incubation with horseradish peroxidase-conjugated secondary antibody (1:500010 000 dilution) for 1 h at room temperature, bands were visualized with the enhanced chemiluminescence system (Amersham, Little Chalfont, UK).
Caspase-3 activity assays
The catalytic activity of caspase-3 was measured using a colorimetric assay according to the manufacturer's instructions (Calbiochem), which is based on spectrophotometric detection of the chromophore p-nitroanilide after cleavage from the labeled substrate DEVD p-nitroanilide. Briefly, after aspirin (1.0 mM) and indomethacin (400 µM) treatment cells were washed twice with ice-cold phosphate-buffered saline and lysed in extraction buffer, containing 50 mM HEPES, 1 mM DTT, 0.1 mM EDTA, 10% glycerol, 0.1% CHAPS, pH 7.4. After centrifugation at 12 000 g at 4°C for 10 min the supernatant (cytosol) was stored at 70°C. The assays were performed in 96-well microtiter plates. Proteolytic reactions were carried out in assay buffer (50 mM HEPES, pH 7.4, 100 mM NaCl, 0.1% CHAPS, 10 mM dithiothreitol, 0.1 mM EDTA, 10% glycerol) containing 20 µg cytosolic protein extract and incubated at 37°C for 10 min. Thereafter, freshly prepared colorimetric substrate was added to the mixtures. The samples were mixed and recorded every 30 min for a total of 4 h at 37°C. Cells without drug treatment were used as controls. Enzyme activity was calculated as pmol/min according to the formula provided by the manufacturer.
Treatment with a general caspase inhibitor and a specific caspase-3 inhibitor
AGS cells were seeded in 6-well plates and incubated for 12 h at 37°C in 5% CO2. Then the cells were pretreated with Z-VAD-fmk (100 µM) and Z-DEVD-fmk (100 µM) for 2 h before addition of aspirin and indomethacin. The cells were evaluated morphologically by acridine orange staining under a fluorescence microscope. At least 300 cells were counted and the percentage of apoptotic cells was determined.
Experimental design and statistical analysis
All experiments were performed in triplicate and were repeated at least three times. Representative experiments or mean values ± SD are shown. Statistical differences were determined by Student's t-test. A P value of <0.05 was considered significant.
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Results
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Aspirin and indomethacin inhibit proliferation of gastric cancer cells
To evaluate the effects of aspirin and indomethacin on the growth of gastric cancer cells, aspirin (1.0 mM) and indomethacin (400 µM) were added to the culture medium for 0, 12, 24, 36, 48 and 60 h. Cell growth was determined by MTT assay. Both aspirin and indomethacin inhibited gastric cancer cell growth in a time-dependent manner (Figure 1
). AGS cells were more sensitive to aspirin and indomethacin growth inhibition compared with MKN-28 cells.

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Fig. 1. Timeresponse characteristics of aspirin and indomethacin effects on proliferation of AGS (A) and MKN-28 (B) cells. The cells were treated with aspirin (1.0 mM) and indomethacin (400 µM) for 60 h. The anti-proliferative effects were measured by MTT assay. The values are expressed as means ± SEM from three independent experiments.
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Aspirin and indomethacin induce apoptosis in AGS and MKN-28 cells
There are several methods to evaluate apoptosis. In this study we used DNA laddering and acridine orange staining to check for apoptosis of AGS and MKN-28 cells after treatment with aspirin and indomethacin. Both aspirin and indomethacin induced apoptosis in AGS and MKN-28 cells, which was characterized by cytoplasmic and nuclear shrinkage, chromatin condensation and fragmentation (Figure 2
).

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Fig. 2. Fluorescence micrographs of AGS (top) and MKN-28 (bottom) cells treated with indomethacin. Cells were treated with 400 µM indomethacin for 24 h. The cells were stained with acridine orange and examined by fluorescence microscopy. (A) Control; (B) treated with indomethacin. Original magnification 400x. Arrow, apoptotic cells.
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Effects of aspirin and indomethacin on the levels of bcl-2, bcl-xL, bax and bak expression in AGS and MKN-28 cells
To further elucidate the mechanisms of aspirin- and indomethacin-induced apoptosis in AGS cells, we evaluated the involvement of bcl-2 family proteins in the apoptotic process by western blot analysis. The bax protein level was increased 6 h after both aspirin and indomethacin treatment and remained elevated up to 48 h (Figure 3
). Similarly, the protein level of bak was also increased 6 h after both aspirin and indomethacin treatment and gradually increased up to a maximum at 48 h. No change was observed in protein expression of bcl-2. The level of bcl-xL protein was undetectable. Similar findings were observed in MKN-28 cells treated with aspirin and indomethacin (Figure 2
).

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Fig. 3. Effect of aspirin and indomethacin on expression of bcl-2 family proteins in (A) AGS and (B) MKN-28 cells. Western blot analysis revealed that aspirin and indomethacin up-regulated bax and bak expression but did not change expression of bcl-2. The figure is representative of three different experiments.
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Effects of aspirin and indomethacin on caspase-3 activity in AGS cells
To assess the role of caspase-3 in apoptosis, we first examined the activities of caspase-3 in AGS cells treated with aspirin and indomethacin. After treatment with 1.0 mM aspirin or 400 µM indomethacin caspase-3 activity started to increase 12 h after treatment and was 5-fold higher than that of control cells 36 h after indomethacin treatment (Figure 4
). The increase in caspase-3 activity was more profound in indomethacin- than aspirin-treated cells.

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Fig. 4. Effect of aspirin and indomethacin on caspase-3 activity of AGS cells. Caspase-3 activity started to increase after 12 h treatment and reached 5-fold of control cells after 36 h indomethacin treatment. All experiments were performed in triplicate.
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Effects of aspirin and indomethacin on caspase-3 and PARP expression and cleavage in AGS cells
The expression of caspase-3 and PARP in AGS cells treated with aspirin and indomethacin were determined by western blotting analysis. As shown in Figure 5
, the 32 kDa proenzyme caspase-3 was cleaved to its active 17 kDa form 12 h after aspirin and indomethacin treatment. The 116 kDa PARP protein was the predominant form in AGS cells during the early treatment phase. The 85 kDa cleavage fragment began to appear 24 h after drug treatment. The cleavage of PARP and caspase-3 were both dose- and time-dependent.

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Fig. 5. Western blot analysis of the effect of aspirin and indomethacin on expression of caspase-3 and PARP in AGS cells. Specific cleavage of caspase-3 and PARP was observed 12 (casapase-3) and 24 h (PARP) after treatment with aspirin and indomethacin. The uncleaved and cleaved proteins are indicated by arrows and sizes of molecular weight markers are shown on the right. The figure is representative of three different experiments.
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Effects of caspase inhibitors on apoptosis of AGS cells
The role of caspase-3 in aspirin- and indomethacin-induced apoptosis in AGS cells was further examined using the general caspase inhibitor Z-VAD-fmk and the specific caspase-3 inhibitor Z-DEVD-fmk. Both tetrapeptides correspond to the sequence at the cleavage site of PARP. During proteolysis Z-VAD-fmk and Z-DEVD-fmk bind irreversibly to caspase-3, thereby inactivating the enzyme. As shown in Figure 6
, 100 µM Z-DEVD-fmk markedly suppressed the apoptotic effects induced by aspirin and indomethacin. The general caspase inhibitor Z-VAD-fmk (100 µM) also significantly suppressed aspirin- and indomethacin-induced apoptosis.

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Fig. 6. The effect of a general caspase inhibitor (z-VAD-fmk) and a specific caspase-3 inhibitor (z-DEVD-fmk) on the extent of apoptosis of AGS cells after treatment with aspirin and indomethacin. AGS cells were exposed to aspirin (1.0 mM) and indomethacin (400 µM) for 24 h. The percentage of apoptotic cells was evaluated by acridine orange staining. (A) Control; (B) Z-VAD-fmk plus aspirin; (C) Z-DEVD-fmk plus aspirin; (D) aspirin only; (E) Z-VAD-fmk plus indomethacin; (F) Z-DEVD-fmk plus indomethacin; (G) indomethacin only. The inhibitors can reverse apoptosis induced by aspirin [(B) and (C) versus (D), P < 0.01] and indomethacin [(E) and (F) versus (G), P < 0.01]. The data represent the means ± SEM of three experiments.
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Discussion
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This study has shown that both aspirin and indomethacin inhibit cell proliferation and induce apoptosis in AGS and MKN-28 gastric cancer cells. AGS cells (wild-type p53) were more sensitive to aspirin and indomethacin compared with MKN-28 cells (mutant type p53). We confirmed that the induction of apoptosis was mediated through bax and bak overexpression, without any change in bcl-2 expression. Increases in caspase-3 activity and cleavage of caspase-3 and PARP were demonstrated. The role of caspase-3 was further confirmed by reversal of apoptosis by a specific caspase-3 inhibitor.
Current evidence indicates that caspases, which are cysteine proteases of the ICE/CED-3 family, are central components of the cell death machinery in various forms of apoptosis (1517). Of these, caspase-3 is the most likely candidate for a mammalian cell death regulator by cleaving as yet unidentified vital cellular proteins. One important substrate is PARP, a nuclear enzyme involved in DNA repair and maintenance of genome integrity and post-translational ribosylation of proteins (18). PARP is specifically cleaved during apoptosis in several model systems (19). In this study we found that the inactive caspase-3 precursor was specifically cleaved to the 17 kDa subunit forming the active protease during aspirin- and indomethacin-induced apoptosis. This occurred in conjunction with specific cleavage of 116 kDa PARP to a 85 kDa proteolytic fragment. The results are consistent with reports in different model systems (13,19,20). Together with the data for caspase-3 inhibitors, it is evident that caspase-3 is the main effector during apoptosis induced by aspirin and indomethacin in gastric cancer cells.
The resistance of cancer cells to chemotherapy-induced apoptosis remains one of the most significant problems in the treatment of cancer, including gastric cancer (21). Resistance may result from a variety of changes that affect the apoptotic pathway, including mutations in the p53 gene, overexpression of bcl-2 and/or bcl-xL or down-regulation or mutations of bax or bak (22,23). Alteration of bcl-2 family proteins is complex but important in gastric cancer. Of gastric cancers, 72% overexpress bcl-2. Inhibition of apoptosis through bcl-2 protein expression appears to be specifically associated with promotion of intestinal-type gastric adenocarcinoma (24). bax frameshift mutations were found in 3340% of gastric cancers with high level microsatellite instability (25,26). Missense Bak gene mutations were also observed in 12.5% of gastric cancers and sequence alterations without amino acid alteration were observed in 4.2% of gastric cancers (27). In gastric cancer cell lines bcl-2 overexpression strongly blocks apoptosis by inhibiting cytochrome c release from mitochondria and caspase-3 activation. On the other hand, bax overexpression sensitizes cells to chemotherapeutic agent-induced apoptosis by enhancing release of cytochrome c from mitochondria ( 28,29). Several potential chemopreventive agents also exert their effect through bax and bak. Sodium butyrate triggers growth arrest and apoptosis in the human gastric cancer SIIA potentially through induction of the pro-apoptotic genes Bax, Bak and Bik (30). Our group has previously reported that induction of apoptosis using protein kinase C inhibitors is also via induction of bax (10).
In this study our data indicate that overexpression of bax and bak is closely involved in apoptosis induced by aspirin and indomethacin, without altering bcl-2 and bcl-xL expression. The increased bax and bak expression was detected just prior to commencement of cell apoptosis, after ~6 h treatment. Therefore, our observation of increased bax and bak expression after NSAID treatment confirms the unique role of these proteins in NSAID-induced apoptosis in gastric cancer. The dose range of aspirin and indomethacin used in the study was higher than those required to inhibit cyclooxygenase enzyme activity. This is observed in various studies, including our previous data, suggesting that a higher dose is required to activate cyclooxygenase-independent pathways (31).
Some studies have shown that bax translocates from a predominantly cytoplasmic location to the mitochondria upon induction of apoptosis (32). Overexpression of bax induces cytochrome c release in vivo and in vitro (33). Cytochrome c released into the cytosol forms a complex with another molecule, Apaf-1, and the unprocessed proform of caspase-9 (34,35). In the presence of dATP or ATP this complex activates the caspases, which in turn can trigger a cascade by activating other caspases (in particular, caspases-3, -6, and -7) (35,36).
In summary, we have shown that aspirin and NSAID induce apoptosis in gastric cancer through up-regulation of bax and bak and activation of caspase-3. Consistent with other potential chemopreventive agents in the gastric cancer model, we believe that bax and bak play a pivotal role in the process of apoptosis. These serve as potential targets for future drug or therapeutic developments for prevention and treatment of gastric cancer.
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Notes
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4 To whom correspondence should be addressed Email: bcywong{at}hku.hk 
* The first two authors contributed equally to this work 
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Acknowledgments
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This study was supported by a grant from the Research Grant Council of the Hong Kong Special Administrative Region, China (HKU 7010/99M) to B.C.Y.W.
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Received January 31, 2001;
revised June 12, 2001;
accepted June 19, 2001.