Activation of the caspase-8/Bid and Bax pathways in aspirin-induced apoptosis in gastric cancer
Qing Gu1,2,
Ji De Wang2,
Harry H.X. Xia2,
Marie C.M. Lin3,
Hua He2,
Bing Zou2,
Shui Ping Tu2,
Yi Yang2,
Xin Guang Liu1,
Shiu Kum Lam2,
Wai Man Wong2,
Annie O.O. Chan2,
Man Fung Yuen2,
Hsiang Fu Kung3 and
Benjamin Chun-Yu Wong2,4
1 Department of Gastroenterology, First Hospital, Peking University, Beijing, People's Republic of China, 2 Department of Medicine, University of Hong Kong, Queen Mary Hospital, Hong Kong and 3 Institute of Molecular Biology, University of Hong Kong, Hong Kong
4 To whom correspondence should be addressed. Tel: +86 852 2855 4541; Fax: +86 852 2872 5828; Email: bcywong{at}hku.hk
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Abstract
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Aspirin-induced apoptosis is one of the important mechanisms for its antitumour effect against gastric cancer. We aimed at investigating the involvement of bcl-2 family members in the apoptotic pathway in gastric cancer. Gastric cancer cell line AGS and MKN-45 were observed as to cell growth inhibition and induction of apoptosis in response to treatment with aspirin. Cell proliferation was measured by MTT assay. Apoptosis was determined by 4'-6-diamidino-2-phenylindole staining. Protein expression was determined by western blotting. We showed that aspirin activated caspase-8, caspase-9 and capase-3, cleaved and translocated Bid, induced a conformational change in and translocation of Bax and cytochrome c release. In addition, suppression of caspase-8 with the specific inhibitor z-IETD-fmk, as well as the pan-caspase inhibitor z-VAD-fmk, prevented Bid cleavage and subsequent apoptosis. The caspase inhibitors failed to abolish the effects on Bax activation. In conclusion, our results identify a role of caspase-8/Bid and activation of Bax as a novel mechanism for aspirin-induced apoptosis in gastric cancer.
Abbreviations: DAPI, 4'-6-diamidino-2-phenylindole; GFP, green fluorescent protein; PARP, poly(ADP-ribose) polymerase; PBS, phosphate-buffered saline; tBid, truncated Bid; TNF, tumor necrosis factor
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Introduction
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Apoptosis is mediated through two major pathways, the death receptor pathway (extrinsic) and the mitochondrial pathway (intrinsic). In the extrinsic pathway, stimulation of death receptors such as Fas and TNFR1 leads to clustering and formation of a death-inducing signaling complex which includes the adapter protein FADD (Fas-associated death domain) and initiator caspases such as caspase-8. Caspase-8 drives its activation through self-cleavage and then activates downstream effector caspases such as caspase-9 and caspase-3 (1). In the intrinsic pathway death receptors transmit the death signals to mitochondria, resulting in the release of several mitochondrial intermembrane space proteins, such as cytochrome c, which associate with Apaf-1 and procaspase-9 to form the apoptosome (2).
The mitochondrial pathway is controlled and regulated by the Bcl-2 family proteins. The anti-apoptotic subfamily comprises Bcl-2 and Bcl-xL. The multidomain pro-apoptotic subfamily consists of Bax and Bak and the BH3 domain-only proteins include Bad and Bim (3). Pro- and anti-apoptotic Bcl-2 family members converge on mitochondria in response to the death signal and compete to regulate the release of cytochrome c (2). The pro-apoptotic proteins Bax and Bak are required for induction of apoptosis by the mitochondrial pathway. Bax is localized in the cytosol of normal cells and translocated to mitochondria after apoptotic stimulation (3,4). Bax-dependent Smac and Omi release plays a pivotal role in thapsigargin-induced apoptosis of human colon cancer (5). Most of the BH3 domain-only proteins are localized outside the mitochondria in living cells and translocate to mitochondria by several different mechanisms after apoptotic stimulation, leading to activation of pro-apoptotic proteins such as Bax and Bak (3). However, It is not clear how the BH3 domain-only proteins (e.g. Bid) transmit the apoptotic signal to the pro-apoptotic family such as Bax and Bak.
Aspirin has potent analgesic, anti-pyretic and anti-inflammatory actions and reduces the risk of gastric and colon cancer (6,7). Some studies have reported that aspirin induces apoptosis with release of cytochrome c from mitochondria (811). However, others suggested that aspirin induces cell cycle arrest and causes necrosis at high concentrations in vitro but does not induce apoptosis (12). Therefore, whether or not aspirin induces apoptosis and the molecular mechanisms involved are not clear.
In the present study we have confirmed that aspirin induces apoptosis in gastric cancer cells and demonstrated that caspase-8/Bid and a conformational change in and translocation of Bax are involved in aspirin-induced apoptosis.
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Materials and methods
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Chemicals
Aspirin and Anti-Bax 6A7 monoclonal antibody were purchased from Sigma (St Louis, MO). Anti-Bax (N-20) polyclonal antibody, anti-caspase3, anti-PARP, anti-cytochrome c were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anticaspase-8 monoclonal antibody and caspase-8 inhibitor (z-IETD-fmk) were purchased from Alexis (Lausen, Switzerland). Anticaspase-9 polyclonal antibody was purchased from Cell Signaling Technology (Beverly, MA). Pan-caspase inhibitor (z-VAD-fmk) and caspase-3 inhibitor (z-DEVD-fmk) were purchased from Calbiochem (San Diego, CA).
Cell culture
Gastric cancer cell lines AGS and MKN-45 were purchased from the American Type Culture Collection (Rockville, MD) and were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum and antibiotics, maintained at 37°C in a humidified atmosphere including 5% CO2.
MTT assay
About 6000 cells/well were grown in 96-well microtiter plates and incubated overnight in 100 µl of culture medium. Cells were then treated with different concentrations of aspirin for fixed time intervals. Ten microliters of MTT (Sigma, St Louis, MO) labeling reagent (final concentration 0.5 mg/ml) was added to each well and the cells were incubated for another 4 h at 37°C. The supernatant was removed and 100 µl of 0.04 mol/l hydrochloric acid in isopropanol was added to each well. A micro ELISA reader (Bio-Rad, Hercules, CA) measured the absorbency at a wavelength of 595 nm.
Apoptosis assay
Cells were seeded in 12-well plates at a density of 5 x 104 cells/well for 24 h and treated with chemicals for 24 or 48 h. Then the cells were washed with ice-cold phosphate-buffered saline (PBS) and stained with 4% PBS containing 10 µg/ml 4'-6-diamidino-2-phenylindole (DAPI). The morphology of the cell was observed under a fluorescence microscope (Olympus, Japan). Apoptotic nuclei can be identified by condensed chromatin gathering at the periphery of the nuclear membrane or totally fragmented nuclear bodies. More than 150 cells/field were counted and the percentage of apoptotic nuclei was determined.
Immunoprecipitation of active Bax
The detection of a conformational change of Bax was performed as described previously (13). In brief, cells were lysed in CHAPS lysis buffer (150 mM Nacl, 10 mM HEPES, pH 7.4, 1% CHAPS) containing protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 10 µg/ml pepstatin A, 10 µg/ml aprotonin). An aliquot of 500 µg total protein was incubated with 2 µg of anti-Bax 6A7 antibody for 2 h at 4°C, followed by 25 µl of protein Gagarose (Roche) for an additional 2 h to precipitate the conformationally changed Bax protein. After extensive washing, the beads were resuspended in Laemmli sample buffer by boiling for 5 min and the eluted proteins were subjected to SDSPAGE immunoblot analysis with anti-Bax N20 polyclonal antibody.
Subcellular fractionation
Mitochondria extraction was as described (14). In brief, cells were lysed in isotonic mitochondria buffer (210 mM mannitol, 70 mM sucrose, 10 mM HEPES, pH 7.4, 1 mM EDTA) containing protease inhibitors. Cells were homogenized with a Dounce homogenizer and centrifuged at 1000 g for 10 min to discard nuclei and unbroken cells. The resulting supernatant was centrifuged at 13 000 g for 15 min to pellet the mitochondria-enriched heavy membrane fraction.
Plasmid construction and immunofluorescence analysis
Human Bax cDNA from AGS cells was isolated by RT-PCR and cloned in the EcoRI and SalI sites of the pEGFP vector (BD Biosciences, Clontech, San Jose, CA). Cells were grown on coverslips, transfected with vector alone or with pEGFP-bax plasmid for 24 h and treated with or without 2.0 mM aspirin for an additional 24 h. After incubation with 25 nM Mito Tracker CMTMRos (Molecular Probes, Eugene, OR) for 30 min cells were fixed with 3.7% formaldehyde. The coverslips were mounted with DAPI-containing mount medium (Vectashield; Burlingame, CA) and analyzed by fluorescence microscopy (Olympus, Tokyo, Japan).
Caspase-3 activity assay
The ApoAlert caspase colorimetric assay kits (Clontech, Palo Alto, CA) was used to detect caspase activation. In brief, cells were cultured in 60 mm dishes and treated with 2.0 mM aspirin for the indicated periods. Cell lysates were prepared in the lysis buffer from the assay kit, normalized for the protein content by BCA assay and incubated with reaction buffer and caspase-3 substrate at 37°C for 1 h. Samples were read at 405 nm by a spectra Max 340 (Molecular Devices, Sunnyvale, CA).
Western blotting
Cells were washed with cold PBS and suspended in the lysis buffer supplied with the protease inhibitors. An aliquot of 30 µg protein was subjected to SDSPAGE and transferred to a PVDF membrane (Millipore, Billerica, MA). After blocking with 5% non-fat milk, the membrane was immunodetected with anti-Bax (1:400), anti-PARP (1:300), anti-bid (1:1000), anti-cytochrome c (1:200), anti-caspase-8 (1:400), anti-caspase-9 (1:500), anti-caspase-3 (1:300) and anti-ß-actin primary antibodies followed by secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA). Immunodetection was performed by the enhanced chemiluminescence assay (Amersham Pharmacia Biotech) following the manufacturer's instructions.
Statistical analysis
The results for determination of proliferation, apoptosis and activity of caspase-3 are expressed as means ± SD. Student's t-test was used to determine the significance of the differences. A P value of <0.05 was considered statistically significant.
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Results
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Caspase inhibitors suppress aspirin-induced apoptosis
As previously described, aspirin induces apoptosis and has anti-proliferative effects in gastric cancer cells (15,16), which was confirmed with AGS and MKN-45 cells in the present study (Figure 1A and B). To demonstrate the involvement of caspases in aspirin-induced apoptosis, we used a pan-caspase inhibitor (z-VAD-fmk), a specific inhibitor for caspase-3 (z-DEVD-fmk) and a specific inhibitor of caspase-8 (z-IETD-fmk). AGS cells were treated with 2 mM aspirin for 48 h in the presence or absence of caspase inhibitors. As shown in Figure 1C, the percentage of apoptotic cells were reduced significantly by all caspase inhibitors, indicating that caspase-3 and caspase-8 were important in aspirin-induced apoptosis.

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Fig. 1. Aspirin-induced apoptosis in gastric cancer cells. (A) AGS and MKN-45 cells were treated with 2 mM aspirin for 13 days. AGS cells were exposed to aspirin at various doses. The anti-proliferative effect was measured by MTT assay. The values are expressed as means ± SD from three independent experiments. (B) AGS and MKN-45 cells were treated with 2 mM aspirin for 24 and 48 h. The percentages of apoptotic cells were significantly increased compared with the control. *P < 0.05. (C) AGS cells were treated with 2 mM aspirin with or without pretreatment with 40 µM z-IETD-fmk (a specific caspase-8 inhibitor), 50 µM z-VAD-fmk (a pancaspase inhibitor) and 50 µM z-DEVD-fmk (a specific caspase-3 inhibitor). *P < 0.05 versus aspirin alone. Apoptotic cells were quantified by DAPI staining. The values are expressed as means ± SD from three independent experiments.
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Effect of aspirin on activation of caspase-3 and cleavage of poly(ADP-ribose) polymerase (PARP)
It is known that caspase-3 and PARP are the hallmarks of apoptosis (17,18). To confirm the involvement of caspase-3 in aspirin-induced apoptosis, we determined the activity of caspase-3. AGS cells were treated with or without 2 mM aspirin for 24 or 48 h. As seen in Figure 2A, the activity of caspase-3 was increased at 24 and 48 h. Expression of caspase-3 and PARP were also detected by western blot analysis. As shown in Figure 2B, aspirin induced cleavage of 32 kDa procaspase-3 into the 17 and 11 kDa subunits. Similarly, the 85 kDa active cleaved unit of PARP was detected after treatment with 2 mM aspirin for 24 and 48 h. Furthermore, after treatment with z-IETD-fmk, z-VAD-fmk or z-DEVD-fmk cleavage of PARP was completely blocked (Figure 2C).

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Fig. 2. Effect of aspirin on caspase-3 and PARP in gastric cancer cells. (A) AGS cells were treated with 2.0 mM aspirin in the presence or absence of caspase-3 inhibitor (z-DEVD-fmk) for 24 or 48 h. Caspase-3 activity was measured. Results are means ± SD of triplicate assays and two experiments. *P < 0.05. (B) AGS cells were treated with 2 mM aspirin for 24 or 48 h. Protein expression was examined by western blot analysis. (C) AGS cells were exposed to 2 mM aspirin with or without capase-8 inhibitor (z-IETD-fmk) or pancaspase inhibitor (z-VAD-fmk) for 48 h. Data are representative of three different experiments.
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Bax conformational change and translocation is involved in apoptosis induced by aspirin
It has been shown that apoptotic stimuli trigger a Bax conformational change and translocation to the mitochondria (4,5,19,20). To determine whether Bax is involved in the apoptosis induced by aspirin we performed immunoprecipitation experiments with anti-Bax 6A7 antibody, which only recognizes the conformationally changed Bax protein. AGS cells were treated with 2.0 mM aspirin and assayed for Bax at different time points. As shown in Figure 3A, the Bax conformational change could be detected at 12 h, and increased time-dependently to 48 h. The conformational change of Bax was also detected in MKN-45 cells (data were not shown). Mitochondria fractionation and western blot analysis showed increased Bax expression at 48 h (Figure 3B). The translocation of Bax was confirmed by transient transfection with a green fluorescent protein (GFP)Bax conjugate (Figure 3C). In normal cells GFPBax has a diffuse distribution in the cytosol. After treatment with aspirin, GFPbax translocated to the mitochondria in apoptotic cells, indicating that aspirin induced a conformational change of bax and translocation from the cytosol to the mitochondria during apoptosis.

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Fig. 3. Aspirin-triggered Bax conformational change and translocation. AGS cells were treated with 2 mM aspirin for up to 48 h. (A) The Bax conformational change was detected by western blot with anti-6A7 antibody. (B) Mitochondrial translocation of Bax was detected by subcellular fractionation and western blot analysis with anti-Bax antibody. (C) AGS cells were transfected with pEGFP-Bax (green) and treated with or without 2 mM aspirin. Nuclei and mitochondria were stained with DAPI (blue) and MitoTracker (red), respectively. Co-localization of green and red fluorescence was indicated by yellow coloration. Two independent experiments were performed, with the same results. Bar 5 µm.
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Effect of aspirin on activation of cytochrome c and caspase-9
Cytochrome c is an important apoptogenic factor in the intrinsic apoptotic pathway, which is released into the cytoplasm and together with caspase-9 forms part of the apoptosome (2,3). To confirm that the apoptosis induced by aspirin involves the mitochondrial pathway, we treated AGS and MKN-45 cells with 2.0 mM aspirin. Aspirin induced cytochrome c release from the mitochondria into the cytosol (Figure 4A) and procaspase-9 was cleaved to the 35 or 37 kDa subunit (Figure 4B), suggesting that the mitochondrial pathway is involved in apoptosis induced by aspirin.

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Fig. 4. Effect of aspirin on cytochrome c and caspase-9. AGS and MKN-45 cells were treated with 2 mM aspirin for the indicated times. (A) Translocation of cytochrome c was determined by cytosolic extraction and western blot analysis. (B) Caspase-9 processing was assessed by western blot analysis. The results are representatives of three separate experiments. Similar results for MKN-45 cells are not shown.
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Effect of aspirin on activation of caspase-8 and truncated Bid (tBid)
It has been reported that Bid is a substrate of caspase-8 and that tBid may translocate to the mitochondria and induce cytochrome c release (21,22). Aspirin also induces caspase-dependent phosphatidylserine externalization (11). Therefore, we determined activation of caspase-8 and Bid in aspirin-induced apoptosis. As shown in Figure 5A, aspirin induced procaspase-8 cleavage into 43 and 41 kDa subunits and Bid cleavage at 24 h in AGS cells. The cleavage of caspase-8 was detected in MKN-45 cells as well (data not shown). In addition, translocation of tBid to the mitochondria was also shown. Thus, the results suggest that aspirin possibily induces apoptosis through caspase-8 and Bid activation. tBid formation was inhibited when AGS cells were treated with z-IETD and z-VAD (Figure 5B), which confirms that caspase-8 is the initiator caspase in aspirin-induced apoptosis.

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Fig. 5. Aspirin-induced caspase-8 cleavage and Bid activation and effect of caspase inhibitors on conformational change of Bax. AGS cells were treated with 2 mM aspirin for up to 48 h. (A) Expression of caspase-8, Bid and tBid were determined by western blot analysis. (B) AGS cells were pretreated with 40 µM capase 8 inhibitor (z-IETD-fmk) or 50 µM pan-caspase inhibitor (z-VAD-fmk), followed by 2 mM aspirin for 48 h. (C) AGS cells were exposed to 2 mM aspirin for 48 h with or without pretreatment with caspase inhibitors. The conformational change of Bax was determined by immunoprecipitation. The western blot results are representative of at least three independent experiments.
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Effect of caspase-8 and Bid on Bax
It has been shown that Bid is the substrate of caspase-8 and that tBid can trigger Bax and Bak activation (23,24). In some studies caspase-8 is the initiator caspase responsible for THG-induced Bax activation and apoptosis in HCT116 cells (5). To examine the effect of caspase-8 and Bid on Bax in aspirin-induced apoptosis, AGS cells were exposed to 2 mM aspirin in the presence or absence of 50 µM z-IETD-fmk or z-VAD-fmk and subjected to western blot analysis with anti-caspase-8 or anti-Bid antibody. As shown in Figure 5C, z-VAD-fmk blocked the conformational change of Bax, however, z-IETD-fmk had little inhibitory effect, suggesting that activation of Bax may be independent of activation of caspase-8.
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Discussion
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It has been reported that non-steroidal anti-inflammatory drugs induce apoptosis in many cells and in response to different stimuli. Proposed mechanisms for the pro-apoptotic effect include activation of caspases (25), induction of cytochrome c release (9), regulation of protein kinase C isoform expression (26), inhibition of NF
B (27,28) and suppression of AP-1 (29). Consistent with a previous study, aspirin induced apoptosis in gastric cancer cells. We have shown that the pan-caspase inhibitor z-VAD-fmk, the caspase-8 inhibitor z-IETD-fmk and the caspase-3 inhibitor z-DEVD-fmk inhibit aspirin-induced apoptosis. Caspase-8 is one major enzyme activating caspase-3. Both TRAIL and tumor necrosis factor (TNF)-
are known to bind to their cell surface receptors, leading to caspase-8 activation. Activated caspase-8 amplifies the apoptotic signal either by directly activating downstream caspases or by cleaving BH3 domain-only proteins such as Bid (30). In the current study suppression of caspase-8 abrogated Bid and PARP cleavage and subsequent apoptosis, suggesting that caspase-8 is required for aspirin-induced apoptosis and that the death receptor pathway (extrinsic) may be one possible mechanism for the pro-apoptotic effects of aspirin.
Bid relays the Fas/TNF apoptotic signal to the mitochondrial pathway (31) and is a specific substrate of caspase-8 in the Fas signaling pathway. Once Bid is cleaved by caspase-8, tBid translocates to the mitochondria from the cytosol to induce mitochondrial damage, cell shrinkage, nuclear condensation and release of cytochrome c independent of caspase activity (21). In addition, tBid is able to activate Bax to form Bax multimers in the mitochondria (32), thereby inducing release of cytochrome c. In the present study we have shown that caspase-8 triggers Bid cleavage in aspirin-induced apoptosis. Furthermore, after treatment with z-IETD-fmk, tBid formation was completely blocked, suggesting that Bid was the substrate of caspase-8 during apoptosis induced by aspirin in AGS cells.
Bax is the other cytosolic protein that can mediate caspase-8-induced mitochondrial changes. In response to apoptotic signals or interaction with pro-apoptotic proteins, Bax undergoes a conformational change whereby it integrates into the mitochondrial membrane inducing the release of cytochrome c (20,33). In addition to Bid, Bax lacks the ability to translocate to the mitochondria and the apoptotic activity depends on mutation or deletion of amino acids in the Bax C-terminal tail (20,34). Bax-deficient HCT116 cells were completely resistant to TRAIL regardless of oxygen content, demonstrating a pivotal role of Bax in TRAIL-induced apoptotic signaling (35). Moreover, movement of Bax from the cytosol to the mitochondria is very important for the death-promoting activity of Bax and GFPBax, as well as for the conformational change of Bax (4,19). In the present study we have demonstrated that aspirin activates Bax by producing a conformational change and translocation from the cytosol to the mitochondria, subsequently inducing release of cytochrome c and activation of caspase-9, suggesting that the mitochondrial pathway may be a potential mechanism for aspirin-induced apoptosis. On the other hand, pretreatment with caspase inhibitors, especially z-IETD-fmk, attenuated the conformational change of Bax but did not completely block it, indicating that Bax may not be regulated by the caspase-8/Bid pathway, supporting possible involvement of the mitochondrial pathway in aspirin-induced apoptosis. Nevertheless, the mechanism by which Bax induces mitochondrial damage and whether the activation of Bax is required for aspirin-induced apoptosis still need to be elucidated.
In conclusion, our results demonstrate that aspirin-induced apoptosis in gastric cancer is coupled to a Bax conformational change and Bax translocation to the mitochondria, as well as activation of caspase-8/Bid and caspase-3. Therefore, cross-talk between the caspase-8/Bid pathway and the mitochondrial pathway may exist in aspirin-induced apoptosis.
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Received July 23, 2004;
revised November 11, 2004;
accepted November 21, 2004.