- Email: [email protected]
Tumor Necrosis Factor-Related Apoptosis Inducing Ligand (TRAIL)-Induced Apoptosis is Dependent on Activation of Cysteine and Serine Proteases
doi:10.1006/cyto.2001.0893, available online at http://www.idealibrary.com on
SHORT COMMUNICATION
TUMOR NECROSIS FACTOR-RELATED APOPTOSIS INDUCING LIGAND (TRAIL)-INDUCED APOPTOSIS IS DEPENDENT ON ACTIVATION OF CYSTEINE AND SERINE PROTEASES In-Chul Park, Myung-Jin Park, Sang-Hyeok Woo, Kyung-Hee Lee, Seung-Hoon Lee, Chang-Hun Rhee, Seok-Il Hong We examined the role of caspases and serine protease(s) in cell death induced by tumour necrosis factor-related apoptosis-inducing ligand (TRAIL). After incubation of adenocarcinoma cells with TRAIL, caspase-3, -8 were activated and the cleavage of Bid induced the release of cytochrome c, from the mitochondria to the cytosol. Tetrapeptide inhibitors of caspase-1, -2, -3, and -8 suppressed DNA fragmentation and attenuated the release of cytochrome c, whereas inhibitors of caspase-5 did not. Interestingly, the general serine protease(s) inhibitor 4-(2aminoethyl)benzylsulfonyl fluoride (AEBSF) resulted in the arrest of apoptosis. However, the AEBSF did not prevent the release of mitochondrial cytochrome c during TRAIL-induced apoptosis. From these results, we postulate that serine protease(s) may be involved in post-mitochondrial apoptotic events, that lead to the activation of the initiator, caspase-9. 2001 Academic Press
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is an apoptosis-inducing member of the tumor necrosis factor (TNF) gene family.1,2 TNF family members transduce the apoptotic signal, by engaging death receptors that belong to a subfamily of the TNF receptor gene superfamily, and include TNFR1, CD95, and death receptor (DR).3–6 The mechanism of Fas-induced apoptosis is fairly well understood, whereas the mechanism of TRAILinduced apoptosis remains to be clarified. In Fasinduced apoptosis, activated caspase-8 cleaves Bid protein, which is subsequently translocated to mitochondria where it can induce mitochondrial dysfunction, such as loss of membrane potential and
From the Laboratory of Cell Biology, Korea Cancer Center Hospital, 215-4 Gongneung-dong, Nowon-ku, 139-240 Seoul, South Korea Correspondence to: Seok-Il Hong, M.D. at the above address. E-mail: [email protected] Received 1 December 2000; received in revised form 10 May 2001; accepted for publication 12 May 2001 2001 Academic Press 1043–4666/01/150166+05 $35.00/0 KEY WORDS: apoptosis/caspases/gastric cancer/serine protease/ TRAIL 166
permeability transition.7–9 Consequently, cytochrome c released from mitochondria to the cytosol activates caspase-3, and activated downstream caspases then cleave the death substrates that are central to apoptotic events.10 Although it is well established that caspases play central roles in apoptotic proteolysis, there exists a body of evidence suggesting that serine proteases may also be involved.11,12 Inhibitors of serine proteases were found to suppress apoptosis,13–15 but the critical steps at which they act remain to be determined. The current study has been undertaken to examine proteolytic events responsible for TRAIL-induced apoptosis. Here, we show that during TRAIL-induced apoptosis in SNU-16 adenocarcinoma cells, caspase-8 and -3 were processed into enzymatically active forms, Bid was cleaved, and cytochrome c was released into the cytosol. Interestingly, the general active sitedirected serine protease inhibitor suppressed apoptotic process, however, the serine protease inhibitors did not prevent the release of mitochondrial cytochrome c. Therefore, these studies suggest that TRAIL-induced apoptosis is dependent on caspases and serine protease, and serine protease(s) have a role in early postmitochondrial steps required to initiate the TRAILinduced apoptotic cascade. CYTOKINE, Vol. 15, No. 3 (7 August), 2001: pp 166–170
TRAIL-induced apoptosis via death proteases / 167
Figure 1.
TRAIL-induced apoptosis in SNU-16 human adenocarcinoma cells.
(A) SNU-16 cells were exposed to various concentrations of TRAIL for 16 h. Cell survival was measured by the tryphan blue exclusion assay. The line graph represents an average and SD of duplicate viable cell counts, expressed as a percentage of the untreated control group normalized to 100%. (B) Agarose gel electrophoresis of isolated DNA from cells treated with various concentrations of TRAIL. (C) Morphological changes in cells treated with 50 ng/ml TRAIL for 4 h. Cells were fixed with 4% formaldehyde and stained with 5 g/ml Hoechst 33258. Arrows indicate nuclear fragmentation and condensed chromatin. (D) Agarose gel electrophoresis of DNA from cells treated with 50 ng/ml of TRAIL for indicated times. After treatment with MMC, the cells were lysed, and the DNA was extracted and submitted to agarose gel electrophoresis as described in Materials and Methods.
RESULTS After cells were treated with various concentrations of TRAIL, 60% of cell cytotoxicity was observed at 50 ng/ml TRAIL, DNA fragmentation, and chromatin condensation. The findings indicated that the exposure of the cells to TRAIL resulted in dose-dependent apoptotic cell death. To determine the kinetics of apoptosis by TRAIL, the cells were sequentially treated with TRAIL at several time points. The fragmentation was observed to occur within 4 h after the treatment (Fig. 1). As shown in Figure 2, TRAIL induced the activation of caspase-8 and -3 in a time-dependent manner. Bid was cleaved into active subunits and cytochrome c was released from mitochondria into the cytoplasm upon treatment with TRAIL, suggesting that caspase-9 was activated during TRAIL-induced apoptosis. To elucidate the role of individual caspases in TRAILmediated apoptosis, we incubated SNU-16 cells with tetrapeptide caspase inhibitors in the presence of TRAIL. As shown in Figure 3, the inhibitors of
Figure 2. Caspase-8, caspase-3 and Bid were activated and cytochrome c was released from mitochondria into the cytosol in TRAILinduced apoptosis. Protein extracts were prepared at the indicated time points after exposure to 50 ng/ml TRAIL. Western blot analyses were performed with antibody against caspase-8, caspase-3, cytochrome c, and Bid.
caspase-1, -2, -3, -8, -9 as well as general inhibitors of caspases completely abrogated the TRAIL-induced DNA fragmentation, whereas inhibitors of caspase-5 and -6 did not. The result indicated that caspase-1, -2, -3, -8, -9 were crucially involved in TRAIL-induced apoptosis. Furthermore, inhibitors of caspase-1, -2, -3,
168 / Park et al.
CYTOKINE, Vol. 15, No. 3 (7 August, 2001: 166–170)
Figure 3. Effects of caspase inhibitors on TRAIL-induced apoptosis in SNU-16 human adenocarcinoma cells. Cells were either untreated (control), or treated with TRAIL (50 ng/ml) in the presence of 100 M Boc-D-FMK (general upstream caspase inhibitor) and the following tetrapeptide inhibitors: Z-YVAD-FMK (caspase-1 inhibitor), Z-VDVAD-FMK (caspase-2 inhibitor), Z-DEVDFMK (caspase-3 inhibitor), Z-WEHD-FMK (caspase-5 inhibitor), Z-VEID-FMK (caspase-6 inhibitor), Z-IETD-FMK (caspase-8 inhibitor), and Z-LEHD-FMK (caspase-9 inhibitor). DNA was extracted and electrophoresed in 1.8% agarose gel, and cytochrome c released from the mitochondria into the cytoplasm was detected by immunoblotting as depicted in Material and Methods. M: 1 kb ladder DNA marker.
-6, and -8 attenuated the release of cytochrome c from mitochondria into the cytoplasm, suggesting that these caspases acted in pre-mitochodria step in TRAILinduced apoptosis. To investigate possible participation of proteolytic enzymes other than caspases, in apoptosis triggered by TRAIL, we examined the effects of an inhibitor that specifically targets serine proteases. As shown in Figure 4, 4-(2-aminoethyl) benzenesulfonye fluoride (AEBSF) significantly suppressed the TRAIL-induced nuclei condensation and DNA fragmentation in adenocarcinoma cells in a dose-dependent manner. These results suggested that TRAIL might have activated a common cell death pathway in the SNU-16 cells, which was blocked by the serine proteases inhibitor AEBSF. To examine that AEBSF might suppress cytochrome c release, we analysed the cytochrome c release by Western blotting. The amount of cytochrome c in the cytosolic fraction increased in cells treated with TRAIL whereas cytochrome c was not detected in untreated cells. Even the use of up to 2 mM AEBSF could not prevent the cytochrome c translocation to cytosol during the apoptosis (Fig. 4B). Based on these results, we postulate that serine proteases may be involved in post-mitochondrial apoptotic events that lead to activation of the initiator caspase, caspase-9.
DISCUSSION In this study, we evaluated the TRAIL-induced apoptotic events and the ability of cysteine and serine proteases to mediate this process. Our data demonstrated that Bid was cleaved into active subunits and cytochrome c was released into the cytoplasm from mitochondria upon treatment with TRAIL. These
results suggested that caspase-9 was activated during TRAIL-induced apoptotic process and were consistent with the notion that caspase-8 is an apical caspase in receptor-mediated apoptosis such as TNF and Fas. TRAIL-induced apoptotic signal has been shown to be mediated through caspases, thus we incubated SNU-16 cells with caspase inhibitors in the presence of TRAIL to determine the role of individual caspases. Inhibitors of caspase-1, -2, -3, -8, and -9 suppressed DNA fragmentation, whereas inhibitors of caspase-5 and -6 did not. The result indicated that caspase-1, -2, -3, -8, -9 were crucially involved in TRAIL-induced apoptosis. Furthermore, inhibitors of caspase-1, -2, -3, -6, and -8 attenuated the release of cytochrome c from mitochondria into the cytosol, suggesting that these caspases acted in pre-mitochondria step in TRAILinduced apoptosis. Since activation of upstream caspases such as caspase-8 and -9 is a prerequisite of TRAIL-induced apoptosis, these inhibitors have substantial effect on the activity of TRAIL. Conversely, it is also possible that caspase-1 and -2 may play a role in TRAIL-mediated apoptosis in adenocarcinoma cells, even though the role of these caspases in other tumour cell types has not yet been reported. Moreover, the fact that procaspase-9 processing is dependent on caspase-3 after cytochrome c stimulation also sheds a new light on the simple hierarchical relationship whereby caspase-9 is situated upstream of caspase-3 in the cytochrome c pathway. Our data indicate that the release of cytochrome c from mitochondria into the cytoplasm occurs downstream of caspase-3. Further study is in need to clarify this relationship. Interestingly, serine protease inhibitor, AEBSF, blocked apoptotic process without affecting the release of mitochondrial cytochrome c during TRAIL-induced apoptosis. Thus, these observations may imply that
TRAIL-induced apoptosis via death proteases / 169
obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA) and anti-caspase-3 antibody from PharMingen (San Diego, CA, USA). SNU-16 cell line was maintained in RPMI 1640 medium (GIBCO, Grand Island, NY, USA) supplemented with heat-inactivated fetal bovine serum, penicillin and streptomycin.
DNA fragmentation and morphological evaluation To assess the effect of TRAIL on the structural integrity of DNA, cells were lysed and then incubated with proteinase K (0.5 mg/ml) for 1 h at 55C. Following phenol/chloroform extraction, the DNA was precipitated following fractionated on 1.8% agarose gel, and the DNA was visualized with ethidium bromide. For the morphological evaluation, cells were fixed with 4% formaldehyde following incubation with Hoechst dye 33258 at 5 g/ml in PBS for 5 min, washed and finally mounted with PBS:glycerol (3:1). Fluorescence was visualized with a fluorescence microscope [photographs were taken using Kodak Elite II-100 slide film (Eastern KODAK company, Rochester, New York, USA).
Western immunoblotting
Figure 4. Inhibition of TRAIL-induced apoptosis and release of cytochrome from mitochondria into the cytosol by the AEBSF. (A) Quantification of apoptosis inhibition by AEBSF. SNU-16 cells were exposed to 50 ng/ml TRAIL for 4 h in the presence of various concentrations of AEBSF. Indicated concentrations of AEBSF were added to the cells 30 min prior to the addition of TRAIL. The apoptotic cells were measured with Hoechst 33258 staining by fluorescence microscopy and apoptotic cell counts expressed as a percentage of total cells normalized to 100%. (B) Agarose gel electrophoresis of cells pretreated with various concentrations of AEBSF before being exposed to 50 ng/ml TRAIL. After incubation for 4 h, DNA was extracted and electrophoresed in 1.8% agarose gel. Cytochrome c in cytosol was detected by immunoblotting. Protein of cytosol fraction was extracted as in Materials and Methods and transferred to a PVDF filter after separation on 12% SDS-PAGE.
multiple serine proteases participate in the proteolytic cascade involved in TRAIL-induced apoptosis execution. In addition, this serine protease inhibitor might interfere with the caspase activation complexes or apoptosome. If so, it would be important to identify inhibitory molecules that could be the target of putative serine proteases that may be involved in apoptosome assembly and/or processing, rather than cytochrome c, caspase-9 or Apaf-1.
Control and treated cells were collected, washed with PBS, and lysed in lysis buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM -glycerolphosphate, 1 mM Na3VO4, 1 g/ml leupeptin, 1 mM PMSF). After brief sonication, the lysates were clarified by centrifugation at 12 000g for 15 min, and protein content was measured by a BCA method. An aliquot of total protein was separated by 10 or 12% sodium dodetyloulphate polyacrylamide gel electrophoresis (SDS-PAGE). Western immunoblotting was carried out with the primary antibody and then with the secondary antibody of HRP-linked IgG, by using an Amersham ECL system. For the detection of cytosolic cytochrome c, the cell pellet was resuspended in 500 l extraction buffer containing 220 mM mannitol, 70 mM sucrose, 50 mM Pipes–KOH, pH 7.4, 50 mM KCl, 5 mM EGTA, 2 mM MgCl2, 1 mM EDTA, 1 mM dithiothreitol, and protease inhibitors. After 30 min incubation on ice, cells were homogenized using a glass dounce and a B pestle. Cell homogenates were spun at 20 000g for 30 min, and supernatants were removed and stored at 70C until analysis by Western blotting.
Acknowledgements We would like to thank Dr. Woon Ki Paik for critical review of this manuscript. This work was partly supported by the National Nuclear R & D program of the Ministry of Science and Technology, Seoul, Korea.
MATERIALS AND METHODS Materials and cell culture Human recombinant TRAIL, AEBSF and caspase inhibitor set II were purchased from Calbiochem Co. (San Diego, CA, USA). Anti-caspase-8 and Bid antibodies were
REFERENCES 1. Wiley SR, Schooley K, Smolak PJ, Din WS, Huang CP, Nicholl JK, Sutherland GR, Smith TD, Rauch C, Smith CA (1995)
170 / Park et al. Identification and characterization of a new member of the TNF family that induces apoptosis. Immunity 3:673–682. 2. Pitti RM, Marsters SA, Ruppert S, Donahue CJ, Moore A, Ashkenazi A (1996) Induction of apoptosis by Apo-2 ligand, a new member of the tumor necrosis factor cytokine family. J Biol Chem 271:12 687–12 690. 3. Sheridan JP, Marsters SA, Pitti RM, Gurney A, Skubatch M, Baldwin D, Ramakrishnan L, Gray CL, Baker K, Wood WI, Goddard AD, Godowski P, Ashkenazi A (1997) Control of TRAILinduced apoptosis by a family of signaling and decoy receptors. Science 277:818–821. 4. Ashkenazi A, Dixit VM (1998) Death receptors: signaling and modulation. Science 28:1305–1308. 5. Pan G, O’Rourke K, Chinnaiyan AM, Gentz R, Ebner R, Ni J, Dixit VM (1997) The receptor for the cytotoxic ligand TRAIL. Science 276:111–113. 6. MacFarlane M, Ahmad M, Srinivasula SM, Fermandes Alnemri T, Cohen GM, Alnemri ES (1997) Identification and molecular cloning of two novel receptors for the cytotoxic ligand TRAIL. J Biol Chem 272:25 417–25 420. 7. Luo X, Budihardjo I, Zou H, Slaughter C, Wang X (1998) Bid, a Bcl2 interacting protein, mediates cytochrome c release from mitochondria in response to activation of cell surface death receptors. Cell 94:481–490. 8. Gross A, Yin XM, Wang K, Wei MC, Jockel J, Milliman C, Erdjument-Bromage H, Tempst P, Korsmeyer SJ (1999) Caspase cleaved BID targets mitochondria and is required for cytochrome c
CYTOKINE, Vol. 15, No. 3 (7 August, 2001: 166–170) release, while BCL-XL prevents this release, but not tumor necrosis factor-R1/Fas death. J Biol Chem 274:1156–1163. 9. Martinou JC (1999) Apoptosis: Key to the mitochondrial gate. Nature 399:411–412. 10. Liu X, Zou H, Slaughter C, Wang X (1997) DFF, a heterodimeric protein that functions downstream of caspase-3 to trigger DNA fragmentation during apoptosis. Cell 89:175–184. 11. Kumar S, Harvey NL (1995) Role of multiple cellular proteases in the execution of programmed cell death. FEBS Lett 375:169–173. 12. Martin SJ, Green DR (1995) Protease activation during apoptosis: death by a thousand cuts? Cell 82:349–352. 13. Weaver VM, Lach B, Walker PR, Sikorska M (1993) Role of proteolysis in apoptosis: involvement of serine proteases in internucleosomal DNA fragmentation in immature thymocytes. Biochem Cell Biol 71:488–500. 14. Shimizu T, Pommier Y (1997) Camptothecin-induced apoptosis in p53-null human leukemia HL60 cells and their isolated nuclei: effects of the protease inhibitors Z-VAD-fmk and dichloroisocoumarin suggest an involvement of both caspases and serine proteases. Leukemia 11:1238–1244. 15. Park IC, Park MJ, Choe TB, Jang JJ, Hong SI, Lee SH (2000) TNF-alpha induces apoptosis mediated by AEBSF-sensitive serine protease(s) that may involve upstream caspase-3/CPP32 protease activation in a human gastric cancer cell line. Int J Oncol 16:1243–1248.
SHORT COMMUNICATION
TUMOR NECROSIS FACTOR-RELATED APOPTOSIS INDUCING LIGAND (TRAIL)-INDUCED APOPTOSIS IS DEPENDENT ON ACTIVATION OF CYSTEINE AND SERINE PROTEASES In-Chul Park, Myung-Jin Park, Sang-Hyeok Woo, Kyung-Hee Lee, Seung-Hoon Lee, Chang-Hun Rhee, Seok-Il Hong We examined the role of caspases and serine protease(s) in cell death induced by tumour necrosis factor-related apoptosis-inducing ligand (TRAIL). After incubation of adenocarcinoma cells with TRAIL, caspase-3, -8 were activated and the cleavage of Bid induced the release of cytochrome c, from the mitochondria to the cytosol. Tetrapeptide inhibitors of caspase-1, -2, -3, and -8 suppressed DNA fragmentation and attenuated the release of cytochrome c, whereas inhibitors of caspase-5 did not. Interestingly, the general serine protease(s) inhibitor 4-(2aminoethyl)benzylsulfonyl fluoride (AEBSF) resulted in the arrest of apoptosis. However, the AEBSF did not prevent the release of mitochondrial cytochrome c during TRAIL-induced apoptosis. From these results, we postulate that serine protease(s) may be involved in post-mitochondrial apoptotic events, that lead to the activation of the initiator, caspase-9. 2001 Academic Press
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is an apoptosis-inducing member of the tumor necrosis factor (TNF) gene family.1,2 TNF family members transduce the apoptotic signal, by engaging death receptors that belong to a subfamily of the TNF receptor gene superfamily, and include TNFR1, CD95, and death receptor (DR).3–6 The mechanism of Fas-induced apoptosis is fairly well understood, whereas the mechanism of TRAILinduced apoptosis remains to be clarified. In Fasinduced apoptosis, activated caspase-8 cleaves Bid protein, which is subsequently translocated to mitochondria where it can induce mitochondrial dysfunction, such as loss of membrane potential and
From the Laboratory of Cell Biology, Korea Cancer Center Hospital, 215-4 Gongneung-dong, Nowon-ku, 139-240 Seoul, South Korea Correspondence to: Seok-Il Hong, M.D. at the above address. E-mail: [email protected] Received 1 December 2000; received in revised form 10 May 2001; accepted for publication 12 May 2001 2001 Academic Press 1043–4666/01/150166+05 $35.00/0 KEY WORDS: apoptosis/caspases/gastric cancer/serine protease/ TRAIL 166
permeability transition.7–9 Consequently, cytochrome c released from mitochondria to the cytosol activates caspase-3, and activated downstream caspases then cleave the death substrates that are central to apoptotic events.10 Although it is well established that caspases play central roles in apoptotic proteolysis, there exists a body of evidence suggesting that serine proteases may also be involved.11,12 Inhibitors of serine proteases were found to suppress apoptosis,13–15 but the critical steps at which they act remain to be determined. The current study has been undertaken to examine proteolytic events responsible for TRAIL-induced apoptosis. Here, we show that during TRAIL-induced apoptosis in SNU-16 adenocarcinoma cells, caspase-8 and -3 were processed into enzymatically active forms, Bid was cleaved, and cytochrome c was released into the cytosol. Interestingly, the general active sitedirected serine protease inhibitor suppressed apoptotic process, however, the serine protease inhibitors did not prevent the release of mitochondrial cytochrome c. Therefore, these studies suggest that TRAIL-induced apoptosis is dependent on caspases and serine protease, and serine protease(s) have a role in early postmitochondrial steps required to initiate the TRAILinduced apoptotic cascade. CYTOKINE, Vol. 15, No. 3 (7 August), 2001: pp 166–170
TRAIL-induced apoptosis via death proteases / 167
Figure 1.
TRAIL-induced apoptosis in SNU-16 human adenocarcinoma cells.
(A) SNU-16 cells were exposed to various concentrations of TRAIL for 16 h. Cell survival was measured by the tryphan blue exclusion assay. The line graph represents an average and SD of duplicate viable cell counts, expressed as a percentage of the untreated control group normalized to 100%. (B) Agarose gel electrophoresis of isolated DNA from cells treated with various concentrations of TRAIL. (C) Morphological changes in cells treated with 50 ng/ml TRAIL for 4 h. Cells were fixed with 4% formaldehyde and stained with 5 g/ml Hoechst 33258. Arrows indicate nuclear fragmentation and condensed chromatin. (D) Agarose gel electrophoresis of DNA from cells treated with 50 ng/ml of TRAIL for indicated times. After treatment with MMC, the cells were lysed, and the DNA was extracted and submitted to agarose gel electrophoresis as described in Materials and Methods.
RESULTS After cells were treated with various concentrations of TRAIL, 60% of cell cytotoxicity was observed at 50 ng/ml TRAIL, DNA fragmentation, and chromatin condensation. The findings indicated that the exposure of the cells to TRAIL resulted in dose-dependent apoptotic cell death. To determine the kinetics of apoptosis by TRAIL, the cells were sequentially treated with TRAIL at several time points. The fragmentation was observed to occur within 4 h after the treatment (Fig. 1). As shown in Figure 2, TRAIL induced the activation of caspase-8 and -3 in a time-dependent manner. Bid was cleaved into active subunits and cytochrome c was released from mitochondria into the cytoplasm upon treatment with TRAIL, suggesting that caspase-9 was activated during TRAIL-induced apoptosis. To elucidate the role of individual caspases in TRAILmediated apoptosis, we incubated SNU-16 cells with tetrapeptide caspase inhibitors in the presence of TRAIL. As shown in Figure 3, the inhibitors of
Figure 2. Caspase-8, caspase-3 and Bid were activated and cytochrome c was released from mitochondria into the cytosol in TRAILinduced apoptosis. Protein extracts were prepared at the indicated time points after exposure to 50 ng/ml TRAIL. Western blot analyses were performed with antibody against caspase-8, caspase-3, cytochrome c, and Bid.
caspase-1, -2, -3, -8, -9 as well as general inhibitors of caspases completely abrogated the TRAIL-induced DNA fragmentation, whereas inhibitors of caspase-5 and -6 did not. The result indicated that caspase-1, -2, -3, -8, -9 were crucially involved in TRAIL-induced apoptosis. Furthermore, inhibitors of caspase-1, -2, -3,
168 / Park et al.
CYTOKINE, Vol. 15, No. 3 (7 August, 2001: 166–170)
Figure 3. Effects of caspase inhibitors on TRAIL-induced apoptosis in SNU-16 human adenocarcinoma cells. Cells were either untreated (control), or treated with TRAIL (50 ng/ml) in the presence of 100 M Boc-D-FMK (general upstream caspase inhibitor) and the following tetrapeptide inhibitors: Z-YVAD-FMK (caspase-1 inhibitor), Z-VDVAD-FMK (caspase-2 inhibitor), Z-DEVDFMK (caspase-3 inhibitor), Z-WEHD-FMK (caspase-5 inhibitor), Z-VEID-FMK (caspase-6 inhibitor), Z-IETD-FMK (caspase-8 inhibitor), and Z-LEHD-FMK (caspase-9 inhibitor). DNA was extracted and electrophoresed in 1.8% agarose gel, and cytochrome c released from the mitochondria into the cytoplasm was detected by immunoblotting as depicted in Material and Methods. M: 1 kb ladder DNA marker.
-6, and -8 attenuated the release of cytochrome c from mitochondria into the cytoplasm, suggesting that these caspases acted in pre-mitochodria step in TRAILinduced apoptosis. To investigate possible participation of proteolytic enzymes other than caspases, in apoptosis triggered by TRAIL, we examined the effects of an inhibitor that specifically targets serine proteases. As shown in Figure 4, 4-(2-aminoethyl) benzenesulfonye fluoride (AEBSF) significantly suppressed the TRAIL-induced nuclei condensation and DNA fragmentation in adenocarcinoma cells in a dose-dependent manner. These results suggested that TRAIL might have activated a common cell death pathway in the SNU-16 cells, which was blocked by the serine proteases inhibitor AEBSF. To examine that AEBSF might suppress cytochrome c release, we analysed the cytochrome c release by Western blotting. The amount of cytochrome c in the cytosolic fraction increased in cells treated with TRAIL whereas cytochrome c was not detected in untreated cells. Even the use of up to 2 mM AEBSF could not prevent the cytochrome c translocation to cytosol during the apoptosis (Fig. 4B). Based on these results, we postulate that serine proteases may be involved in post-mitochondrial apoptotic events that lead to activation of the initiator caspase, caspase-9.
DISCUSSION In this study, we evaluated the TRAIL-induced apoptotic events and the ability of cysteine and serine proteases to mediate this process. Our data demonstrated that Bid was cleaved into active subunits and cytochrome c was released into the cytoplasm from mitochondria upon treatment with TRAIL. These
results suggested that caspase-9 was activated during TRAIL-induced apoptotic process and were consistent with the notion that caspase-8 is an apical caspase in receptor-mediated apoptosis such as TNF and Fas. TRAIL-induced apoptotic signal has been shown to be mediated through caspases, thus we incubated SNU-16 cells with caspase inhibitors in the presence of TRAIL to determine the role of individual caspases. Inhibitors of caspase-1, -2, -3, -8, and -9 suppressed DNA fragmentation, whereas inhibitors of caspase-5 and -6 did not. The result indicated that caspase-1, -2, -3, -8, -9 were crucially involved in TRAIL-induced apoptosis. Furthermore, inhibitors of caspase-1, -2, -3, -6, and -8 attenuated the release of cytochrome c from mitochondria into the cytosol, suggesting that these caspases acted in pre-mitochondria step in TRAILinduced apoptosis. Since activation of upstream caspases such as caspase-8 and -9 is a prerequisite of TRAIL-induced apoptosis, these inhibitors have substantial effect on the activity of TRAIL. Conversely, it is also possible that caspase-1 and -2 may play a role in TRAIL-mediated apoptosis in adenocarcinoma cells, even though the role of these caspases in other tumour cell types has not yet been reported. Moreover, the fact that procaspase-9 processing is dependent on caspase-3 after cytochrome c stimulation also sheds a new light on the simple hierarchical relationship whereby caspase-9 is situated upstream of caspase-3 in the cytochrome c pathway. Our data indicate that the release of cytochrome c from mitochondria into the cytoplasm occurs downstream of caspase-3. Further study is in need to clarify this relationship. Interestingly, serine protease inhibitor, AEBSF, blocked apoptotic process without affecting the release of mitochondrial cytochrome c during TRAIL-induced apoptosis. Thus, these observations may imply that
TRAIL-induced apoptosis via death proteases / 169
obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA) and anti-caspase-3 antibody from PharMingen (San Diego, CA, USA). SNU-16 cell line was maintained in RPMI 1640 medium (GIBCO, Grand Island, NY, USA) supplemented with heat-inactivated fetal bovine serum, penicillin and streptomycin.
DNA fragmentation and morphological evaluation To assess the effect of TRAIL on the structural integrity of DNA, cells were lysed and then incubated with proteinase K (0.5 mg/ml) for 1 h at 55C. Following phenol/chloroform extraction, the DNA was precipitated following fractionated on 1.8% agarose gel, and the DNA was visualized with ethidium bromide. For the morphological evaluation, cells were fixed with 4% formaldehyde following incubation with Hoechst dye 33258 at 5 g/ml in PBS for 5 min, washed and finally mounted with PBS:glycerol (3:1). Fluorescence was visualized with a fluorescence microscope [photographs were taken using Kodak Elite II-100 slide film (Eastern KODAK company, Rochester, New York, USA).
Western immunoblotting
Figure 4. Inhibition of TRAIL-induced apoptosis and release of cytochrome from mitochondria into the cytosol by the AEBSF. (A) Quantification of apoptosis inhibition by AEBSF. SNU-16 cells were exposed to 50 ng/ml TRAIL for 4 h in the presence of various concentrations of AEBSF. Indicated concentrations of AEBSF were added to the cells 30 min prior to the addition of TRAIL. The apoptotic cells were measured with Hoechst 33258 staining by fluorescence microscopy and apoptotic cell counts expressed as a percentage of total cells normalized to 100%. (B) Agarose gel electrophoresis of cells pretreated with various concentrations of AEBSF before being exposed to 50 ng/ml TRAIL. After incubation for 4 h, DNA was extracted and electrophoresed in 1.8% agarose gel. Cytochrome c in cytosol was detected by immunoblotting. Protein of cytosol fraction was extracted as in Materials and Methods and transferred to a PVDF filter after separation on 12% SDS-PAGE.
multiple serine proteases participate in the proteolytic cascade involved in TRAIL-induced apoptosis execution. In addition, this serine protease inhibitor might interfere with the caspase activation complexes or apoptosome. If so, it would be important to identify inhibitory molecules that could be the target of putative serine proteases that may be involved in apoptosome assembly and/or processing, rather than cytochrome c, caspase-9 or Apaf-1.
Control and treated cells were collected, washed with PBS, and lysed in lysis buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM -glycerolphosphate, 1 mM Na3VO4, 1 g/ml leupeptin, 1 mM PMSF). After brief sonication, the lysates were clarified by centrifugation at 12 000g for 15 min, and protein content was measured by a BCA method. An aliquot of total protein was separated by 10 or 12% sodium dodetyloulphate polyacrylamide gel electrophoresis (SDS-PAGE). Western immunoblotting was carried out with the primary antibody and then with the secondary antibody of HRP-linked IgG, by using an Amersham ECL system. For the detection of cytosolic cytochrome c, the cell pellet was resuspended in 500 l extraction buffer containing 220 mM mannitol, 70 mM sucrose, 50 mM Pipes–KOH, pH 7.4, 50 mM KCl, 5 mM EGTA, 2 mM MgCl2, 1 mM EDTA, 1 mM dithiothreitol, and protease inhibitors. After 30 min incubation on ice, cells were homogenized using a glass dounce and a B pestle. Cell homogenates were spun at 20 000g for 30 min, and supernatants were removed and stored at 70C until analysis by Western blotting.
Acknowledgements We would like to thank Dr. Woon Ki Paik for critical review of this manuscript. This work was partly supported by the National Nuclear R & D program of the Ministry of Science and Technology, Seoul, Korea.
MATERIALS AND METHODS Materials and cell culture Human recombinant TRAIL, AEBSF and caspase inhibitor set II were purchased from Calbiochem Co. (San Diego, CA, USA). Anti-caspase-8 and Bid antibodies were
REFERENCES 1. Wiley SR, Schooley K, Smolak PJ, Din WS, Huang CP, Nicholl JK, Sutherland GR, Smith TD, Rauch C, Smith CA (1995)
170 / Park et al. Identification and characterization of a new member of the TNF family that induces apoptosis. Immunity 3:673–682. 2. Pitti RM, Marsters SA, Ruppert S, Donahue CJ, Moore A, Ashkenazi A (1996) Induction of apoptosis by Apo-2 ligand, a new member of the tumor necrosis factor cytokine family. J Biol Chem 271:12 687–12 690. 3. Sheridan JP, Marsters SA, Pitti RM, Gurney A, Skubatch M, Baldwin D, Ramakrishnan L, Gray CL, Baker K, Wood WI, Goddard AD, Godowski P, Ashkenazi A (1997) Control of TRAILinduced apoptosis by a family of signaling and decoy receptors. Science 277:818–821. 4. Ashkenazi A, Dixit VM (1998) Death receptors: signaling and modulation. Science 28:1305–1308. 5. Pan G, O’Rourke K, Chinnaiyan AM, Gentz R, Ebner R, Ni J, Dixit VM (1997) The receptor for the cytotoxic ligand TRAIL. Science 276:111–113. 6. MacFarlane M, Ahmad M, Srinivasula SM, Fermandes Alnemri T, Cohen GM, Alnemri ES (1997) Identification and molecular cloning of two novel receptors for the cytotoxic ligand TRAIL. J Biol Chem 272:25 417–25 420. 7. Luo X, Budihardjo I, Zou H, Slaughter C, Wang X (1998) Bid, a Bcl2 interacting protein, mediates cytochrome c release from mitochondria in response to activation of cell surface death receptors. Cell 94:481–490. 8. Gross A, Yin XM, Wang K, Wei MC, Jockel J, Milliman C, Erdjument-Bromage H, Tempst P, Korsmeyer SJ (1999) Caspase cleaved BID targets mitochondria and is required for cytochrome c
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