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Differential roles for Bak in Triton X-100- and deoxycholate-induced apoptosis
Biochemical and Biophysical Research Communications 378 (2009) 529–533
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Differential roles for Bak in Triton X-100- and deoxycholate-induced apoptosis Hirofumi Sawai *, Naochika Domae Department of Internal Medicine, Osaka Dental University, 8-1 Kuzuhahanazonocho, Hirakata, Osaka 573-1121, Japan
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Article history: Received 14 November 2008 Available online 28 November 2008
Keywords: Apoptosis Detergent Triton X-100 Deoxycholate Bak Bax Caspase
a b s t r a c t We recently reported that Bax activation occurs downstream of caspase activation in Triton X-100 (TX)induced apoptosis. Here, Bak was found to be activated in TX-induced apoptosis. Although z-VAD-fmk completely suppressed Bax activation, it only partially attenuated TX-induced Bak activation. Moreover, activation of both Bak and Bax was detected in apoptosis induced by deoxycholate, a physiological detergent in bile. z-VAD-fmk completely suppressed deoxycholate-induced Bak as well as Bax activation. Furthermore, Bak siRNA attenuated TX- but not deoxycholate-induced caspase activation. These results suggest that Bak activation may occur upstream of caspase activation in TX- but not deoxycholateinduced apoptosis and that the mechanism of TX-induced apoptosis may differ from that of deoxycholate-induced apoptosis at least with regard to the role for Bak. Ó 2008 Elsevier Inc. All rights reserved.
Apoptosis plays a crucial role in various physiological and pathological phenomena including development and immune responses [1–3]. The mechanism of apoptosis has been extensively investigated in the last two decades. Extracellular stimuli such as TNF, Fas ligand, radiation, ultraviolet and anti-cancer drugs activate caspases, which in turn activate specific enzymes to degrade nuclei [4,5]. Two major pathways to caspase activation have been proposed: the death-receptor pathway and the mitochondrial pathway [5,6]. Bcl-2 family proteins including anti-apoptotic Bcl2 and Bcl-xL and pro-apoptotic Bax and Bak play crucial roles in the latter pathway [7,8]. Among various stimuli to induce apoptosis, sublytic concentrations (below the critical micellar concentrations) of detergents such as Triton X-100 (TX) and deoxycholate have been reported to induce apoptosis [9,10]. For the clinical application of detergents as anti-cancer drugs understanding the mechanism of detergentinduced apoptosis is fundamental. It has been reported that apoptosis induced by deoxycholate, a physiological detergent in bile, plays a role in cholestatic liver disease [11], and the mechanisms of deoxycholate-induced apoptosis including protein kinase C, Fas, and MAP kinases have been extensively investigated in hepatocytes [12–15]. However, the involvement of Bax or Bak in detergent-induced apoptosis or the mechanisms of detergent-induced apoptosis in non-hepatic cells have not been reported in detail. It has been reported that detergents including TX induced a conformational change (exposure of N-terminal epitope) of Bax
* Corresponding author. Fax: +81 72 864 3179. E-mail address: [email protected] (H. Sawai). 0006-291X/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2008.11.073
in vitro [16,17], and the similar conformational change has been reported in apoptosis induced by various stimuli including staurosporine, anti-Fas antibody, tumor necrosis factor-a and ultraviolet, suggesting that the conformational change indicates activation of pro-apoptotic property of Bax [18–21]. We have recently reported that the rapid activation of caspases concomitant with the conformational activation of Bax was observed in TX-induced apoptosis in U-937 leukemia cells [22]. Bax activation in TX-induced apoptosis was completely inhibited by caspase inhibitors, suggesting that sublytic concentrations of TX do not directly induce the conformational change of Bax in cells and that Bax activation occurs downstream of caspase activation in TX-induced apoptosis [22]. To investigate the upstream target of TX-induced apoptosis, we focused on Bak in this study. Activation of Bak by a conformational change similar to Bax has been reported in apoptosis induced by various stimuli including staurosporine, etoposide, cisplatin, and anti-Fas antibody [23–25]. We demonstrate that the active conformation of Bak is induced in TX- and deoxycholate-induced apoptosis and that Bak activation occurs upstream of caspase activation in TX- but not deoxycholate-induced apoptosis. Materials and methods Reagents. Triton X-100 was purchased from Sigma; Sodium deoxycholate from Wako Pure Chemical Industries (Japan); AntiCaspase-3 (clone 19) and anti-Bax (clone 6A7) mouse monoclonal antibodies from BD Biosciences; Anti-Caspase-8 (clone 1C12) mouse monoclonal antibody from Cell Signaling Technology;
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H. Sawai, N. Domae / Biochemical and Biophysical Research Communications 378 (2009) 529–533
Anti-Caspase-9 (clone 5B4) mouse monoclonal antibody from Medical & Biological Laboratories (Japan); Anti-Bak (clone TC100) mouse monoclonal antibody from Calbiochem; Anti-Bax (clone 2D2) mouse monoclonal antibody, anti-ICAD (FL-331) and anti-Actin rabbit polyclonal antibodies, anti-mouse or anti-rabbit IgG goat secondary antibodies conjugated with horseradish peroxidase, and anti-mouse IgG goat secondary antibody conjugated with FITC from Santa Cruz Biotechnology; Cy3-conjugated antimouse IgG goat secondary antibody from Chemicon; DEVD-MCA and z-VAD-fmk from Peptide Institute (Japan). Other reagents were purchased from Sigma unless otherwise indicated. Cell culture. Human monoblastic leukemia cell line U-937 and human T-lymphocytic leukemia cell line Jurkat were maintained in RPMI1640 medium containing 10% fetal bovine serum with 100 U/ml penicillin and 100 lg/ml streptomycin in a humidified 5% CO2 incubator. Caspase activity assay. Caspase activity was measured as described previously [22]. Western blot analysis. Western blot analysis was performed as described [26]. Immunostaining with FACS analysis. Immunostaining with antiBak or Bax antibody was performed as described with modifications [25]. The cells were fixed with 10% formalin solution in PBS on ice for 10 min and permeabilized in PBS containing 0.1% saponin for 5 min at room temperature. Then the cells were incubated with 1 lg/ml anti-Bak (clone TC-100) or anti-Bax (clone 6A7) mouse monoclonal antibody for 30 min. After washing with PBS, the cells were incubated for 30 min with FITC-conjugated antimouse IgG goat secondary antibody. After washing with PBS, the cells were analyzed by FACSCalibur (BD Biosciences). siRNA transfection. Bak siRNA (ON-TARGETplus SMARTpool) and control siRNA (ON-TARGETplus siCONTROL Non-targeting Pool) were purchased from Dharmacon (Thermo Fisher Scientific). siRNA was transfected into Jurkat cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. Briefly, 0.5–50 pmol siRNA or 1 ll Lipofectamine 2000 was diluted in 50 ll OPTI-MEM I Reduced Serum Medium (Gibco). After 5 min incubation, siRNA
was mixed with Lipofectamine 2000 and incubated for 20 min. The mixture (total 100 ll) of siRNA and Lipofactamine 2000 was added to the cells (2.5 105/0.4 ml OPTI-MEM I) in 24 well plates. After 24 h incubation, 0.5 ml RPMI medium containing 10% serum was added to each well and the cells were further incubated for 24 h. Transfection efficiency was assessed using BLOCK-iT Fluorescent Oligo (Invitrogen) and fluorescence microscopy, and it was more than 50% when Jurkat cells were used. Statistical analysis. Statistical analysis was performed by oneway ANOVA and post-hoc test. Results and discussion We previously reported that the active conformation of Bax, a pro-apoptotic member of Bcl-2 family proteins, was induced downstream of caspase activation in TX-induced apoptosis [22]. To investigate the mechanism of TX-induced apoptosis upstream of caspase activation, we focused on Bak, another pro-apoptotic member of Bcl-2 family. As we expected, the active conformation of Bak was detected in TX-induced apoptosis by immunostaining with anti-Bak antibody clone TC-100, which was previously reported to preferentially react with the active conformation of Bak [23–25], and confocal microscopy in U-937 cells (data not shown). To quantitatively detect TX-induced Bak activation, FACS analysis was performed as described in ‘‘Material and methods”. As shown in Fig. 1A, a slight but significant increase of the cells with the active conformation of Bak was detected at 5 min, and approximately 40% of the cells was positive for the active Bak between 15 and 30 min. As we previously reported [22], the cells with the active conformation of Bax, detected by anti-Bax antibody clone 6A7, slightly increased at 10 min and peaked at 30 min (Fig. 1B). Next, we investigated whether activation of Bak and Bax is specific to TX among detergents or not. Deoxycholate, a physiological detergent in bile, was previously shown to induce apoptosis in not only hepatocytes but also lymphocytes [10,11]. In our hands, sublytic concentrations (0.2–0.5 mM) of deoxycholate induced typical apoptotic morphological changes including membrane blebbing,
Fig. 1. Activation of Bak and Bax in TX- and deoxycholate-induced apoptosis in U-937 cells. (A and B) U-937 cells were treated with 0.01% TX for the indicated minutes, and the active conformation of Bak (A) or Bax (B) was detected using FACS analysis as described in Materials and methods. The values indicate % of the cells with the active conformation of Bak (A) or Bax (B). The data are means ± SD of at least three independent experiments. (C and D) U-937 cells were treated with 0.5 mM deoxycholate for the indicated minutes, and caspase activity assay (C, the values indicate% of the caspase activity compared with that at 0 min) and Western blot analysis for Caspases-3, -8, -9, ICAD, and Actin (D) were performed as described in Materials and methods. (E and F) U-937 cells were treated with 0.5 mM deoxycholate for the indicated minutes, and the active conformation of Bak (E) or Bax (F) was detected as in Fig. 1A and B.
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nuclear condensation and fragmentation, whereas more than 1 mM deoxycholate induced immediate membrane lysis in U-937 cells (data not shown). A concentration of 0.5 mM of deoxycholate was used to maximally induce apoptosis in the following experiments. Deoxycholate-induced caspase activity was first detected at 5 min and peaked at 15–30 min in U-937 cells (Fig. 1C). Western blot analysis showed proteolytic activation of procaspases-3, -8, and -9 (Fig. 1D). Procaspase-3 decreased at 5 min and completely disappeared at 15 min, suggesting proteolytic activation of caspase-3. The cleaved products of procaspase-8 were first detected at 10 min concomitantly with reduction of procaspase-8, whereas those of procaspase-9 with its reduction were first detected at 5 min, suggesting that activation of caspase-9 may occur upstream of caspase-8 activation. Similar results were previously obtained in TX-induced apoptosis [22]. Proteolysis of ICAD, inhibitor of caspase-activated DNase (CAD), was also detected (Fig. 1D), indicating activation of CAD, which can induce DNA fragmentation [4]. Furthermore, the active conformations of both Bak and Bax were detected in deoxycholate-induced apoptosis in U-937 cells. An increase of the cells with the active Bak was first detected at 10 min and approximately 30% of the cells were positive for the active Bak between 15 and 30 min (Fig. 1E), whereas an increase of the cells with the active Bax was first detected at 15 min and peaked at 30 min (Fig. 1F). These results suggest that activation of Bak and Bax is not specific to TX-induced apoptosis and may play a role in deoxycholate-induced apoptosis. We previously reported that activation of Bax in TX-induced apoptosis was completely inhibited by pretreatment with caspase inhibitors [22]. The effect of z-VAD-fmk, an irreversible pan-caspase inhibitor, on Bak activation in TX-induced apoptosis was examined. Although pretreatment with z-VAD-fmk completely inhibited TX-induced caspase activation (Fig. 2A), z-VAD-fmk only partially inhibited (approximately by 50%) TX-induced Bak activation (Fig. 2B). In contrast, z-VAD-fmk almost completely suppressed TX-induced Bax activation (Fig. 2C) as we previously reported [22]. These results suggest that Bak may be activated upstream of caspase activation by a caspase-independent mechanism whereas Bax activation is totally caspase-dependent in TX-induced apoptosis in U-937 cells. Effects of z-VAD-fmk on deoxycholate-induced apoptosis were also examined. As expected, deoxycholate-induced caspase activation was completely inhibited by z-VAD-fmk (Fig. 2A). In contrast with only partial inhibition of TX-induced Bak activation, deoxycholate-induced Bak activation was completely suppressed by zVAD-fmk (Fig. 2B). Consistent with TX-induced Bax activation, deoxycholate-induced Bax activation was completely inhibited by z-VAD-fmk (Fig. 2C). These results indicate that Bak activation occurs downstream of caspase activation in deoxycholate-induced apoptosis and suggest that mechanism of deoxycholate-induced apoptosis may differ from that of TX-induced apoptosis in U-937 cells. To investigate whether Bak activation in TX- or deoxycholateinduced apoptosis is specific to U-937 cells or not, Jurkat cells were used in the following experiments. As previously reported [10], apoptotic cell death was induced by treatment with sublytic concentrations of TX in Jurkat cells. A slight but significant increase of the caspase activity was first detected at 60 min, and the caspase activity further increased thereafter up to 120 min (Fig. 3A). Deoxycholate-induced caspase activation was first detected at 30 min, and approximately 5-fold increase of the caspase activity was detected between 60 and 120 min (Fig. 3A). Expression of Bak but not Bax protein was detected in Jurkat cells by Western blot analysis (data not shown), as it was previously reported that frameshift mutation existed in Bax gene in Jurkat cells [27]. Next, activation of Bak in TX- or deoxycholate-induced apoptosis in Jurkat cells was investigated by FACS analysis. A significant increase
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Fig. 2. Differential effects of z-VAD-fmk on TX- and deoxycholate-induced activation of Bak and Bax. U-937 cells were pretreated with 50 lM z-VAD-fmk (V) for 30 min, and then treated with 0.1% TX or 0.5 mM deoxycholate (DC) for 30 min. Caspase activity assay (A) and FACS analysis for active Bak (B) or Bax (C) were performed as in Fig. 1. (A) The values indicate % of the caspase activity compared with that of control ( ). (B and C) The values indicate % of the cells with the active conformation of Bak (B) or Bax (C).
of the cells with the active conformation of Bak by TX was detected at 60 min (Fig. 3B), when a slight increase of the caspase activity was detected (Fig. 3A). Similarly, a significant increase of the cells with the active Bak by deoxycholate was detected at 30 min (Fig. 3B), when the caspase activity was significantly increased (Fig. 3A). These results demonstrate that activation of Bak in TXor deoxycholate-induced apoptosis is not specific to U-937 cells, and also suggest that Bax may not be required for TX- or deoxycholate-induced apoptosis at least in Jurkat cells. Furthermore, effects of caspase inhibition on TX- or deoxycholate-induced Bak activation in Jurkat cells were investigated. As expected, treatment with z-VAD-fmk completely inhibited caspase activation in TX- or deoxycholate-induced apoptosis in Jurkat cells (Fig. 3C). FACS analysis showed that TX-induced Bak activation was not apparently inhibited by z-VAD-fmk, whereas deoxycholate-induced Bak activation was completely inhibited by z-VAD-fmk in Jurkat cells (Fig. 3D). These results suggest that Bak activation may occur upstream of caspase activation in TX-induced apoptosis in contrast with that deoxycholate-induced Bak activation is dependent on caspase activation in Jurkat cells as well as in U937 cells. To investigate whether Bak is required for TX- or deoxycholateinduced apoptosis, knock-down experiments using Bak siRNA were performed. Preliminary experiments showed that the transfection efficiency using FITC-labeled control siRNA was more than 50% in Jurkat cells, whereas it was less than 10% in U-937 cells. Therefore, Jurkat cells were used in the following experiments. As shown in
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Fig. 3. TX- and deoxycholate-induced activation of caspase and Bak in Jurkat cells. (A and B) Jurkat cells were treated with 0.01% TX or 0.5 mM deoxycholate (DC) for the indicated minutes, and caspase activity assay (A) and FACS analysis for active Bak (B) were performed as in Fig. 1. (C and D) Differential effects of z-VAD-fmk on TX- and deoxycholate-induced Bak activation. (C) Jurkat cells were pretreated with 50 lM z-VAD-fmk for 30 min and then treated with 0.01% TX for 120 min or 0.5 mM deoxycholate (DC) for 60 min. Caspase activity assay was performed as in Fig. 1. (D) Jurkat cells were pretreated with 50 lM z-VAD-fmk for 30 min and then treated with 0.01% TX or 0.5 mM deoxycholate (DC) for 60 min. FACS analysis for active Bak was performed as in Fig. 1.
Fig. 4A, the expression of Bak protein was reduced by transfection with Bak siRNA in a dose-dependent manner between 1 and 100 nM, whereas transfection with control siRNA did not affect the level of Bak protein (Fig. 4A). Similarly, TX-induced caspase activation was significantly suppressed by transfection with Bak siRNA compared with control siRNA in a dose-dependent manner between 1 and 100 nM (Fig. 4B). Bak siRNA at a concentration of 0.1 nM did not reduce the expression of Bak protein or suppress TX-induced caspase activation (data not shown). On the other hand, deoxycholate-induced caspase activation was not inhibited by Bak siRNA between 1 and 100 nM (Fig. 4C). These results suggest that Bak activation may play a crucial role in TX- but not deoxycholate-induced caspase activation at least in Jurkat cells, and also suggest that the mechanism of TX-induced apoptosis may differ from that of deoxycholate-induced apoptosis. Activation of Bak by a conformational change in apoptosis induced by various stimuli including staurosporine, etoposide, cisplatin and anti-Fas antibody has been reported [23–25]. It has been reported that the active conformation of Bak, in which a concealed N-terminal epitope is exposed, can be detected using antibodies against N-terminus of Bak such as clone TC-100 used in this study. Furthermore, it was previously shown that caspase inhibition by z-VAD-fmk did not attenuate Bak activation induced by various stimuli, suggesting that Bak activation occurs upstream of caspase activation [23,25]. In this study an increase of the cells with the active conformation of Bak was induced by sublytic concentrations of TX or deoxycholate. Although caspase inhibition by z-VAD-fmk completely inhibited Bax activation induced by TX or deoxycholate, z-VAD-fmk only partially attenuated TX-induced Bak activation, whereas it completely inhibited deoxycholate-induced Bak activation. These results suggest that Bak activation may occur upstream of caspase activation in TX-induced apoptosis whereas it occurs downstream of caspase activation in deoxycholate-induced apoptosis. Inhibition of deoxycholate-induced Bak activation by z-VAD-fmk in this study is different from the previous reports showing that z-VAD-fmk did not inhibit Bak activation induced by other stimuli including Fas and anti-cancer drugs [23,25], implying that the mechanism of deoxycholate-induced
apoptosis might be distinct from that of apoptosis induced by other stimuli. The mechanisms of differential roles for Bak in TX- and deoxycholate-induced apoptosis remain to be determined. However, it has been reported that Bax binds to Bcl-XL in the presence of sodium cholate (the oxidized form of deoxycholate) but not TX, suggesting that the different detergents might induce the different conformational changes of Bax [17]. It is speculated that Bak might bind to anti-apoptotic Bcl-2 family members and not be fully activated in deoxycholate-induced apoptosis. Since Bak is a membrane protein in contrast with Bax, which is at least partly a soluble protein, it would be difficult to directly demonstrate the effects of detergents on the conformational change and heterodimerization of Bak in vitro. Bid is a BH3-only protein of Bcl-2 family, and its truncated form (tBid), cleaved by caspase-8 [28,29], was reported to bind Bak and Bax to release cytochrome c from mitochondria [18,30]. As we previously reported [22], z-VAD-fmk inhibited the cleavage of Bid in TX-induced apoptosis, indicating that Bid cleavage occurs downstream of caspase activation. These results suggest that the conformational change of Bak is not dependent on tBid in TX-induced apoptosis. Involvement of bile salt-induced apoptosis in cholestatic liver injury has been proposed [11]. The mechanisms of apoptosis induced by bile salts including deoxycholate have been extensively studied, and various signaling molecules including protein kinase C, Fas, and mitogen-activated protein kinases have been reported to be involved in bile salt-induced apoptosis [12–15]. The relationship between Bak and these molecules remains to be determined and is currently under investigation in our laboratory. In conclusion, our results show that activation of Bak may play a crucial role in TX- but not deoxycholate-induced apoptosis and that the mechanism of TX-induced apoptosis may differ from that of deoxycholate-induced apoptosis. Since both TX and deoxycholate are capable of inducing extremely rapid apoptosis, both detergents might work as anti-cancer drugs with properties distinct from each other. For example, direct injection of detergents into solid tumors would rapidly induce apoptosis, which might be more beneficial than necrosis. Conjugation of monoclonal antibodies
H. Sawai, N. Domae / Biochemical and Biophysical Research Communications 378 (2009) 529–533
Fig. 4. Inhibition of TX-induced caspase activation by Bak siRNA. Jurkat cells were transfected with the indicated concentrations of control (c) or Bak (k) siRNA as described in Materials and methods. (A) Western blot analysis for Bak and Actin was performed as in Fig. 1. Density ratios of Bak versus Actin are indicated. The data are representative of two similar experiments. (B and C) After siRNA transfection for 48 h, Jurkat cells were treated with 0.01% TX for 90 min (B) or 0.5 mM deoxycholate for 30 min (C) and caspase activity assay was performed as in Fig. 1. The values indicate % of the caspase activity compared with that of no treatment with TX or deoxycholate (N). The data are means ± SD of a triplicate experiment and representative of three similar experiments. * Indicates p < 0.01.
(such as anti-CD20) with detergents might augment their apoptosis-inducing activity. Further studies will be required for the clinical application of these detergents as anti-cancer drugs. Acknowledgments This work was supported by ‘‘High-Tech Research Center” Project for Private Universities: matching fund subsidy from Ministry of Education, Culture, Sports, Science and Technology (2007–2011). References [1] H. Steller, Mechanisms and genes of cellular suicide, Science 267 (1995) 1445– 1449. [2] C.B. Thompson, Apoptosis in the pathogenesis and treatment of disease, Science 267 (1995) 1456–1462. [3] S. Nagata, Apoptosis by death factor, Cell 88 (1997) 355–365. [4] S. Nagata, Apoptotic DNA fragmentation, Exp. Cell Res. 256 (2000) 12–18.
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Differential roles for Bak in Triton X-100- and deoxycholate-induced apoptosis Hirofumi Sawai *, Naochika Domae Department of Internal Medicine, Osaka Dental University, 8-1 Kuzuhahanazonocho, Hirakata, Osaka 573-1121, Japan
a r t i c l e
i n f o
Article history: Received 14 November 2008 Available online 28 November 2008
Keywords: Apoptosis Detergent Triton X-100 Deoxycholate Bak Bax Caspase
a b s t r a c t We recently reported that Bax activation occurs downstream of caspase activation in Triton X-100 (TX)induced apoptosis. Here, Bak was found to be activated in TX-induced apoptosis. Although z-VAD-fmk completely suppressed Bax activation, it only partially attenuated TX-induced Bak activation. Moreover, activation of both Bak and Bax was detected in apoptosis induced by deoxycholate, a physiological detergent in bile. z-VAD-fmk completely suppressed deoxycholate-induced Bak as well as Bax activation. Furthermore, Bak siRNA attenuated TX- but not deoxycholate-induced caspase activation. These results suggest that Bak activation may occur upstream of caspase activation in TX- but not deoxycholateinduced apoptosis and that the mechanism of TX-induced apoptosis may differ from that of deoxycholate-induced apoptosis at least with regard to the role for Bak. Ó 2008 Elsevier Inc. All rights reserved.
Apoptosis plays a crucial role in various physiological and pathological phenomena including development and immune responses [1–3]. The mechanism of apoptosis has been extensively investigated in the last two decades. Extracellular stimuli such as TNF, Fas ligand, radiation, ultraviolet and anti-cancer drugs activate caspases, which in turn activate specific enzymes to degrade nuclei [4,5]. Two major pathways to caspase activation have been proposed: the death-receptor pathway and the mitochondrial pathway [5,6]. Bcl-2 family proteins including anti-apoptotic Bcl2 and Bcl-xL and pro-apoptotic Bax and Bak play crucial roles in the latter pathway [7,8]. Among various stimuli to induce apoptosis, sublytic concentrations (below the critical micellar concentrations) of detergents such as Triton X-100 (TX) and deoxycholate have been reported to induce apoptosis [9,10]. For the clinical application of detergents as anti-cancer drugs understanding the mechanism of detergentinduced apoptosis is fundamental. It has been reported that apoptosis induced by deoxycholate, a physiological detergent in bile, plays a role in cholestatic liver disease [11], and the mechanisms of deoxycholate-induced apoptosis including protein kinase C, Fas, and MAP kinases have been extensively investigated in hepatocytes [12–15]. However, the involvement of Bax or Bak in detergent-induced apoptosis or the mechanisms of detergent-induced apoptosis in non-hepatic cells have not been reported in detail. It has been reported that detergents including TX induced a conformational change (exposure of N-terminal epitope) of Bax
* Corresponding author. Fax: +81 72 864 3179. E-mail address: [email protected] (H. Sawai). 0006-291X/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2008.11.073
in vitro [16,17], and the similar conformational change has been reported in apoptosis induced by various stimuli including staurosporine, anti-Fas antibody, tumor necrosis factor-a and ultraviolet, suggesting that the conformational change indicates activation of pro-apoptotic property of Bax [18–21]. We have recently reported that the rapid activation of caspases concomitant with the conformational activation of Bax was observed in TX-induced apoptosis in U-937 leukemia cells [22]. Bax activation in TX-induced apoptosis was completely inhibited by caspase inhibitors, suggesting that sublytic concentrations of TX do not directly induce the conformational change of Bax in cells and that Bax activation occurs downstream of caspase activation in TX-induced apoptosis [22]. To investigate the upstream target of TX-induced apoptosis, we focused on Bak in this study. Activation of Bak by a conformational change similar to Bax has been reported in apoptosis induced by various stimuli including staurosporine, etoposide, cisplatin, and anti-Fas antibody [23–25]. We demonstrate that the active conformation of Bak is induced in TX- and deoxycholate-induced apoptosis and that Bak activation occurs upstream of caspase activation in TX- but not deoxycholate-induced apoptosis. Materials and methods Reagents. Triton X-100 was purchased from Sigma; Sodium deoxycholate from Wako Pure Chemical Industries (Japan); AntiCaspase-3 (clone 19) and anti-Bax (clone 6A7) mouse monoclonal antibodies from BD Biosciences; Anti-Caspase-8 (clone 1C12) mouse monoclonal antibody from Cell Signaling Technology;
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Anti-Caspase-9 (clone 5B4) mouse monoclonal antibody from Medical & Biological Laboratories (Japan); Anti-Bak (clone TC100) mouse monoclonal antibody from Calbiochem; Anti-Bax (clone 2D2) mouse monoclonal antibody, anti-ICAD (FL-331) and anti-Actin rabbit polyclonal antibodies, anti-mouse or anti-rabbit IgG goat secondary antibodies conjugated with horseradish peroxidase, and anti-mouse IgG goat secondary antibody conjugated with FITC from Santa Cruz Biotechnology; Cy3-conjugated antimouse IgG goat secondary antibody from Chemicon; DEVD-MCA and z-VAD-fmk from Peptide Institute (Japan). Other reagents were purchased from Sigma unless otherwise indicated. Cell culture. Human monoblastic leukemia cell line U-937 and human T-lymphocytic leukemia cell line Jurkat were maintained in RPMI1640 medium containing 10% fetal bovine serum with 100 U/ml penicillin and 100 lg/ml streptomycin in a humidified 5% CO2 incubator. Caspase activity assay. Caspase activity was measured as described previously [22]. Western blot analysis. Western blot analysis was performed as described [26]. Immunostaining with FACS analysis. Immunostaining with antiBak or Bax antibody was performed as described with modifications [25]. The cells were fixed with 10% formalin solution in PBS on ice for 10 min and permeabilized in PBS containing 0.1% saponin for 5 min at room temperature. Then the cells were incubated with 1 lg/ml anti-Bak (clone TC-100) or anti-Bax (clone 6A7) mouse monoclonal antibody for 30 min. After washing with PBS, the cells were incubated for 30 min with FITC-conjugated antimouse IgG goat secondary antibody. After washing with PBS, the cells were analyzed by FACSCalibur (BD Biosciences). siRNA transfection. Bak siRNA (ON-TARGETplus SMARTpool) and control siRNA (ON-TARGETplus siCONTROL Non-targeting Pool) were purchased from Dharmacon (Thermo Fisher Scientific). siRNA was transfected into Jurkat cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. Briefly, 0.5–50 pmol siRNA or 1 ll Lipofectamine 2000 was diluted in 50 ll OPTI-MEM I Reduced Serum Medium (Gibco). After 5 min incubation, siRNA
was mixed with Lipofectamine 2000 and incubated for 20 min. The mixture (total 100 ll) of siRNA and Lipofactamine 2000 was added to the cells (2.5 105/0.4 ml OPTI-MEM I) in 24 well plates. After 24 h incubation, 0.5 ml RPMI medium containing 10% serum was added to each well and the cells were further incubated for 24 h. Transfection efficiency was assessed using BLOCK-iT Fluorescent Oligo (Invitrogen) and fluorescence microscopy, and it was more than 50% when Jurkat cells were used. Statistical analysis. Statistical analysis was performed by oneway ANOVA and post-hoc test. Results and discussion We previously reported that the active conformation of Bax, a pro-apoptotic member of Bcl-2 family proteins, was induced downstream of caspase activation in TX-induced apoptosis [22]. To investigate the mechanism of TX-induced apoptosis upstream of caspase activation, we focused on Bak, another pro-apoptotic member of Bcl-2 family. As we expected, the active conformation of Bak was detected in TX-induced apoptosis by immunostaining with anti-Bak antibody clone TC-100, which was previously reported to preferentially react with the active conformation of Bak [23–25], and confocal microscopy in U-937 cells (data not shown). To quantitatively detect TX-induced Bak activation, FACS analysis was performed as described in ‘‘Material and methods”. As shown in Fig. 1A, a slight but significant increase of the cells with the active conformation of Bak was detected at 5 min, and approximately 40% of the cells was positive for the active Bak between 15 and 30 min. As we previously reported [22], the cells with the active conformation of Bax, detected by anti-Bax antibody clone 6A7, slightly increased at 10 min and peaked at 30 min (Fig. 1B). Next, we investigated whether activation of Bak and Bax is specific to TX among detergents or not. Deoxycholate, a physiological detergent in bile, was previously shown to induce apoptosis in not only hepatocytes but also lymphocytes [10,11]. In our hands, sublytic concentrations (0.2–0.5 mM) of deoxycholate induced typical apoptotic morphological changes including membrane blebbing,
Fig. 1. Activation of Bak and Bax in TX- and deoxycholate-induced apoptosis in U-937 cells. (A and B) U-937 cells were treated with 0.01% TX for the indicated minutes, and the active conformation of Bak (A) or Bax (B) was detected using FACS analysis as described in Materials and methods. The values indicate % of the cells with the active conformation of Bak (A) or Bax (B). The data are means ± SD of at least three independent experiments. (C and D) U-937 cells were treated with 0.5 mM deoxycholate for the indicated minutes, and caspase activity assay (C, the values indicate% of the caspase activity compared with that at 0 min) and Western blot analysis for Caspases-3, -8, -9, ICAD, and Actin (D) were performed as described in Materials and methods. (E and F) U-937 cells were treated with 0.5 mM deoxycholate for the indicated minutes, and the active conformation of Bak (E) or Bax (F) was detected as in Fig. 1A and B.
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nuclear condensation and fragmentation, whereas more than 1 mM deoxycholate induced immediate membrane lysis in U-937 cells (data not shown). A concentration of 0.5 mM of deoxycholate was used to maximally induce apoptosis in the following experiments. Deoxycholate-induced caspase activity was first detected at 5 min and peaked at 15–30 min in U-937 cells (Fig. 1C). Western blot analysis showed proteolytic activation of procaspases-3, -8, and -9 (Fig. 1D). Procaspase-3 decreased at 5 min and completely disappeared at 15 min, suggesting proteolytic activation of caspase-3. The cleaved products of procaspase-8 were first detected at 10 min concomitantly with reduction of procaspase-8, whereas those of procaspase-9 with its reduction were first detected at 5 min, suggesting that activation of caspase-9 may occur upstream of caspase-8 activation. Similar results were previously obtained in TX-induced apoptosis [22]. Proteolysis of ICAD, inhibitor of caspase-activated DNase (CAD), was also detected (Fig. 1D), indicating activation of CAD, which can induce DNA fragmentation [4]. Furthermore, the active conformations of both Bak and Bax were detected in deoxycholate-induced apoptosis in U-937 cells. An increase of the cells with the active Bak was first detected at 10 min and approximately 30% of the cells were positive for the active Bak between 15 and 30 min (Fig. 1E), whereas an increase of the cells with the active Bax was first detected at 15 min and peaked at 30 min (Fig. 1F). These results suggest that activation of Bak and Bax is not specific to TX-induced apoptosis and may play a role in deoxycholate-induced apoptosis. We previously reported that activation of Bax in TX-induced apoptosis was completely inhibited by pretreatment with caspase inhibitors [22]. The effect of z-VAD-fmk, an irreversible pan-caspase inhibitor, on Bak activation in TX-induced apoptosis was examined. Although pretreatment with z-VAD-fmk completely inhibited TX-induced caspase activation (Fig. 2A), z-VAD-fmk only partially inhibited (approximately by 50%) TX-induced Bak activation (Fig. 2B). In contrast, z-VAD-fmk almost completely suppressed TX-induced Bax activation (Fig. 2C) as we previously reported [22]. These results suggest that Bak may be activated upstream of caspase activation by a caspase-independent mechanism whereas Bax activation is totally caspase-dependent in TX-induced apoptosis in U-937 cells. Effects of z-VAD-fmk on deoxycholate-induced apoptosis were also examined. As expected, deoxycholate-induced caspase activation was completely inhibited by z-VAD-fmk (Fig. 2A). In contrast with only partial inhibition of TX-induced Bak activation, deoxycholate-induced Bak activation was completely suppressed by zVAD-fmk (Fig. 2B). Consistent with TX-induced Bax activation, deoxycholate-induced Bax activation was completely inhibited by z-VAD-fmk (Fig. 2C). These results indicate that Bak activation occurs downstream of caspase activation in deoxycholate-induced apoptosis and suggest that mechanism of deoxycholate-induced apoptosis may differ from that of TX-induced apoptosis in U-937 cells. To investigate whether Bak activation in TX- or deoxycholateinduced apoptosis is specific to U-937 cells or not, Jurkat cells were used in the following experiments. As previously reported [10], apoptotic cell death was induced by treatment with sublytic concentrations of TX in Jurkat cells. A slight but significant increase of the caspase activity was first detected at 60 min, and the caspase activity further increased thereafter up to 120 min (Fig. 3A). Deoxycholate-induced caspase activation was first detected at 30 min, and approximately 5-fold increase of the caspase activity was detected between 60 and 120 min (Fig. 3A). Expression of Bak but not Bax protein was detected in Jurkat cells by Western blot analysis (data not shown), as it was previously reported that frameshift mutation existed in Bax gene in Jurkat cells [27]. Next, activation of Bak in TX- or deoxycholate-induced apoptosis in Jurkat cells was investigated by FACS analysis. A significant increase
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Fig. 2. Differential effects of z-VAD-fmk on TX- and deoxycholate-induced activation of Bak and Bax. U-937 cells were pretreated with 50 lM z-VAD-fmk (V) for 30 min, and then treated with 0.1% TX or 0.5 mM deoxycholate (DC) for 30 min. Caspase activity assay (A) and FACS analysis for active Bak (B) or Bax (C) were performed as in Fig. 1. (A) The values indicate % of the caspase activity compared with that of control ( ). (B and C) The values indicate % of the cells with the active conformation of Bak (B) or Bax (C).
of the cells with the active conformation of Bak by TX was detected at 60 min (Fig. 3B), when a slight increase of the caspase activity was detected (Fig. 3A). Similarly, a significant increase of the cells with the active Bak by deoxycholate was detected at 30 min (Fig. 3B), when the caspase activity was significantly increased (Fig. 3A). These results demonstrate that activation of Bak in TXor deoxycholate-induced apoptosis is not specific to U-937 cells, and also suggest that Bax may not be required for TX- or deoxycholate-induced apoptosis at least in Jurkat cells. Furthermore, effects of caspase inhibition on TX- or deoxycholate-induced Bak activation in Jurkat cells were investigated. As expected, treatment with z-VAD-fmk completely inhibited caspase activation in TX- or deoxycholate-induced apoptosis in Jurkat cells (Fig. 3C). FACS analysis showed that TX-induced Bak activation was not apparently inhibited by z-VAD-fmk, whereas deoxycholate-induced Bak activation was completely inhibited by z-VAD-fmk in Jurkat cells (Fig. 3D). These results suggest that Bak activation may occur upstream of caspase activation in TX-induced apoptosis in contrast with that deoxycholate-induced Bak activation is dependent on caspase activation in Jurkat cells as well as in U937 cells. To investigate whether Bak is required for TX- or deoxycholateinduced apoptosis, knock-down experiments using Bak siRNA were performed. Preliminary experiments showed that the transfection efficiency using FITC-labeled control siRNA was more than 50% in Jurkat cells, whereas it was less than 10% in U-937 cells. Therefore, Jurkat cells were used in the following experiments. As shown in
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Fig. 3. TX- and deoxycholate-induced activation of caspase and Bak in Jurkat cells. (A and B) Jurkat cells were treated with 0.01% TX or 0.5 mM deoxycholate (DC) for the indicated minutes, and caspase activity assay (A) and FACS analysis for active Bak (B) were performed as in Fig. 1. (C and D) Differential effects of z-VAD-fmk on TX- and deoxycholate-induced Bak activation. (C) Jurkat cells were pretreated with 50 lM z-VAD-fmk for 30 min and then treated with 0.01% TX for 120 min or 0.5 mM deoxycholate (DC) for 60 min. Caspase activity assay was performed as in Fig. 1. (D) Jurkat cells were pretreated with 50 lM z-VAD-fmk for 30 min and then treated with 0.01% TX or 0.5 mM deoxycholate (DC) for 60 min. FACS analysis for active Bak was performed as in Fig. 1.
Fig. 4A, the expression of Bak protein was reduced by transfection with Bak siRNA in a dose-dependent manner between 1 and 100 nM, whereas transfection with control siRNA did not affect the level of Bak protein (Fig. 4A). Similarly, TX-induced caspase activation was significantly suppressed by transfection with Bak siRNA compared with control siRNA in a dose-dependent manner between 1 and 100 nM (Fig. 4B). Bak siRNA at a concentration of 0.1 nM did not reduce the expression of Bak protein or suppress TX-induced caspase activation (data not shown). On the other hand, deoxycholate-induced caspase activation was not inhibited by Bak siRNA between 1 and 100 nM (Fig. 4C). These results suggest that Bak activation may play a crucial role in TX- but not deoxycholate-induced caspase activation at least in Jurkat cells, and also suggest that the mechanism of TX-induced apoptosis may differ from that of deoxycholate-induced apoptosis. Activation of Bak by a conformational change in apoptosis induced by various stimuli including staurosporine, etoposide, cisplatin and anti-Fas antibody has been reported [23–25]. It has been reported that the active conformation of Bak, in which a concealed N-terminal epitope is exposed, can be detected using antibodies against N-terminus of Bak such as clone TC-100 used in this study. Furthermore, it was previously shown that caspase inhibition by z-VAD-fmk did not attenuate Bak activation induced by various stimuli, suggesting that Bak activation occurs upstream of caspase activation [23,25]. In this study an increase of the cells with the active conformation of Bak was induced by sublytic concentrations of TX or deoxycholate. Although caspase inhibition by z-VAD-fmk completely inhibited Bax activation induced by TX or deoxycholate, z-VAD-fmk only partially attenuated TX-induced Bak activation, whereas it completely inhibited deoxycholate-induced Bak activation. These results suggest that Bak activation may occur upstream of caspase activation in TX-induced apoptosis whereas it occurs downstream of caspase activation in deoxycholate-induced apoptosis. Inhibition of deoxycholate-induced Bak activation by z-VAD-fmk in this study is different from the previous reports showing that z-VAD-fmk did not inhibit Bak activation induced by other stimuli including Fas and anti-cancer drugs [23,25], implying that the mechanism of deoxycholate-induced
apoptosis might be distinct from that of apoptosis induced by other stimuli. The mechanisms of differential roles for Bak in TX- and deoxycholate-induced apoptosis remain to be determined. However, it has been reported that Bax binds to Bcl-XL in the presence of sodium cholate (the oxidized form of deoxycholate) but not TX, suggesting that the different detergents might induce the different conformational changes of Bax [17]. It is speculated that Bak might bind to anti-apoptotic Bcl-2 family members and not be fully activated in deoxycholate-induced apoptosis. Since Bak is a membrane protein in contrast with Bax, which is at least partly a soluble protein, it would be difficult to directly demonstrate the effects of detergents on the conformational change and heterodimerization of Bak in vitro. Bid is a BH3-only protein of Bcl-2 family, and its truncated form (tBid), cleaved by caspase-8 [28,29], was reported to bind Bak and Bax to release cytochrome c from mitochondria [18,30]. As we previously reported [22], z-VAD-fmk inhibited the cleavage of Bid in TX-induced apoptosis, indicating that Bid cleavage occurs downstream of caspase activation. These results suggest that the conformational change of Bak is not dependent on tBid in TX-induced apoptosis. Involvement of bile salt-induced apoptosis in cholestatic liver injury has been proposed [11]. The mechanisms of apoptosis induced by bile salts including deoxycholate have been extensively studied, and various signaling molecules including protein kinase C, Fas, and mitogen-activated protein kinases have been reported to be involved in bile salt-induced apoptosis [12–15]. The relationship between Bak and these molecules remains to be determined and is currently under investigation in our laboratory. In conclusion, our results show that activation of Bak may play a crucial role in TX- but not deoxycholate-induced apoptosis and that the mechanism of TX-induced apoptosis may differ from that of deoxycholate-induced apoptosis. Since both TX and deoxycholate are capable of inducing extremely rapid apoptosis, both detergents might work as anti-cancer drugs with properties distinct from each other. For example, direct injection of detergents into solid tumors would rapidly induce apoptosis, which might be more beneficial than necrosis. Conjugation of monoclonal antibodies
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Fig. 4. Inhibition of TX-induced caspase activation by Bak siRNA. Jurkat cells were transfected with the indicated concentrations of control (c) or Bak (k) siRNA as described in Materials and methods. (A) Western blot analysis for Bak and Actin was performed as in Fig. 1. Density ratios of Bak versus Actin are indicated. The data are representative of two similar experiments. (B and C) After siRNA transfection for 48 h, Jurkat cells were treated with 0.01% TX for 90 min (B) or 0.5 mM deoxycholate for 30 min (C) and caspase activity assay was performed as in Fig. 1. The values indicate % of the caspase activity compared with that of no treatment with TX or deoxycholate (N). The data are means ± SD of a triplicate experiment and representative of three similar experiments. * Indicates p < 0.01.
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