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Characterization of TNF-induced caspase-independent necroptosis
Leukemia Research 38 (2014) 706–713
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Characterization of TNF-induced caspase-independent necroptosis Hirofumi Sawai ∗ Department of Internal Medicine, Osaka Dental University, Osaka, Japan
a r t i c l e
i n f o
Article history: Received 17 October 2013 Received in revised form 21 January 2014 Accepted 1 February 2014 Available online 10 February 2014 Keywords: Necroptosis Apoptosis TNF Caspase ATP
a b s t r a c t Caspase-independent programmed necrotic cell death (necroptosis) has recently been described. Previously described models of necroptosis required 16 h or more of induction, which made the interpretation of findings somewhat difficult. In human monocytic leukemia cell line U937 necroptosis could be induced within 6 h by combination of TNF and Z-VAD-fmk. Here we show that the reduction in intracellular ATP levels may not be the sole determinant of necroptosis, and that necroptosis is associated with the loss of mitochondrial membrane potential, but not the activation of Bak/Bax or calcineurin. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction Apoptosis and necrosis are two major forms of cell death with distinguishing morphological features [1–3]. Apoptosis is characterized by cell shrinkage, fragmented nuclei with condensed chromatin, and formation of apoptotic bodies, whereas necrosis is characterized by swelling of cells and organelles and loss of plasma membrane integrity. The mechanisms of apoptosis have been investigated extensively. The two major pathways leading to apoptosis have been established: the death-receptor (or extrinsic) pathway and mitochondrial (or intrinsic) pathway [4,5]. The death-receptor pathway was shown to be triggered by death ligands including Fas (CD95) ligand. Binding of Fas ligand to its receptor Fas induces the formation of death-inducing signaling complex composed of FADD and procaspase-8, resulting in caspase-8 activation through induced proximity. Previous studies demonstrated that active caspase-8 either directly cleaved procaspase-3 to generate active caspase-3 or indirectly activated caspase-3 via the mitochondrial pathway by cleaving Bid [6,7]. The mitochondrial pathway is generally used in response to genotoxic stresses including irradiation and anticancer drugs. The activation of proapoptotic Bcl-2 family members including Bax and Bak was reported to lead to the release of cytochrome c from the intermembrane space of mitochondria to the cytosol. Cytochrome c associates with Apaf-1 and procaspase-9 to form
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the complex termed an apoptosome, resulting in the activation of caspase-9, which proteolytically activates executioner caspases including caspase-3, -6, and -7. In addition to post-apoptotic secondary necrosis, caspaseindependent necrosis has also been reported [3,8]. The involvement of RIP1 kinase in Fas-induced caspase-8-independent necrotic cell death has been demonstrated [9]. Specific inhibitors of caspaseindependent necrosis, necrostatins, have recently been identified, and necrostatin-inhibitable caspase-independent necrosis was referred to as necroptosis [10]. Necrostatins have been shown to inhibit the kinase activity of RIP1 [11], which interacts with RIP3 [12,13]. The signaling pathways leading to necroptosis downstream of RIP1 and RIP3 have recently been elucidated. The RIP1/RIP3 complex activates MLKL, which subsequently activates PGAM5 by phosphorylation, resulting in the dephosphorylation of Drp1. Dephosphorylated Drp1 promotes mitochondrial fission, leading to necrotic cell death [14,15]. Although the enzymes involved in energy metabolism have been suggested to play a crucial role in necroptosis [16], the precise relationship between mitochondrial fission and necroptosis remains to be determined. In previous reports necroptosis was induced at 16 h or later [10,11], which made the interpretation of the data somewhat difficult. In the present study, we investigated various molecular events including a reduction in intracellular ATP levels, conformational changes in Bax/Bak, and the loss of mitochondrial membrane potential (m) during necroptosis in comparison with apoptosis using U937 human monocytic leukemia cell line, in which necroptosis could be induced within 6 h by TNF in the presence of Z-VAD-fmk.
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Fig. 1. TNF-induced caspase-independent necroptosis in U937 cells. (A and B) U937 cells were treated with 10 ng/ml TNF (T) and 10 g/ml cycloheximide for the indicated times, and dual staining with Annexin V-FITC/PI was performed. As indicated, cells were treated in the presence of 10 M Z-VAD-fmk (V) or 50 M Z-Asp-CH2 -DCB (Asp). Representative dot plots are shown in (A). (C) When U937 cells were treated with 10 M necrostatin-1 (N), TNF/ZVAD-induced necrosis was completely inhibited whereas TNF-induced apoptosis was only slightly affected. The data shown are the average of three independent experiments. The bars indicate standard deviations. AnV-PI+, Annexin V− /PI+ (late necrosis); AnV + PI+, Annexin V+ /PI+ (necrosis); AnV + PI-, Annexin V+ /PI− (early apoptosis or necrosis).
2. Materials and methods 2.1. Cell culture Human monocytic leukemia cell line U937 cells and human T cell leukemia cell line Jurkat cells were purchased from RIKEN Cell Bank (Japan), and human myelocytic leukemia cell line HL60 cells were purchased from JCRB Cell Bank (Japan). Caspase-8-deficient Jurkat (JB6) cells were provided by Dr. S. Nagata [17]. Cells were maintained in RPMI1640 medium supplemented with 10% heat-inactivated fetal bovine serum and 100 U/ml penicillin/100 g/ml streptomycin in a humidified 5% CO2 incubator at 37 ◦ C. 2.2. Assessment of apoptosis and necroptosis To induce caspase-independent necroptosis, U937 cells were pretreated with 10 M Z-VAD-fmk (Peptide Institute, Japan) for 30 min and were then treated with 10 ng/ml TNF (Wako Pure Chemical Industries, Japan) plus 10 g/ml cycloheximide (Sigma) for the indicated times. Cells were incubated with 2.5 l FITC-conjugated Annexin V (MBL, Japan) and 2.5 g/ml propidium iodide (Sigma) in 100 l binding
buffer containing 10 mM Hepes/KOH (pH 7.4), 140 mM NaCl, and 2.5 mM CaCl2 for 30 min at 4 ◦ C, and were then analyzed using FACS Calibur (BD Biosciences). We recently reported that early necroptotic cells were Annexin V+ /PI− before becoming PI+ , which could be almost completely inhibited by necrostatin-1 [18,19]. On the other hand, Annexin V+ /PI− early apoptotic cells were only slightly affected by necrostatin-1. Agonistic anti-Fas antibody (clone CH-11) (MBL, Japan) was used instead of TNF in some experiments. 2.3. ATP depletion Cells for ATP depletion experiments were transferred to RPMI1640 medium without glucose (GIBCO) supplemented with 0.5% bovine albumin instead of fetal bovine serum, and 10 g/ml oligomycin (Wako Pure Chemical Industries, Japan) was then added to these cells [20]. 2.4. Inhibition of necroptosis by necrostatin-1 To inhibit necroptosis, 10 M necrostatin-1 (Sigma) was added to cells 30 min before the addition of TNF.
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Fig. 2. Induction of necroptosis in U937, Jurkat, and HL60 cells. (A) U937 cells were treated with 100 ng/ml agonistic anti-Fas antibody (F) plus 10 g/ml cycloheximide for 8 h. As indicated, cells were treated in the presence of 10 M Z-VAD-fmk (V) or 10 M necrostatin-1 (N). (B) HL60 cells were treated with 20 ng/ml TNF (T) plus 10 g/ml cycloheximide for 8 h. As indicated, cells were treated in the presence of 10 M Z-VAD-fmk (V) or 10 M necrostatin-1 (N). (C) Jurkat cells were treated with agonistic 100 ng/ml agonistic anti-Fas antibody (F) or 20 ng/ml TNF (T) plus 10 g/ml cycloheximide for 8 h. As indicated, cells were treated in the presence of 10 M Z-VAD-fmk (V) or 10 M necrostatin-1 (N). (D) Caspase-8-deficient Jurkat (JB6) cells were treated with 20 ng/ml TNF (T) plus 10 g/ml cycloheximide for 8 h. As indicated, cells were treated in the presence of 10 M Z-VAD-fmk (V) or 10 M necrostatin-1 (N). Dual staining with Annexin V-FITC/PI was performed as described above. The data shown are the average of three independent experiments. The bars indicate standard deviations. AnV-PI+, Annexin V− /PI+ (late necrosis); AnV + PI+, Annexin V+ /PI+ (necrosis); AnV + PI-, Annexin V+ /PI− (early apoptosis or necrosis).
2.5. Inhibition of mitochondrial fission by mdivi-1 Mdivi-1 (Sigma) was used as an inhibitor of Drp1, which plays a crucial role in mitochondrial fission. To inhibit mitochondrial fission, 50 or 100 M mdivi-1 was added to cells 30 min before the addition of TNF.
2.6. Inhibition of calcineurin To inhibit calcineurin, 10 M cyclosporine A (Wako Pure Chemical Industries, Japan) or 1 M FK506 (Alexis) was added to cells simultaneously with the addition of TNF.
Measurement Reagent for Cells for 10 min at room temperature. Luminescence was measured using SpectraMax M5 (Molecular Devices). 2.8. FACS analysis for conformational changes in Bak and Bax Conformational changes in Bak and Bax were analyzed as previously described [21,22]. Briefly, cells were fixed in 10% paraformaldehyde in PBS for 10 min at 4 ◦ C followed by permeabilization with 0.1% saponin in PBS for 5 min at room temperature. Cells were then incubated with anti-Bak (TC100) (Calbiochem) and anti-Bax (6E7) (Calbiochem) antibody, which reacts with the active conformation of Bak and Bax, respectively, for 30 min at 4 ◦ C. After washing with PBS, cells were incubated with FITC-conjugated anti-mouse IgG antibody (Santa Cruz biotechnology) for 30 min at 4 ◦ C. These cells were then analyzed using FACS Calibur (BD Biosciences).
2.7. Measurement of intracellular ATP levels 2.9. Analysis of m and externalization of phosphatidylserine Intracellular ATP levels were measured using ATP Measurement Reagent for Cells (TOYO Ink, Japan), which contains luciferin and luciferase. According to the manufacturer’s instructions, cells (50 l) were incubated with 50 l ATP
Cells were incubated with 100 nM CMXRos (Molecular Probes) for the last 15 min of the treatment at 37 ◦ C. Cells were then incubated with 2.5 l FITC-conjugated
H. Sawai / Leukemia Research 38 (2014) 706–713
2.10. Statistical analysis One-way ANOVA and post hoc analysis was performed using IBM SPSS Statistics Version 21.0.
3. Results
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Annexin V (MBL, Japan) in 100 l binding buffer for 30 min at 4 ◦ C, and were analyzed using FACS Calibur (BD Biosciences).
3.1. Induction of caspase-independent necrostatin-inhibitable necroptosis in U937 as well as Jurkat and HL60 cells
3.2. Induction of necroptosis in caspase-8-deficient Jurkat (JB6) cells We previously reported that the inhibition of caspase-8 by ZVAD may play a crucial role in necroptosis [19]. To further confirm this, we used caspase-8 deficient Jurkat (JB6) cells [17]. When JB6 cells were treated with TNF/ZVAD, necroptosis was induced in JB6 cells similar to that in Jurkat cells (Fig. 2D). In contrast to Jurkat cells, in which apoptosis was induced by TNF, necroptosis was induced by TNF in JB6 cells. 3.3. Necrostatin-1 did not inhibit ATP-depletion-induced necrosis We investigated the role of ATP depletion in apoptosis and necroptosis in U937 cells. In TNF-induced apoptosis, a slight and an approximately 80% reduction in intracellular ATP levels was detected at 2 h and 4 h, respectively (Fig. 3A). In contrast, while no reduction in intracellular ATP levels was detected at 2 h, a slight
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As we recently reported [19,22], caspase-independent necrosis was induced when human monocytic leukemia U937 cells were treated with TNF in the presence of Z-VAD-fmk (ZVAD), a pan-caspase inhibitor (Fig. 1A and B). Although an increase in Annexin V− or PI+ cells was not detected at 2 h, approximately 20% and 50% of cells became PI+ at 4 h and 6 h, respectively. In contrast, when U937 cells were treated with TNF in the presence of Z-Asp-CH2 -DCB (ZAsp), a caspase-3-specific inhibitor [19], no increase in Annexin V+ cells was observed at 4 h and only a slight increase was detected at 6 h. As shown in Fig. 1C, the pretreatment with 10 M necrostatin-1 completely inhibited TNF/ZVAD-induced necrotic cell death, which indicated that necroptosis was induced by TNF/ZVAD in U937 cells. On the other hand, TNF-induced cell death was genuine apoptosis since it was not apparently inhibited by necrostatin-1. We then investigated whether necroptosis could be induced by treating U937 cells with agonistic anti-Fas antibody instead of TNF. When cells were treated for 8 h with agonistic anti-Fas antibody (clone CH-11) in the presence of ZVAD, approximately 20% of cells became PI+ , which were then completely inhibited by necrostain-1 (Fig. 2A). We then investigated whether necroptosis could be induced in cells other than U937 cells. When human myelocytic leukemia HL60 cells were treated with TNF/ZVAD, an increase of PI+ cells was observed within 8 h, which was inhibited by necrostatin1 (Fig. 2B). In contrast, necrostain-1 did not apparently inhibit TNF-induced apoptosis in HL60 cells (data not shown). When human T cell leukemia Jurkat cells were treated with agonistic anti-Fas antibody, typical apoptotic morphological changes were induced within 2 h, which was completely inhibited by pretreatment with ZVAD at 2 h. However, necrotic changes were observed in Fas/ZVAD-treated Jurkat cells after 8 h, which was attenuated by necrostatin-1 (Fig. 2C). Necroptosis was also induced by TNF/ZVAD in Jurkat cells (Fig. 2C). Necrostatin-1 did not apparently inhibit Fasor TNF-induced apoptosis in Jurkat cells (data not shown).
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Fig. 3. Reduction in intracellular ATP levels in TNF-induced apoptosis and necroptosis. (A) U937 cells were treated with 10 ng/ml TNF (T) plus 10 g/ml cycloheximide in the presence or absence of 10 M Z-VAD-fmk (V) or 50 M Z-Asp-CH2 -DCB (Asp) for the indicated times. Intracellular ATP levels were measured as described in Section 2. The data shown are the average of a triplicate experiment. The bars indicate standard deviations. Similar results were obtained in three independent experiments. (B) U937 cells were pretreated with or without 10 M necrostatin-1 for 30 min. Cells were then treated with 10 ng/ml TNF (T) plus 10 g/ml cycloheximide in the presence or absence of 10 M Z-VAD-fmk (V) or 50 M Z-Asp-CH2 -DCB (Asp) for 6 h, and intracellular ATP levels were measured. The data shown are the average of a triplicate experiment. The bars indicate standard deviations.
and a more than 80% reduction was observed at 4 h and 6 h, respectively, in TNF/ZVAD-induced necroptosis (Fig. 3A). In the presence of ZAsp, no reduction in ATP levels was observed within 4 h, and a slight reduction was detected at 6 h (Fig. 3A). Necrostatin-1 only slightly attenuated the reduction in ATP levels in TNF-induced apoptosis, whereas necrostatin-1 almost completely suppressed this reduction in TNF/ZVAD-induced necroptosis (Fig. 3B). We then examined whether ATP depletion itself could induce necroptosis. As was previously reported [20], treating U937 cells with oligomycin, an inhibitor of F0/F1 ATP synthase, in the absence of glucose rapidly reduced intracellular ATP levels to approximately 20% at 0.5 h and less than 10% after 2 h (Fig. 4A). Under these conditions approximately 20% and 60% of cells became PI+ at 5 h and 6 h, respectively (Fig. 4B). Necrostatin-1 did not affect necrotic cell death induced by ATP depletion (Fig. 4C).
3.4. Conformational changes in Bak and Bax during apoptosis and necroptosis We investigated the involvement of Bak and Bax in necroptosis. As we and others previously reported [21,22], conformational changes in Bak and Bax were induced during TNF-induced apoptosis, while a conformational change in Bak was detected in approximately 60% of cells and that in Bax was detected in 10% only (Fig. 5). ZVAD significantly (p < 0.05) inhibited TNF-induced conformational changes in Bak and Bax. In contrast, ZAsp significantly (p < 0.05) augmented a conformational change in Bax and also slightly (but not significantly) augmented that in Bak.
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3.6. Effects of calcineurin inhibitors on TNF/ZVAD-induced necroptosis
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Fig. 5. Conformational changes in Bak and Bax in TNF-induced apoptosis and necroptosis. U937 cells were treated with 10 ng/ml TNF (T) plus 10 g/ml cycloheximide in the presence or absence of 10 M Z-VAD-fmk (V) or 50 M Z-Asp-CH2 -DCB (Asp) for 2 h. Conformational changes in Bak and Bax were examined as described in Section 2. The data shown are the average of three independent experiments. The bars indicate standard deviations. *, p < 0.05; ns, no significance compared with TNF only.
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Calcineurin was previously shown to be involved in Drp1 activation and mitochondrial fission induced by mitochondrial depolarization [24]. To investigate the involvement of calcineurin in TNF/ZVAD-induced necroptosis, the effects of calcineurin inhibitors (cyclosporine A and FK506) on TNF/ZVAD-induced necroptosis were examined (Fig. 7). Cyclosporine A, which binds to cyclophilin and thereby inhibits calcineurin, significantly (p < 0.05) reduced TNF/ZVAD-induced Annexin V− /PI+ (late necroptotic) cells. However, FK506, another calcineurin inhibitor, did not apparently inhibit necroptosis. These results indicated that calcineurin may not play an important role in TNF/ZVAD-induced necroptosis, and suggest that other mechanisms such as the loss of m might be involved in necroptosis. 3.7. Loss of m in TNF/ZVAD-induced necroptosis
Fig. 4. Induction of necrosis by ATP depletion. (A) U937 cells were incubated in serum-free medium with or without glucose (G) in the presence or absence of 10 g/ml oligomycin (O) for the indicated times. Intracellular ATP levels were measured as described above. The data shown are the average of a triplicate experiment. The bars indicate standard deviations. Similar results were obtained in three independent experiments. (B) U937 cells were incubated in serum-free medium without glucose in the presence of 10 g/ml oligomycin for the indicated times. Dual staining with Annexin V-FITC/PI was performed. (C) U937 cells were incubated in serumfree medium with or without glucose (G) in the presence or absence of 10 g/ml oligomycin (O) or 10 M necrostatin-1 (N) for 6 h, and dual staining with Annexin V-FITC/PI was performed. The data shown are the average of three independent experiments. The bars indicate standard deviations. AnV-PI+, Annexin V− /PI+ (late necrosis); AnV + PI+, Annexin V+ /PI+ (necrosis); AnV + PI-, Annexin V+ /PI− (early apoptosis or necrosis).
3.5. Effects of mdivi-1, an inhibitor of Drp1, on apoptosis and necroptosis
We examined the changes in m compared with the externalization of phosphatidylserine (PS) during apoptosis and necroptosis
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Mdivi-1, an inhibitor of Drp1, was recently reported to protect cells from necroptosis [15]. Since mitochondrial fission was also shown to be involved in apoptosis [23], we investigated the effects of mdivi-1 on TNF-induced apoptosis and TNF/ZVADinduced necroptosis in U937 cells (Fig. 6). Although mdivi-1 did not apparently affect TNF-induced apoptosis at 50 M, it markedly inhibited TNF-induced apoptosis at 100 M. In contrast, mdivi1 markedly suppressed TNF/ZVAD-induced necroptosis even at 50 M and almost completely inhibited necroptosis at 100 M.
Fig. 6. Effects of mdivi-1 on TNF-induced apoptosis and necroptosis. U937 cells were pretreated with 50 or 100 M mdivi-1 (m) for 30 min. Cells were then treated with 10 ng/ml TNF (T) plus 10 g/ml cycloheximide in the presence or absence of 10 M Z-VAD-fmk (V) for 6 h. Dual staining with Annexin V-FITC/PI was performed. The data shown are the average of three independent experiments. The bars indicate standard deviations. AnV-PI+, Annexin V− /PI+ (late necrosis); AnV + PI+, Annexin V+ /PI+ (necrosis); AnV + PI-, Annexin V+ /PI− (early apoptosis or necrosis).
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Fig. 7. Effects of calcineurin inhibitors on TNF-induced necroptosis. U937 cells were treated with 10 ng/ml TNF (T) plus 10 g/ml cycloheximide in the presence or absence of 10 M Z-VAD-fmk (V) for 6 h. As indicated, cells were treated with 10 M cyclosporine A (Cs) or 1 M FK506 (F). Dual staining with Annexin V-FITC/PI was performed. The data shown are the average of four independent experiments. The bars indicate standard deviations. AnV-PI+, Annexin V− /PI+ (late necrosis); AnV + PI+, Annexin V+ /PI+ (necrosis); AnV + PI-, Annexin V+ /PI− (early apoptosis or necrosis). *, p < 0.05; ns, no significance compared with TNF plus Z-VAD-fmk.
using dual staining with Annexin V-FITC/CMXRos [25]. As previously reported [26], the loss of m was detected in TNF-induced apoptosis as well as TNF/ZVAD-induced necroptosis (Fig. 8A). Kinetic studies revealed that the loss of m was induced almost in parallel to PS externalization with slower kinetics in TNF/ZVAD-induced necroptosis than in TNF-induced apoptosis (Fig. 8B). Thus, early apoptotic and necroptotic cells became Annexin V+ /CMXRoslow (the lower right quadrant), whereas late necroptotic cells became Annexin V− /CMXRoslow (the lower left
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quadrant). Necrostatin-1 almost completely attenuated the loss of m in TNF/ZVAD-induced necroptosis, whereas it only slightly affected in TNF-induced apoptosis (Fig. 8C). Since cyclosporine A inhibited TNF/ZVAD-induced late necroptosis (Fig. 7), we investigated the effect of cyclosporine A on m in necroptosis. As shown in Fig. 8D, cyclosporine A significantly (p < 0.05) reduced Annexin V− /CMXRoslow late necroptotic cells. 4. Discussion Necroptosis has recently been established owing to the identification of necrostatins as its specific inhibitors. However, necroptosis was observed at 16 h or later in previous reports, which made the interpretation of the data somewhat difficult. For example, necrosis secondary to apoptosis may have contributed to delayed necrotic cell death. In the present study, we used U937 cells, in which TNF-induced caspase-independent necrostatininhibitable necroptosis could be induced within 6 h. As shown in Fig. 2B and C, cell lines other than U937 were markedly less sensitive to necroptosis. More than 50% of U937 cells became PI+ after being treated with TNF/ZVAD for 6 h, whereas less than 20% of HL60 and Jurkat cells became PI+ 8 h after treatment with Fas/ZVAD or TNF/ZVAD. Agonistic anti-Fas antibody was less potent than TNF in inducing necroptosis in U937 cells since less than 20% of cells became PI+ when treated with Fas/ZVAD for 8 h (Fig. 2A). These results indicated that U937 cells were very sensitive to TNF/ZVADinduced necroptosis. We previously reported that the inhibition of caspase-8 by ZVAD may play a crucial role in necroptosis, whereas necroptosis was not induced by the inhibition of caspase-3 by ZAsp [18,19]. This may have been because caspase-8 cleaves and inhibits RIP1, thereby preventing necroptosis. Caspase-8-deficient Jurkat (JB6) cells were used to confirm that caspase-8 played an inhibitory
Fig. 8. Loss of m in TNF-induced apoptosis and necroptosis. (A and B) U937 cells were treated with 10 ng/ml TNF (T) plus 10 g/ml cycloheximide in the presence or absence of 10 M Z-VAD-fmk (V) for the indicated times. Dual staining with Annexin V-FITC/CMXRos was performed as described in Section 2. Representative dot plots are shown in (A). (C) U937 cells were treated with 10 ng/ml TNF (T) plus 10 g/ml cycloheximide in the presence or absence of 10 M Z-VAD-fmk (V) or 10 M necrostatin-1 (N) for 6 h, and dual staining with Annexin V-FITC/CMXRos was performed. (D) U937 cells were treated with 10 ng/ml TNF (T) plus 10 g/ml cycloheximide in the presence of 10 M Z-VAD-fmk (V) for 6 h. As indicated, cells were treated with 10 M cyclosporine A (Cs) or 1 M FK506 (F). Dual staining with Annexin V-FITC/CMXRos was performed. *, p < 0.05; ns, no significance compared with TNF plus Z-VAD-fmk. The data shown are the average of at least three independent experiments. The bars indicate standard deviations. AnV-Xros-, Annexin V− /CMXRoslow (late necrosis); AnV + XRos-, Annexin V+ /CMXRoslow (early apoptosis or necrosis); AnV + XRos+, Annexin V+ /CMXRoshigh (very early apoptosis or necrosis); AnV-XRos+, Annexin V− /CMXRoshigh (normal).
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role in necroptosis (Fig. 2D). Necroptosis was induced by TNF even in the absence of ZVAD in JB6 cells, which further suggested that caspase-8 may inhibit TNF-induced necroptosis. ATP has been reported to play a crucial role in determining whether cells die by apoptosis or necrosis [20]. As shown in Fig. 3A, the reduction in intracellular ATP levels was more rapidly induced in apoptosis than in necroptosis. Although intracellular ATP levels were almost the same between apoptosis and necroptosis at 6 h, approximately 50% of cells became PI+ in necroptosis at 6 h, while less than 5% became PI+ in apoptosis (Fig. 1B). Furthermore, incubating cells in glucose-free medium in the presence of oligomycin reduced intracellular ATP levels to less than 10% at 2 h, although an increase in PI+ cells was not detected within 3 h (Fig. 4A and B). As shown in Fig. 4C, necrosis induced by ATP depletion was not affected by necrostatin-1, which indicated that ‘classical’ necrotic cell death, but not necroptosis was induced by ATP depletion. These results suggest that intracellular ATP levels may not be the sole determinant of necroptosis. Bak and Bax, proapoptotic Bcl-2 family members, have been shown to play crucial roles in the mitochondrial apoptotic pathway [4,5]. The activation of Bak and Bax with conformational changes in apoptosis induced by various stimuli including staurosporine, agonistic anti-Fas antibody, and anticancer drugs has been reported previously [21,22]. As shown in Fig. 5, conformational changes in Bak and, to a lesser extent, Bax were detected in TNF-induced apoptosis. Conformational changes in Bak and Bax were attenuated in the presence of ZVAD, which suggested that the activation of Bak/Bax may not play a crucial role in TNF/ZVAD-induced necroptosis. Moreover, conformational changes in Bak/Bax were augmented in the presence of ZAsp, indicating that ZAsp may inhibit apoptosis downstream of Bak/Bax activation. Since ZAsp is a caspase-3specific inhibitor while ZVAD inhibits both caspase-8 and caspase-3 [19], these results suggest that caspase-8 and caspase-3 may play a stimulatory and inhibitory role, respectively, in Bak/Bax activation. In accordance with these results, tBid, the cleavage product of Bid by caspase-8, was shown to induce Bak/Bax activation with conformational changes [27,28]. The RIP1/RIP3 complex was recently shown to activate MLKL/PGAM5 in necroptosis, resulting in the dephosphorylation of Drp1, which subsequently promoted mitochondrial fission [14,15]. Mdivi-1 has been identified as a specific Drp1 inhibitor [23]. As previously reported, mdivi-1 inhibited both TNF-induced apoptosis and TNF/ZVAD-induced necroptosis (Fig. 6). Since TNF/ZVADinduced necroptosis was almost completely inhibited whereas TNF-induced apoptosis was only partially inhibited by 100 M mdivi-1, mitochondrial fission may play a more important role in necroptosis than in apoptosis. Calcineurin has been reported to be involved in Drp1 activation and mitochondrial fission induced by mitochondrial depolarization [24]; therefore, we investigated whether calcineurin may play a role in necroptosis. As shown in Fig. 7, cyclosporine A slightly inhibited TNF/ZVAD-induced necroptosis, whereas FK506 did not apparently affect necroptosis. Since cyclosporine A binds to cyclophilin and thereby inhibits calcineurin, it may block the loss of m, in which cyclophilin D is involved [29]. In contrast, FK506 inhibits calcineurin by binding to FKBP12 and has no effect on m [24]. These results indicate that calcineurin may not play an important role in TNF/ZVAD-induced necroptosis, and suggest that cyclosporine A might partly inhibit necroptosis by blocking the loss of m. Therefore, we examined the changes in m in TNF-induced apoptosis and necroptosis (Fig. 8A and B). The results indicated that although the loss of m was detected in both apoptosis and necroptosis, it occurred with slower kinetics in necroptosis than in apoptosis. Necrostatin-1 almost completely attenuated the loss of m induced by TNF/ZVAD (Fig. 8C), which confirmed that the
Fig. 9. Mechanisms of TNF-induced apoptosis and necroptosis. TNF-induced apoptosis inhibits the necroptotic signaling pathway through cleavage of RIP1 by the active caspase-8. Z-VAD-fmk inhibits caspase-8, thereby preventing apoptosis and facilitating necroptosis. Z-Asp-CH2 -DCB prevents apoptosis without inducing necroptosis by inhibiting caspase-3, but not caspase-8. Necrostatin-1 prevents necroptosis by inhibiting kinase activity of RIP1. Bak/Bax activation is inhibited by Z-VAD-fmk, but not by Z-Asp-CH2 -DCB. Mdivi-1, an inhibitor of Drp1, prevents necroptosis more effectively than apoptosis. Cyclosporine A partly prevents late necroptosis by attenuating the loss of m. The reduction in intracellular ATP levels seems to be a late event in both apoptosis and necroptosis.
loss of m was induced in necroptosis and not by TNF per se. Furthermore, we investigated the effect of cyclosporine A on the loss of m in necroptosis. As shown in Fig. 8D, cyclosporine A partially attenuated the loss of m in necroptosis, suggesting that cyclosporine A may partly inhibit TNF/ZVAD-induced necroptosis by blocking the loss of m. In conclusion, we investigated various molecular events during necroptosis in comparison with apoptosis using U937 human monocytic leukemia cell line, in which necroptosis could be induced within several hours by TNF in the presence of ZVAD. The results obtained suggest that a reduction in intracellular ATP levels may not be the sole determinant of necroptosis, and that the loss of m, but not activation of Bak/Bax or calcineurin may be involved in necroptosis (Fig. 9). Conflict of interest statement The author declares that he has no competing interests. Acknowledgment Author’s contributions: H.S. designed and performed experiments, analyzed data and wrote the paper. Funding: This work was supported in part by Grant-in-Aid for Scientific Research (C) (25462941) from Japan Society for the Promotion of Science. References [1] Kerr JFR, Wyllie AH, Currie AR. Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer 1972;26:239–57. [2] Shimizu S, Eguchi Y, Kamiike W, Waguri S, Uchiyama Y, Matsuda H, et al. Retardation of chemical hypoxia-induced necrotic cell death by Bcl-2 and ICE inhibitors: possible involvement of common mediators in apoptotic and necrotic signal transductions. Oncogene 1996;12:2045–50. [3] Vercammen D, Brouckaert G, Denecker G, Van de Craen M, Declercq W, Fiers W, et al. Dual signaling of the Fas receptor: initiation of both apoptotic and necrotic cell death pathways. J Exp Med 1998;188:919–30. [4] Scaffidi C, Fulda S, Srinivasan A, Friesen C, Li F, Tomaselli KJ, et al. Two CD95 (APO-1/Fas) signaling pathways. EMBO J 1998;17:1675–87. [5] Hengartner MO. The biochemistry of apoptosis. Nature 2000;407:770–6. [6] Luo X, Budihardjo I, Zou H, Slaughter C, Wang X. Bid, a Bcl2 interacting protein, mediates cytochrome c release from mitochondria in response to activation of cell surface death receptors. Cell 1998;94:481–90.
H. Sawai / Leukemia Research 38 (2014) 706–713 [7] Li H, Zhu H, Xu CJ, Yuan J. Cleavage of BID by caspase 8 mediates the mitochondrial damage in the Fas pathway of apoptosis. Cell 1998;94:491–501. [8] Vercammen D, Beyaert R, Denecker G, Goossens V, Van Loo G, Declercq W, et al. Inhibition of caspases increases the sensitivity of L929 cells to necrosis mediated by tumor necrosis factor. J Exp Med 1998;187:1477–85. [9] Holler N, Zaru R, Micheau O, Thome M, Attinger A, Valitutti S, et al. Fas triggers an alternative, caspase-8-independent cell death pathway using the kinase RIP as effector molecule. Nat Immunol 2000;1:489–95. [10] Degterev A, Huang Z, Boyce M, Li Y, Jagtap P, Mizushima N, et al. Chemical inhibitor of nonapoptotic cell death with therapeutic potential for ischemic brain injury. Nat Chem Biol 2005;1:112–9. [11] Degterev A, Hitomi J, Germscheid M, Ch’en IL, Korkina O, Teng X, et al. Identification of RIP1 kinase as a specific cellular target of necrostatins. Nat Chem Biol 2008;4:313–21. [12] He S, Wang L, Miao L, Wang T, Du F, Zhao L, et al. Receptor interacting protein kinase-3 determines cellular necrotic response to TNF-alpha. Cell 2009;137:1100–11. [13] Cho YS, Challa S, Moquin D, Genga R, Ray TD, Guildford M, et al. Phosphorylation-driven assembly of the RIP1-RIP3 complex regulates programmed necrosis and virus-induced inflammation. Cell 2009;137:1112–23. [14] Sun L, Wang H, Wang Z, He S, Chen S, Liao D, et al. Mixed lineage kinase domain-like protein mediates necrosis signaling downstream of RIP3 kinase. Cell 2012;148:213–27. [15] Wang Z, Jiang H, Chen S, Du F, Wang X. The mitochondrial phosphatase PGAM5 functions at the convergence point of multiple necrotic death pathways. Cell 2012;148:228–43. [16] Zhang DW, Shao J, Lin J, Zhang N, Lu BJ, Lin SC, et al. RIP3, an energy metabolism regulator that switches TNF-induced cell death from apoptosis to necrosis. Science 2009;325:332–6. [17] Kawahara A, Ohsawa Y, Matsumura H, Uchiyama Y, Nagata S. Caspaseindependent cell killing by Fas-associated protein with death domain. J Cell Biol 1998;143:1353–60. [18] Sawai H, Domae N. Discrimination between primary necrosis and apoptosis by necrostatin-1 in Annexin V-positive/propidium iodide-negative cells. Biochem Biophys Res Commun 2011;411:569–73.
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[19] Sawai H. Differential effects of caspase inhibitors on TNF-induced necroptosis. Biochem Biophys Res Commun 2013;432:451–5. [20] Eguchi Y, Shimizu S, Tsujimoto Y. Intracellular ATP levels determine cell death fate by apoptosis or necrosis. Cancer Res 1997;57:1835–40. [21] Zhang L, Shimizu S, Sakamaki K, Yonehara S, Tsujimoto Y. A caspase-8independent signaling pathway activated by Fas ligation leads to exposure of the Bak N terminus. J Biol Chem 2004;279:33865–74. [22] Sawai H, Domae N. Differential roles for Bak in Triton X-100- and deoxycholateinduced apoptosis. Biochem Biophys Res Commun 2009;378:529–33. [23] Cassidy-Stone A, Chipuk JE, Ingerman E, Song C, Yoo C, Kuwana T, et al. Chemical inhibition of the mitochondrial division dynamin reveals its role in Bax/Bak-dependent mitochondrial outer membrane permeabilization. Dev Cell 2008;14:193–204. [24] Cereghetti GM, Stangherlin A, Martins de Brito O, Chang CR, Blackstone C, Bernardi P, et al. Dephosphorylation by calcineurin regulates translocation of Drp1 to mitochondria. Proc Natl Acad Sci U S A 2008;105:15803–8. [25] Denecker G, Dooms H, Van Loo G, Vercammen D, Grooten J, Fiers W, et al. Phosphatidyl serine exposure during apoptosis precedes release of cytochrome c and decrease in mitochondrial transmembrane potential. FEBS Lett 2000;465:47–52. [26] Bossy-Wetzel E, Newmeyer DD, Green DR. Mitochondrial cytochrome c release in apoptosis occurs upstream of DEVD-specific caspase activation and independently of mitochondrial transmembrane depolarization. EMBO J 1998;17:37–49. [27] Desagher S, Osen-Sand A, Nichols A, Eskes R, Montessuit S, Lauper S, et al. Bid-induced conformational change of Bax is responsible for mitochondrial cytochrome c release during apoptosis. J Cell Biol 1999;144: 891–901. [28] Wei MC, Lindsten T, Mootha VK, Weiler S, Gross A, Ashiya M, et al. tBID, a membrane-targeted death ligand, oligomerizes BAK to release cytochrome c. Genes Dev 2000;14:2060–71. [29] Nakagawa T, Shimizu S, Watanabe T, Yamaguchi O, Otsu K, Yamagata H, et al. Cyclophilin D-dependent mitochondrial permeability transition regulates some necrotic but not apoptotic cell death. Nature 2005;434:652–8.
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Characterization of TNF-induced caspase-independent necroptosis Hirofumi Sawai ∗ Department of Internal Medicine, Osaka Dental University, Osaka, Japan
a r t i c l e
i n f o
Article history: Received 17 October 2013 Received in revised form 21 January 2014 Accepted 1 February 2014 Available online 10 February 2014 Keywords: Necroptosis Apoptosis TNF Caspase ATP
a b s t r a c t Caspase-independent programmed necrotic cell death (necroptosis) has recently been described. Previously described models of necroptosis required 16 h or more of induction, which made the interpretation of findings somewhat difficult. In human monocytic leukemia cell line U937 necroptosis could be induced within 6 h by combination of TNF and Z-VAD-fmk. Here we show that the reduction in intracellular ATP levels may not be the sole determinant of necroptosis, and that necroptosis is associated with the loss of mitochondrial membrane potential, but not the activation of Bak/Bax or calcineurin. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction Apoptosis and necrosis are two major forms of cell death with distinguishing morphological features [1–3]. Apoptosis is characterized by cell shrinkage, fragmented nuclei with condensed chromatin, and formation of apoptotic bodies, whereas necrosis is characterized by swelling of cells and organelles and loss of plasma membrane integrity. The mechanisms of apoptosis have been investigated extensively. The two major pathways leading to apoptosis have been established: the death-receptor (or extrinsic) pathway and mitochondrial (or intrinsic) pathway [4,5]. The death-receptor pathway was shown to be triggered by death ligands including Fas (CD95) ligand. Binding of Fas ligand to its receptor Fas induces the formation of death-inducing signaling complex composed of FADD and procaspase-8, resulting in caspase-8 activation through induced proximity. Previous studies demonstrated that active caspase-8 either directly cleaved procaspase-3 to generate active caspase-3 or indirectly activated caspase-3 via the mitochondrial pathway by cleaving Bid [6,7]. The mitochondrial pathway is generally used in response to genotoxic stresses including irradiation and anticancer drugs. The activation of proapoptotic Bcl-2 family members including Bax and Bak was reported to lead to the release of cytochrome c from the intermembrane space of mitochondria to the cytosol. Cytochrome c associates with Apaf-1 and procaspase-9 to form
∗ Tel.: +81 72 864 3079; fax: +81 72 864 3179. E-mail address: [email protected] http://dx.doi.org/10.1016/j.leukres.2014.02.002 0145-2126/© 2014 Elsevier Ltd. All rights reserved.
the complex termed an apoptosome, resulting in the activation of caspase-9, which proteolytically activates executioner caspases including caspase-3, -6, and -7. In addition to post-apoptotic secondary necrosis, caspaseindependent necrosis has also been reported [3,8]. The involvement of RIP1 kinase in Fas-induced caspase-8-independent necrotic cell death has been demonstrated [9]. Specific inhibitors of caspaseindependent necrosis, necrostatins, have recently been identified, and necrostatin-inhibitable caspase-independent necrosis was referred to as necroptosis [10]. Necrostatins have been shown to inhibit the kinase activity of RIP1 [11], which interacts with RIP3 [12,13]. The signaling pathways leading to necroptosis downstream of RIP1 and RIP3 have recently been elucidated. The RIP1/RIP3 complex activates MLKL, which subsequently activates PGAM5 by phosphorylation, resulting in the dephosphorylation of Drp1. Dephosphorylated Drp1 promotes mitochondrial fission, leading to necrotic cell death [14,15]. Although the enzymes involved in energy metabolism have been suggested to play a crucial role in necroptosis [16], the precise relationship between mitochondrial fission and necroptosis remains to be determined. In previous reports necroptosis was induced at 16 h or later [10,11], which made the interpretation of the data somewhat difficult. In the present study, we investigated various molecular events including a reduction in intracellular ATP levels, conformational changes in Bax/Bak, and the loss of mitochondrial membrane potential (m) during necroptosis in comparison with apoptosis using U937 human monocytic leukemia cell line, in which necroptosis could be induced within 6 h by TNF in the presence of Z-VAD-fmk.
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Fig. 1. TNF-induced caspase-independent necroptosis in U937 cells. (A and B) U937 cells were treated with 10 ng/ml TNF (T) and 10 g/ml cycloheximide for the indicated times, and dual staining with Annexin V-FITC/PI was performed. As indicated, cells were treated in the presence of 10 M Z-VAD-fmk (V) or 50 M Z-Asp-CH2 -DCB (Asp). Representative dot plots are shown in (A). (C) When U937 cells were treated with 10 M necrostatin-1 (N), TNF/ZVAD-induced necrosis was completely inhibited whereas TNF-induced apoptosis was only slightly affected. The data shown are the average of three independent experiments. The bars indicate standard deviations. AnV-PI+, Annexin V− /PI+ (late necrosis); AnV + PI+, Annexin V+ /PI+ (necrosis); AnV + PI-, Annexin V+ /PI− (early apoptosis or necrosis).
2. Materials and methods 2.1. Cell culture Human monocytic leukemia cell line U937 cells and human T cell leukemia cell line Jurkat cells were purchased from RIKEN Cell Bank (Japan), and human myelocytic leukemia cell line HL60 cells were purchased from JCRB Cell Bank (Japan). Caspase-8-deficient Jurkat (JB6) cells were provided by Dr. S. Nagata [17]. Cells were maintained in RPMI1640 medium supplemented with 10% heat-inactivated fetal bovine serum and 100 U/ml penicillin/100 g/ml streptomycin in a humidified 5% CO2 incubator at 37 ◦ C. 2.2. Assessment of apoptosis and necroptosis To induce caspase-independent necroptosis, U937 cells were pretreated with 10 M Z-VAD-fmk (Peptide Institute, Japan) for 30 min and were then treated with 10 ng/ml TNF (Wako Pure Chemical Industries, Japan) plus 10 g/ml cycloheximide (Sigma) for the indicated times. Cells were incubated with 2.5 l FITC-conjugated Annexin V (MBL, Japan) and 2.5 g/ml propidium iodide (Sigma) in 100 l binding
buffer containing 10 mM Hepes/KOH (pH 7.4), 140 mM NaCl, and 2.5 mM CaCl2 for 30 min at 4 ◦ C, and were then analyzed using FACS Calibur (BD Biosciences). We recently reported that early necroptotic cells were Annexin V+ /PI− before becoming PI+ , which could be almost completely inhibited by necrostatin-1 [18,19]. On the other hand, Annexin V+ /PI− early apoptotic cells were only slightly affected by necrostatin-1. Agonistic anti-Fas antibody (clone CH-11) (MBL, Japan) was used instead of TNF in some experiments. 2.3. ATP depletion Cells for ATP depletion experiments were transferred to RPMI1640 medium without glucose (GIBCO) supplemented with 0.5% bovine albumin instead of fetal bovine serum, and 10 g/ml oligomycin (Wako Pure Chemical Industries, Japan) was then added to these cells [20]. 2.4. Inhibition of necroptosis by necrostatin-1 To inhibit necroptosis, 10 M necrostatin-1 (Sigma) was added to cells 30 min before the addition of TNF.
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Fig. 2. Induction of necroptosis in U937, Jurkat, and HL60 cells. (A) U937 cells were treated with 100 ng/ml agonistic anti-Fas antibody (F) plus 10 g/ml cycloheximide for 8 h. As indicated, cells were treated in the presence of 10 M Z-VAD-fmk (V) or 10 M necrostatin-1 (N). (B) HL60 cells were treated with 20 ng/ml TNF (T) plus 10 g/ml cycloheximide for 8 h. As indicated, cells were treated in the presence of 10 M Z-VAD-fmk (V) or 10 M necrostatin-1 (N). (C) Jurkat cells were treated with agonistic 100 ng/ml agonistic anti-Fas antibody (F) or 20 ng/ml TNF (T) plus 10 g/ml cycloheximide for 8 h. As indicated, cells were treated in the presence of 10 M Z-VAD-fmk (V) or 10 M necrostatin-1 (N). (D) Caspase-8-deficient Jurkat (JB6) cells were treated with 20 ng/ml TNF (T) plus 10 g/ml cycloheximide for 8 h. As indicated, cells were treated in the presence of 10 M Z-VAD-fmk (V) or 10 M necrostatin-1 (N). Dual staining with Annexin V-FITC/PI was performed as described above. The data shown are the average of three independent experiments. The bars indicate standard deviations. AnV-PI+, Annexin V− /PI+ (late necrosis); AnV + PI+, Annexin V+ /PI+ (necrosis); AnV + PI-, Annexin V+ /PI− (early apoptosis or necrosis).
2.5. Inhibition of mitochondrial fission by mdivi-1 Mdivi-1 (Sigma) was used as an inhibitor of Drp1, which plays a crucial role in mitochondrial fission. To inhibit mitochondrial fission, 50 or 100 M mdivi-1 was added to cells 30 min before the addition of TNF.
2.6. Inhibition of calcineurin To inhibit calcineurin, 10 M cyclosporine A (Wako Pure Chemical Industries, Japan) or 1 M FK506 (Alexis) was added to cells simultaneously with the addition of TNF.
Measurement Reagent for Cells for 10 min at room temperature. Luminescence was measured using SpectraMax M5 (Molecular Devices). 2.8. FACS analysis for conformational changes in Bak and Bax Conformational changes in Bak and Bax were analyzed as previously described [21,22]. Briefly, cells were fixed in 10% paraformaldehyde in PBS for 10 min at 4 ◦ C followed by permeabilization with 0.1% saponin in PBS for 5 min at room temperature. Cells were then incubated with anti-Bak (TC100) (Calbiochem) and anti-Bax (6E7) (Calbiochem) antibody, which reacts with the active conformation of Bak and Bax, respectively, for 30 min at 4 ◦ C. After washing with PBS, cells were incubated with FITC-conjugated anti-mouse IgG antibody (Santa Cruz biotechnology) for 30 min at 4 ◦ C. These cells were then analyzed using FACS Calibur (BD Biosciences).
2.7. Measurement of intracellular ATP levels 2.9. Analysis of m and externalization of phosphatidylserine Intracellular ATP levels were measured using ATP Measurement Reagent for Cells (TOYO Ink, Japan), which contains luciferin and luciferase. According to the manufacturer’s instructions, cells (50 l) were incubated with 50 l ATP
Cells were incubated with 100 nM CMXRos (Molecular Probes) for the last 15 min of the treatment at 37 ◦ C. Cells were then incubated with 2.5 l FITC-conjugated
H. Sawai / Leukemia Research 38 (2014) 706–713
2.10. Statistical analysis One-way ANOVA and post hoc analysis was performed using IBM SPSS Statistics Version 21.0.
3. Results
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3.1. Induction of caspase-independent necrostatin-inhibitable necroptosis in U937 as well as Jurkat and HL60 cells
3.2. Induction of necroptosis in caspase-8-deficient Jurkat (JB6) cells We previously reported that the inhibition of caspase-8 by ZVAD may play a crucial role in necroptosis [19]. To further confirm this, we used caspase-8 deficient Jurkat (JB6) cells [17]. When JB6 cells were treated with TNF/ZVAD, necroptosis was induced in JB6 cells similar to that in Jurkat cells (Fig. 2D). In contrast to Jurkat cells, in which apoptosis was induced by TNF, necroptosis was induced by TNF in JB6 cells. 3.3. Necrostatin-1 did not inhibit ATP-depletion-induced necrosis We investigated the role of ATP depletion in apoptosis and necroptosis in U937 cells. In TNF-induced apoptosis, a slight and an approximately 80% reduction in intracellular ATP levels was detected at 2 h and 4 h, respectively (Fig. 3A). In contrast, while no reduction in intracellular ATP levels was detected at 2 h, a slight
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As we recently reported [19,22], caspase-independent necrosis was induced when human monocytic leukemia U937 cells were treated with TNF in the presence of Z-VAD-fmk (ZVAD), a pan-caspase inhibitor (Fig. 1A and B). Although an increase in Annexin V− or PI+ cells was not detected at 2 h, approximately 20% and 50% of cells became PI+ at 4 h and 6 h, respectively. In contrast, when U937 cells were treated with TNF in the presence of Z-Asp-CH2 -DCB (ZAsp), a caspase-3-specific inhibitor [19], no increase in Annexin V+ cells was observed at 4 h and only a slight increase was detected at 6 h. As shown in Fig. 1C, the pretreatment with 10 M necrostatin-1 completely inhibited TNF/ZVAD-induced necrotic cell death, which indicated that necroptosis was induced by TNF/ZVAD in U937 cells. On the other hand, TNF-induced cell death was genuine apoptosis since it was not apparently inhibited by necrostatin-1. We then investigated whether necroptosis could be induced by treating U937 cells with agonistic anti-Fas antibody instead of TNF. When cells were treated for 8 h with agonistic anti-Fas antibody (clone CH-11) in the presence of ZVAD, approximately 20% of cells became PI+ , which were then completely inhibited by necrostain-1 (Fig. 2A). We then investigated whether necroptosis could be induced in cells other than U937 cells. When human myelocytic leukemia HL60 cells were treated with TNF/ZVAD, an increase of PI+ cells was observed within 8 h, which was inhibited by necrostatin1 (Fig. 2B). In contrast, necrostain-1 did not apparently inhibit TNF-induced apoptosis in HL60 cells (data not shown). When human T cell leukemia Jurkat cells were treated with agonistic anti-Fas antibody, typical apoptotic morphological changes were induced within 2 h, which was completely inhibited by pretreatment with ZVAD at 2 h. However, necrotic changes were observed in Fas/ZVAD-treated Jurkat cells after 8 h, which was attenuated by necrostatin-1 (Fig. 2C). Necroptosis was also induced by TNF/ZVAD in Jurkat cells (Fig. 2C). Necrostatin-1 did not apparently inhibit Fasor TNF-induced apoptosis in Jurkat cells (data not shown).
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Fig. 3. Reduction in intracellular ATP levels in TNF-induced apoptosis and necroptosis. (A) U937 cells were treated with 10 ng/ml TNF (T) plus 10 g/ml cycloheximide in the presence or absence of 10 M Z-VAD-fmk (V) or 50 M Z-Asp-CH2 -DCB (Asp) for the indicated times. Intracellular ATP levels were measured as described in Section 2. The data shown are the average of a triplicate experiment. The bars indicate standard deviations. Similar results were obtained in three independent experiments. (B) U937 cells were pretreated with or without 10 M necrostatin-1 for 30 min. Cells were then treated with 10 ng/ml TNF (T) plus 10 g/ml cycloheximide in the presence or absence of 10 M Z-VAD-fmk (V) or 50 M Z-Asp-CH2 -DCB (Asp) for 6 h, and intracellular ATP levels were measured. The data shown are the average of a triplicate experiment. The bars indicate standard deviations.
and a more than 80% reduction was observed at 4 h and 6 h, respectively, in TNF/ZVAD-induced necroptosis (Fig. 3A). In the presence of ZAsp, no reduction in ATP levels was observed within 4 h, and a slight reduction was detected at 6 h (Fig. 3A). Necrostatin-1 only slightly attenuated the reduction in ATP levels in TNF-induced apoptosis, whereas necrostatin-1 almost completely suppressed this reduction in TNF/ZVAD-induced necroptosis (Fig. 3B). We then examined whether ATP depletion itself could induce necroptosis. As was previously reported [20], treating U937 cells with oligomycin, an inhibitor of F0/F1 ATP synthase, in the absence of glucose rapidly reduced intracellular ATP levels to approximately 20% at 0.5 h and less than 10% after 2 h (Fig. 4A). Under these conditions approximately 20% and 60% of cells became PI+ at 5 h and 6 h, respectively (Fig. 4B). Necrostatin-1 did not affect necrotic cell death induced by ATP depletion (Fig. 4C).
3.4. Conformational changes in Bak and Bax during apoptosis and necroptosis We investigated the involvement of Bak and Bax in necroptosis. As we and others previously reported [21,22], conformational changes in Bak and Bax were induced during TNF-induced apoptosis, while a conformational change in Bak was detected in approximately 60% of cells and that in Bax was detected in 10% only (Fig. 5). ZVAD significantly (p < 0.05) inhibited TNF-induced conformational changes in Bak and Bax. In contrast, ZAsp significantly (p < 0.05) augmented a conformational change in Bax and also slightly (but not significantly) augmented that in Bak.
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Calcineurin was previously shown to be involved in Drp1 activation and mitochondrial fission induced by mitochondrial depolarization [24]. To investigate the involvement of calcineurin in TNF/ZVAD-induced necroptosis, the effects of calcineurin inhibitors (cyclosporine A and FK506) on TNF/ZVAD-induced necroptosis were examined (Fig. 7). Cyclosporine A, which binds to cyclophilin and thereby inhibits calcineurin, significantly (p < 0.05) reduced TNF/ZVAD-induced Annexin V− /PI+ (late necroptotic) cells. However, FK506, another calcineurin inhibitor, did not apparently inhibit necroptosis. These results indicated that calcineurin may not play an important role in TNF/ZVAD-induced necroptosis, and suggest that other mechanisms such as the loss of m might be involved in necroptosis. 3.7. Loss of m in TNF/ZVAD-induced necroptosis
Fig. 4. Induction of necrosis by ATP depletion. (A) U937 cells were incubated in serum-free medium with or without glucose (G) in the presence or absence of 10 g/ml oligomycin (O) for the indicated times. Intracellular ATP levels were measured as described above. The data shown are the average of a triplicate experiment. The bars indicate standard deviations. Similar results were obtained in three independent experiments. (B) U937 cells were incubated in serum-free medium without glucose in the presence of 10 g/ml oligomycin for the indicated times. Dual staining with Annexin V-FITC/PI was performed. (C) U937 cells were incubated in serumfree medium with or without glucose (G) in the presence or absence of 10 g/ml oligomycin (O) or 10 M necrostatin-1 (N) for 6 h, and dual staining with Annexin V-FITC/PI was performed. The data shown are the average of three independent experiments. The bars indicate standard deviations. AnV-PI+, Annexin V− /PI+ (late necrosis); AnV + PI+, Annexin V+ /PI+ (necrosis); AnV + PI-, Annexin V+ /PI− (early apoptosis or necrosis).
3.5. Effects of mdivi-1, an inhibitor of Drp1, on apoptosis and necroptosis
We examined the changes in m compared with the externalization of phosphatidylserine (PS) during apoptosis and necroptosis
(%) 100 80 60
AnV-PI+ AnV+PI+ AnV+PI-
40 20 0
Mdivi-1, an inhibitor of Drp1, was recently reported to protect cells from necroptosis [15]. Since mitochondrial fission was also shown to be involved in apoptosis [23], we investigated the effects of mdivi-1 on TNF-induced apoptosis and TNF/ZVADinduced necroptosis in U937 cells (Fig. 6). Although mdivi-1 did not apparently affect TNF-induced apoptosis at 50 M, it markedly inhibited TNF-induced apoptosis at 100 M. In contrast, mdivi1 markedly suppressed TNF/ZVAD-induced necroptosis even at 50 M and almost completely inhibited necroptosis at 100 M.
Fig. 6. Effects of mdivi-1 on TNF-induced apoptosis and necroptosis. U937 cells were pretreated with 50 or 100 M mdivi-1 (m) for 30 min. Cells were then treated with 10 ng/ml TNF (T) plus 10 g/ml cycloheximide in the presence or absence of 10 M Z-VAD-fmk (V) for 6 h. Dual staining with Annexin V-FITC/PI was performed. The data shown are the average of three independent experiments. The bars indicate standard deviations. AnV-PI+, Annexin V− /PI+ (late necrosis); AnV + PI+, Annexin V+ /PI+ (necrosis); AnV + PI-, Annexin V+ /PI− (early apoptosis or necrosis).
H. Sawai / Leukemia Research 38 (2014) 706–713
(%) 100
∗
80
ns
60
AnV-PI+ AnV+PI+
40
AnV+PI-
20 0 (-)
Cs
F
TV
CsTV FTV
Fig. 7. Effects of calcineurin inhibitors on TNF-induced necroptosis. U937 cells were treated with 10 ng/ml TNF (T) plus 10 g/ml cycloheximide in the presence or absence of 10 M Z-VAD-fmk (V) for 6 h. As indicated, cells were treated with 10 M cyclosporine A (Cs) or 1 M FK506 (F). Dual staining with Annexin V-FITC/PI was performed. The data shown are the average of four independent experiments. The bars indicate standard deviations. AnV-PI+, Annexin V− /PI+ (late necrosis); AnV + PI+, Annexin V+ /PI+ (necrosis); AnV + PI-, Annexin V+ /PI− (early apoptosis or necrosis). *, p < 0.05; ns, no significance compared with TNF plus Z-VAD-fmk.
using dual staining with Annexin V-FITC/CMXRos [25]. As previously reported [26], the loss of m was detected in TNF-induced apoptosis as well as TNF/ZVAD-induced necroptosis (Fig. 8A). Kinetic studies revealed that the loss of m was induced almost in parallel to PS externalization with slower kinetics in TNF/ZVAD-induced necroptosis than in TNF-induced apoptosis (Fig. 8B). Thus, early apoptotic and necroptotic cells became Annexin V+ /CMXRoslow (the lower right quadrant), whereas late necroptotic cells became Annexin V− /CMXRoslow (the lower left
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quadrant). Necrostatin-1 almost completely attenuated the loss of m in TNF/ZVAD-induced necroptosis, whereas it only slightly affected in TNF-induced apoptosis (Fig. 8C). Since cyclosporine A inhibited TNF/ZVAD-induced late necroptosis (Fig. 7), we investigated the effect of cyclosporine A on m in necroptosis. As shown in Fig. 8D, cyclosporine A significantly (p < 0.05) reduced Annexin V− /CMXRoslow late necroptotic cells. 4. Discussion Necroptosis has recently been established owing to the identification of necrostatins as its specific inhibitors. However, necroptosis was observed at 16 h or later in previous reports, which made the interpretation of the data somewhat difficult. For example, necrosis secondary to apoptosis may have contributed to delayed necrotic cell death. In the present study, we used U937 cells, in which TNF-induced caspase-independent necrostatininhibitable necroptosis could be induced within 6 h. As shown in Fig. 2B and C, cell lines other than U937 were markedly less sensitive to necroptosis. More than 50% of U937 cells became PI+ after being treated with TNF/ZVAD for 6 h, whereas less than 20% of HL60 and Jurkat cells became PI+ 8 h after treatment with Fas/ZVAD or TNF/ZVAD. Agonistic anti-Fas antibody was less potent than TNF in inducing necroptosis in U937 cells since less than 20% of cells became PI+ when treated with Fas/ZVAD for 8 h (Fig. 2A). These results indicated that U937 cells were very sensitive to TNF/ZVADinduced necroptosis. We previously reported that the inhibition of caspase-8 by ZVAD may play a crucial role in necroptosis, whereas necroptosis was not induced by the inhibition of caspase-3 by ZAsp [18,19]. This may have been because caspase-8 cleaves and inhibits RIP1, thereby preventing necroptosis. Caspase-8-deficient Jurkat (JB6) cells were used to confirm that caspase-8 played an inhibitory
Fig. 8. Loss of m in TNF-induced apoptosis and necroptosis. (A and B) U937 cells were treated with 10 ng/ml TNF (T) plus 10 g/ml cycloheximide in the presence or absence of 10 M Z-VAD-fmk (V) for the indicated times. Dual staining with Annexin V-FITC/CMXRos was performed as described in Section 2. Representative dot plots are shown in (A). (C) U937 cells were treated with 10 ng/ml TNF (T) plus 10 g/ml cycloheximide in the presence or absence of 10 M Z-VAD-fmk (V) or 10 M necrostatin-1 (N) for 6 h, and dual staining with Annexin V-FITC/CMXRos was performed. (D) U937 cells were treated with 10 ng/ml TNF (T) plus 10 g/ml cycloheximide in the presence of 10 M Z-VAD-fmk (V) for 6 h. As indicated, cells were treated with 10 M cyclosporine A (Cs) or 1 M FK506 (F). Dual staining with Annexin V-FITC/CMXRos was performed. *, p < 0.05; ns, no significance compared with TNF plus Z-VAD-fmk. The data shown are the average of at least three independent experiments. The bars indicate standard deviations. AnV-Xros-, Annexin V− /CMXRoslow (late necrosis); AnV + XRos-, Annexin V+ /CMXRoslow (early apoptosis or necrosis); AnV + XRos+, Annexin V+ /CMXRoshigh (very early apoptosis or necrosis); AnV-XRos+, Annexin V− /CMXRoshigh (normal).
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H. Sawai / Leukemia Research 38 (2014) 706–713
role in necroptosis (Fig. 2D). Necroptosis was induced by TNF even in the absence of ZVAD in JB6 cells, which further suggested that caspase-8 may inhibit TNF-induced necroptosis. ATP has been reported to play a crucial role in determining whether cells die by apoptosis or necrosis [20]. As shown in Fig. 3A, the reduction in intracellular ATP levels was more rapidly induced in apoptosis than in necroptosis. Although intracellular ATP levels were almost the same between apoptosis and necroptosis at 6 h, approximately 50% of cells became PI+ in necroptosis at 6 h, while less than 5% became PI+ in apoptosis (Fig. 1B). Furthermore, incubating cells in glucose-free medium in the presence of oligomycin reduced intracellular ATP levels to less than 10% at 2 h, although an increase in PI+ cells was not detected within 3 h (Fig. 4A and B). As shown in Fig. 4C, necrosis induced by ATP depletion was not affected by necrostatin-1, which indicated that ‘classical’ necrotic cell death, but not necroptosis was induced by ATP depletion. These results suggest that intracellular ATP levels may not be the sole determinant of necroptosis. Bak and Bax, proapoptotic Bcl-2 family members, have been shown to play crucial roles in the mitochondrial apoptotic pathway [4,5]. The activation of Bak and Bax with conformational changes in apoptosis induced by various stimuli including staurosporine, agonistic anti-Fas antibody, and anticancer drugs has been reported previously [21,22]. As shown in Fig. 5, conformational changes in Bak and, to a lesser extent, Bax were detected in TNF-induced apoptosis. Conformational changes in Bak and Bax were attenuated in the presence of ZVAD, which suggested that the activation of Bak/Bax may not play a crucial role in TNF/ZVAD-induced necroptosis. Moreover, conformational changes in Bak/Bax were augmented in the presence of ZAsp, indicating that ZAsp may inhibit apoptosis downstream of Bak/Bax activation. Since ZAsp is a caspase-3specific inhibitor while ZVAD inhibits both caspase-8 and caspase-3 [19], these results suggest that caspase-8 and caspase-3 may play a stimulatory and inhibitory role, respectively, in Bak/Bax activation. In accordance with these results, tBid, the cleavage product of Bid by caspase-8, was shown to induce Bak/Bax activation with conformational changes [27,28]. The RIP1/RIP3 complex was recently shown to activate MLKL/PGAM5 in necroptosis, resulting in the dephosphorylation of Drp1, which subsequently promoted mitochondrial fission [14,15]. Mdivi-1 has been identified as a specific Drp1 inhibitor [23]. As previously reported, mdivi-1 inhibited both TNF-induced apoptosis and TNF/ZVAD-induced necroptosis (Fig. 6). Since TNF/ZVADinduced necroptosis was almost completely inhibited whereas TNF-induced apoptosis was only partially inhibited by 100 M mdivi-1, mitochondrial fission may play a more important role in necroptosis than in apoptosis. Calcineurin has been reported to be involved in Drp1 activation and mitochondrial fission induced by mitochondrial depolarization [24]; therefore, we investigated whether calcineurin may play a role in necroptosis. As shown in Fig. 7, cyclosporine A slightly inhibited TNF/ZVAD-induced necroptosis, whereas FK506 did not apparently affect necroptosis. Since cyclosporine A binds to cyclophilin and thereby inhibits calcineurin, it may block the loss of m, in which cyclophilin D is involved [29]. In contrast, FK506 inhibits calcineurin by binding to FKBP12 and has no effect on m [24]. These results indicate that calcineurin may not play an important role in TNF/ZVAD-induced necroptosis, and suggest that cyclosporine A might partly inhibit necroptosis by blocking the loss of m. Therefore, we examined the changes in m in TNF-induced apoptosis and necroptosis (Fig. 8A and B). The results indicated that although the loss of m was detected in both apoptosis and necroptosis, it occurred with slower kinetics in necroptosis than in apoptosis. Necrostatin-1 almost completely attenuated the loss of m induced by TNF/ZVAD (Fig. 8C), which confirmed that the
Fig. 9. Mechanisms of TNF-induced apoptosis and necroptosis. TNF-induced apoptosis inhibits the necroptotic signaling pathway through cleavage of RIP1 by the active caspase-8. Z-VAD-fmk inhibits caspase-8, thereby preventing apoptosis and facilitating necroptosis. Z-Asp-CH2 -DCB prevents apoptosis without inducing necroptosis by inhibiting caspase-3, but not caspase-8. Necrostatin-1 prevents necroptosis by inhibiting kinase activity of RIP1. Bak/Bax activation is inhibited by Z-VAD-fmk, but not by Z-Asp-CH2 -DCB. Mdivi-1, an inhibitor of Drp1, prevents necroptosis more effectively than apoptosis. Cyclosporine A partly prevents late necroptosis by attenuating the loss of m. The reduction in intracellular ATP levels seems to be a late event in both apoptosis and necroptosis.
loss of m was induced in necroptosis and not by TNF per se. Furthermore, we investigated the effect of cyclosporine A on the loss of m in necroptosis. As shown in Fig. 8D, cyclosporine A partially attenuated the loss of m in necroptosis, suggesting that cyclosporine A may partly inhibit TNF/ZVAD-induced necroptosis by blocking the loss of m. In conclusion, we investigated various molecular events during necroptosis in comparison with apoptosis using U937 human monocytic leukemia cell line, in which necroptosis could be induced within several hours by TNF in the presence of ZVAD. The results obtained suggest that a reduction in intracellular ATP levels may not be the sole determinant of necroptosis, and that the loss of m, but not activation of Bak/Bax or calcineurin may be involved in necroptosis (Fig. 9). Conflict of interest statement The author declares that he has no competing interests. Acknowledgment Author’s contributions: H.S. designed and performed experiments, analyzed data and wrote the paper. Funding: This work was supported in part by Grant-in-Aid for Scientific Research (C) (25462941) from Japan Society for the Promotion of Science. References [1] Kerr JFR, Wyllie AH, Currie AR. Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer 1972;26:239–57. [2] Shimizu S, Eguchi Y, Kamiike W, Waguri S, Uchiyama Y, Matsuda H, et al. Retardation of chemical hypoxia-induced necrotic cell death by Bcl-2 and ICE inhibitors: possible involvement of common mediators in apoptotic and necrotic signal transductions. Oncogene 1996;12:2045–50. [3] Vercammen D, Brouckaert G, Denecker G, Van de Craen M, Declercq W, Fiers W, et al. Dual signaling of the Fas receptor: initiation of both apoptotic and necrotic cell death pathways. J Exp Med 1998;188:919–30. [4] Scaffidi C, Fulda S, Srinivasan A, Friesen C, Li F, Tomaselli KJ, et al. Two CD95 (APO-1/Fas) signaling pathways. EMBO J 1998;17:1675–87. [5] Hengartner MO. The biochemistry of apoptosis. Nature 2000;407:770–6. [6] Luo X, Budihardjo I, Zou H, Slaughter C, Wang X. Bid, a Bcl2 interacting protein, mediates cytochrome c release from mitochondria in response to activation of cell surface death receptors. Cell 1998;94:481–90.
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