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DISULFIRAM INHIBITS TNF-α-INDUCED CELL DEATH
doi:10.1006/cyto.2000.0725, available online at http://www.idealibrary.com on
DISULFIRAM INHIBITS TNF--INDUCED CELL DEATH Aiping Zhao, Zheng-Qi Wu,* Matthew Pollack, Florence M. Rollwagen,* Przemyslaw Hirszel, Xiaoming Zhou Disulfiram, a clinically employed alcohol deterrent, was recently discovered to inhibit caspase-3 and DNA fragmentation. Using LLC-PK1 cells and murine liver as models, we examined if the drug inhibited TNF--induced cell death. Disulfiram produced dose-dependent inhibition of TNF--induced cell death as well as caspase-3-like activity. Disulfiram retained 80% of its effect when added 4 h after TNF-. Disulfiram protected the cells from cytokine-induced death for at least 6 days. The cells rescued by the drug preserved the ability to proliferate. The cells died spontaneously after exposure to TNF- for just 70 min. Co-administration of 15 M disulfiram and TNF- for 70 min prior to their removal abolished TNF--induced killing, and this was associated with restoration of mitochondrial membrane potential and suppression of reactive oxygen species. Treatment of mice with TNF- and D-galactosamine for 5 h markedly increased hepatic DNA fragmentation and caspase-3-like activity. Disulfiram at 0.6 mmol/kg abolished these effects. We conclude that disulfiram is a potent inhibitor of TNF--induced cell death in vitro. The underlying mechanisms include stabilization of mitochondrial membrane potential, suppression of reactive oxygen species, and inhibition of caspase-3-like activity. We further conclude that disulfiram inhibits DNA fragmentation in vivo in association with the blockade of caspase-3-like activity.
As the molecular mechanisms underlying apoptosis are unraveled, it becomes increasingly clear that caspases play a fundamental role in this type of programmed cell death. The caspases comprise a growing family of aspartic acid-specific, cysteine proteases.1,2 The caspases are activated through a proteolytic cascade that serves to transmit and amplify death signals. This family of proteases can be divided into apoptosis initiators such as caspase-8, 9 and 10 that participate in transduction of death signals, and apoptosis effectors such as caspase-3, 6 and 7, or caspase-3-like proteases, From the Departments of Medicine and *Pathology, Uniformed Services University, Bethesda, MD 20814, USA Correspondence to: Dr Xiaoming Zhou, Department of Medicine, Uniformed Services University, 4301 Jones Bridge Road, Bethesda, MD 20814, USA. E-mail: [email protected] Received 8 February 2000; received in revised form 6 April 2000; accepted for publication 24 April 2000 1043–4666/00/091356+12 $35.00/0 KEY WORDS: caspase-3/reactive oxygen species/apoptosis/LLCPK1 cells/liver Abbreviations: Ac-DEVD-AMC: Ac-Asp-Glu-Val-Asp-7-amino-4methylcoumarin; BAF: boc-aspartyl (OMe)-fluoromethylketone; DCFH-DA: 2 , 7 - dichlorofluorescin diacetate; JC-1: 5, 5 , 6, 6 -tetrachloro-1, 1 , 3, 3 -tetraethylbenzimidazolyl-carbocyanine iodide; MTT: 3-(4,5)-dimethylthiazol-2-yl)-2,5-diphenyltetrozolium bromide; TUNEL: terminal deoxynucleotidyl transferasemediated dUTP nick end labeling; ZVAD.fmk: Z-Val-Ala-Asp (OMe)-fluoromethylketone 1356
which execute death signals. Among the apoptotic effectors, caspase-3 is the best characterized. The proteolytic functions of this enzyme are largely responsible for classical manifestation of apoptosis. Inhibition of caspase-3 usually abrogates apoptosis.1–6 Due to the complex relationship between apoptosis and cell death, however, a caspase inhibitor often fails to block and sometimes even enhances cell death.7,8 For example, Vercammen et al. have demonstrated that inhibition of caspases increased the sensitivity of a murine fibrosarcoma cell line to TNF--induced death by 1000-fold.9 This was due mainly to an increase in the production of reactive oxygen species. The similar phenomenon was also observed in the human neutrophils,10 NIH 3T3 fibroblasts and U937 leukemic cells.11 Free oxygen radicals are generated continuously as side products of electron transfer reactions. Superoxide and hydroxyl radicals, collectively called reactive oxygen species, are the major free radical species involved in biological processes. Cells have a welldeveloped antioxidant system, thus minimizing the detrimental effects of free radicals. Apoptosis or necrosis occurs only when the increased generation of free radicals overwhelms the capacity of the antioxidant mechanisms, and/or when the defense mechanisms are compromised. TNF-, for example, stimulates CYTOKINE, Vol. 12, No. 9 (September), 2000: pp 1356–1367
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Disulfiram inhibits TNF--induced death / 1357
Figure 1. Disulfiram inhibited TNF- (30 ng/ml)-induced caspase-3-like activity (A) and cell death (B) in a dose-dependent manner. Disulfiram (15 M) was still able to rescue LLC-PK1 cells even when it was added after TNF- (C). Disulfiram up to, at least, 80 M had no significant effect on cell viability and did not stimulate the caspase-3-like activity (D). The caspase-3-like activity was measured 2 h post treatment by DEVD-CHO-sensitive hydrolysis of Ac-DEVD-AMC. The cell viability was measured by crystal violet assays 24 h post treatment. Disulfiram was added either simultaneously with TNF- (30 ng/ml) or hours after TNF-, and the cells were incubated up to 24 h (C). Each point represents the average of four (A) or three (B and C) experiments. *P<0.01 as compared with no disulfiram (control) (ANOVA).
caspase-8, which in turn cleaves protein Bid resulting in functional damage to mitochondria and overproduction of reactive oxygen species.12,13 Most, but not all in vitro data are consistent with the notion that TNF- activates caspase-3, at least in part, via increases in the levels of reactive oxygen species. The inhibition of caspase-3 stimulates the production of free radicals through a yet defined mechanism.9–11 Disulfiram is a member of the dithiocarbamate family comprising a broad class of molecules possessing an R1R2NC(S)SR3 functional group, which enables them to react with sulfhydryl groups. Recent biochemi-
cal studies have demonstrated that disulfiram inhibits processing of pro-caspase-3 and -1, and suppresses active caspase-3 through a direct thiol–disulfide interaction with the cysteine residues in these caspases. The blockade of caspase-3 by disulfiram in Jurkat T lymphocytes and rat thymocytes is associated with inhibition of DNA fragmentation, a hallmark of apoptosis.14,15 It is unknown, however, whether the abrogation of apoptosis translates to an increase in cell survival. In the present in vitro and in vivo study we sought to ascertain whether disulfiram would decrease cell death induced by TNF-, a cytokine
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pathogenetically linked to a variety of diseases and disease models,16 and whether the drug expresses other cytoprotective mechanisms in addition to inhibition of caspase-3.
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Disulfiram is a potent inhibitor of caspase-3-like activity and cell death in vitro Disulfiram inhibited TNF--induced caspase-3like activity in a dose-dependent manner (Fig. 1A). The drug at 5 M completely suppressed caspase-3like activity (9.61.3 pmol/min/105 cells, TNF-; 0.80.1 pmol/min/105 cells, disulfiram; and 1.0 0.2 pmol/min/105 cells, no treatment). The inhibition of the enzyme by disulfiram was associated with a dosedependent increase in cell survival rate. Disulfiram at as little as 10 M completely blocked the killing effect of TNF- (Fig. 1B). Moreover, disulfiram when added 4 h after TNF- still retained 80% of its effect (Fig. 1C). Disulfiram, up to at least 80 M, had no significant effect on cell viability and did not stimulate the caspase-3-like activity (Fig. 1D). Disulfiram at 15 M protected the cells from death at least for six days as determined by MTT assays (Fig. 2A). Without disulfiram, only 20.00.1% of the cells remained viable 12 h post treatment (Fig. 2B). The potent effect exerted by disulfiram led us to examine whether the cells saved by disulfiram were still capable of proliferation. Indeed, cell cycle and grow rate analyses revealed there was virtually no difference in cell growth between control which received no treatment and the cells rescued by the drug (Fig. 3A, C and D). Disulfiram had no significant effect on cell growth (Fig. 3B).
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In contrast to the potent cytoprotection promoted by disulfiram, ZVAD.fmk, a well known general inhibitor of caspases, failed to block cell death induced by TNF- (30 ng/ml) after incubation for 24 h (Fig. 4). This observation is consistent with our previous study that neither Ac-DEVD.fmk, a specific inhibitor of caspase-3-like proteases, nor boc-aspartylfluoromethylzone, a general inhibitor of caspases inhibited cell death under the same condition even when their concentrations reached 200 M and 100 M, respectively.17 These data suggest that disulfiram may have additional cytoprotective mechanisms. Mitochondria and reactive oxygen species also play a critical role in TNF--induced apoptosis. In fact, collapse of mitochondrial membrane potential and generation of reactive oxygen species occur before
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Figure 2. Disulfiram at 15 µM protected LLC-PK1 cells from TNF- (30 ng/ ml)-induced death for 6 days (A). Without disulfiram most of the cells became non-viable within 12 h (B). The medium was changed every 24 h. The cell viability was examined by MTT assays (A) or crystal violet assays (B). *P<0.01 as compared with 0 h time point (ANOVA, n=3).
caspase-3 is activated.12,13 To determine how long the treatment was sufficient for the damage to take place, we briefly pulsed the cells with TNF- and then switched to regular growth medium up to 24 h. Although the cells were still attached to the dishes after exposure to the cytokine for 70 min, the cells died spontaneously in the regular growth medium with only 38.22.1% survived following incubation in the growth medium for additional 22 h plus 50 min (Fig. 5A). This survival rate was virtually indistinguishable
Disulfiram inhibits TNF--induced death / 1359
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Figure 3. The cells rescued by disulfiram retained their proliferative capability. After treatment with TNF- (30 ng/ml) and disulfiram (15 M) for 48 h, the cells were trypsinized and plated down at subconfluence. The cells were counted daily. Square: no TNF-; Triangle: TNF- and disulfiram (A, n=5). Disulfiram did not significantly affect cell growth (B, n=5). The cell cycle was analyzed with propidium iodide-based flow cytometry. Data are the representatives of four independent experiments (C and D). C: No TNF-; G1, 47.4%, S, 38.4%, G2/M, 14.2%. D: TNF- and disulifiram; G1, 46.3%; S, 37.8%, G2/M, 15.9%.
from that treated continuously for 24 h (33.02.9%; Fig. 5B). Coadministration of 15 M disulfiram with TNF- followed by their removal 70 min later completely abolished the TNF--induced cell death (982% viable, Fig. 5C). The effect of disulfiram appears not to be significantly related to inhibition of caspase-3-like activity, as the increase in this activity just barely reached the level of significance (P=0.048,
Fig. 5D). In support of this notion, boc-aspartylfluoromethylzone had no significant effect (Fig. 5C). In contrast to BAF, cyclosporine, a better known stabilizer of mitochondrial membrane potential, and N-acetylcysteine, an antioxidant, abrogated cell death, suggesting that disulfiram may prevent collapse of mitochondrial membrane potential and suppress reactive oxygen species (Fig. 5C). Treatment with 2 M
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of the levels of intracellular reactive oxygen species inaccurate in this group. To determine if the inhibition of reactive oxygen species by disulfiram contributed to its long-term cytoprotection, we treated the cells with TNF- and N-acetylcysteine for 24 h and found that N-acetylcysteine protected the cells from death in a dose-dependent manner (Fig. 8A). N-acetylcysteine abrogated TNF--induced caspase-3-like activity, suggesting that disulfiram may also inhibit caspase-3like activity via suppression of reactive oxygen species (Fig. 8B).
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Disulfiram inhibits TNF--induced apoptosis and caspase-3-like activity in the livers of mice
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The potent cytoprotection promoted by disulfiram via multiple mechanisms prompted us to examine if the drug inhibited apoptosis and caspase-3-like activity in vivo as well. We adopted a liver model of caspase-3mediated apoptosis.4 We were able to repeat the previous finding that treatment with TNF- and D-galactosamine for 5 h caused dramatic increases in DNA fragmentation (from 92.87.2% Vmax to 4220.0922.1% Vmax, P<0.05). The increment in DNA fragmentation was accompanied with an increase in caspase-3-like activity (from 14.48.6 pmol/min/mg protein to 475.38.6 pmol/min/mg protein, P<0.05, Fig. 9). More importantly, disulfiram inhibited DNA fragmentation in a dose-dependent manner, which was associated in parallel with inhibition of caspase-3-like activity. Disulfiram at 0.6 mmol/ kg dramatically inhibited the effects of TNF- on DNA fragmentation (412.0210.2% Vmax, P<0.05) and caspase-3-like activity (43.613.9 pmol/min/mg protein, P<0.05, Fig. 9). The inhibition of TNF-induced DNA fragmentation by disulfiram was also reproduced with TUNEL assays (Fig. 10). Disulfiram itself at 1 mmol/kg had no effect on DNA fragmentation and caspase-3-like activity under the same condition (data not shown).
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cyclosporine and 10 mM N-acetylcysteine in the same way as disulfiram rescued 858% and 985% of the cells, respectively. Interestingly, direct measurement of the mitochondrial membrane potential with JC-1 dye revealed that mitochondrial membrane potential did not decrease until 2 h after treatment with TNF-, because the number of the cells containing the aggregates of the dye did not decrease until this time point. Disulfiram restored the membrane potential at this point as evidenced by more cells containing JC-1 aggregates (Fig. 6). Cyclosporine also restored the membrane potential (data not shown). To determine if the stabilization of mitochondrial membrane potential by disulfiram was related to its potent cytoprotection, we extended the treatment with TNF- and cyclosporine to 8 and 24 h, respectively, and found that cyclosporine did not inhibit cell death (data not shown), suggesting that prevention of collapse of mitochondrial membrane potential by disulfiram can only protect the cells from short-term challenge of TNF-, but is not sufficient enough to sustain the cells against the continuous insult from the cytokine. To examine if disulfiram acted as an antioxidant, the intracellular levels of reactive oxygen species were measured with flow cytometry at 70 min or 24 h after treatment. Disulfiram reduced the levels of reactive oxygen species induced by the cytokine, and even under the control conditions as the positions of curves shifted toward left, although the effect of the drug became weak after long time of incubation (Fig. 7). After treated with the cytokine for 24 h, most of the cells were dead, which made the measurement
DISCUSSION Disulfiram inhibits aldehyde dehydrogenase, which is thought to represent its mechanism of action in the treatment of alcoholism (under the trade name of Antabus or Aversan).18 Disulfiram also affects metal ion distribution in vivo,19,20 regulates cytochrome P-45021 and suppresses lipid peroxidation.22 Our previous study has demonstrated that TNF--induced apoptosis was mediated via caspase-3-like proteases and preceded cell death in LLC-PK1 cells.17 Using this model, we have now shown that disulfiram prevented TNF--induced cell death. We have also documented that the cytoprotective mechanisms afforded by disulfiram are not limited to inhibition of caspase-3 but
Disulfiram inhibits TNF--induced death / 1361
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Figure 5. The treatment with TNF- (30 ng/ml) for a brief period prior to incubation with the regular growth medium up to 24 h (A) or continuously for 24 h (B) decreased cell viability. Coadministration with disulfiram (DSF, P<0.01, Student’s t-test) or cyclosporine (Cyx, P<0.01, ANOVA) or N-acetylcysteine (NAC, P<0.01 Student’s t-test) but not BAF for 70 min prior to switch to the regular growth medium inhibited the cell death (C). The treatment with TNF- for 70 min barely significantly increased caspase-3-like activity as measured by DEVD-CHO-sensitive hydrolysis of Ac-DEVD-AMC (D). *P<0.01 as compared with the groups that received no TNF- (A and B) or TNF- alone (C; n=3).
include stabilization of mitochondrial membrane potential and suppression of reactive oxygen species (Figs 6 and 7). Caspase-8 and 10 also play an important role in TNF--induced cell death. In view of that disulfiram inhibits caspase-3 through thiol-oxidation and caspase-8 and 10 have thiol groups, it is likely that disulfiram also inhibits the initiator caspases as well. This effect could contribute disulfiram cytoprotection. In fact disulfiram has been shown to inhibit caspase-1, one of the initiator caspases.14,15 Caspase-3 acts at a relatively late stage of apoptosis. Some cell damage apparently precedes the activation of the enzyme. Examples include collapse of mitochondrial membrane potential, release of cytochrome c into cytosol and generation of reactive oxy-
gen species.12,13 Rarely addressed are how long a caspase inhibitor can protect cells from death and whether cells rescued by a caspase inhibitor retain their proliferative function. In fact, the present and previous studies have demonstrated that cell death occurred in the presence of a caspase inhibitor (Fig. 4),7,8,17 raising the question of whether or not caspase activation is just a consequence of the death pathway but is not actively involved in the death process. It has been proposed that the cell death is mediated by two pathways, nucleic and cytosolic. The nucleic pathway like DNA fragmentation, chromatin condensation and hypodiploid nuclei is caspase-dependent, whereas cytosolic mechanism like mitochondria damage and lipid peroxidation is
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Figure 6. Disulfiram (15 µM) prevented the collapse of mitochondrial membrane potential induced by TNF- (30 ng/ml). After treatment for 2 h, the cells were loaded with 3 g/ml JC-1 for 30 min and analyzed with flow cytometry. Each panel is a representative of four independent experiments. FL2 AGG: JC-1 aggregates. FL-1 MONO: JC-1 monomers.
caspase-independent.7,8 Many publications have reported the effect of ZVAD or BAF on apoptosis usually characterized by DNA fragmentation. In this sense the caspase is involved in the death process. However, blockade of caspase-3 or apoptosis does not
necessarily lead to abrogation of cell death because of the presence of a caspase-independent pathway that is likely involved with reactive oxygen species.7–11 Disulfiram in fact not only protected the cells from TNF--induced death for at least 6 days (Fig. 2A), but 70 min
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Figure 7. Disulfiram (15 µM) suppressed TNF- (30 ng/ml)-induced reactive oxygen species. The cells were loaded with DCFH-DA (20 M) for 70 min or at last 70 min before analyzed with flow cytometry. Each panel is a representative of four independent experiments.
Disulfiram inhibits TNF--induced death / 1363
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N-acetylcysteine (NAC) inhibited TNF- (30 ng/ml)-induced cell death (A) and caspase-3-like activity (B). The cell viability was measured by crystal violet assays 24 h post treatment (A). The caspase-3-like activity was measured 2 (B, 1–3) or 4 h (B, 4–6) post treatment by DEVD-CHO-sensitive hydrolysis of Ac-DEVD-AMC. Cells were treated with vehicles (1,4), TNF- alone (2,5), or TNF- and 10 µM NAC (3,6). *P<0.01 as compared with no N-acetylcysteine (ANOVA, n=3). †P<0.0001 as compared with the groups treated with TNF- alone (Student’s t-test, n=3).
Disulfiram (DSF) inhibited TNF- (10 mg/kg)-induced DNA fragmentation (A) and caspase-3-like activity (B) in the livers of mice in a dose-dependent manner. Mice were injected with vehicles (1), TNF- (2), TNF- and 0.2 mmol/kg DSF (3), TNF- and 0.6 mmol/ kg DSF (4), or TNF- and 1.0 mmol/kg DSF (5). Caspase-3-like activity was measured with DEVD-CHO-sensitive hydrolysis of Ac-DEVD-AMC. DNA fragmentation was quantified with a ELISA method. Mice were sacrificed at 5 h. Each group had five mice except the group treated with TNF- alone that had six mice. *P<0.05 as compared with the group that received vehicles only (non-parametric test).
the cells were able to proliferate as rapidly as control cells (Fig. 3). Since ZVAD did not block cell death (Fig. 4) whereas N-acetylcysteine did (Fig. 8), one would argue that disulfiram promoted cell survival through the reduction of the levels of free radicals in a caspase-independent manner. However, an antioxidant usually needs to be pre-incubated before an
insult starts or simultaneously added with a death inducer in order to observe its cytoprotection. Disulfiram retained 80% of its effect when it was added fours after TNF- (Fig. 1C). This effect appears to be derived from the inhibition of both caspases and free radicals. It is certainly possible that a unique combination of blockade of caspase-3 activity, stabilization
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Figure 10. Disulfiram reduced TNF--induced DNA fragmentation as measured by TUNEL assays. Mice were injected either with vehicles (a), or with 10 g/kg TNF- (b), or 10 g/kg TNF- and 0.6 mmol/ kg disulfiram (c). The doses of disulfiram were divided by two injections. One was given simultaneously with TNF- and another one was given every 2.5 h later. Mice were sacrificed at 5 h. Each panel is a representative of four independent experiments.
of mitochondrial membrane potential, suppression of reactive oxygen species, and other yet to be defined functions of disulfiram make it more effective in inhibition of TNF--induced cell death than known caspase inhibitors. It is noteworthy, however, that disulfiram, at much higher concentration (300 M), has been shown to induce apoptosis in a hepatoma cell
CYTOKINE, Vol. 12, No. 9 (September, 2000: 1356–1367)
line.23 Similarly, the chronic administration of the drug in high doses may prove toxic.24 Disulfiram is a homodimer of the antioxidant, diethyldithiocarbamate, connected through a disulfide bond. Disulfiram chelates metal ions including copper which is important for superoxide dismutase and depletes the intracellular reduced form of glutathione.24–26 These activities have led to categorization of the drug as an oxidant. Although disulfiram has been shown to inhibit lipid peroxidation, this effect has been ascribed to its attenuation of generation of oxidized metabolites of toxins by inhibition of cytochrome P-450.22,26 Using flow cytometry, we have now demonstrated that at low concentration disulfiram is a potent antioxidant (Fig. 7). Inside a cell disulfiram is quickly reduced to diethyldithiocarbamate.27 The latter blocks the generation of reactive oxygen species possibly by inhibiting the Fenton reaction and scavenging reactive oxygen and nitrogen species.28,29 It is speculated that disulfiram may exert its antioxidant effect via diethyldithiocarbamate. Mitochondria are one of the main targets of reactive oxygen species. It is also possible that the stabilization of mitochondria membrane potential by disulfiram is benefited from its reducing effect. Given the critical roles apoptosis plays in the pathogenesis of a variety of diseases, attention has been directed toward exploration of apoptosis inhibitors, mainly caspase-3 inhibitors, as potential therapeutic agents. For example, ZVAD, a general inhibitor of caspases, has been shown to reduce ischemiainduced cardiomyocyte apoptosis30 and endotoxemiainflicted hepatic injury;4,5 DEVD attenuates neuronal death induced by ischemia/reperfusion injury;3 and YVAD, an inhibitor of caspase-1 and -3, slows nigral transplant rejection, 31 and inhibits TNF-- and endotoxemia-induced hepatic apoptosis, necrosis and lethality.32 TNF- is a potent inducer of apoptosis. TNF-induced apoptosis and cell death have been implicated in the pathogeneses of a variety of diseases and disease models such as acquired immune-deficiency syndrome, acute pancreatitis, sepsis, and Group B streptococcal meningitis.16 Disulfiram inhibited TNF--induced DNA fragmentation in association with the blockade of caspase-3-like activity in the mouse liver (Figs 9 and 10). However, DNA fragmentation is not specific to apoptosis, but also to necrosis.33 Further study is needed to determine whether disulfiram inhibits TNF-induced hepatic apoptosis, or necrosis, or both; whether the effect of disulfiram is also mediated by the suppression of free radicals; whether disulfiram improves the survival rate as a result of the inhibition of hepatic injury and the ‘‘window of opportunity’’ of disulfiram. Nevertheless, the present study has documented that disulfiram inhibits cell death in vivo and
Disulfiram inhibits TNF--induced death / 1365
opens the possibility of using disulfiram in the treatment of the diseases associated with TNF--induced apoptosis. Disulfiram has been used clinically as an alcohol deterrent since 1948. The previous safety record gives disulfiram an advantage over other caspase inhibitors that are untested in humans.
MATERIALS AND METHODS Animals Male C3Heb/FeJ mice (18 to 25 g) were purchased from Jackson Laboratory (Bar Harbor, ME, USA). Recombinant murine TNF- (10 g/kg, Roche Molecular Biochemicals, Indianapolis, IN, USA) dissolved in 0.9% NaCl plus 0.1% BSA was injected intravenously. D-galactosamine (700 mg/ kg, to enhance the effect of TNF-) dissolved in pyrogen-free 0.9% NaCl and disulfiram which was dissolved in paraffin oil (Difco Laboratories, Detroit, MI, USA) were given intraperitoneally in separate syringes. The dose of disulfiram was divided by two injections. One was given at the same time when TNF- was injected. Another one was given 2.5 h later. Control animals were given vehicles only via appropriate routes. The animals were sacrificed at 5 h after injections. Livers were sectioned transversely across the midportion of each lobe. The tissues were preserved either on histology slides or in liquid nitrogen as appropriate. The experimental protocols followed the criteria of the Uniformed Services University and of the National Research Council for the care and use of laboratory animals in research.
Cells, cell viable assays and materials The LLC-PK1 cells (American Tissue Culture Collections, Manassas, VA, USA) were grown in Medium 199 plus 3% fetal bovine serum at 37C with 5% CO2. Only confluent cells were used except in the cases of growth assays. The cell death was induced by 30 ng/ml TNF- in the presence of 100 ng/ml gliotoxin, a toxic epipolythiodioxopiperazine metabolite from the pathogenic fungi as previously described.17,34 The use of gliotoxin is to potentiate the effect of TNF-. The cells were pretreated with gliotoxin for 30 min prior to addition of TNF-. Cell viability was examined by crystal violet dye assays. The absorption was measured at 550 nm by a multi-well plate reader.35 Since the cell viability varied day by day, the same set of experiments were performed at the same day. Cell viability was also examined by conversion of the dye 3-(4,5)-Dimethylthiazol-2-yl-2,5diphenyltetrozolium bromide (MTT) to formazan dye crystals according to the manufacturer’s instructions (Boehringer Mannheim, Gaithersburg, MD, USA). Briefly, the cells were incubated with 0.5 mM MTT at 37C for 4 h and then dissolved in 10% SDS (pH 3.0). The absorption was measured at 550 nm in reference to 690 nm with a multi-well plate reader. All chemicals were purchased from Sigma (St. Louis, MO, USA) unless indicated. ZVAD was purchased from BIOMOL (Plymouth Meeting, PA, USA).
Caspase activity assays The activity of caspase-3-like proteases was measured as increases in DEVD-CHO-sensitive hydrolysis of fluoro-
genic tetrapeptide substrate, Ac-DEVD-7-amino-4methylcoumarin (Ac-DEVD-AMC), according to the manufacturer’s instruction (BIOMOL, Plymouth Meeting, PA, USA). Since caspase-7 also hydrolyzes the substrate, the activity is referred as caspase-3-like activity. The cells were lysed in 25 mM HEPES buffer (pH 7.5) containing 5 mM EDTA, 2 mM DTT, 0.1% CHAPS (3[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate), and 0.1% Triton X-100 at 22C for 10 min, then the supernatants were taken for measurement of hydrolysis of Ac-DEVD-AMC as a function of time at 22C. Each experiment was performed in duplicate. To reduce the variations among each assay, all experiments were performed in the same day. For assays of caspase-3-like activity in livers, the freshly excised or liquid nitrogen preserved livers were homogenized in the HEPES buffer (pH 7.5) described above. After centrifuged at 11 000g for 30 min, the protein contents in the supernatant were measured by the Protein Assay Reagent (BioRad, Richmond, CA, USA). The caspase-3-like activity was assayed with 12 g protein.
Mitochondrial membrane potential measurement The membrane potential of energized mitochondria (negative inside) promotes a directional uptake of 5, 5 , 6, 6 -tetrachloro-1, 1 , 3, 3 -tetraethylbenzimidazolylcarbocyanine iodide (JC-1) dye into the matrix, also with subsequent formation of aggregates. The aggregate fluorescence is sensitive to transient membrane potential changes. Briefly, the cells were loaded with 3 g/ml of JC-1 (Molecular Probes, Eugene, OR, USA) at 37C for 30 min. After gated out small-sized debris, the red (aggregates) and green (monomer) emitted fluorescence was collected through 575/30 nm (FL2) and 525/40 nm (FL1) bandpass filters, respectively.36 At least 20 000 cells were analyzed in each experiment.
Cytofluorometric analyses of cell cycles After treatment with TNF- and disulfiram for 48 h, the cells were plated at subconfluence in a 6-well plate and grew for 48 h. The cells were collected and incubated in a hypotonic fluorochrome solution (Propidium iodide 0.5 mg/ml in 0.1% sodium citrate plus 0.1% Triton X-100) overnight at 4C. The propidium iodide fluorescence of each individual nucleus was measured with excitation of 488 nm and emission of 620 nm.37 At least 20 000 cells were analyzed in each experiment.
Reactive oxygen species measurement The cells were placed at 1106/well in a 6-well plate. The cells were loaded with 20 M DCFH-DA in the culture medium at 37C for 70 min and analyzed by flow cytometry.38 Forward scatter, side scatter, and FL1 were collected for all events falling in established gates. At least 20 000 cells were analyzed in each experiment.
DNA fragmentation assays DNA fragmentation in the livers was determined by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method according to the manufacturer’s procedures (Boehringer Mannheim, Gaithersburg, MD, USA). Briefly, livers were fixed with 10% formalin and
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embedded in paraffin. A 5-m-thick section was cut. Sections were deparaffinated and rehydrated and then assayed for direct immunoperoxidase detection of digoxigenin-labeled genomic DNA. DNA fragmentation was quantified by a ELISA kit according to the manufacturer’s procedures (Boehringer Mannheim, Gaithersburg, MD, USA). Briefly, the cytoplasma extract containing 1 g of protein was taken for measure of the kinetics of product generation (Vmax) which was used as an index of DNA fragmentation. The assay allows the specific quantitation of histone-associated DNA fragments (mono- and oligonucleosomes) in the cytoplasmic fraction of cell lysates and has been used extensively to demonstrate apoptosis in vivo.
Statistics Numerical data are expressed as meansstandard errors. Each experiment was performed in duplicate or triplicate. Statistical analyses were performed with Student’s t-test, or one-way analysis of variance (ANOVA), or nonparametric test as appropriate. Post hoc multiple comparisons were made by Dunnett test (ANOVA) or Dunn’s test (non-parametric). Null hypotheses were rejected at the level of 0.05.
Acknowledgements Authors thank Ms Karen Wolcott for her assistance in flow cytometry, Dr Elliot Kagan for allowing us to use the multi-well reader, Dr Ajay Verma for permitting us to use the fluorometer, Dr Karen Gray for her suggestions during the early phase of the study, and Dr Ying-Yue Li for her assistance in animal experiments. The study was supported in large part by a Grant-in-Aid from The National Kidney Foundation/The National Capital Area, Inc (to XZ). 2000 Academic Press
REFERENCES 1. Kumar S (1999) Regulation of caspase activation in apoptosis: implications in pathogenesis and treatment of disease. Clin Exp Pharmacol Physiol 26:295–303. 2. Stennicke HR, Salvesen GS (1998) Properties of the caspases. Biochim Biophys Acta 1387:17–31– 3. Endres M, Namura S, Shimizu-Sasamata M, Waeber C, Zhang L, Gomez-Isla T, Hyman BT, Moskowitz MA (1998) Attenuation of delayed neuronal death after mild focal ischemia in mice by inhibition of the caspase family. J Cereb Blood Flow Metab 18: 238–247. 4. Jaeschke H, Fisher MA, Lawson JA, Simmons CA, Farhood A, Jones DA (1998) Activation of caspase 3 (CPP32)-like proteases is essential for TNF--induced hepatic parenchymal cell apoptosis and neutrophil-mediated necrosis in a murine endotoxin shock model. J Immunol 160:3480–3486. 5. Lawson JA, Fisher MA, Simmons CA, Farhood A, Jaeschke H (1998) Parenchymal cell apoptosis as a signal for sinusoidal sequestration and transendothelial migration of neutrophils in murine models of endotoxin and Fas-Antibody-induced liver injury. Hepatology 28:761–767.
CYTOKINE, Vol. 12, No. 9 (September, 2000: 1356–1367) 6. Nicholson DW, Ali A, Thornberry NA, Vaillancourt JP, Ding CK, Gallant M, Gareau Y, Griffin PR, Labelle M, Lazebnik YA, Munday NA, Raju SR, Smulson ME, Yamin TT, Yu VL, Miller DK (1995) Identification and inhibition of the ICE/ CED-3 protease necessary for mammalian apoptosis. Nature 376:37–43. 7. Deas O, Dumont C, MacFarlane M, Rouleau M, Hebib C, Harper F, Hirsch F, Charpentier B, Cohen GM, Senik A (1998) Caspase-independent cell death induced by anti-CD2 or staurosporine in activated human peripheral T lymphocytes. J Immunol 161:3375–3383. 8. Trapani JA, Jans DA, Jans PJ, Smyth MJ, Browne KA, Sutton VR (1998) Efficient nuclear targeting of granzyme B and the nuclear consequences of apoptosis induced by granzyme B and perforin are caspase-dependent, but cell death is caspaseindependent. J Biol Chem 273:27934–27938. 9. Vercammen D, Beyaert R, Denecker G, Goossens V, Van Loo G, Declercq W, Grooten J, Fiers W, Vandenabeele P (1998) Inhibition of caspases increases the sensitivity of L929 cells to necrosis mediated by tumor necrosis factor. J Exp Med 187: 1477–1485. 10. Yamashita K, Takahashi A, Kobayashi S, Hirata H, Mesner PW Jr, Kaufmann SH, Yonehara S, Yamamoto K, Uchiyama T, Sasada M (1999) Caspases mediate tumor necrosis factor-alpha-induced neutrophil apoptosis and downregulation of reactive oxygen production. Blood 93:674–685. 11. Khwaja A, Tatton L (1999) Resistance to the cytotoxic effects of tumor necrosis factor alpha can be overcome by inhibition of a FADD/Caspase-dependent signaling pathway. J Biol Chem 1274:36817–36823. 12. Lemasters JJ, Qian T, Elmore SP, Trost LC, Nishimura Y, Herman B, Bradham CA, Brenner DA, Nieminen AL (1998) Confocal microscopy of the mitochondrial permeability transition in necrotic cell killing, apoptosis and autophagy. Biofactors 8:283–285. 13. Wallach D, Kovalenko AV, Varfolomeev EE, Boldin MP (1998) Death-inducing functions of ligands of the tumor necrosis factor family: a Sanhedrin verdict. Curr Opin Immunol 10: 279–288. 14. Nobel CSI, Burgess DH, Zhivotovsky B, Burkitt MJ, Orrenius S, Slater AFG (1997) Mechanism of dithiocarbamate inhibition of apoptosis: Thiol oxidation by dithiocarbamate disulfides directly inhibits processing of the caspase-3 proenzyme. Chem Res Toxicol 10:636–643. 15. Nobel CSI, Kimland M, Nicholson DW, Orrenius S, Slater AFG (1997) Disulfiram is a potent inhibitor of proteases of the caspase family. Chem Res Toxicol 10:1319–1324. 16. Beutler BA (1999) The role of tumor necrosis factor in health and disease. J Rheumatol Suppl 57:16–21. 17. Zhou X, Zhao A, Goping G, Hirszel P (2000) Gliotoxininduced cytotoxicity proceeds via apoptosis and is mediated by caspases and reactive oxygen species in LLC-PK1 cells. Toxicol Sci 54:194–202. 18. Veverka KA, Johnson KL, Mays DC, Lipsky JJ, Naylor S (1997) Inhibition of aldehyde dehydrogenase by disulfiram and its metabolite methyl diethylthiocarbamoyl-sulfoxide. Biochem Pharmacol 53:511–518. 19. Sorensen JA, Andersen O (1989) Effects of diethyldithiocarbamate and tetraethylthiuram disulfide on zinc metabolism in mice. Pharmacol Toxicol 65:209–213. 20. Ono H, Funakoshi T, Shimada H, Kojima S (1997) Comparative effects of disulfiram and diethyldithiocarbamate against testicular toxicity in rats caused by acute exposure to cadmium. J Toxicol Environ Health 50:389–399. 21. Martini R, Ingelman-Sundberg M, Murray M (1997) Pretranslational and posttranslational regulation of rat hepatic CYPs 3A2 and 2E1 by disulfiram. Biochem Pharmacol 54:1323–1329. 22. Kyle ME, Serroni A, Farber JL (1989) The inhibition of lipid peroxidation by disulfiram prevents the killing of cultured hepatocytes by allyl alcohol, tert-butyl hydroperoxide, hydrogen peroxide and diethyl maleate. Chem Biol Interact 72:269–275. 23. Liu GY, Frank N, Bartsch H, Lin JK (1998) Induction of apoptosis by thiuramdisulfides, the reactive metabolites of dithiocarbamates, through coordinative modulation of NFkappaB, c-fos/cjun, and p53 proteins. Mol Carcinog 22:235–246.
Disulfiram inhibits TNF--induced death / 1367 24. Delmaestro E, Trombetta LD (1995) The effects of disulfiram on the hippocampus and cerebellum of the rat brain: a study on oxidative stress. Toxicol Lett 75:235–243. 25. Burkitt MJ, Bishop HS, Milne L, Tsang SY, Provan GJ, Nobel CS, Orrenius S, Slater AF (1998) Dithiocarbamate toxicity toward thymocytes involves their copper-catalyzed conversion to thiuram disulfides, which oxidize glutathione in a redox cycle without the release of reactive oxygen species. Arch Biochem Biophys 353:73–84. 26. Dogterom P, Mulder GJ, Nagelkerke JF (1989) Lipid peroxidation-dependent and -independent protein thiol modifications in isolated rat hepatocytes: differential effects of vitamin E and disulfiram. Chem Biol Interact 71:291–306. 27. Johansson B (1992) A review of the pharmacokinetics and pharmacodynamics of disulfiram and its metabolites. Acta Psychiatr Scand Suppl 369:15–26. 28. Liu J, Shigenaga MK, Yan LJ, Mori A, Ames BN (1996) Antioxidant activity of diethyldithiocarbamate. Free Radic Res 24:461–472. 29. Mankhetkorn S, Abedinzadeh Z, Houee-Levin C (1994) Antioxidant action of sodium diethyldithiocarbamate: reaction with hydrogen peroxide and superoxide radical. Free Radic Biol Med 17:517–527. 30. Yaoita H, Ogawa K, Maehara K, Maruyama Y (1998) Attenuation of ischemia/reperfusion injury in rats by a caspase inhibitor. Circulation 97:276–281. 31. Schierle GS, Hansson O, Leist M, Nicotera P, Widner H, Brundin P (1999) Caspase inhibition reduces apoptosis and increases survival of nigral transplants. Nat Med 5:97–100.
32. Mignon A, Rouquet N, Fabre M, Martin S, Pages JC, Dhainaut JF, Kahn A, Briand P, Joulin V (1999) LPS challenge in D-galactosamine-sensitized mice accounts for caspase-dependent fulminant hepatitis, not for septic shock. Am J Respir Crit Care Med 159:1308–1315. 33. Bohlinger I, Leist M, Gantner F, Angermuller S, Tiegs G, Wendel A (1996) DNA fragmentation in mouse organs during endotoxic shock. Am J Path 149:1381–1393. 34. Waring P, Beaver J (1996) Gliotoxin and related epipolythiodioxopiperazines. Gen Pharmacol 27:1311–1316. 35. Wang C, Mayo MW, Baldwin AS Jr (1996) TNF- and cancer therapy-induced apoptosis: potentiation by inhibition of NF-B. Science 274:784–787. 36. Salvioli S, Ardizzoni A, Franceschi C, Cossarizza A (1997) JC-1, but not DiOC6(3) or rhodamine 123, is a reliable fluorescent probe to assess delta psi changes in intact cells: implications for studies on mitochondrial functionality during apoptosis. FEBS Lett 411:77–82. 37. Nicoletti I, Migliorati G, Pagliacci MC, Grignani F, Riccardi C (1991) A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. J Immunol Methods 139:271–279. 38. Robinson P (1997) Oxadative metabolism. In: Robinson JP, Darzynkiewicz Z, Dean PN, Dressler LG, Rabinovitch PS, Stewart CC, Tanke HJ, Wheeless LL (eds) Current Protocols in Cytometry. John Wiley & Sons, Inc, New York, pp 9.7.1–9.7.3.
DISULFIRAM INHIBITS TNF--INDUCED CELL DEATH Aiping Zhao, Zheng-Qi Wu,* Matthew Pollack, Florence M. Rollwagen,* Przemyslaw Hirszel, Xiaoming Zhou Disulfiram, a clinically employed alcohol deterrent, was recently discovered to inhibit caspase-3 and DNA fragmentation. Using LLC-PK1 cells and murine liver as models, we examined if the drug inhibited TNF--induced cell death. Disulfiram produced dose-dependent inhibition of TNF--induced cell death as well as caspase-3-like activity. Disulfiram retained 80% of its effect when added 4 h after TNF-. Disulfiram protected the cells from cytokine-induced death for at least 6 days. The cells rescued by the drug preserved the ability to proliferate. The cells died spontaneously after exposure to TNF- for just 70 min. Co-administration of 15 M disulfiram and TNF- for 70 min prior to their removal abolished TNF--induced killing, and this was associated with restoration of mitochondrial membrane potential and suppression of reactive oxygen species. Treatment of mice with TNF- and D-galactosamine for 5 h markedly increased hepatic DNA fragmentation and caspase-3-like activity. Disulfiram at 0.6 mmol/kg abolished these effects. We conclude that disulfiram is a potent inhibitor of TNF--induced cell death in vitro. The underlying mechanisms include stabilization of mitochondrial membrane potential, suppression of reactive oxygen species, and inhibition of caspase-3-like activity. We further conclude that disulfiram inhibits DNA fragmentation in vivo in association with the blockade of caspase-3-like activity.
As the molecular mechanisms underlying apoptosis are unraveled, it becomes increasingly clear that caspases play a fundamental role in this type of programmed cell death. The caspases comprise a growing family of aspartic acid-specific, cysteine proteases.1,2 The caspases are activated through a proteolytic cascade that serves to transmit and amplify death signals. This family of proteases can be divided into apoptosis initiators such as caspase-8, 9 and 10 that participate in transduction of death signals, and apoptosis effectors such as caspase-3, 6 and 7, or caspase-3-like proteases, From the Departments of Medicine and *Pathology, Uniformed Services University, Bethesda, MD 20814, USA Correspondence to: Dr Xiaoming Zhou, Department of Medicine, Uniformed Services University, 4301 Jones Bridge Road, Bethesda, MD 20814, USA. E-mail: [email protected] Received 8 February 2000; received in revised form 6 April 2000; accepted for publication 24 April 2000 1043–4666/00/091356+12 $35.00/0 KEY WORDS: caspase-3/reactive oxygen species/apoptosis/LLCPK1 cells/liver Abbreviations: Ac-DEVD-AMC: Ac-Asp-Glu-Val-Asp-7-amino-4methylcoumarin; BAF: boc-aspartyl (OMe)-fluoromethylketone; DCFH-DA: 2 , 7 - dichlorofluorescin diacetate; JC-1: 5, 5 , 6, 6 -tetrachloro-1, 1 , 3, 3 -tetraethylbenzimidazolyl-carbocyanine iodide; MTT: 3-(4,5)-dimethylthiazol-2-yl)-2,5-diphenyltetrozolium bromide; TUNEL: terminal deoxynucleotidyl transferasemediated dUTP nick end labeling; ZVAD.fmk: Z-Val-Ala-Asp (OMe)-fluoromethylketone 1356
which execute death signals. Among the apoptotic effectors, caspase-3 is the best characterized. The proteolytic functions of this enzyme are largely responsible for classical manifestation of apoptosis. Inhibition of caspase-3 usually abrogates apoptosis.1–6 Due to the complex relationship between apoptosis and cell death, however, a caspase inhibitor often fails to block and sometimes even enhances cell death.7,8 For example, Vercammen et al. have demonstrated that inhibition of caspases increased the sensitivity of a murine fibrosarcoma cell line to TNF--induced death by 1000-fold.9 This was due mainly to an increase in the production of reactive oxygen species. The similar phenomenon was also observed in the human neutrophils,10 NIH 3T3 fibroblasts and U937 leukemic cells.11 Free oxygen radicals are generated continuously as side products of electron transfer reactions. Superoxide and hydroxyl radicals, collectively called reactive oxygen species, are the major free radical species involved in biological processes. Cells have a welldeveloped antioxidant system, thus minimizing the detrimental effects of free radicals. Apoptosis or necrosis occurs only when the increased generation of free radicals overwhelms the capacity of the antioxidant mechanisms, and/or when the defense mechanisms are compromised. TNF-, for example, stimulates CYTOKINE, Vol. 12, No. 9 (September), 2000: pp 1356–1367
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Figure 1. Disulfiram inhibited TNF- (30 ng/ml)-induced caspase-3-like activity (A) and cell death (B) in a dose-dependent manner. Disulfiram (15 M) was still able to rescue LLC-PK1 cells even when it was added after TNF- (C). Disulfiram up to, at least, 80 M had no significant effect on cell viability and did not stimulate the caspase-3-like activity (D). The caspase-3-like activity was measured 2 h post treatment by DEVD-CHO-sensitive hydrolysis of Ac-DEVD-AMC. The cell viability was measured by crystal violet assays 24 h post treatment. Disulfiram was added either simultaneously with TNF- (30 ng/ml) or hours after TNF-, and the cells were incubated up to 24 h (C). Each point represents the average of four (A) or three (B and C) experiments. *P<0.01 as compared with no disulfiram (control) (ANOVA).
caspase-8, which in turn cleaves protein Bid resulting in functional damage to mitochondria and overproduction of reactive oxygen species.12,13 Most, but not all in vitro data are consistent with the notion that TNF- activates caspase-3, at least in part, via increases in the levels of reactive oxygen species. The inhibition of caspase-3 stimulates the production of free radicals through a yet defined mechanism.9–11 Disulfiram is a member of the dithiocarbamate family comprising a broad class of molecules possessing an R1R2NC(S)SR3 functional group, which enables them to react with sulfhydryl groups. Recent biochemi-
cal studies have demonstrated that disulfiram inhibits processing of pro-caspase-3 and -1, and suppresses active caspase-3 through a direct thiol–disulfide interaction with the cysteine residues in these caspases. The blockade of caspase-3 by disulfiram in Jurkat T lymphocytes and rat thymocytes is associated with inhibition of DNA fragmentation, a hallmark of apoptosis.14,15 It is unknown, however, whether the abrogation of apoptosis translates to an increase in cell survival. In the present in vitro and in vivo study we sought to ascertain whether disulfiram would decrease cell death induced by TNF-, a cytokine
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pathogenetically linked to a variety of diseases and disease models,16 and whether the drug expresses other cytoprotective mechanisms in addition to inhibition of caspase-3.
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Disulfiram is a potent inhibitor of caspase-3-like activity and cell death in vitro Disulfiram inhibited TNF--induced caspase-3like activity in a dose-dependent manner (Fig. 1A). The drug at 5 M completely suppressed caspase-3like activity (9.61.3 pmol/min/105 cells, TNF-; 0.80.1 pmol/min/105 cells, disulfiram; and 1.0 0.2 pmol/min/105 cells, no treatment). The inhibition of the enzyme by disulfiram was associated with a dosedependent increase in cell survival rate. Disulfiram at as little as 10 M completely blocked the killing effect of TNF- (Fig. 1B). Moreover, disulfiram when added 4 h after TNF- still retained 80% of its effect (Fig. 1C). Disulfiram, up to at least 80 M, had no significant effect on cell viability and did not stimulate the caspase-3-like activity (Fig. 1D). Disulfiram at 15 M protected the cells from death at least for six days as determined by MTT assays (Fig. 2A). Without disulfiram, only 20.00.1% of the cells remained viable 12 h post treatment (Fig. 2B). The potent effect exerted by disulfiram led us to examine whether the cells saved by disulfiram were still capable of proliferation. Indeed, cell cycle and grow rate analyses revealed there was virtually no difference in cell growth between control which received no treatment and the cells rescued by the drug (Fig. 3A, C and D). Disulfiram had no significant effect on cell growth (Fig. 3B).
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In contrast to the potent cytoprotection promoted by disulfiram, ZVAD.fmk, a well known general inhibitor of caspases, failed to block cell death induced by TNF- (30 ng/ml) after incubation for 24 h (Fig. 4). This observation is consistent with our previous study that neither Ac-DEVD.fmk, a specific inhibitor of caspase-3-like proteases, nor boc-aspartylfluoromethylzone, a general inhibitor of caspases inhibited cell death under the same condition even when their concentrations reached 200 M and 100 M, respectively.17 These data suggest that disulfiram may have additional cytoprotective mechanisms. Mitochondria and reactive oxygen species also play a critical role in TNF--induced apoptosis. In fact, collapse of mitochondrial membrane potential and generation of reactive oxygen species occur before
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caspase-3 is activated.12,13 To determine how long the treatment was sufficient for the damage to take place, we briefly pulsed the cells with TNF- and then switched to regular growth medium up to 24 h. Although the cells were still attached to the dishes after exposure to the cytokine for 70 min, the cells died spontaneously in the regular growth medium with only 38.22.1% survived following incubation in the growth medium for additional 22 h plus 50 min (Fig. 5A). This survival rate was virtually indistinguishable
Disulfiram inhibits TNF--induced death / 1359
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Figure 3. The cells rescued by disulfiram retained their proliferative capability. After treatment with TNF- (30 ng/ml) and disulfiram (15 M) for 48 h, the cells were trypsinized and plated down at subconfluence. The cells were counted daily. Square: no TNF-; Triangle: TNF- and disulfiram (A, n=5). Disulfiram did not significantly affect cell growth (B, n=5). The cell cycle was analyzed with propidium iodide-based flow cytometry. Data are the representatives of four independent experiments (C and D). C: No TNF-; G1, 47.4%, S, 38.4%, G2/M, 14.2%. D: TNF- and disulifiram; G1, 46.3%; S, 37.8%, G2/M, 15.9%.
from that treated continuously for 24 h (33.02.9%; Fig. 5B). Coadministration of 15 M disulfiram with TNF- followed by their removal 70 min later completely abolished the TNF--induced cell death (982% viable, Fig. 5C). The effect of disulfiram appears not to be significantly related to inhibition of caspase-3-like activity, as the increase in this activity just barely reached the level of significance (P=0.048,
Fig. 5D). In support of this notion, boc-aspartylfluoromethylzone had no significant effect (Fig. 5C). In contrast to BAF, cyclosporine, a better known stabilizer of mitochondrial membrane potential, and N-acetylcysteine, an antioxidant, abrogated cell death, suggesting that disulfiram may prevent collapse of mitochondrial membrane potential and suppress reactive oxygen species (Fig. 5C). Treatment with 2 M
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of the levels of intracellular reactive oxygen species inaccurate in this group. To determine if the inhibition of reactive oxygen species by disulfiram contributed to its long-term cytoprotection, we treated the cells with TNF- and N-acetylcysteine for 24 h and found that N-acetylcysteine protected the cells from death in a dose-dependent manner (Fig. 8A). N-acetylcysteine abrogated TNF--induced caspase-3-like activity, suggesting that disulfiram may also inhibit caspase-3like activity via suppression of reactive oxygen species (Fig. 8B).
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The potent cytoprotection promoted by disulfiram via multiple mechanisms prompted us to examine if the drug inhibited apoptosis and caspase-3-like activity in vivo as well. We adopted a liver model of caspase-3mediated apoptosis.4 We were able to repeat the previous finding that treatment with TNF- and D-galactosamine for 5 h caused dramatic increases in DNA fragmentation (from 92.87.2% Vmax to 4220.0922.1% Vmax, P<0.05). The increment in DNA fragmentation was accompanied with an increase in caspase-3-like activity (from 14.48.6 pmol/min/mg protein to 475.38.6 pmol/min/mg protein, P<0.05, Fig. 9). More importantly, disulfiram inhibited DNA fragmentation in a dose-dependent manner, which was associated in parallel with inhibition of caspase-3-like activity. Disulfiram at 0.6 mmol/ kg dramatically inhibited the effects of TNF- on DNA fragmentation (412.0210.2% Vmax, P<0.05) and caspase-3-like activity (43.613.9 pmol/min/mg protein, P<0.05, Fig. 9). The inhibition of TNF-induced DNA fragmentation by disulfiram was also reproduced with TUNEL assays (Fig. 10). Disulfiram itself at 1 mmol/kg had no effect on DNA fragmentation and caspase-3-like activity under the same condition (data not shown).
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Figure 4. ZVAD failed to block cell death. The cells were preincubated with ZVAD for 30 min prior to addition of TNF-. After treatment for 24 h, the cell viability was measured by crystal violet assays (n=3).
cyclosporine and 10 mM N-acetylcysteine in the same way as disulfiram rescued 858% and 985% of the cells, respectively. Interestingly, direct measurement of the mitochondrial membrane potential with JC-1 dye revealed that mitochondrial membrane potential did not decrease until 2 h after treatment with TNF-, because the number of the cells containing the aggregates of the dye did not decrease until this time point. Disulfiram restored the membrane potential at this point as evidenced by more cells containing JC-1 aggregates (Fig. 6). Cyclosporine also restored the membrane potential (data not shown). To determine if the stabilization of mitochondrial membrane potential by disulfiram was related to its potent cytoprotection, we extended the treatment with TNF- and cyclosporine to 8 and 24 h, respectively, and found that cyclosporine did not inhibit cell death (data not shown), suggesting that prevention of collapse of mitochondrial membrane potential by disulfiram can only protect the cells from short-term challenge of TNF-, but is not sufficient enough to sustain the cells against the continuous insult from the cytokine. To examine if disulfiram acted as an antioxidant, the intracellular levels of reactive oxygen species were measured with flow cytometry at 70 min or 24 h after treatment. Disulfiram reduced the levels of reactive oxygen species induced by the cytokine, and even under the control conditions as the positions of curves shifted toward left, although the effect of the drug became weak after long time of incubation (Fig. 7). After treated with the cytokine for 24 h, most of the cells were dead, which made the measurement
DISCUSSION Disulfiram inhibits aldehyde dehydrogenase, which is thought to represent its mechanism of action in the treatment of alcoholism (under the trade name of Antabus or Aversan).18 Disulfiram also affects metal ion distribution in vivo,19,20 regulates cytochrome P-45021 and suppresses lipid peroxidation.22 Our previous study has demonstrated that TNF--induced apoptosis was mediated via caspase-3-like proteases and preceded cell death in LLC-PK1 cells.17 Using this model, we have now shown that disulfiram prevented TNF--induced cell death. We have also documented that the cytoprotective mechanisms afforded by disulfiram are not limited to inhibition of caspase-3 but
Disulfiram inhibits TNF--induced death / 1361
140
140 A
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40
*
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120 Cell viability (%)
TNF-α
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2 C *
*
60 40 20
0 TNF-α BAF (µM) DSF (µM) Cyx (µM) NAC (mM)
*
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+ 0 0 0 0
+ 0 15 0 0
+ 0 0 0.5 0
+ 0 0 1 0
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Figure 5. The treatment with TNF- (30 ng/ml) for a brief period prior to incubation with the regular growth medium up to 24 h (A) or continuously for 24 h (B) decreased cell viability. Coadministration with disulfiram (DSF, P<0.01, Student’s t-test) or cyclosporine (Cyx, P<0.01, ANOVA) or N-acetylcysteine (NAC, P<0.01 Student’s t-test) but not BAF for 70 min prior to switch to the regular growth medium inhibited the cell death (C). The treatment with TNF- for 70 min barely significantly increased caspase-3-like activity as measured by DEVD-CHO-sensitive hydrolysis of Ac-DEVD-AMC (D). *P<0.01 as compared with the groups that received no TNF- (A and B) or TNF- alone (C; n=3).
include stabilization of mitochondrial membrane potential and suppression of reactive oxygen species (Figs 6 and 7). Caspase-8 and 10 also play an important role in TNF--induced cell death. In view of that disulfiram inhibits caspase-3 through thiol-oxidation and caspase-8 and 10 have thiol groups, it is likely that disulfiram also inhibits the initiator caspases as well. This effect could contribute disulfiram cytoprotection. In fact disulfiram has been shown to inhibit caspase-1, one of the initiator caspases.14,15 Caspase-3 acts at a relatively late stage of apoptosis. Some cell damage apparently precedes the activation of the enzyme. Examples include collapse of mitochondrial membrane potential, release of cytochrome c into cytosol and generation of reactive oxy-
gen species.12,13 Rarely addressed are how long a caspase inhibitor can protect cells from death and whether cells rescued by a caspase inhibitor retain their proliferative function. In fact, the present and previous studies have demonstrated that cell death occurred in the presence of a caspase inhibitor (Fig. 4),7,8,17 raising the question of whether or not caspase activation is just a consequence of the death pathway but is not actively involved in the death process. It has been proposed that the cell death is mediated by two pathways, nucleic and cytosolic. The nucleic pathway like DNA fragmentation, chromatin condensation and hypodiploid nuclei is caspase-dependent, whereas cytosolic mechanism like mitochondria damage and lipid peroxidation is
1362 / Zhao et al.
CYTOKINE, Vol. 12, No. 9 (September, 2000: 1356–1367)
FL2 AGG
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Figure 6. Disulfiram (15 µM) prevented the collapse of mitochondrial membrane potential induced by TNF- (30 ng/ml). After treatment for 2 h, the cells were loaded with 3 g/ml JC-1 for 30 min and analyzed with flow cytometry. Each panel is a representative of four independent experiments. FL2 AGG: JC-1 aggregates. FL-1 MONO: JC-1 monomers.
caspase-independent.7,8 Many publications have reported the effect of ZVAD or BAF on apoptosis usually characterized by DNA fragmentation. In this sense the caspase is involved in the death process. However, blockade of caspase-3 or apoptosis does not
necessarily lead to abrogation of cell death because of the presence of a caspase-independent pathway that is likely involved with reactive oxygen species.7–11 Disulfiram in fact not only protected the cells from TNF--induced death for at least 6 days (Fig. 2A), but 70 min
TNF-α
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DCFH-DA
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DCFH-DA
Figure 7. Disulfiram (15 µM) suppressed TNF- (30 ng/ml)-induced reactive oxygen species. The cells were loaded with DCFH-DA (20 M) for 70 min or at last 70 min before analyzed with flow cytometry. Each panel is a representative of four independent experiments.
Disulfiram inhibits TNF--induced death / 1363
6000
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12 B 10
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Figure 8.
1
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Figure 9.
N-acetylcysteine (NAC) inhibited TNF- (30 ng/ml)-induced cell death (A) and caspase-3-like activity (B). The cell viability was measured by crystal violet assays 24 h post treatment (A). The caspase-3-like activity was measured 2 (B, 1–3) or 4 h (B, 4–6) post treatment by DEVD-CHO-sensitive hydrolysis of Ac-DEVD-AMC. Cells were treated with vehicles (1,4), TNF- alone (2,5), or TNF- and 10 µM NAC (3,6). *P<0.01 as compared with no N-acetylcysteine (ANOVA, n=3). †P<0.0001 as compared with the groups treated with TNF- alone (Student’s t-test, n=3).
Disulfiram (DSF) inhibited TNF- (10 mg/kg)-induced DNA fragmentation (A) and caspase-3-like activity (B) in the livers of mice in a dose-dependent manner. Mice were injected with vehicles (1), TNF- (2), TNF- and 0.2 mmol/kg DSF (3), TNF- and 0.6 mmol/ kg DSF (4), or TNF- and 1.0 mmol/kg DSF (5). Caspase-3-like activity was measured with DEVD-CHO-sensitive hydrolysis of Ac-DEVD-AMC. DNA fragmentation was quantified with a ELISA method. Mice were sacrificed at 5 h. Each group had five mice except the group treated with TNF- alone that had six mice. *P<0.05 as compared with the group that received vehicles only (non-parametric test).
the cells were able to proliferate as rapidly as control cells (Fig. 3). Since ZVAD did not block cell death (Fig. 4) whereas N-acetylcysteine did (Fig. 8), one would argue that disulfiram promoted cell survival through the reduction of the levels of free radicals in a caspase-independent manner. However, an antioxidant usually needs to be pre-incubated before an
insult starts or simultaneously added with a death inducer in order to observe its cytoprotection. Disulfiram retained 80% of its effect when it was added fours after TNF- (Fig. 1C). This effect appears to be derived from the inhibition of both caspases and free radicals. It is certainly possible that a unique combination of blockade of caspase-3 activity, stabilization
1364 / Zhao et al.
Figure 10. Disulfiram reduced TNF--induced DNA fragmentation as measured by TUNEL assays. Mice were injected either with vehicles (a), or with 10 g/kg TNF- (b), or 10 g/kg TNF- and 0.6 mmol/ kg disulfiram (c). The doses of disulfiram were divided by two injections. One was given simultaneously with TNF- and another one was given every 2.5 h later. Mice were sacrificed at 5 h. Each panel is a representative of four independent experiments.
of mitochondrial membrane potential, suppression of reactive oxygen species, and other yet to be defined functions of disulfiram make it more effective in inhibition of TNF--induced cell death than known caspase inhibitors. It is noteworthy, however, that disulfiram, at much higher concentration (300 M), has been shown to induce apoptosis in a hepatoma cell
CYTOKINE, Vol. 12, No. 9 (September, 2000: 1356–1367)
line.23 Similarly, the chronic administration of the drug in high doses may prove toxic.24 Disulfiram is a homodimer of the antioxidant, diethyldithiocarbamate, connected through a disulfide bond. Disulfiram chelates metal ions including copper which is important for superoxide dismutase and depletes the intracellular reduced form of glutathione.24–26 These activities have led to categorization of the drug as an oxidant. Although disulfiram has been shown to inhibit lipid peroxidation, this effect has been ascribed to its attenuation of generation of oxidized metabolites of toxins by inhibition of cytochrome P-450.22,26 Using flow cytometry, we have now demonstrated that at low concentration disulfiram is a potent antioxidant (Fig. 7). Inside a cell disulfiram is quickly reduced to diethyldithiocarbamate.27 The latter blocks the generation of reactive oxygen species possibly by inhibiting the Fenton reaction and scavenging reactive oxygen and nitrogen species.28,29 It is speculated that disulfiram may exert its antioxidant effect via diethyldithiocarbamate. Mitochondria are one of the main targets of reactive oxygen species. It is also possible that the stabilization of mitochondria membrane potential by disulfiram is benefited from its reducing effect. Given the critical roles apoptosis plays in the pathogenesis of a variety of diseases, attention has been directed toward exploration of apoptosis inhibitors, mainly caspase-3 inhibitors, as potential therapeutic agents. For example, ZVAD, a general inhibitor of caspases, has been shown to reduce ischemiainduced cardiomyocyte apoptosis30 and endotoxemiainflicted hepatic injury;4,5 DEVD attenuates neuronal death induced by ischemia/reperfusion injury;3 and YVAD, an inhibitor of caspase-1 and -3, slows nigral transplant rejection, 31 and inhibits TNF-- and endotoxemia-induced hepatic apoptosis, necrosis and lethality.32 TNF- is a potent inducer of apoptosis. TNF-induced apoptosis and cell death have been implicated in the pathogeneses of a variety of diseases and disease models such as acquired immune-deficiency syndrome, acute pancreatitis, sepsis, and Group B streptococcal meningitis.16 Disulfiram inhibited TNF--induced DNA fragmentation in association with the blockade of caspase-3-like activity in the mouse liver (Figs 9 and 10). However, DNA fragmentation is not specific to apoptosis, but also to necrosis.33 Further study is needed to determine whether disulfiram inhibits TNF-induced hepatic apoptosis, or necrosis, or both; whether the effect of disulfiram is also mediated by the suppression of free radicals; whether disulfiram improves the survival rate as a result of the inhibition of hepatic injury and the ‘‘window of opportunity’’ of disulfiram. Nevertheless, the present study has documented that disulfiram inhibits cell death in vivo and
Disulfiram inhibits TNF--induced death / 1365
opens the possibility of using disulfiram in the treatment of the diseases associated with TNF--induced apoptosis. Disulfiram has been used clinically as an alcohol deterrent since 1948. The previous safety record gives disulfiram an advantage over other caspase inhibitors that are untested in humans.
MATERIALS AND METHODS Animals Male C3Heb/FeJ mice (18 to 25 g) were purchased from Jackson Laboratory (Bar Harbor, ME, USA). Recombinant murine TNF- (10 g/kg, Roche Molecular Biochemicals, Indianapolis, IN, USA) dissolved in 0.9% NaCl plus 0.1% BSA was injected intravenously. D-galactosamine (700 mg/ kg, to enhance the effect of TNF-) dissolved in pyrogen-free 0.9% NaCl and disulfiram which was dissolved in paraffin oil (Difco Laboratories, Detroit, MI, USA) were given intraperitoneally in separate syringes. The dose of disulfiram was divided by two injections. One was given at the same time when TNF- was injected. Another one was given 2.5 h later. Control animals were given vehicles only via appropriate routes. The animals were sacrificed at 5 h after injections. Livers were sectioned transversely across the midportion of each lobe. The tissues were preserved either on histology slides or in liquid nitrogen as appropriate. The experimental protocols followed the criteria of the Uniformed Services University and of the National Research Council for the care and use of laboratory animals in research.
Cells, cell viable assays and materials The LLC-PK1 cells (American Tissue Culture Collections, Manassas, VA, USA) were grown in Medium 199 plus 3% fetal bovine serum at 37C with 5% CO2. Only confluent cells were used except in the cases of growth assays. The cell death was induced by 30 ng/ml TNF- in the presence of 100 ng/ml gliotoxin, a toxic epipolythiodioxopiperazine metabolite from the pathogenic fungi as previously described.17,34 The use of gliotoxin is to potentiate the effect of TNF-. The cells were pretreated with gliotoxin for 30 min prior to addition of TNF-. Cell viability was examined by crystal violet dye assays. The absorption was measured at 550 nm by a multi-well plate reader.35 Since the cell viability varied day by day, the same set of experiments were performed at the same day. Cell viability was also examined by conversion of the dye 3-(4,5)-Dimethylthiazol-2-yl-2,5diphenyltetrozolium bromide (MTT) to formazan dye crystals according to the manufacturer’s instructions (Boehringer Mannheim, Gaithersburg, MD, USA). Briefly, the cells were incubated with 0.5 mM MTT at 37C for 4 h and then dissolved in 10% SDS (pH 3.0). The absorption was measured at 550 nm in reference to 690 nm with a multi-well plate reader. All chemicals were purchased from Sigma (St. Louis, MO, USA) unless indicated. ZVAD was purchased from BIOMOL (Plymouth Meeting, PA, USA).
Caspase activity assays The activity of caspase-3-like proteases was measured as increases in DEVD-CHO-sensitive hydrolysis of fluoro-
genic tetrapeptide substrate, Ac-DEVD-7-amino-4methylcoumarin (Ac-DEVD-AMC), according to the manufacturer’s instruction (BIOMOL, Plymouth Meeting, PA, USA). Since caspase-7 also hydrolyzes the substrate, the activity is referred as caspase-3-like activity. The cells were lysed in 25 mM HEPES buffer (pH 7.5) containing 5 mM EDTA, 2 mM DTT, 0.1% CHAPS (3[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate), and 0.1% Triton X-100 at 22C for 10 min, then the supernatants were taken for measurement of hydrolysis of Ac-DEVD-AMC as a function of time at 22C. Each experiment was performed in duplicate. To reduce the variations among each assay, all experiments were performed in the same day. For assays of caspase-3-like activity in livers, the freshly excised or liquid nitrogen preserved livers were homogenized in the HEPES buffer (pH 7.5) described above. After centrifuged at 11 000g for 30 min, the protein contents in the supernatant were measured by the Protein Assay Reagent (BioRad, Richmond, CA, USA). The caspase-3-like activity was assayed with 12 g protein.
Mitochondrial membrane potential measurement The membrane potential of energized mitochondria (negative inside) promotes a directional uptake of 5, 5 , 6, 6 -tetrachloro-1, 1 , 3, 3 -tetraethylbenzimidazolylcarbocyanine iodide (JC-1) dye into the matrix, also with subsequent formation of aggregates. The aggregate fluorescence is sensitive to transient membrane potential changes. Briefly, the cells were loaded with 3 g/ml of JC-1 (Molecular Probes, Eugene, OR, USA) at 37C for 30 min. After gated out small-sized debris, the red (aggregates) and green (monomer) emitted fluorescence was collected through 575/30 nm (FL2) and 525/40 nm (FL1) bandpass filters, respectively.36 At least 20 000 cells were analyzed in each experiment.
Cytofluorometric analyses of cell cycles After treatment with TNF- and disulfiram for 48 h, the cells were plated at subconfluence in a 6-well plate and grew for 48 h. The cells were collected and incubated in a hypotonic fluorochrome solution (Propidium iodide 0.5 mg/ml in 0.1% sodium citrate plus 0.1% Triton X-100) overnight at 4C. The propidium iodide fluorescence of each individual nucleus was measured with excitation of 488 nm and emission of 620 nm.37 At least 20 000 cells were analyzed in each experiment.
Reactive oxygen species measurement The cells were placed at 1106/well in a 6-well plate. The cells were loaded with 20 M DCFH-DA in the culture medium at 37C for 70 min and analyzed by flow cytometry.38 Forward scatter, side scatter, and FL1 were collected for all events falling in established gates. At least 20 000 cells were analyzed in each experiment.
DNA fragmentation assays DNA fragmentation in the livers was determined by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method according to the manufacturer’s procedures (Boehringer Mannheim, Gaithersburg, MD, USA). Briefly, livers were fixed with 10% formalin and
1366 / Zhao et al.
embedded in paraffin. A 5-m-thick section was cut. Sections were deparaffinated and rehydrated and then assayed for direct immunoperoxidase detection of digoxigenin-labeled genomic DNA. DNA fragmentation was quantified by a ELISA kit according to the manufacturer’s procedures (Boehringer Mannheim, Gaithersburg, MD, USA). Briefly, the cytoplasma extract containing 1 g of protein was taken for measure of the kinetics of product generation (Vmax) which was used as an index of DNA fragmentation. The assay allows the specific quantitation of histone-associated DNA fragments (mono- and oligonucleosomes) in the cytoplasmic fraction of cell lysates and has been used extensively to demonstrate apoptosis in vivo.
Statistics Numerical data are expressed as meansstandard errors. Each experiment was performed in duplicate or triplicate. Statistical analyses were performed with Student’s t-test, or one-way analysis of variance (ANOVA), or nonparametric test as appropriate. Post hoc multiple comparisons were made by Dunnett test (ANOVA) or Dunn’s test (non-parametric). Null hypotheses were rejected at the level of 0.05.
Acknowledgements Authors thank Ms Karen Wolcott for her assistance in flow cytometry, Dr Elliot Kagan for allowing us to use the multi-well reader, Dr Ajay Verma for permitting us to use the fluorometer, Dr Karen Gray for her suggestions during the early phase of the study, and Dr Ying-Yue Li for her assistance in animal experiments. The study was supported in large part by a Grant-in-Aid from The National Kidney Foundation/The National Capital Area, Inc (to XZ). 2000 Academic Press
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32. Mignon A, Rouquet N, Fabre M, Martin S, Pages JC, Dhainaut JF, Kahn A, Briand P, Joulin V (1999) LPS challenge in D-galactosamine-sensitized mice accounts for caspase-dependent fulminant hepatitis, not for septic shock. Am J Respir Crit Care Med 159:1308–1315. 33. Bohlinger I, Leist M, Gantner F, Angermuller S, Tiegs G, Wendel A (1996) DNA fragmentation in mouse organs during endotoxic shock. Am J Path 149:1381–1393. 34. Waring P, Beaver J (1996) Gliotoxin and related epipolythiodioxopiperazines. Gen Pharmacol 27:1311–1316. 35. Wang C, Mayo MW, Baldwin AS Jr (1996) TNF- and cancer therapy-induced apoptosis: potentiation by inhibition of NF-B. Science 274:784–787. 36. Salvioli S, Ardizzoni A, Franceschi C, Cossarizza A (1997) JC-1, but not DiOC6(3) or rhodamine 123, is a reliable fluorescent probe to assess delta psi changes in intact cells: implications for studies on mitochondrial functionality during apoptosis. FEBS Lett 411:77–82. 37. Nicoletti I, Migliorati G, Pagliacci MC, Grignani F, Riccardi C (1991) A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. J Immunol Methods 139:271–279. 38. Robinson P (1997) Oxadative metabolism. In: Robinson JP, Darzynkiewicz Z, Dean PN, Dressler LG, Rabinovitch PS, Stewart CC, Tanke HJ, Wheeless LL (eds) Current Protocols in Cytometry. John Wiley & Sons, Inc, New York, pp 9.7.1–9.7.3.