Biochemical and Biophysical Research Communications 264, 622– 629 (1999) Article ID bbrc.1999.1576, available online at http://www.idealibrary.com on
The Antioxidant 4b,5,9b,10-Tetrahydroindeno[1,2-b]indole Inhibits Apoptosis by Preventing Caspase Activation Following Mitochondrial Depolarization G. P. Devitt, E. M. Creagh, and T. G. Cotter 1 Tumour Biology Laboratory, Department of Biochemistry, University College Cork, Cork, Ireland
Received September 24, 1999
Oxidative stress appears to have a central role in the induction of apoptosis following the exposure of cells to a range of cytotoxic insults. The modulation of apoptosis by a diverse range of antioxidants has been reported in many systems. We demonstrate, for the first time, the anti-apoptotic properties of the antioxidant, 4b,5,9b,10tetrahydroindeno[1,2-b]indole (THII), in Jurkat T cells subjected to a number of cytotoxic insults. THII was found to inhibit the morphological features of apoptosis in cells treated with the cytotoxic agents camptothecin, actinomycin D and ultraviolet (UV) irradiation. However, THII was unable to inhibit apoptosis induced by anti-Fas IgM. Peroxide and superoxide anion production following UV treatment was monitored, and THII was found to only partially inhibit superoxide anion production. THII was unable to inhibit mitochondrial depolarization in UV, Camptothecin or anti-Fas-treated cells. Further downstream, THII exibited strong inhibition of caspase-3 activation in UV, but not in anti-Fastreated cells. These results suggest that THII may exert its effects downstream of mitochondrial depolarization, but upstream of caspase-3 activation. © 1999 Academic Press
Apoptosis is a genetically programmed form of cell death which involves the activation of an endogenous suicide programme (1). Morphological characteristics of apoptosis include cell shrinkage, membrane blebbing, chromatin condensation and apoptotic body formation. Apoptosis is vital for normal physiological processes such as deletion of auto-reactive thymocytes (2), tissue homeostasis (3) and developmental limb sculpting (4). The process is also inAbbrevations used: Act D, actinomycin D; AIDS, acquired immune deficiency syndrome; Camp, camptothecin; DPI, diphenylene iodonium; GSH, glutathione; PDTC, pyrollinedithiocarbamate; Phen, phenanthroline; ROS, reactive oxygen species; THII, 4b,5,9b,10tetrahydroindeno[1,2-b]indole; UV, ultraviolet. 1 To whom correspondence should be addressed. Fax: 353-21904259. E-mail:
[email protected]. 0006-291X/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
volved in a number of pathological conditions such as autoimmune diseases, cancer, neurodegenerative diseases and AIDS (5, 6). The induction of apoptosis by diverse cytotoxic stimuli has been shown to result in oxidative stress within the cell. Oxidative stress is a term given to the generation of reactive oxygen species (ROS), which is a collective term for a number of oxygen containing free radicals, including the superoxide anion (O 22), hydroxyl radicals (OH •) and some nonradical derivatives of oxygen, such as hydrogen peroxide (H 2O 2). A number of varying antioxidants have been shown to be potent inhibitors of apoptosis e.g. butylated hydroxyanisole (BHA), cysteamine, pyrrolidinedithiocarbamate (PDTC), phenanthroline (Phen), diphenylene iodonium (DPI) etc. (7, 8, 9). They all have different modes of action but are classed as antioxidants for their ability to reduce the oxidative stress of a cell. They may do this by directly scavenging ROS or by blocking the effect of ROS production, thus implicating ROS as common mediators of apoptosis. The mitochondria are thought to be major sites of ROS production in the cell (10). A reduction of the mitochondrial transmembrane potential during cell death has been established in many cell systems and this disruption promotes the generation of ROS (11). The activation of a family of proteases, now known as caspases, has been found to be a common event in apoptosis (12, 13). Caspases are a family of cysteine proteases that cleave their substrates after specific aspartic acid residues (14). Caspase-3 is an effector caspase and is responsible for cleavage of many proteins including poly (ADP-ribose) polymerase (PARP) (15), U1-70kD (16), DFF (17) and other caspases (18). Two caspase-3 proenzymes of 32 kDa are proteolytically processed into an active tetramer containing two p17 and two p12 subunits. Caspase-3 is found to be activated in apoptosis induced by a wide range of stimuli including etoposide, staurosporine, camptothecin (Camp), ultraviolet irradiation (UV), actinomycin D (Act D) and the anti-Fas IgM (19 –24).
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The effects of three diverse antioxidants on apoptosis were examined during this study, dipenylene iodonium (DPI), phenanthroline (Phen) and 4b,5,9b,10-tetrahydroindeno[1,2-b]indole (THII). DPI is a NADPH oxidase inhibitor and prevents conversion of O 2 to O 22 by this enzyme (9). Phen is a metal ion chelator and functions by removing free transition metal ions, thereby minimizing the amplification of ROS toxicity within the cell (25). THII, a recently identified antioxidant, has been shown to inhibit lipid peroxidation and is thought to act as a free radical scavenger (26). THII has not yet been identified as an inhibitor of apoptosis. Apoptosis induced by UV and the cytotoxic agents, Camp and Act D all involve the production of ROS (27, 28). Therefore, apoptosis induced by these agents should be inhibitable by antioxidants. Induction of apoptosis by cross-ligation of Fas, however, does not involve production of ROS and is not normally inhibitable by antioxidants (29). Recently it has been demonstrated that anti-Fas induced apoptosis results in the rapid loss of reduced glutathione (GSH) (30), which may constitute an oxidative stress. This study identifies the antioxidant compound THII as an inhibitor of oxidant-induced apoptosis. We demonstrate its ability to inhibit apoptosis induced by UV, Camp and Act D but not anti-Fas IgM. THII demonstrates no effect on the production of peroxide by any of these cytotoxic treatments and only a partial reduction of superoxide production following UV treatment. Results indicate that THII does not inhibit mitochondrial depolarisation (Dcm) but does inhibit the activation of caspase-3, suggesting that this antioxidant compound is inhibiting apoptosis at a point downstream of the mitochondrion. MATERIALS AND METHODS Cell culture. The human leukaemic T cell lymphoblast line, Jurkat (31), was maintained in RPMI-1640 medium supplemented with 10% FCS, 2 mM glutamine and 1% penicillin-streptomycin. Cells were maintained at 37°C in a humidified 5% CO 2 atmosphere. All reagents were obtained from Gibco (Paisley, UK). Induction of apoptosis. Jurkat cells (5 3 10 5/ml) were seeded in 24 well plates (Nunc, Paisley, U.K.) and exposed to various cytotoxic agents: Camptothecin (50 mg/ml) (Sigma, Poole, U.K.), Actinomycin D (5 mg/ml) (Sigma) and the anti-Fas IgM antibody (300 ng/ml) (Upstate Biotech., Lake Placid, NY). Alternatively, cells were exposed from below to a 302 nm UV transilluminator source at a distance of 2.5 cm for 10 min at room temperature. Cells were then incubated at 37°C for the indicated times. Inhibition of apoptosis. THII (4b,5,9b,10-tetrahydroindeno[1,2b]indole) (Dr. M. Sainsbury, University of Bath) was prepared as a 1 mg/ml stock solution in DMSO. DPI (Diphenyleneiodonium) (Sigma) was prepared as a 10 mM stock in DMSO. Phen (Phenanthroline) (Reidel de Haen, Seelze, Germany) was prepared in ethanol as a 1 M stock and was stored at 220°C. The inhibitors were added 10 min before cytotoxic insult. Cell viability and morphology. Cell number was assessed using a Neubauer haemocytometer, and viability was determined by the ability to exclude trypan blue. Cell morphology was evaluated by
cytospin (100 ml, 5 3 10 5/ml), in their suspension media, onto slides using a Shandon Cytospin 2 (Cheshire, U.K.) at 500 rpm for 2 min. Slides were air dried, fixed and stained using the Rapi-Diff II staining kit (Paramount reagents Ltd., U.K.). Apoptotic cells were identified as described previously (32). Measurement of intracellular peroxide levels. Peroxide levels were assessed using a method previously described (33). Briefly, cells (5 3 10 5/ml) were incubated with 5 mM DCFH/DA (Molecular Probes, Leiden, The Netherlands), made as 10 mM Stock in DMSO, for 1 h at 37°C. Cells were incubated with both apoptosis inducing and inhibiting agents either before or during this 1 h incubation period (depending on the timepoint at which the peroxide measurement was to be made). Peroxide levels were measured using a FACScan flow cytometer (Becton Dickinson, CA) with excitation and emission settings of 488 and 530 nm, respectively (FL-1 channel). Measurement of intracellular superoxide anion levels. Superoxide anion levels were measured using an adaptation of the method employed by (34). The dye hydroethidine (Molecular Probes) was used, which is oxidised by the superoxide anion within the cell to produce ethidium bromide which fluoresces when it intercalates into DNA. Cells (5 3 10 5 /ml) were incubated with 10 mM hydroethidine (made as 10 mM stock in DMSO) for 15 min at 37°C. Cells were incubated with both apoptosis inducing and inhibiting agents either before or during this 15 min incubation period. Intracellular superoxide anion levels were assessed by measuring the fluorescence due to ethidium bromide using a FACScan flow cytometer with excitation and emission settings of 488 and 600 nm, respectively (FL-2 channel). Measurement of mitochondrial membrane potential. Mitochondrial membrane potential was analyzed using the fluorochrome JC-1, which incorporates into mitochondria and forms monomers (green fluorescence at 527 nm; FL-1) or aggregates at high transmembrane potential (red fluorescence at 590 nm; FL-2) (35). JC-1 was prepared as a 5 mg/ml stock solution in DMSO and used at 5 mg/ml. JC-1 was added for 15 min at 37°C after a 4 h incubation period with cytotoxic agents. Mitochondrial membrane potential was assessed by measuring the fluorescence due to JC-1 monomers and aggregates using a FACScan flow cytometer. Western blot analysis. Cells (2 3 10 6 ) were centrifuged at 200 3 g for 5 min, washed once in PBS, and lysed in 30 ml lysis buffer (0.15 M NaCl, 1% NP-40, 0.25% Sodium deoxycholate, 50 mM Tris-HCl (pH 8.0), 1 mM Na 3 VO 4 , 1 mM NaF, 1 mM EGTA, 0.1 mM phenylmethylsulfonyl fluoride (PMSF) and protease inhibitor cocktail (1 mg/ml antipain, 1 mg/ml aprotonin, 1 mg/ml chymostatin, 0.1 mg/ml leupeptin and 1 mg/ml pepstatin) for 30 min then centrifuged at 14,000 rpm (Eppendorf Centrifuge 5417 C) for 15 min. An equal volume of 23 SDS gel loading buffer (100 mM Tris-HCl (pH 6.8), 200 mM DTT, 4% SDS, 0.2% bromophenol blue and 20% glycerol) was added to samples, boiled for 5 min and loaded on a 15% SDS-polyacrylamide gel, electrophoresed and blotted onto nitrocellulose membrane (Schleicher and Schuell Dassel, Germany). After neutralization with 5% non-fat dried milk (NFDM) in PBS, the membranes were incubated for 18 h with the rabbit anti human caspase-3 polyclonal IgG (Upstate Biotech.), diluted 1:1000 in 5% NFDM, at 4°C with gentle agitation. The secondary antibody used was horseradish peroxidaseconjugated goat anti-rabbit (Dako, Glostrup, Denmark), at a 1:1000 dilution, for 1 h at room temperature. Membranes incubated with mouse anti-b-Actin (Sigma) at a 1:5000 dilution were incubated for 1 h, washed and were further incubated with horseradish peroxidase-conjugated anti-mouse monoclonal antibody (Sigma) at a 1:1000 dilution for 1 h. Bands were revealed using enhanced chemoluminescence detection kit (Amersham, Buckinghamshire, U.K.) and exposed to autoradiographic film.
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RESULTS THII inhibits the morphological features of apoptosis. To establish whether the antioxidant compound THII is capable of inhibiting apoptosis, the morphological features of Jurkat cells treated with UV, Camp or Act D in the presence of THII were observed. THII was found to reduce apoptosis in a dose dependent manner (Figs. 1A and 1C). However, THII was incapable of inhibiting apoptosis induced by anti-Fas IgM, suggesting that the compound is mediating its anti-apoptotic effects via its antioxidant properties, as the anti-Fas IgM has been reported to induce apoptosis through a ROS-independent pathway (29). THII alone has little effect on background levels of apoptosis over four hours. The NADPH oxidase inhibitor DPI also inhibited UV, Camp and Act D induced apoptosis in a dose dependent manner (Figs. 1B and 1C). Interestingly, both THII and DPI seemed to enhance anti-Fas induced apoptosis. THII partially inhibits ROS production. Whether ROSs play a role as initiators or effectors of apoptosis is unknown. Some antioxidants have been shown to directly inhibit the production of ROS (10). Therefore, we sought to determine if THII could inhibit ROS production during apoptosis. Peroxide and superoxide anion production levels in UV and Camp treated Jurkat cells were monitored for 1 hour after the initial insult using the fluorescent probes DCFH-DA and DHE, respectively. In UV treated cells DPI, but not THII, inhibited peroxide production (Figs. 2A and 2B). Peroxide production was not inhibited by either DPI or THII in Camp treated cells (Figs. 2C and 2D). Superoxide anion levels in UV treated cells were analyzed and it was found that both THII and DPI partially inhibited its production (Figs. 2E and 2F). Superoxide anion production in Camp treated cells was no different to untreated cells. Peroxide and superoxide anion production in anti-Fas treated cells was also no different than untreated, as has been reported previously (36). THII does not inhibit loss of Dcm. Following the observation that THII does not appear to inhibit the majority of ROS production by Camp and UV treatment, the protective effect of THII on the mitchondria was investigated. The mitochondria are thought to be central mediators of apoptosis and a major site of ROS production. Disruption of Dcm occurs as a result of many apoptotic stimuli (10, 11). We found that loss of Dcm occurred between four and six hours after treatment with UV, Camp or anti-Fas. Neither DPI nor THII were able to inhibit the loss of Dcm in any of the cytotoxic treatments (Fig. 3). However, Phen (a known metal ion chelator) was found to be quite effective in inhibiting loss of Dcm. This result places the effects of THII downstream of loss of Dcm.
FIG. 1. THII reduces oxidant-induced apoptosis in a dose dependent manner. (A) Dose response curve for THII mediated protection against cytotoxic agents in Jurkat cells. (B) Dose response curve for DPI mediated protection against cytotoxics in Jurkat cells. Apoptosis was measured by morphological assessment of cytospins taken four hours after incubation with cytotoxic agents. THII and DPI were added ten minutes prior to cytotoxic agent addition. Results are means 6 SE values from three independent experiments. (C) Morphology of Jurkat cells treated with cytotoxics and allowed to recover for four hours. (I) Control cells, (II) 10 min UV, (III) 10 min UV plus 10 mg/ml THII, (IV) 10 min UV plus 10 mM DPI, (V) 300 ng/ml Fas, (VI) 300 ng/ml Fas plus 10 mg/ml THII.
THII inhibits caspase-3 activation. Caspase-3 is an effector caspase which lies downstream of cytochrome C release from the mitochondria in the apoptotic cascade. Caspase-3 has been shown to be activated in both ROS-dependent and -independent apoptosis (13, 15, 20, 24). Therefore, the activation of caspase-3 in the presence of THII and DPI was examined. Results revealed that both THII and DPI were found to inhibit caspase-3 activation in UV treated cells, as can be seen
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FIG. 1—Continued
from the decreased levels of the p17 and p12 subunits in the UV plus THII/DPI treated cells compared to UV alone (Fig. 4A). Similar results were obtained with Camp treated cells (data not shown). As expected, caspase-3 activation in anti-Fas treated cells is not inhibited by pretreatment with THII (Fig. 4B). Neither is it inhibitable by DPI (data not shown). In addition,
there appears to be a slight increase in caspase-3 activation in the antioxidant plus anti-Fas treated cells. DISCUSSION The inhibition of apoptosis by antioxidants suggests that oxidative stress plays a central role in apoptosis
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FIG. 2. THII has no effect on peroxide production and a partial reducing effect on the production of superoxide following cytotoxic treatment to cells. Histogram analysis of peroxide and superoxide production following 10 min UV or 50 mg/ml Camp as measured by DCFH-DA in FL-1 and DHE in FL-2, respectively. Cells treated with UV and (A) 10 mg/ml THII, (B) 10 mM DPI. Cells treated with Camp and (C) 10 mg/ml THII, (D) 10 mM DPI. Cells treated with UV and (E) 10 mg/ml THII, (F) 10 mM DPI. Peroxide readings were taken thirty minutes after treatment with UV or Camp, which was equivalent to peak peroxide production. Superoxide readings were taken immediately after treatment with UV which was equivalent to peak superoxide anion production. Results are representative of three independent experiments.
induced by a wide range of cytotoxic agents (37). However, apoptosis has been shown to occur in an anaerobic environment (38, 39), although this does not neces-
sarily prevent the production of ROS (40) or creation of an oxidative environment in the cell. Cross-ligation of the Fas receptor induces apoptosis without the produc-
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FIG. 3. Mitochondrial depolarisation is not influenced by pre-incubation with THII. Contour plots of mitochondrial depolarisation (Dcm) as measured by JC-1 on a FACScan in FL-1 and FL-2. Readings were taken four hours after initial insult with cytotoxic agent. (A) Control cells, (B) 10 min UV (C) 100 nM Valinomycin, (D) 10 min UV plus 10 mg/ml THII, (E) 10 min UV plus 10 mM DPI, (F) 10 min UV plus 300 mM Phenanthroline.
tion of ROS and many antioxidants have proved to be ineffective against Fas induced apoptosis (29). Our results show that THII is capable of inhibiting apoptosis induced by a number of cytotoxic agents
FIG. 4. THII inhibits oxidant-induced activation of caspase 3. (A) Western blot of caspase-3 activation. Lane 1: Control cells, 2: UV treated cells, 3: UV plus 10 mg/ml THII, 4: UV plus 5 mg/ml THII, 5: UV plus 1 mg/ml THII, 6: UV plus 10 mM DPI, 7: UV plus 5 mM DPI, 8: UV plus 1 mM DPI. (B) Lane 1: Control cells, 2: Fas treated cells, 3: Fas plus 20 mg/ml THII, 4: Fas plus 10 mg/ml THII, 5: Fas plus 5 mg/ml THII.
known to involve ROS production. Pretreatment with THII is able to inhibit the characteristic morphological features of apoptosis in cells treated with UV, Camp or Act D. However, THII is not able to inhibit apoptosis induced by anti-Fas IgM, thus implicating ROS as the target for the protective function of THII. Further evidence supporting ROS as the target for THII’s mode of action is its ability to inhibit superoxide anion production in UV treated cells. However, the fact that THII is effective against Camp induced apoptosis, which does not involve any increase in superoxide anion levels, suggests that the antioxidant effects of THII are not solely due to the partial inhibition of superoxide anion production observed in the results. One of the major sites for ROS production are the mitochondria (10). The mitochondria are thought to be central regulators of the apoptotic pathway. The disruption of the mitochondrial membrane potential (Dcm) appears to precede nuclear related events (41). This loss of Dcm is indicative of the opening of a large conductance channel known as the mitochondrial Permeability Transition (PT) pore (42). The release of cytochrome C from mitochondria, however, seems to precede PT pore opening (43). Cytosolic cytochrome C is essential for activation of caspase-9 via association with Apaf-1 (44). Active caspase-9 then proceeds to activate other caspases including Caspase-3 (19). Loss of Dcm in Jurkat cells treated with Camp, Fas or UV occurred between 4 and 6 hours after initial insult. This precludes the generation of ROS due to depolarization of the mitochondria in this cell line, as ROS
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production precedes this by at least 3 h. Neither THII nor DPI were capable of inhibiting loss of Dcm, suggesting that they exert their anti-apoptotic effects either on a mitochondrial independent pathway or else downstream of loss of Dcm. Furthermore, the antioxidant phenanthroline was capable of inhibiting loss of Dcm, suggesting that it exerts its effects upstream of mitochondrial membrane depolarization. Activation of Caspase-3 is common to both ROSdependent and -independent apoptosis (13, 19, 20, 24). Our results show the activation of caspase-3 by treatment with UV and Fas. Both THII and DPI were capable of inhibiting activation of caspase-3 in UV treated Jurkats. However, neither of them could inhibit activation of caspase-3 in anti-Fas induced apoptosis. The antioxidant PDTC has previously been shown to inhibit anti-Fas induced apoptosis but not by an antioxidant mechanism (45). The fact that THII is capable of inhibiting caspase-3 activation in UV treated (ROS-dependent) but not in anti-Fas IgM treated (ROS-independent) cells further supports the previous evidence suggesting ROS as the target for THII’s mode of action. This result also suggests that THII does not exert its effects by direct modification of caspases as was shown for PDTC and other dithiocarbamates (45, 46). Furthermore, it has been reported that in certain cell types anti-Fas can signal through a mitochondria-independent apoptosis pathway. Fas receptor engagement causes rapid activation of caspases by a death-inducing signalling complex (DISC) (47). Interestingly, the activation of caspase-3 in anti-Fas treated Jurkats seems to be increased when pretreated with THII. This result correlates well with the increased levels of apoptosis as determined by cytospin. The activity of caspases is optimal in a reducing environment (45, 46). Perhaps THII operates by reducing the general redox status of the cell. In the case of apoptosis involving oxidative stress, THII possibly scavenges the ROS produced to reduce this stress to normal or tolerable levels thereby inhibiting apoptosis. However, in the case of anti-Fas induced apoptosis, THII reduces the redox status of the cell, creating a reducing environment in which the activity of caspases is optimal. Taken together our results indicate that THII is capable of inhibiting apoptosis induced by a wide range of stimuli that involve the induction of oxidative stress. THII may exert its effects somewhere between loss of Dcm and the activation of caspase-3. Alternatively, it may act generally to reduce the redox status of the cell. ACKNOWLEDGMENTS This work was supported by the Health Research Board of Ireland and Forbairt, Ireland. The authors are grateful to Dr. M. Sainsbury, Department of Chemistry, University of Bath, UK, for supplying the THII compound.
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