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Cardiovascular Research 2003 57(1):119-128; doi:10.1016/S0008-6363(02)00646-6
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Copyright © 2003, European Society of Cardiology

Reactive oxygen species regulate FLICE inhibitory protein (FLIP) and susceptibility to Fas-mediated apoptosis in cardiac myocytes

Joji Nitobea,*, Seiji Yamaguchia, Masaki Okuyamaa, Naoki Nozakia, Masataka Satab, Takuya Miyamotoa, Yasuchika Takeishia, Isao Kubotaa and Hitonobu Tomoikea

aFirst Department of Internal Medicine, Yamagata University School of Medicine, 2-2-2 Iida-Nishi, Yamagata 990-9585, Japan
bDepartment of Cardiovascular Medicine, University of Tokyo, Tokyo, Japan

* Corresponding author. Tel.: +81-23-628-5302; fax: +81-23-628-5305. jnitobe{at}med.id.yamagata-u.ac.jp

Received 3 January 2002; accepted 22 August 2002


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: Fas ligand (FasL) is a key cytokine which initiates apoptosis when FasL binds to its receptor, Fas. Cardiac myocytes are generally resistant to Fas-induced apoptosis. However, sublethal dose of doxorubicin (Dox) can sensitize cardiac myocytes to Fas-induced apoptosis. We investigated the molecular mechanism by which Dox sensitizes cardiac myocytes to Fas-induced apoptosis. FLICE inhibitory protein (FLIP) is a key molecule for blocking Fas-induced apoptosis by functioning as a caspase-8 dominant negative. Methods and results: FLIP was constitutively expressed in cultured neonatal rat cardiac myocytes. FLIP protein levels were markedly down-regulated by Dox in a time-dependent and dose-dependent manner. Next, we examined the relation of reactive oxygen species (ROS) by Dox to the expression of FLIP. Both of N-acetylcysteine (NAC) and the combination of superoxide dismutase and catalase restored the decreased FLIP in Dox-treated cardiac myocytes to the basal level. NAC also restored the increased formation of thiobarbituric acid-reactive substance after Dox-treatment. Concurrently, the susceptibility to Fas-mediated apoptosis disappeared with the treatments of the antioxidant agents. Hydrogen peroxide down-regulated FLIP in a dose-dependent fashion and also sensitized cardiac myocytes to Fas-induced apoptosis. Conclusions: FLIP, an inhibitor of apoptosis induced by cytokines of TNF family, contributes at least partly to Dox-induced sensitization to Fas-mediated apoptosis in cardiac myocytes. The expression of FLIP in cardiac myocytes is regulated by ROS.

KEYWORDS Apoptosis; Cardiomyopathy; Cytokines; Free radicals; Myocytes


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Doxorubicin, an anthracycline antibiotics, has been used as a powerful drug in the fight against neoplastic diseases. However, its practical use is sometimes limited by acute and chronic cardiotoxicities [1]. The cardiotoxicity is dose-dependent and causes irreversible myocardial damage, resulting in dilated cardiomyopathy with fatal congestive heart failure [1]. The exact causal mechanism of doxorubicin-induced cardiomyopathy remains unclear, but most of the evidence indicates that free radicals are involved [2,3]. The chemical structure of doxorubicin is prone to the generation of free radicals, and the oxidative stress correlates with cellular injury [4]. Increased oxidative stress may lead to a variety of subcellular changes in the myocardium.

Apoptosis plays a pivotal role in loss of cells not only during physiological phenomena such as normal turnover of tissues, but also in many pathophysiological phenomena. Evidence is accumulating that the apoptotic mechanism is involved in various heart disorders [5–8]. Fas/Fas ligand system is one of the key regulators of apoptosis [9]. Fas/CD95 is the cell-surface receptor expressed in many cell types including cardiac myocytes [10]. Fas/CD 95 ligand (FasL) is expressed on the membrane of cells, such as activated T cells [11], NK cells [12], endothelial cells [13] or immune privileged sites [14]. Both the membrane-bound FasL and its cleaved form, soluble FasL, can induce apoptosis in vitro and in vivo [15–17], while the lethal ability is more potent in the membrane-bound FasL [18]. The Fas/FasL system has been reported to be activated in human heart failure [19–21]. Doxorubicin-induced deterioration in left ventricular performance in rat was inhibited by neutralizing anti-FasL antibody [22], suggesting an important role of Fas/FasL system in doxorubicin cardiomyopathy. However, it is obscure how administration of doxorubicin renders the myocardium prone to Fas-mediated apoptosis. We previously observed that cardiac myocytes were generally resistant to Fas-mediated apoptosis in vitro. However, after treatment of sublethal dose of doxorubicin, cardiac myocyte apoptosis was dramatically facilitated by recombinant FasL [23]. This finding is intriguing, because the doxorubicin-induced sensitivity to Fas in cardiomyocytes can be induced by a molecule which may be a target for treating doxorubicin-associated cardiomyopathy.

Recently, FLICE-inhibitory protein (FLIP), a molecule with sequence homology to caspase-8 (FLICE), was identified as an anti-apoptotic protein [24]. FLIP inhibits Fas- and TNF-mediated apoptosis [24,25]. When FasL binds to Fas, Fas-associated death domain (FADD) is activated and then, caspase-8 is cleaved and activated, leading to subsequent activations of caspase cascades. FLIP is capable of binding FADD, thereby preventing cleavage and activation of caspase-8, thus shutting off the initiation of the death pathway [24–26]. Although high levels of transcripts of FLIP are expressed in the heart compared to other organs [26], little is known about the basic mechanisms controlling the expression of FLIP. In this study, we investigated whether FLIP levels are related to the doxorubicin-induced sensitivity to Fas in cultured neonatal rat cardiac myocytes. Moreover, we examined the hypothesis that the expression of FLIP was down-regulated by oxidative stress.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1 Materials
Human soluble recombinant FasL (rFasL) [27] and human Fas chimeric protein (Fas-Fc) [28] were provided by Bioscience Research Laboratory, Mochida Pharmaceutical Co. (Tokyo, Japan). A polyclonal anti-FLIP antibody was generated in a Japanese white rabbit according to the protocol previously reported [26,29,30]. Anti-Fas and -FADD antibodies were purchased from Transduction Laboratory (Lexington, KY, USA). Doxorubicin, propidium iodide (PI), trypsin EDTA, superoxide dismutase, catalase and N-acetylcysteine (NAC) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Caspase-8 inhibitor (Z-lle-Glu-Thr-Asp-CH2F or Z-IETD-FMK) was purchased from Medical and Biological Laboratories Co. Ltd. (Nagoya, Japan). F-12 powder and fetal bovine serum (FBS) were from Cosmo Bio Co. Ltd. (Tokyo, Japan). Dulbecc’os phosphate-buffered saline, transferrin, and insulin-selenite were from Gibco BRL (Grand Island, NY, USA). Collagenase and hydrogen peroxide (H2O2) were from Wako Pure Chemical Industry (Osaka, Japan).

2.2 Cell culture
The animals were handled according to the animal welfare regulations of Yamagata University, and the study protocol was approved by the Animal Subjects Committee of Yamagata University. The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health (NIH Publication No. 85-23, revised 1996). Cardiac myocytes were isolated from 0- to 1-day-old Wister–Imamichi rats (Imamichi Institute for Animal Reproduction, Japan) by the methods previously described [23]. In brief, the ventricles were minced and digested with collagenase. After washing with MSS buffer (30 mmol/l HEPES, 4.1 mmol/l dextrose, 2 mmol/l KCl, 120 mmol/l NaCl, and 1 mmol/l KH2PO4), the digested fractions were filtered through a mesh. The pooled cells were resuspended in F-12 medium (10.62 g/l F-12 mixture, 15 mmol/l HEPES, 22 mmol/l NaHCO3, 5 µg/ml transferrin) with supplements of 5 µg/ml insulin-selenite, 50 U/ml penicillin, 50 µg/ml streptomycin and 10% FBS. After preplating in another flask to minimize non-myocyte contamination, cardiac myocytes were seeded onto fibronectin-coated dishes (5x105 cells/ml). Four days after seeding, the culture medium was changed to serum free F-12 medium containing transferrin, insulin and selenite. Doxorubicin treatment was started immediately after medium change. After treatment with or without doxorubicin (0.5 µmol/l) for 12 h, rFasL was added onto the cell culture, and again incubated for 12 h.

2.3 Detection of apoptosis
TUNEL staining was performed with a commercially available kit for detection of end-labeled DNA according to the manufactures instructions (Mebstain Apoptosis Kit Direct, MBL, Nagoya, Japan). The cardiac myocytes were fixed, washed, and incubated with distilled water, and then incubated with TdT and FITC-dUTP. Counter staining was performed with propidium iodide (PI) by diluting with PBS to obtain a concentration of 1 µg/ml. Approximately 800–1000 nuclei from random fields were analyzed for each sample. The apoptotic index was calculated as apoptotic nuclei/total nucleix100 (%). The presence of apoptotic profiles was also confirmed by staining with Hoechst dye #33342.

2.4 Cell viability assay
Cell viability was analyzed by MTT assay [31]. Equal numbers of cardiomyocytes were plated on 96-well plates. MTT (3-(4,5-dimethyl-thiazole-2-yl)-2,5-diphenyl tetrazolium bromide; 0.625 mg/ml) was added to each well. The plates were then analyzed with a multiwell plate reader at 590 nm.

2.5 Western blot analysis
Cultured cardiac myocytes were lysed for 30 min on ice in a lysis buffer containing 2% NP-40, 0.5% sodium deoxycholate, 0.2% SDS, and protease inhibitor mixture, and then centrifuged at 12 000 rpm for 10 min at 4 °C. The protein concentration of each sample was measured using BCA protein assay reagents (Pierce Chemical Co., Rockford, IL, USA). BSA was used as a protein assay standard. For SDS–PAGE, 80 µg of protein of each sample was added on 10% polyacrylamide gel and then electrophoretically transferred to PVDF membrane. After blocking with a buffer containing 5% non-fat milk and 0.2% Tween-20 for 1 h at room temperature, the membrane was incubated with either anti-FLIP, anti-Fas, or anti-FADD antibody for 3 h at room temperature. After washing three times with PBS containing 5% non-fat milk, the membranes were incubated with horseradish peroxidase-conjugated anti-rabbit IgG, anti-mouse IgG or anti-goat IgG for 1 h at room temperature. The membrane was washed again and the signals were visualized by enhanced chemiluminescence (Amersham Pharmacia Biotech., Japan).

2.6 Measurement of lipid peroxidation
The extent of lipid peroxidation was determined by using a thiobarbituric acid reactive substance (TBARS) assay in cultured rat neonatal cardiac myocytes. Malondialdehyde (MDA) was used as standard and the results were expressed as nanomol MDA per milligram protein [32].

2.7 Caspase-8 protease assay
Activation of caspase-8 was detected by the caspase-8 colorimetric protease assay kit (MBL, Nagoya, Japan). The assay is based on spectrophotometric detection of the chromophore p-nitroanilide (pNa) after cleavage from the labeled substrate IETD-pNa. IETD-pNa is a specific substrate for activated caspase-8, which has Ile-Glu-The-Asp amino acid sequence labeled with p-nitroanilide. The pNa light emission was quantified using a microtiter plate at 405 nm.

2.8 Data and statistical analysis
Data were expressed as mean±S.E. The values were tested by one-way analysis of variance (ANOVA), and was followed by the Scheffe's F-test. Differences were considered statistically significant at P<0.05.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1 Doxorubicin-induced sensitization to Fas-mediated apoptosis in neonatal rat cardiac myocytes
The apoptotic index had no significant change from control to treatments of recombinant human FasL (FasL) in neonatal rat cardiac myocytes (Fig. 1A). However, 12 h after pretreatment of sublethal dose of doxorubicin (0.5 µmol/l), FasL markedly increased the apoptotic index in a dose-dependent manner. The FasL-induced apoptosis was completely blocked by anti-Fas antibody and caspase-8 inhibitor (Z-IETD-FMK), indicating that Fas-mediated signaling was activated by FasL after pretreatment of doxorubicin. Similarly, although FasL alone did not affect the viability of cardiac myocytes, FasL reduced the viability of cardiac myocytes in the presence of sublethal dose of doxorubicin (Fig. 1B). Cardiac myocytes were stained with Hoechst dye (Fig. 1C,D). With 1 µg/ml of FasL after treatment of doxorubicin, nuclei of cells showed typical morphology of apoptosis such as nuclear condensation and fragmentation (Fig. 1D).


Figure 1
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  		Fig. 1 Human recombinant Fas ligand (FasL) induced apoptosis in neonatal rat cardiac myocytes after treatment of doxorubicin. In the absence and presence of doxorubicin (Dox: 0.5 µmol/l) for 12 h, cardiac myocytes were incubated with various concentrations of FasL for additional 12 h. (A) Apoptotic index was calculated as a ratio of apoptotic cells to total cell number by in situ TUNEL staining. (B) Cell viability was determined by MTT assay. * P<0.01, ** P<0.001 vs. untreated cells; § P<0.05, §§ P<0.001 vs. FasL 1 µg/ml with doxorubicin. Results are expressed as mean±S.E.M. of five separate experiments. (C,D) Nuclear morphology of cardiac myocytes were stained with Hoechst 33248. Nuclei of untreated cardiac myocytes were shown (C). Cardiomyocytes were incubated with 1 µg/ml of FasL after treatment of doxorubicin (D). Arrows indicate fragmented chromatine in nuclei of apoptotic cells.

 
3.2 Down-regulation of FLIP protein levels by doxorubicin
FasL-induced activation of caspase-8 occurred only after treatment of sublethal dose of doxorubicin in cardiac myocytes [23] (also shown in Fig. 4). Fas protein was constitutively expressed at basal condition and slightly up-regulated by 24 h treatment of doxorubicin (data not shown). FADD protein levels were unchanged by the treatment (data not shown). We assessed expression levels of FLIP, a molecule with sequence homology to caspase-8 and capacity of preventing proteolytic activation of caspase-8. Immunoblotting revealed that untreated cardiac myocytes express a 55-kDa immunoreactive protein that has an identical mobility to the positive control, an extract from COS cells transfected with the FLIPL expression plasmid [29]. Doxorubicin (0.5 µmol/l) strongly down-regulated the expression of the FLIP-immunoreactive protein in a time-dependent manner (Fig. 2A). When cardiac myocytes were treated with various concentrations of doxorubicin for 24 h, expression of FLIP protein was down-regulated in a dose-dependent manner (Fig. 2B).


Figure 4
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  		Fig. 4 Enzymatic activities of caspase-8 in cardiac myocytes. Caspase 8 activity was dramatically enhanced after both treatments of recombinant Fas ligand (FasL: 1 µg/ml) and doxorubicin (Dox: 0.5 µM). N-acetylcysteine (NAC) and the combination of superoxide dismutase (SOD) and catalase (CAT) abolished the enhancement of caspase-8 activity after both treatments of FasL and doxorubicin. Concentrations were as follows: SOD 500 U/ml, CAT 500 U/ml, and NAC 50 µmol/l. Results are expressed as mean±S.E.M. of three separate experiments.

 

Figure 2
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  		Fig. 2 Expression of FLIP on cardiac myocytes treated with doxorubicin. (A) The expression of FLIPL (55 kDa) was determined by Western blot analysis in cardiac myocytes incubated with and without doxorubicin (Dox; 0.5 µmol/l) for the times indicated. FLIPL expression was quantified by densitometry and presented as mean±S.E.M. from three independent experiments; * P<0.05 vs. 0 h without Dox. (B) FLIPL expressions of cardiac myocytes which were incubated for 24 h with various doses of doxorubicin. FLIPL expression was quantified by densitometry and presented as mean±S.E.M. from three independent experiments; * P<0.01 vs. serum free (SF).

 
3.3 Antioxidants decrease doxorubicin-induced sensitivity to Fas and recover down-regulation of FLIP levels by doxorubicin
Antioxidants were used to determine whether reactive oxygen species can contribute to the sensitization to Fas after the treatment of doxorubicin in neonatal rat cardiac myocytes. In the presence of antioxidants, SOD and CAT, the apoptotic index was markedly reduced despite both treatments of doxorubicin and FasL (Fig. 3A). Similarly a scavenger of reactive oxygen species, N-acetylcysteine (NAC), decreased the apoptotic index induced by treatments of doxorubicin and FasL. The SOD and CAT recovered the decreased cell viability by doxorubicin and FasL, and NAC also restored the cell viability (Fig. 3B). The effects of these antioxidants indicate that reactive oxygen species are closely related to the doxorubicin-induced susceptibility to Fas in neonatal rat cardiac myocytes. Next, we examined whether the down-regulated FLIP levels by doxorubicin can be restored by the antioxidants (Fig. 3C). In the presence of NAC and the combination of SOD and CAT, the decreased levels of FLIP by doxorubicin were restored to the untreated levels. As expected with these findings, doxorubicin increased TBARS formation in the cultured cardiac myocytes, and NAC lowered the increased formation of TBARS with doxorubicin (Fig. 3D).


Figure 3
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  		Fig. 3 Antioxidants abolished apoptosis of cardiac myocytes induced by recombinant Fas ligand (FasL) after treatment of doxorubicin (Dox; 0.5 µmol/l). (A) Cardiomyocyte apoptosis was estimated by TUNEL staining in the presence of N-acetylcysteine (NAC) and the combination of superoxide dismutase (SOD) and catalase (CAT). Apoptotic index was calculated as a ratio of apoptotic cells to total cell number. (B) Cell viability was determined by MTT assay in the presence of NAC and the combination of SOD and CAT. Results are expressed as mean±S.E.M. of three separate experiments. (C) Antioxidants restored the expression of FLIPL (55 kDa) in cardiac myocytes treated with Dox (0.5 µmol/l). FLIPL levels were determined by Western blot analysis in cardiomyocytes incubated with doxorubicin in the presence of NAC and the combination of SOD and CAT. FLIPL expression was quantified by densitometry and presented as mean±S.E.M. from three independent experiments. (D) Oxidative stress by Dox was assessed by TBARS assay. NAC abolished the increase of MDA formation by Dox. Concentrations were as follows: SOD 500 U/ml, CAT 500 U/ml, and NAC 50 µmol/l.

 
Activity of caspase-8 increased with the addition of FasL after pretreatments of doxorubicin. However, FasL or doxorubicin alone could not activate caspase-8 (Fig. 4). The SOD and CAT abolished the caspase-8 activation induced by FasL and doxorubicin. NAC also abolished the increased caspase-8 activity.

3.4 Hydrogen peroxide down-regulates FLIP levels and increases Fas-mediated apoptosis
To determine whether H2O2 inhibits the expression of FLIP, cardiac myocytes were incubated with exogenously administered H2O2. As shown in Fig. 5, the levels of FLIP were decreased by incubation with H2O2 (50 µmol/l for 12 h) in a dose-dependent manner. Moreover, FasL after pretreatment of H2O2 significantly decreased the cell viability of cardiac myocytes, whereas FasL or H2O2 alone had no significant change in the cell viability of cardiac myocytes (Fig. 5B).


Figure 5
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  		Fig. 5 Hydrogen peroxide (H2O2) down-regulated the expression of FLIPL and sensitized cardiac myocytes to Fas. (A) The expression of FLIPL (55 kDa) was determined by Western blot analysis in cardiomyocytes incubated with various doses of hydrogen peroxide for 24 h. FLIPL expression was quantified by densitometry. (B) Cardiac myocytes were incubated with human recombinant Fas ligand (FasL: 1 µg/ml) for 12 h after 12 h treatment with H2O2 (50 µmol/l). Cell viability was determined by MTT assay. * P<0.01 vs. untreated cells. Results are expressed as mean±S.E.M. of three separate experiments.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
The mechanisms by which doxorubicin causes cardiotoxicity have been the subject of extensive investigation and debate for more than two decades [1,2]. It has been suggested that apoptosis of cardiac myocytes is involved in the doxorubicin-induced loss of contractile function [22,33,34]. Recently, neutralizing anti-FasL antibody was reported to recover the doxorubicin-induced decrease of left ventricular performance in rat [22] suggesting that doxorubicin-induced apoptosis of cardiac myocytes may be executed through Fas-mediated pathway. Whereas cardiac myocytes were generally resistant to Fas-mediated apoptosis, cardiac myocytes became susceptible to Fas-mediated apoptosis after treatment of sublethal dose of doxorubicin [23]. Caspase-8 activity was markedly increased by FasL only after the pretreatment of doxorubicin indicating that doxorubicin modulates the signal from its receptor Fas to caspase-8. We focused on the behavior of FLIP, an anti-apoptotic molecule by inhibiting activation of caspase-8. Rat neonatal cardiac myocytes constitutively expressed FLIP protein, which is consistent with the resistance of cardiac myocytes to Fas-mediated apoptosis despite Fas expression in the untreated cardiac myocytes [10]. However, treatment of doxorubicin down-regulated the protein level of FLIP in neonatal rat cardiac myocytes in a time- and dose-dependent manner. Doxorubicin-induced downregulation of FLIP is one possible mechanism by which cardiomyocytes are prone to Fas-mediated apoptosis.

Doxorubicin has been reported to produce reactive oxygen intermediates including hydroxyl radicals and superoxide as well as hydrogen peroxide [4]. Doxorubicin also decreased protein levels of intracellular antioxidant enzymes, glutathione peroxidase (GSH) and manganese SOD [35]. The myocardial redox balance is markedly impaired by doxorubicin. Reactive oxygen species produced by doxorubicin play a role in cell death of cardiac myocytes [34]. Lower concentrations of doxorubicin produced apoptotic cell death of cultured cardiac myocytes [33,34,36] and there appears to be a switch from apoptosis to necrosis at doxorubicin concentrations of 2–10 µmol/l [33,34]. In addition to doxorubicin concentration, cultured conditions, species and aging are important factors of cell survival of cardiac myocytes. In this study, 0.5 µmol/l of doxorubicin did not induce an apparent decrease in cell viability of neonatal rat cardiac myocytes. Nevertheless, 0.5 µmol/l doxorubicin down-regulated FLIP expression to a critical level at which cardiac myocytes became susceptible to FasL-induced apoptosis.

Although valuable information regarding signal transduction pathways leading to apoptosis has been obtained from the study using neonatal cardiac myocytes, there is little information regarding the mechanisms for apoptosis wherein this process occurs in the adult heart. Results from the in vitro study using neonatal cardiomyocytes can not always be applied to the adult intact heart.

One of the most important findings in the present study is the profound inhibition of FLIP expression by reactive oxygen species. Combination of superoxide dismutase (SOD) and catalase, which are antioxidant enzymes hydrolyzing superoxide and hydrogen peroxide, respectively, prevented the doxorubicin-induced decrease of FLIP protein expression. Likewise, the antioxidant N-acetylcysteine (NAC) also restored the down-regulation of FLIP with doxorubicin treatment. Conversely, hydrogen peroxide, which is the most diffusible reactive oxygen species, itself decreases FLIP expression in a dose dependent manner. These results indicate that reactive oxygen species mediate the inhibitory action of doxorubicin on FLIP expression in cardiac myocytes. Following the reversal to the baseline level of FLIP by treatments with antioxidants, cardiac myocytes became resistant to Fas-mediated apoptosis again. Levels of FLIP have been demonstrated to play a pivotal role in the Fas-mediated apoptosis in various cell types [24–26,37]. Thus, reactive oxygen species produced by doxorubicin sensitize rat neonatal cardiac myocytes to Fas-mediated apoptosis at least partly by the way of down-regulating FLIP. In human Jurkat T cell lines, doxorubicin-induced apoptosis was reported to be independent of Fas signaling [38,39]. However in leukaemic cells [40], incubation with anti-Fas IgM antibody and doxorubicin augmented apoptotic responses as we demonstrated using neonatal rat cardiomyocytes in the present study. These data implicate that doxorubicin- and Fas-mediated apoptosis is cell specific events and may share some common components in signaling pathways leading to apoptosis. To our knowledge, precise mechanisms for the downregulation of FLIP by reactive oxygen species are still unknown. Transcriptional regulations and the protein stability or degradation of FLIP are suggested as possible mechanisms [41,42].

Besides doxorubicin-induced heart failure, reactive oxygen species have been shown to cause contractile failure and structural damage in various types of heart failure [43]. There may be a possibility that FLIP levels are down-regulated by reactive oxygen species in the myocardium of the failing heart. Recently, Imanishi et al. [44] observed that FLIP-positive cells rarely had fragmented DNA, while TUNEL-positive cells rarely contained FLIP in end-stage human hearts. Cardiac myocytes of the failing heart may be primed for apoptotic signal by FasL which has been reported to be elevated in patients with congestive heart failure [21] (Fig. 6). In the transgenic mouse overexpressing FasL in the heart, neither myocardial apoptosis nor necrosis occurred in the heart despite expression of Fas [45]. However, since the FLIP level should be unchanged in the transgenic mouse, the absence of apoptosis in the FasL-enforced myocardium might be due to relatively high FLIP levels.


Figure 6
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  		Fig. 6 One of proposed mechanism for doxorubicin-induced cardiomyopathy. Administration of doxorubicin produces reactive oxygen species (ROS) in cardiac myocytes and the ROS down-regulates the expression of FLIP that acts in a dominant-negative manner to inhibit caspase-8. In addition to increased level of Fas, the down-regulated FLIP renders cardiac myocytes Fas-sensitive, resulting in apoptosis. Fas ligand (FasL) may be expressed by leukocytes, cardiac myocytes and endothelial cells following administration of doxorubicin. FADD, Fas-associated death domain.

 
In conclusion, FLIP contributes, at least in part, to doxorubicin-induced susceptibility to Fas-mediated apoptosis in cardiac myocytes. Importantly, the expression of FLIP is regulated by reactive oxygen species in cardiac myocytes.

Time for primary review 25 days.


   Acknowledgements
 
This study was supported by grants from the Ministry of Education, Science and Culture, Japan (10307016 and 12670645).


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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