ACTIVATION OF NEUTRAL SPHINGOMYELINASE PARTICIPATES IN ETHANOL-INDUCED APOPTOSIS IN HEP G2 CELLS

Jian-Jun Liu, Ji-Yao Wang, Erik Hertervig1, Yajun Cheng2, Åke Nilsson and Rui-Dong Duan2,*

Department of Gastroenterology, Zhongshan Hospital, Shanghai Medical University, Shanghai, People's Republic of China,
1 Department of Internal Medicine and
2 Cell Biology Department 1, Lund University Hospital, S–22185 Lund, Sweden

Received 13 January 2000; in revised form 19 May 2000; accepted 7 June 2000


    ABSTRACT
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
The mechanism underlying ethanol-induced apoptosis in liver cells is not clear. Sphingomyelin (SM) metabolism is a novel signal transduction pathway that has an impact on apoptosis in many cell types. We investigated whether the SM pathway is involved in ethanol-induced apoptosis in the liver. Hep G2 cells were treated with ethanol followed by assaying apoptosis, sphingomyelinase (SMase) activity, caspase-3 activity, and the changes of SM content in the cells. We found that ethanol dose-dependently increased apoptosis and the effect was accompanied by increases of caspase-3 activity and neutral SMase activity. At concentrations of 80 and 160 mM, ethanol significantly increased caspase-3 activity by 120% and neutral SMase activity by 24%. The activity of acid SMase was only slightly increased without statistical significance. C2-ceramide, the exogenous SM metabolite, mimicked the effects of ethanol on apoptosis and caspase-3 activation. When the SM content was determined 24 h after treatment with ethanol, its level was 15% lower than that of controls. The results indicate that metabolism of SM triggered by neutral SMase participates in ethanol-induced apoptosis in Hep G2 cells and activation of caspase-3 is involved in the apoptotic pathway.


    INTRODUCTION
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
Previous studies have demonstrated that ethanol is able to induce apoptosis in liver cells both in vivo and in vitro (Benedetti et al., 1988Go; Goldin et al., 1993Go; Higuchi et al., 1996Go). The mechanism underlying this effect is not clear. Sphingomyelin (SM) is a constituent of the eukaryotic membrane and the metabolism of SM has emerged as a novel signal transduction pathway. The hydrolysis of SM catalysed by sphingomyelinase (SMase) generates ceramide, which is closely associated with apoptosis in many cell types (Hannun and Bell, 1989Go; Kolesnick, 1991Go). We investigated the potential involvement of SM metabolism in ethanol-induced apoptosis in Hep G2 cells. We found that neutral SMase was stimulated by ethanol in a dose-dependent manner, concomitant with an increase in caspase-3 activity, a key factor in the execution phase of apoptosis.


    MATERIALS AND METHODS
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
Materials
The hepatoblastoma cell line Hep G2 was obtained from American Tissue Culture Collection (Rockville, MD, USA). RMPI-1640 medium, heat-inactivated fetal calf serum (FCS) and 0.05% trypsin:0.02% EDTA were from Life Technologies AB (Stockholm, Sweden). [14C]CH3-choline-labelled SM was obtained from Astra Draco (Lund, Sweden) and its specific activity was 56 µCi/mg. Authentic SM was obtained from Dr Lena Nyberg at the Swedish Dairies' Association and had a purity of over 95%. C2-ceramide (C2-Cer) was from Calbiochem (La Jolla, CA, USA). The Cell Death Detection kit was purchased from Boehringer Mannheim (Bromma, Sweden). Caspase-3 substrate Asp-Glu-Val-Asp-p-nitroanilide (DEVD-pNA) was from Oncogene Research Products (Cambridge, MA, USA).

Cell culture
The cells were cultured in RMPI-1640 medium containing 100 IU/ml penicillin, 10 µg/ml streptomycin, 10% heat-inactivated FCS, and maintained at 37°C in a humidified incubator with 95% air and 5% CO2. For the following experiments, cells were harvested with 0.05% trypsin: 0.02% EDTA at about 80% confluence and subcultured in RMPI-1640 medium overnight for attachment. According to previous studies (Neuman et al., 1993Go, 1998Go), the optimal concentration of ethanol to induce apoptosis in Hep G2 cells is about 80 mM; we therefore chose ethanol at concentrations ranging from 40–160 mM in this study.

Apoptosis detection
Apoptosis was detected by the Cell Death Detection ELISA kit (Veldman et al., 1998Go). Briefly, 104 cells were seeded in a 96-well plate in 200 µl of RMPI-1640 medium. After incubation with different concentrations of ethanol or C2-Cer, cells were lysed in the lysis buffer and centrifuged. The supernatant containing the cytoplasmic histone-associated DNA fragments was reacted with the anti-histone and anti-DNA antibodies. Apoptosis was evaluated by the enrichment of the levels of mono- and oligonucleosomes released into the cytoplasm, according to the instructions of the manufacturer.

SMase activity assay
The method for the SMase activity assay has been described elsewhere (Duan and Nilsson, 1999Go). Briefly, cells were harvested, washed in phosphate-buffered saline (PBS), and sonicated for 10 s in the homogenization buffer containing 0.25 M sucrose, 5 mM MgCl2, 0.15 M KCl, 50 mM KH2PO4, 1 mM PMSF, 1 mM benzamidine, and 10 mM taurocholate. Five-µl samples were added to a 75-µl acid assay buffer or neutral assay buffer, respectively. The acid buffer contained 30 mM Tris-maleate, 0.15 M NaCl, and 0.16% bile salt mixture (taurocholate:taurodeoxycholate: glycocholate: glycodeoxycholate 42:26:22:10, w/w) at pH 5.0, whereas the neutral buffer contained 30 mM Tris-HCl, 0.15 M NaCl, 4 mM MgCl2, and the bile salt mixture at pH 7.4. The reaction was started by adding 8000 dpm [14C]SM in 20 µl of 0.15 M NaCl containing 0.16% bile salt mixture, followed by incubation at 37°C for 30 min. After incubation, the reaction was stopped by adding 0.4 ml chloroform/methanol (2:1) and phase partition was carried out by centrifugation at 10 000 rpm for 3 s. An aliquot of 100 µl of the upper phase containing cleaved [14C]phosphocholine was taken and the radioactivity was assayed by liquid scintillation counting.

Determination of SM content
The measurement of SM was according to Hedlund et al. (1998). Briefly, 3 x 106 cells were seeded in 25 cm2 flasks in RMPI-1640 medium and labelled with 3H-choline chloride (1 µCi/ml) for 48 h. The medium was then replaced with fresh RMPI-1640 medium containing ethanol, followed by incubation for 24 h. Total lipids were extracted according to Bligh and Dyer (1959) and applied on silica gel plates (60 F, 0.25 mm) for thin-layer chromatography. After developing with chloroform/methanol/30% ammonia (65:25:4, by vol), the SM band was visualized by iodine vapour and scraped according to the position of authentic SM. The radioactivity in the band was measured by liquid scintillation counting.

Caspase-3 activity
Caspase-3 activity was assayed using DEVD-pNA as substrate (Cheng et al., 1999Go). Briefly, 3 x 106 Hep G2 cells were incubated for 24 h in 3 ml of RMPI-1640 medium containing ethanol or C2-Cer. After incubation, cells were scraped and washed three times in PBS. The cell pellet was incubated in 80 µl of ice-cold lysis buffer containing 50 mM HEPES, 100 mM NaCl, 0.1% CHAPS, 1 mM DTT, and 0.1 mM EDTA, pH 7.4 for 5 min, followed by centrifugation at 10 000 g for 10 min at 4°C. The aliquots of the extract were added to 50 mM HEPES buffer, pH 7.4 containing 100 mM NaCl, 0.1% CHAPS, 10 mM DTT, 0.1 mM EDTA, 10% glycerol, and 200 µM DEVD-pna for caspase-3 assay. Caspase-3 activity was determined every 15 min by a plate reader and the activity was calculated according to the instructions of the manufacturer.

Statistical analysis
Results are expressed as means ± SEM. The data were analysed by the non-paired Wilcoxon test. P < 0.05 was considered statistically significant.


    RESULTS
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 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
Apoptosis induced by ethanol
After incubating Hep G2 cells with ethanol for 24 h, the apoptosis rate increased, as indicated by the elevation of the content of DNA–histone complex in the cytoplasm (Fig. 1Go). At concentrations of 80 and 160 mM, ethanol increased the enrichment factor by 15 and 20%, respectively, compared with the control value (P < 0.05).



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Fig. 1. Apoptosis induced by ethanol. Hep G2 cells were incubated with different concentrations of ethanol for 24 h. Apoptosis was determined by the Cell Death Detection ELISA kit. Experiments were performed in duplicate and the results are from three independent experiments. *P < 0.05 compared with control.

 
Changes in caspase-3 activity induced by ethanol
As shown in Fig. 2Go, the activity of caspase-3 increased following treatment of the cells with ethanol. The stimulatory effect of ethanol started at 40 mM, with 80 to 160 mM increasing the activity of caspase-3 by about twofold over the control level.



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Fig. 2. Increase in caspase-3 activity by ethanol. Hep G2 cells were incubated with different concentrations of ethanol for 24 h. Cells were lysed and caspase-3 activity was then determined. Experiments were performed in triplicate and the results are from three independent experiments. *P < 0.05 compared with control.

 
Changes in SMase activity
After incubation of the cells with ethanol for 24 h, neutral SMase activity increased significantly in a dose-dependent manner (Fig. 3aGo). At 80 and 160 mM, ethanol increased neutral SMase by 24% and 33% respectively (P < 0.05). The activity of acid SMase showed an increase with increasing ethanol concentrations, but statistical significance was not reached (Fig. 3bGo).



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Fig. 3. Effects of ethanol on SMase activity. Hep G2 cells were incubated with different concentrations of ethanol for 24 h. The cells were lysed and the activities of neutral (a) and acid (b) SMases were then determined. All assays were done in triplicate and the results are from three independent experiments. *P < 0.05 compared with the control.

 
Effect of C2-Cer on apoptosis and caspase-3 activity
Hydrolysis of SM generates ceramide. In order to confirm the participation of the SMase pathway, the effects of ceramide on apoptosis and caspase-3 in Hep G2 cells were examined. As shown in Fig. 4Go, treating the cells for 24 h with 40 µM of cell membrane permeable C2-Cer increased the apoptosis rate by 20% (P < 0.05) and caspase-3 by 30% (P < 0.05), compared with controls.



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Fig. 4. Apoptosis and caspase-3 activity after C2-ceramide (C2-Cer) treatment. Hep G2 cells were incubated with 40 mM C2-Cer for 24 h and apoptosis (upper panel) and caspase-3 activity (lower panel) were determined. The results are from three separate experiments. *P < 0.05 compared with control.

 
Changes in SM in the cells after ethanol treatment
At the end of incubation with ethanol, the levels of SM in the cells were determined. As shown in Fig. 5Go, the content of SM in Hep G2 cells was decreased by 12% and 15% after incubation with 80 and 160 mM ethanol respectively. However, due to the great variations, none of the changes were statistically significant.



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Fig. 5. Changes of SM content after ethanol treatment. Hep G2 cells were labelled with [14C]choline chloride for 48 h, followed by treatment with ethanol for 24 h. The total lipids were extracted and separated by thin-layer chromatography. The radioactivities in SM bands were determined. Experiments were performed in triplicate and the results are from three independent experiments.

 

    DISCUSSION
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
Previous studies have established that ethanol can induce apoptosis in liver cells (Benedetti et al., 1988Go; Neuman et al., 1993Go; Higuchi et al., 1996Go). However, the mechanism of this effect has not been elucidated. In the present work, we found that the induction of apoptosis by ethanol in Hep G2 cells was accompanied by a significant increase in neutral, but not acid, SMase activity, and by an activation of caspase-3. C2-Cer, the species of ceramide that can penetrate the cell membrane, also activated caspase-3 and induced apoptosis in Hep G2 cells. Our data therefore indicate that the SM-metabolic pathway and caspase-3 activation participate in ethanol-induced apoptosis in liver cells.

SM metabolism is considered a novel signal transduction pathway, which generates multiple lipid messengers, such as ceramide, sphingosine, and sphingosine-1-phosphate (Kolesnick, 1991Go). Of the biologically active molecules derived from SM hydrolysis, ceramide is the most important; it can arrest the cell cycle, inhibit cell proliferation, and induce apoptosis (Spiegel and Merrill, 1996Go). SM can be hydrolysed by different types of SMase and different SMases have been found to be activated in response to different stimuli (Okazaki et al., 1998Go). In the present study, ethanol significantly increased the activity of neutral SMase in a dose-dependent manner. The activity of acid SMase tended to increase with the dose of ethanol, but statistical significance was not reached, indicating the role of acid SMase is less important than neutral SMase in mediating the effect of ethanol.

Caspase-3 is a key enzyme in the execution phase of apoptosis (Stennicke and Salvesen, 1998Go) and a close link between ceramide and caspase-3 has been demonstrated (Yoshimura et al., 1998Go; Takeda et al., 1999Go). The interaction of ceramide and caspase-3 varies with the cell type. In PC12 cells and CHP-100 cells, ceramide formation leads to caspase-3 activation (Yoshimura et al., 1998Go; Spinedi et al., 1999Go), whereas in human T and B lymphocytes, Fas-induced generation of ceramide is activated by caspase-3 (Genestier et al., 1998Go). In the present study, we found that C2-Cer increased the activity of caspase-3, indicating that caspase-3 in Hep G2 cells can be a downstream target of ceramide.

Although the results described above favour the role of SMase in ethanol-induced apoptosis, we only found a small reduction (12–15%) of SM after treatment with ethanol, which was not statistically significant. It is known that the kinetic changes in SM hydrolysis in response to stimuli vary from minutes to hours, depending on the type of both cells and agonists. Due to the existence of the SM cycle, the ceramide formed can be recycled to SM and the SM levels can be more or less restored (Okazaki et al., 1989Go; Riboni et al., 1997Go). The levels of SM in this study were determined after a 24-h incubation with ethanol; whether or not the recycling mechanism had been activated at this point is unknown. Kinetic studies in the future may help clarify this.

The mechanism by which ethanol activates neutral SMase has not been explored. It is known that generation of ceramide from SM hydrolysis can be increased by numerous factors, including various cytokines and stress situations (Kolesnick 1991Go; Higuchi et al., 1996Go; Pena et al., 1997Go). From the present study, two factors are worthy of discussion. First, ethanol was found to induce the secretion and expression of several cytokines including tumour necrosis factor-{alpha} (TNF-{alpha}) (Neuman et al., 1998Go), which is a well known activator of neutral SMase (Kim et al., 1991Go; Ballou et al., 1992Go). The sensitivity of the liver cells to TNF-{alpha} can also be enhanced by ethanol (Colell et al., 1998Go). Second, it is well known that ethanol may induce oxidative stress, especially depletion of glutathione and production of reactive oxygen species, such as hydrogen peroxide (Higuchi et al., 1996Go). Recently glutathione was found to be an inhibitor of neutral SMase (Liu and Hannun, 1997Go), and hydrogen peroxide may induce hydrolysis of SM and apoptosis in bronchial epithelial cells (Goldkorn et al., 1998Go). Taken together, the generation of cytokines, the depletion of glutathione, and the production of reactive oxidants may be responsible for the enhanced neutral SMase activity induced by ethanol, resulting in caspase-3 activation and apoptosis.


    ACKNOWLEDGEMENTS
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
This work was supported by grants from the Swedish Medical Research Council (12156 and 03696), Swedish Cancerfonden (4246-B99-01XAA), the Albert Påhlsson Foundation, Crafoordiska Foundation, Swedish Medicine Association, and funds from the University Hospital of Lund, Sweden.


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 INTRODUCTION
 MATERIALS AND METHODS
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 ACKNOWLEDGEMENTS
 REFERENCES
 
* Author to whom correspondence should be addressed. Back


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 ACKNOWLEDGEMENTS
 REFERENCES
 
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