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, S22185 Lund, Sweden
Received 13 January 2000; in revised form 19 May 2000; accepted 7 June 2000
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ABSTRACT |
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INTRODUCTION |
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MATERIALS AND METHODS |
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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., 1993, 1998
), the optimal concentration of ethanol to induce apoptosis in Hep G2 cells is about 80 mM; we therefore chose ethanol at concentrations ranging from 40160 mM in this study.
Apoptosis detection
Apoptosis was detected by the Cell Death Detection ELISA kit (Veldman et al., 1998). 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, 1999). 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., 1999). 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.
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RESULTS |
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DISCUSSION |
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SM metabolism is considered a novel signal transduction pathway, which generates multiple lipid messengers, such as ceramide, sphingosine, and sphingosine-1-phosphate (Kolesnick, 1991). 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, 1996
). 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., 1998
). 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, 1998) and a close link between ceramide and caspase-3 has been demonstrated (Yoshimura et al., 1998
; Takeda et al., 1999
). 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., 1998
; Spinedi et al., 1999
), whereas in human T and B lymphocytes, Fas-induced generation of ceramide is activated by caspase-3 (Genestier et al., 1998
). 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 (1215%) 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., 1989; Riboni et al., 1997
). 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 1991; Higuchi et al., 1996
; Pena et al., 1997
). 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-
(TNF-
) (Neuman et al., 1998
), which is a well known activator of neutral SMase (Kim et al., 1991
; Ballou et al., 1992
). The sensitivity of the liver cells to TNF-
can also be enhanced by ethanol (Colell et al., 1998
). 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., 1996
). Recently glutathione was found to be an inhibitor of neutral SMase (Liu and Hannun, 1997
), and hydrogen peroxide may induce hydrolysis of SM and apoptosis in bronchial epithelial cells (Goldkorn et al., 1998
). 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.
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ACKNOWLEDGEMENTS |
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FOOTNOTES |
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REFERENCES |
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Benedetti, A., Brunelli, E., Risicato, R., Cilluffo, T., Jezequel, A. M. and Orlandi, F. (1988) Subcellular changes and apoptosis induced by ethanol in rat liver. Journal of Hepatology 6, 137143.[ISI][Medline]
Bligh, E. G. and Dyer, W. J. (1959) A rapid method of total lipid extraction and purification. Canadian Journal of Biochemistry and Physiology 37, 911917.
Cheng, Y., Tauschel, H.-D., Nilsson, Å. and Duan, R.-D. (1999) Ursodeoxycholic acid increases the activities of alkaline sphingomyelinase and caspase-3 in the rat colon. Scandinavian Journal of Gastroenterology 34, 915920.[ISI][Medline]
Colell, A., Garcia-Ruiz, C., Miranda, M., Ardite, E., Mari, M., Maoales, A., Corrales, F., Kaplowitz, N. and Fernandez-Checa, J. C. (1998) Selective glutathione depletion of mitochondria by ethanol sensitises hepatocytes to tumour necrosis factor. Gastroenterology 118, 15411551.
Duan, R.-D. and Nilsson, Å. (1999) Sphingolipids hydrolyzing enzymes in the gastrointestinal tract. Methods in Enzymology 311, 276286.[ISI]
Genestier, L., Prigent, A. F., Paillot, R., Quemeneur, L., Durand, I., Banchereau, J., Revillard, J. R. and Bonnefoy-Berard, N. (1998) Caspase-dependent ceramide production in Fas- and HLA class 1-mediated peripheral T cell apoptosis. Journal of Biological Chemistry 273, 50605066.
Goldin, R. D., Hunt, N. C., Clark, J. and Wickramasinghe, S. N. (1993) Apoptotic bodies in a murine model of alcoholic liver diseases: reversibility of ethanol-induced changes. Journal of Pathology 171, 7376.[ISI][Medline]
Goldkorn, T., Balaban, N., Shannon, M., Chea, V., Matsukuma, K., Gilchrist, D., Wang, H. and Chan, C. (1998) H2O2 acts on cellular membranes to generate ceramide signalling and initiate apoptosis in tracheobronchial epithelial cells. Journal of Cell Science 111, 32093220.
Hannun, Y. A. and Bell, R. M. (1989) Functions of sphingolipids and sphingolipid breakdown products in cellular regulation. Science 243, 500507.[ISI][Medline]
Hedlund, M., Duan, R.-D., Nilsson, Å. and Svanborg, C. (1998) Sphingomyelin, glycosphingolipids, and ceramide signalling in cells exposed to p-fimbriated Escherichia coli. Molecular Microbiology 29, 12971306.[ISI][Medline]
Higuchi, H., Kurose, I., Kato, S., Miura, S. and Ishii, H. (1996) Ethanol-induced apoptosis and oxidative stress in hepatocytes. Alcoholism: Clinical and Experimental Research 20, 340A 346A.
Kim, M. Y., Linardic, C., Obeid, L. and Hannun, Y. A. (1991) Identification of sphingomyelin turnover as an effector mechanism for the action of tumour necrosis factor- and
-interferon. Specific role in cell differentiation. Journal of Biological Chemistry 266, 484489.
Kolesnick, R. N. (1991) Sphingomyelin derivatives as cellular signals. Progress on Lipid Research 30, 138.
Liu, B. and Hannun, Y. A. (1997) Inhibition of the neutral magnesium-dependent sphingomyelinase by glutathione. Journal of Biological Chemistry 272, 1628116287.
Neuman, M. G., Koren, G. and Tiribelli, C. (1993) In vitro assessment of the ethanol-induced hepatotoxicity on Hep G2 cell line. Biochemical and Biophysical Research Communications 197, 932941.[ISI][Medline]
Neuman, M. G., Shear, N. H., Bellentani, S. B. and Tiribelli, C. (1998) Role of cytokines in ethanol-induced cytotoxicity in vitro in Hep G2 cells. Gastroenterology 115, 157166.[ISI][Medline]
Okazaki, T., Bell, R. M. and Hannun, Y. A. (1989) Sphingomyelin turnover induced by vitamin D3 in HL-60 cells. Role in cell differentiation. Journal of Biological Chemistry 264, 1907619080.
Okazaki, T., Kondo, T., Kitano, T. and Tashima, M. (1998) Diversity and complexity of ceramide signalling in apoptosis. Cell Signal 10, 685692.[ISI][Medline]
Pena, L. A., Fuks, Z. and Kolesnick, R. (1997) Stress-induced apoptosis and the sphingomyelin pathway. Biochemical Pharmacology 53, 615621.[ISI][Medline]
Riboni, L., Viani, P., Bassi, R., Prinetti, A. and Tettamanti, G. (1997). The role of sphingolipids in the process of signal transduction. Progress in Lipid Research 36, 153195.[ISI][Medline]
Spiegel, S. and Merrill, A. H. Jr (1996) Sphingolipid metabolism and cell growth regulation. FASEB Journal 10, 13881397.
Spinedi, A., Di Bartolomeo, S., Di Sano, F., Rodolfo, C., Ambrosino, A. and Piacentini, M. (1999) Ceramide accumulation precedes caspase-dependent apoptosis in CHP-100 neuroepithelioma cells exposed to the protein phosphatase inhibitor okadaic acid. Cell Death and Differentiation 6, 618623.[ISI][Medline]
Stennicke, H. R. and Salvesen, G. S. (1998) Properties of the caspases. Biochimica et Biophysica Acta 1387, 1731.[ISI][Medline]
Takeda, Y., Tashima, M., Takahashi, A., Uchiyama, T. and Okazaki, T. (1999) Ceramide generation in nitric oxide-induced apoptosis. Activation of magnesium-dependent neutral sphingomyelinase via caspase-3 Journal of Biological Chemistry 274, 1065410660.
Veldman, R. J., Klappe, K., Hoekstra, D. and Kok, J. W. (1998) Metabolism and apoptotic properties of elevated ceramide in HT29rev cells. Biochemical Journal 331, 563569.[ISI][Medline]
Yoshimura, S., Banno, Y., Nakashima, S., Takanaka, K., Sakai, H., Nishimura, Y., Sakai, N., Shimizu, S., Eguchi, Y., Tsujimoto, Y. and Nozawa, Y. (1998) Ceramide formation leads to caspase-3 activation during hypoxic PC12 cell death: Inhibitory effects of Bcl-2 on ceramide formation and caspase-3 activation. Journal of Biological Chemistry 273, 69216927.