Sphingolipids suppress preneoplastic rat hepatocytes in vitro and in vivo

Ilona Silins, Mariann Nordstrand, Johan Högberg and Ulla Stenius1

Institute of Environmental Medicine, Karolinska Institutet, Box 210, S-17177 Stockholm, Sweden

1 To whom correspondence should be addressed Email: ulla.stenius{at}imm.ki.se


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Sphingolipids can modulate cell growth, differentiation and apoptosis. In the present investigation, selective death of hepatocytes localized in enzyme-altered foci (EAF hepatocytes) was shown to be induced by sphingolipids. Sphingosine (20 µM) caused rapid cell death predominantly of EAF hepatocytes in vitro. During 4 h of such exposure, cytochrome c was released from the mitochondria into the cytoplasm and the number of cells demonstrating cleaved caspase-9 activity increased. The selective sensitivity of EAF cells to sphingolipid-induced death was attenuated by tumor necrosis factor-{alpha}. In previous studies we have demonstrated that EAF hepatocytes are resistant to Fas-mediated apoptosis, a resistance shown here to be reversed by low concentrations of sphingosine. Immunohistological staining revealed higher levels of glucosylated ceramide in EAF than in the surrounding tissue. Furthermore, an inhibitor of glucosylation enhanced the toxicity of ceramide towards EAF cells. TLC analysis suggested low levels of sphingosine in preneoplastic lesions. In in vivo experiments EAF-bearing rats were fed a diet supplemented with 0.1% sphingomyelin for 2 weeks. Sphingolipid feeding reduced the number of EAF and EAF area in the liver by 40–50% as compared with rats fed a control diet. These studies indicate that the turnover of sphingolipids in preneoplastic EAF hepatocytes is altered. This alteration may explain not only the increased sensitivity of EAF cells towards sphingolipid-induced cell death, but also the resistance of these hepatocytes to cell death involving sphingolipids as second messengers. Furthermore, sphingomyelin in the diet may prevent EAF development. It is suggested that the altered turnover of sphingolipids might be a target for chemoprevention of hepatocellular carcinoma.

Abbreviations: DEN, diethylnitrosamine; DMS, N,N-dimethylsphingosine; EAF, enzyme-altered foci; GCDC, glucochenodeoxycholic acid; GST-P, glutathione S-transferase pi; PDMP, D-threo-1-phenyl-2-decanoylamino-1-propanol; TNF{alpha}, tumor necrosis factor-{alpha}.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Resistance to apoptosis characterizes many different types of cancer cells. Such resistance can be acquired via various mechanisms and appears to be essential for the development of cancer. Pre-neoplastic lesions in rat liver, so-called enzyme-altered foci (EAF), are also resistant to apoptosis (13). The growth of these foci is induced by carcinogens and they appear to be resistant, e.g. to apoptosis mediated by Fas or triggered by genotoxicity (4). Furthermore, in many cases carcinogens increase the mass of EAF by inhibiting apoptosis (5) and withdrawal of the carcinogen leads to enhanced apoptosis in EAF and many EAF may thereby disappear (5,6). Although the underlying mechanisms are not fully understood, these observations suggest that regulation of apoptosis is altered in EAF.

Sphingolipids have been demonstrated to play important roles in signal transduction involved in various responses such as differentiation, proliferation and apoptosis (7). In a variety of cell types, ceramide is generated in response to stress caused, e.g. by exposure to irradiation, UV-light or chemotherapeutic agents and this substance may function as a second messenger in connection with cell death pathways (8,9). One of the several lines of evidence supporting this conclusion is that knockout mice lacking acid sphingomyelinase exhibit defects in liver cell apoptosis (10). In addition, the resistance of acid sphingomyelinase –/– hepatocytes to Fas-mediated apoptosis is reversed by ceramide (11).

Drugs, which elevate intracellular levels of sphingolipids and ceramide have been found to induce apoptosis in transformed cells in culture (7). Dysfunctional metabolism of ceramide and other sphingolipids in cancer cells may also give rise to multi-drug resistance (12). As well as inducing apoptosis in cancer cells in vitro, sphingolipids have been reported to inhibit carcinogenesis in vivo. For instance, dietary sphingolipids inhibit the growth of different stages of 1,2-dimethylhydrazine-induced colon tumors (1315). Recently, dietary sphingolipids have also been shown to reduce the frequency of intestinal tumors in Min mice (16). Furthermore, ceramide analogs can prevent tumor growth and liver metastases in nude mice (17). Interestingly, in this same study the level of ceramide in human colon cancer was observed to be decreased.

In the present study our major question was whether preneoplastic cells are particularly sensitive to cell death induced by sphingolipids. In order to test the hypothesis, preneoplastic glutathione S-transferase (GST-P)-positive hepatocytes isolated from DEN-treated rats were exposed to ceramide, sphingosine or inhibitors of enzymes involved in sphingolipid metabolism and effects on cell death subsequently monitored in vitro. Furthermore, the effect of sphingolipid feeding on EAF development was studied in vivo.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Donor animals and primary cell cultures
Female Sprague–Dawley rats were injected i.p. with diethylnitrosamine (DEN) (0.3 mmol/kg body wt; Sigma, St Louis, MO) dissolved in 0.15 M NaCl within 24 h after birth. At 3 weeks of age, these rats were weaned and injected thereafter with the same dose of DEN once each week (18). One week following the final of seven to 11 such injections, hepatocytes were isolated from these animals.

Hepatocytes were isolated employing collagenase perfusion and then seeded onto collagen-coated plates (2 x 105 cells/35 mm plate; Sarstedt, Sweden). The cells were cultured in complete medium for 1.5 h and thereafter in serum-free RPMI 1640 medium (Life Technologies, Paisley, UK). To this latter medium various concentrations of the test substances were added, i.e. D-sphingosine, N-acyl-D-sphingosine (C2-ceramide), N-stearoyl-D-sphingosine (C18-ceramide) N,N-dimethylsphingosine, glucochenodeoxy-cholic acid (sodium salt; GCDC) or fumonisin B1 (dissolved in DMSO; Sigma) or tumor necrosis factor-{alpha} (TNF{alpha}, Biosource International, Camarillo, CA). D-Threo-1-phenyl-2-decanoylamino-1-propanol (PDMP, Sigma) was added to the culture medium 4 h prior to the addition of the test substance. After completion of treatment, the cells were washed and fixed in 3.7% formaldehyde for 1.5 h.

Immunocytochemistry
After fixation, the cells were stained with rabbit polyclonal anti-GST-P antibodies (BIO23 Yp, Biotrin, Ireland) (4) or with rabbit polyclonal anti-cleaved caspase-9 antibodies (#9507, Cell Signaling Technology, Beverly, MA). In order to detect cleaved caspase-9, the plates were pre-treated with saponin (Sigma) (18) and, following incubation with the primary antibodies, stained using the labeled streptavidin–biotin procedure (LSAB 2 kit, DAKO, Denmark). Peroxidase-conjugated secondary anti-rabbit antibodies (P217, DAKO) were utilized for revealing GST-P. Peroxidase activity was visualized with 3,3-diaminobenzidine tetrahydrochloride (DAB) as substrate.

Percentage of marker (GST-P or caspase-9)-positive cells were determined by examining at least 500 cells exhibiting characteristic hepatocyte morphology and located in several different randomly selected regions on each plate. All experiments were repeated at least three times employing different batches of cells. Statistical significance was evaluated utilizing the Mann–Whitney U-test. P value <0.05 was considered statistically significant.

Glucosylceramide immunohistochemistry
The livers of rats receiving one dose of DEN within 24 h after birth and seven additional doses of DEN (0.3 mmol/kg body wt) were used for glucosyl-ceramide immunohistochemistry. One week following the final injection, the liver was fixed in formaldehyde as described previously (19). Appropriate sections were stained overnight by incubation with rabbit anti-glucosyl-ceramide antibodies (20) (RAS 0010, GlycoTech Produktions und Handelsgesellschaft mbH, Germany), followed by secondary anti-rabbit peroxidase antibodies (P217, DAKO). Peroxidase activity was visualized using DAB as substrate.

Western blotting
Cells (6 x 106/10 cm plate) were cultured in the same manner as for immunocytochemistry. For detection of cytochrome c, the cells were washed with phosphate-buffered saline and thereafter scraped off the plates and homogenized on ice in 50 mM HEPES, pH 7.4, 220 mM mannitol, 68 mM sucrose, 50 mM KCl, 5 mM EGTA, 2 mM MgCl2, 1 mM DTT and 1 mM PMSF. The resulting homogenate was centrifuged, the high-speed supernantant thus obtained subjected to SDS–polyacrylamide gel and thereafter transferred to a PVDF membrane (Bio-Rad, Hercules, CA). Protein bands were identified and detected employing monoclonal mouse anti-cytochrome c antibodies (#556433, BD Pharmingen, San Diego, CA), followed by secondary anti-mouse antibodies (sc-2031, Santa Cruz Biotechnology).

For detection of p27, cyclin D2 and cyclin E, the plates were washed with phosphate-buffered saline and the cells then removed by scraping and homogenized in IPB7 buffer (20 mM triethanolamine–Cl, 0.7 M NaCl, 0.5% Nonidet P-40, 0.2% sodium deoxycholate, 0.1 mg trypsin inhibitor II-T/ml and 1 mM PMSF) in order to lyse them. The resulting suspension was subjected to SDS–polyacrylamide gel electrophoresis and thereafter blotted onto a PVDF membrane. Appropriate primary polyclonal rabbit antibodies (sc-528, sc-593, sc-481, Santa Cruz Biotechnology), followed by secondary anti-rabbit antibodies (P217, DAKO) were utilized to detect the protein bands. The blots were monitored with an enhanced chemiluminescence (ECL) detection system (Amersham Pharmacia Biotech, UK).

Thin-layer chromatography
Rats were treated using the same protocol as for the in vitro donor animals. One week after the last injection, hepatic nodules and surrounding tissue were dissected out separately and homogenized in a buffer containing 0.25 M sucrose, 25 mM KCl, 50 mM Tris and 0.5 mM EDTA pH 7.4. Lipids were extracted from each sample by chloroform:methanol and subsequently mixing and vortexing with 1 ml chloroform and 1 ml water. After centrifugation, filtration and drying under nitrogen, the samples were resuspended in chloroform. Samples (equalized for protein content) were spotted on to thin-layer chromatography silica plates (Merck, Germany) and developed in chloroform/methanol/25%NH3 (20:5:0.2, vol/vol/vol) to one-third of the total plate length. After drying, the plate was rechromatographed in heptane/diisopropylether/acetic acid (60:40:3, vol/vol/vol) (21). Lipids were visualized by spraying with cupric sulphate in aqueous phosphoric acid and drying for 15 min in 150°C. D-Sphingosine, C18 ceramide and sphingosine-1-phosphate standards were obtained from Sigma.

Diet experiments
Female Sprague–Dawley rats (180 g), fed a commercial standard pellet diet (RM3, SDS, UK), were injected i.p. with DEN (0.3 mmol/kg body wt) dissolved in 0.15 M NaCl once a week for 6 weeks. The rats were randomly divided into two groups and 4 days after the last DEN injection one group of animals was fed the standard diet supplemented with sphingomyelin (99% from chicken egg yolk from Sigma) 0.1 g/100 g diet (14), for 2 weeks. Control animals received the same diet without supplementation. All rats were observed daily and weighed twice a week to register any signs of sickness or weight loss. Liver sections were stained with polyclonal rabbit antibodies directed towards GST-P (Biotrin). Calculations of GST-P-positive area were performed using a software program based on the procedure of Pugh et al. (22). All experiments involving animals were approved by the local ethical committee according to the guidelines of the Swedish National Board of Laboratory Animals. Two experiments with similar design were performed. The number of animals receiving control diet was three and five in two different experiments and the number of rats receiving sphingomyelin diet was five and five. Statistical analyses were conducted using two-way ANOVA analysis and Mann–Whitney U-test. P value <0.05 was considered statistically significant.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Primary cultures of hepatocytes isolated from DEN-treated rats containing both EAF and non-EAF hepatocytes were exposed to ceramide. GST-P-positive hepatocytes are derived from EAF and GST-P-negative hepatocytes are derived from surrounding hepatic tissue and we have described previously a number of indications that the GST-P-positive cells in these cultures originate from EAF (18,23,24). The initial GST-P-% varied from cell batch to cell batch, but under control conditions the number of GST-P-positive cells did not change during the culture period (18). In cultures exposed to ceramide (75 µM) a loss of cells was observed within 24 h (Table I). As can be seen from Table I, such treatment also resulted in a decrease in the percentage of the remaining cells that were GST-P-positive, from 9.9 ± 4.6% to 1.4 ± 2.2%. This indicates that GST-P-positive hepatocytes were lost selectively. Exposure of cells to a long chain ceramide, C-18, also resulted in a selective loss of GST-P-positive cells (Table I). As expected (25) a lower concentration was needed to induce selection. When similar exposure to sphingosine, a metabolite of ceramide, was carried out for 24 h, the percentage of GST-P-positive cells decreased in a dose-dependent fashion (Table I). A 10 µM sample of sphingosine reduced the survival of GST-P-positive cells significantly, and this was associated with decreased number of cells on the plates.


View this table:
[in this window]
[in a new window]
 
Table I. Selective induction of death of GST-P-positive hepatocytes by C2-ceramide, C18-ceramide, sphingosine and an inhibitor of sphingosine kinase, DMS

 
The enzyme sphingosine kinase converts sphingosine to sphingosine-1-phosphate and inhibition of this enzyme by N,N-dimethylsphingosine (DMS) has been demonstrated to increase the level of ceramide in various types of cells (26). Treatment of hepatocyte cultures from DEN-treated rats with DMS was observed here to decrease total cell survival and to selectively decrease the survival of GST-P-positive cells, i.e. GST-P-negative cells were relatively resistant to this effect (Table I). A 20 µM aliquot of DMS reduced the relative number of GST-P-positive cells from ~10 to 3%.

In an effort to elucidate whether sphingosine and DMS exerts the effect on GST-P-positive cells by increasing the level of ceramide, an inhibitor of ceramidase, fumonisin B1 (9) was employed. However, fumonisin B1 (25 µM) treatment did not inhibit the death of GST-P-positive cells induced by a combination of sphingosine (10 µM) and DMS (10 µM), indicating a direct effect of sphingosine. In one typical experiment in fumonisin B1 treated cells the combination of sphingosine and DMS reduced the relative number of GST-P-positive cells from 7.0 ± 1.4 to 0.75 ± 0.70% (mean ± SD of three different plates).

In certain experiments, the percentage of GST-P-positive cells among all cells that detached from the plates was determined. It was found that the amount of GST-P-positive cells in the supernatant increased in proportion to the decrease in GST-P-% among cells remaining on the plates (data not shown). In other control experiments, sphingosine and ceramide were shown to induce cell death in hepatocyte cultures from untreated rats at concentrations similar to those required to cause death of hepatocytes isolated from DEN-treated rats (data not shown).

In general, the extent of cell death caused by exposure to sphingolipids was found to be dependent on cell density (data not shown). Because of a certain variability in the density of the primary cultures of hepatocytes, minor differences in responses to the same concentration were observed in different experiments. Variation within single experiments was small (compare with the effect of sphingosine in Table I). This suggests that the amount of e.g. sphingosine per cell, rather than the concentration in the medium, determined the responses.

Figure 1A documents the time-course of these effects of sphingosine on GST-P-positive cells during 4 h of incubation. A significant decrease in GST-P-% was induced already after 2 h incubation, a reduction associated with decreased total cell survival. The percentage of cells remaining on the plates decreased from 61 ± 1.7% after 1 h exposure to 36 ± 4% after 4 h of exposure. These effects of sphingosine rapidly reached their maximal level, being only slightly greater after 24 h of incubation. In order to characterize the mechanism(s) involved in the cell death observed here, we examined the possible effects of sphingolipids on mediators of apoptosis signals. Release of mitochondrial cytochrome c was enhanced after 1–4 h of exposure to sphingosine (Figure 1B). Caspase-9 activity was monitored employing immunocytochemical staining with antibodies directed specifically towards cleaved caspase-9. A significant increase of activated caspase-9 expressing cells after 1 h of such exposure was monitored (Figure 1C). Primary cultures of hepatocytes have recently been shown to be resistant to ceramide-induced apoptosis (27). However, similar concentrations of ceramide did, indeed, induce cell death in our cultures. The reason for this apparent discrepancy is not known, but may involve the use of different conditions for cell culturing.



View larger version (13K):
[in this window]
[in a new window]
 
Fig. 1. Time-course of the selective induction of the death of GST- P-positive hepatocytes by sphingosine; release of cytochrome c into the cytosol and cleavage of caspase-9. Hepatocyte cultures from DEN-treated rats were exposed to sphingosine for the periods indicated. (A) The results are expressed as the percentage of GST-P-positive cells remaining on the plates in the presence of sphingosine (20–25 µM). (B) Western blot analysis of the release of cytochrome c into the cytosol. (C) Immunocytochemical staining of cells for cleaved caspase-9. The results are expressed as the percentage of the total number of cells which stained positively in the absense (open columns) or presence (filled columns) of 30 µM sphingosine. The bars represent the means ± SD of three different experiments. *P < 0.05 compared with unexposed cells.

 
We have demonstrated previously that GST-P-positive cells from DEN-treated rats are resistant to Fas-mediated apoptosis (4), a process shown by others to involve sphingolipids (11). Consequently, the effect of sphingosine on the resistance of GST-P-positive cells towards Fas-mediated apoptosis was examined here by using glycochenodeoxycholic acid (GCDC), a cholic acid known to activate Fas in hepatocytes (28). As depicted in Figure 2A, the resistance of GST-P-positive cells against GCDC was reversed by sphingosine, 10 µM of this compound abolishing the effect of GCDC on the percent of GST-P-positive cells totally. In contrast, sphingosine had no effect on the total cell death caused by GCDC, as reflected by the number of cells remaining on the plates (Figure 2B). In this experiment 10 µM sphingosine alone had no effect on the percent of GST-P-positive cells (Figure 2A) or on the number of cells remaining on the plates (data not shown).



View larger version (15K):
[in this window]
[in a new window]
 
Fig. 2. Elimination by sphingosine of the resistance of GST-P-positive cells to Fas-mediated apoptosis induced by GCDC. Hepatocyte cultures from DEN-treated rats exposed to 50 µM GCDC and with or without 10 µM sphingosine for 24 h. The results are expressed as (A) the percentage of GST-P-positive cells and (B) the number of cells remaining per unit area on the plates. The bars represent the means ± SD of three different plates. *P < 0.05 compared to cells exposed to GCDC.

 
The cytokine TNF-{alpha} exerts a number of biological effects, including activation of several key enzymes involved in sphingolipid/ceramide metabolism (29,30). Thus, TNF-{alpha} has been demonstrated to activate sphingomyelinase and sphingosine kinase and thereby increase the level of sphingosine-1-phosphate in cells (30). Figure 3A documents the ability of TNF-{alpha} to protect against the decrease in the relative number of GST-P-positive cells induced by sphingosine. This protective effect apparently did not extend to GST-P-negative cells, as the total numbers of cells on the plates were not significantly affected by the presence of TNF-{alpha} (Figure 3B).



View larger version (14K):
[in this window]
[in a new window]
 
Fig. 3. Protection by TNF-{alpha} against the death of GST-P-positive cells caused by sphingosine. Hepatocyte cultures from DEN-treated rats were treated with sphingosine at the concentrations indicated with (filled columns) or without (open columns) 10 ng TNF-{alpha}/ml for 24 h. The results are expressed as (A) the percentage of GST-P-positive cells and (B) the number of cells remaining per unit area on the plates. The bars represent the means ± SD of three different plates. *P < 0.05 compared with cells exposed to sphingosine.

 
Sphingolipids have been shown to influence cell proliferation primarily by increasing the intracellular level of sphingosine-1-phosphate, which apparently serves as a second messenger in connection with this process (7). Therefore, the effect of sphingosine on the levels of p27, cyclin E and D2, which all vary during the cell cycle, were monitored. Exposure to this substance for 4 h was associated with a concentration-dependent decrease in p27 expression and an unchanged level of cyclin E (Figure 4), whereas the level of cyclin D2 was increased by 24 h of exposure. Higher concentrations of sphingosine were used in these studies due to the need for high cell density for western blot analysis. Together with the effects obtained with DMS (Table I) and TNF{alpha} (Figure 3) the data suggest that added sphingosine might modulate signal transduction, influencing both cell death and proliferation.



View larger version (34K):
[in this window]
[in a new window]
 
Fig. 4. Effects on p27, cyclin E and cyclin D2 expression upon exposure to sphingosine. Hepatocyte cultures from DEN-treated rats were exposed to various concentrations of sphingosine for the periods indicated. Western blot analysis of the expression of p27, cyclin E and cyclin D2 in the total cell culture (i.e. GST-P-positive and -negative cells combined).

 
Glucosylation of ceramide confers resistance to apoptosis on many types of cancer cells (31). As shown in Figure 5, cell death induced by ceramide was potentiated by PDMP, an inhibitor of such glucosylation (32), which decreased the relative number of GST-P-positive cells from 2.5 ± 0.2 to 0.2 ± 0.2%. No significant effect of PDMP on total cell survival was observed (data not shown). These findings prompted us to examine the level of glucosylceramide (GlcCer) in EAF tissue. An immunohistological study indicated the presence of higher levels of GlcCer in many EAF compared with the surrounding tissue, which exhibited only weak staining (Figure 6A). It was seen that the staining in EAF tissue was concentrated to cytoplasmic vesicular structures.



View larger version (10K):
[in this window]
[in a new window]
 
Fig. 5. Inhibition of glucosylceramide synthase potentiates ceramide-induced death of GST-P-positive cells. Hepatocytes cultures from DEN-treated rats were exposed to the concentrations of PDMP indicated with (filled columns) or without (open columns) 30 µM C2-ceramide for 17 h. The results are expressed as the percentage of GST-P-positive cells. The bars represent the means ± SD of three different plates. *P < 0.05 compared with cells exposed to PDMP alone.

 


View larger version (62K):
[in this window]
[in a new window]
 
Fig. 6. Enhanced immunohistochemical staining for glucosylceramide and sphingosine analysis in preneoplastic nodules in the liver of a DEN-treated rat. (A) The liver section, including an EAF, was stained for glucosylceramide as described under Materials and methods. (B) TLC analysis of C18 ceramide, sphingosine and sphingosine-1-phosphate in nodules and surrounding tissue from the livers of DEN-treated rats.

 
Lipid extracts from nodules and surrounding tissue were separated by TLC. As can be seen, in Figure 6B nodular extracts gave smaller dots with the retention time corresponding to sphingosine than extracts from surrounding tissue. For sphingosine-1-phosphate smaller dots were obtained in two of these extracts. No marked difference of C18 ceramide was detected. The data suggest alterations in sphingosine content of EAF tissue. However, a more definitive analysis is needed to confirm this and to elucidate possible other changes in sphingolipid content.

Earlier studies have shown that sphingomyelin feeding reduced the number of 1,2-dimethylhydrazine-induced colon tumors (1315). The effect of sphingomyelin in diet (0.1%; the same concentration as used in ref. 14) on DEN-induced EAF is shown in Figure 7. Sphingomyelin reduced the number of EAF by 40%. Thus, rats fed the control diet exibited 74 ± 7.4 EAF/cm2 liver tissue and rats fed the sphingomyelin diet exhibited 44.6 ± 6.4 EAF/cm2 (Figure 7A). EAF area fraction (EAF area/liver section area) was reduced by 50% (Figure 7B). To study the effect on the smallest EAF the effect of sphingomyelin on the number of single- and double GST-P-positive hepatocytes (33) was monitored. Also, this parameter was reduced by sphingomyelin diet, from 2.3 ± 0.4 to 1.3 ± 0.3 positive cells/mm2, a reduction by 43% (Figure 7C). No effect on liver morphology or growth of the animals was detected (data not shown).



View larger version (15K):
[in this window]
[in a new window]
 
Fig. 7. Sphingolipid feeding reduced the number of EAF and EAF area in the liver. Female rats were treated with i.p. injections of DEN once weekly for 6 weeks. One week after the last treatment the rats were fed a diet without supplements (control diet) or with 0.1% of sphingomyelin. After 14 days of feeding, the rats were killed and EAF parameters analyzed. Data are presented as mean ± SE. In (A) and (B) the number of rats receiving control diet was eight and sphingomyelin diet was 10 (see Materials and methods). In (C) the number of rats receiving both control and sphingomyelin diet was five. Statistical analyses were performed using two-way ANOVA (A and B) or Mann–Whitney U-test (C). *P < 0.05.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In the present study we have demonstrated that sphingolipids selectively induce cell death in preneoplastic EAF hepatocytes in vitro and that sphingomyelin in diet can reduce the number of EAF in vivo. Ceramide, sphingosine and an inhibitor of sphingosine kinase all activate a cell death pathway in such GST-P-positive cells in vitro. We also provide evidence suggesting that sphingolipids play a role in the resistance of these hepatocytes to apoptosis. An altered regulation of sphingolipid metabolism may explain both the increased sensitivity of EAF cells to death induced by sphingolipids and the resistance to cell death pathways in which sphingolipids act as second messengers.

It is well established that addition of sphingolipids to cells in culture or elevation of endogenous ceramide levels by, e.g. inhibition of sphingosine kinase gives rise to apoptosis in many types of cells (9). Accumulation of ceramide and sphingosine appears also to be involved in hepatocyte apoptosis in vivo (34). In this study the death induced selectively by various sphingolipids in GST-P-positive cells in vitro occurred rapidly. During 4 h of exposure, cytochrome c was released into the cytoplasm and the number of cells staining positively for activated caspase-9 increased. These observations indicate that apoptosis was induced in at least a fraction of affected cells, but it is not clear whether the death of all cells can be explained by apoptosis. For instance, sphingosine has been reported to induce both apoptosis and necrosis in a dose-dependent fashion, with partial overlap of the dose–response curves for these two effects (35).

In a previous study we demonstrated that GST-P-positive hepatocytes are resistant to Fas-mediated apoptosis induced by GCDC (4). Here, we found that treatment with low concentrations of sphingosine abolishes this resistance of GST-P-positive cells, rendering them susceptible to GCDC-induced apoptosis. These observations are in agreement with a growing body of evidence indicating the involvement of sphingolipids in Fas-mediated apoptosis. Recently, sphingolipids were reported to be required for the clustering and capping of Fas receptors (3638).

Several of the in vitro experiments described here (i.e. Figures 1 GoGo4) suggest that sphingolipids interfere with signaling pathways, specifically targeting GST-P-positive cells. For example, the experiment shown in Figure 3 suggests that the effect of sphingosine is modulated by TNF-{alpha} in GST-P-positive cells. These results together with the effects of sphingosine on cell cycle markers lend support to the conclusion that sphingosine added exogenously may influence signaling pathways in a selective way.

It is well established that sphingolipids inhibit colon carcinogenesis in different models (1317). The finding that dietary sphingomyelin reduces EAF in liver suggests that sphingolipids might have chemopreventive properties in this organ. EAF are the earliest preneoplastic lesions in rodent liver and the 40–50% reduction of EAF area fraction indicates that these lesions were suppressed. It remains to be elucidated whether the findings presented here are typical for all types of EAF.

Sphingolipids are prominent among components of food (39). Dietary sphingomyelin is slowly hydrolyzed in the intestine and it has been demonstrated in rat experiments that 2–5% of sphingomyelin administered by gavage is recovered in the liver within 2 h (40,41). An interesting question is whether, e.g. sphingosine, apparently taken up more efficiently than sphingomyelin (13), is more effective than sphingomyelin as chemopreventive agent.

Although the mechanism for the reduction of EAF remains to be elucidated, a low level of sphingosine in EAF tissue would provide a good explanation for the effects demonstrated here. The intracellular concentration of sphingolipids can be reduced by, e.g. enhanced glucosylation (42). Our immunohistological data are compatible with the finding that the constitutive rate of glucosylation of ceramide in EAF hepatocytes is higher than in surrounding non-EAF tissue. This finding was further supported from the pronounced potentiation of the effect of ceramide treatment in vitro observed here upon exposure to PDMP, a potent inhibitor of ceramide glucosylation (32). The mechanism underlying the enhanced glucosylation is of interest to elucidate in greater detail, especially in light of the elevated levels of glucosylceramide detected in many types of cancer cells (42).

Altogether, the present investigation indicates that the turnover of sphingolipids in preneoplastic EAF hepatocytes from DEN-treated rats is altered. This alteration may play a role in the adaptation of EAF to stress, conferring resistance to apoptosis induced by various extracellular stimuli. However, this altered turnover might at the same time give rise to increased sensitivity to sphingolipid-induced cell death, creating a possibility for eliminating preneoplastic cells. Additional studies, including in vivo studies with cancer endpoints, may provide a new strategy for chemoprevention of hepatocellular carcinoma.


    Acknowledgments
 
Supported by National Institute for Working Life, Sweden.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

  1. Schulte-Hermann,R., Bursch,W., Grasl-Kraupp,B., Müllauer,L. and Ruttkay-Nedecky,B. (1995) Apoptosis and multistage carcinogenesis in rat liver. Mutat. Res. Fundam. Molec. Mech. Mutat., 333, 81–87.
  2. Pitot,H.C. (1990) Altered hepatic foci: their role in murine hepatocarcinogenesis. Annu. Rev. Pharmacol. Toxicol., 30, 465–500.[CrossRef][ISI][Medline]
  3. Pitot,H.C. (1998) Hepatocyte death in hepatocarcinogenesis. Hepatology, 28, 1–5.[CrossRef][ISI][Medline]
  4. Nordstrand,M. and Stenius,U. (1999) Fas-mediated apoptosis is attenuated in preneoplastic GST-P-positive hepatocytes isolated from diethylnitrosamine-treated rats. Cell Biol. Toxicol., 15, 239–247.[CrossRef][ISI][Medline]
  5. Stinchcombe,S., Buchmann,A., Bock,K.W. and Schwarz,M. (1995) Inhibition of apoptosis during 2,3,7,8-tetrachlorodibenzo-p-dioxin-mediated tumour promotion in rat liver. Carcinogenesis, 16, 1271–1275.[Abstract]
  6. Bursch,W., Lauer,B., Timmermann-Trosiener,I., Barthel,G., Schuppler,J. and Schulte-Hermann,R. (1984) Controlled death (apoptosis) of normal and putative preneoplastic cells in rat liver following withdrawal of tumor promoters. Carcinogenesis, 5, 453–458.[Abstract]
  7. Hannun,Y.A. (1996) Functions of ceramide in coordinating cellular responses to stress. Science, 274, 1855–1859.[Abstract/Free Full Text]
  8. Chatterjee,M. and Wu,S. (2001) Cell line dependent involvement of ceramide in ultraviolet light-induced apoptosis. Mol. Cell. Biochem., 219, 21–27.[CrossRef][ISI][Medline]
  9. Kolesnick,R.N. and Krönke,M. (1998) Regulation of ceramide production and apoptosis. Annu. Rev. Physiol., 60, 643–665.[CrossRef][ISI][Medline]
  10. Santana,P., Pena,L.A., Haimovitz-Friedman,A., Martin,S., Green,D., McLoughlin,M., Cordon-Cardo,C., Schuchman,E.H., Fuks,Z. and Kolesnick,R. (1996) Acid sphingomyelinase-deficient human lymphoblasts and mice are defective in radiation-induced apoptosis. Cell, 86, 189–199.[ISI][Medline]
  11. Kirschnek,S., Paris,F., Weller,M., Grassmé,H., Ferlinz,K., Riehle,A., Fuks,Z., Kolesnick,R. and Gulbins,E. (2000) CD95-mediated apoptosis in vivo involves acid sphingomyelinase. J. Biol. Chem., 275, 27316–27323.[Abstract/Free Full Text]
  12. Senchenkov,A., Litvak,D.A. and Cabot,M.C. (2001) Targeting ceramide metabolism—a strategy for overcoming drug resistance. J. Natl Cancer Inst., 93, 347–356.[Abstract/Free Full Text]
  13. Dillehay,D.L., Webb,S.K., Schmelz,E.-M. and Merrill,A.H. (1994) Dietary sphingomyelin inhibits 1,2-dimethylhydrazine-induced colon cancer in CF1 mice. J. Nutr., 124, 615–620.[ISI][Medline]
  14. Schmelz,E.M., Dillehay,D.L., Webb,S.K., Schmelz,E.-M. and Merrill,A.H. (1996) Sphingomyelin consumption suppresses aberrant colonic crypt foci and increases the proportion of adenomas versus adenocarcinomas in CF1 mice treated with 1,2-dimethylhydrazine: implications for dietary sphingolipids and colon carcinogenesis. Cancer Res., 56, 4936–4941.[Abstract]
  15. Schmelz,E.M., Sullards,M.C., Dillehay,D.L. and Merrill,A.H. (2000) Colonic cell proliferation and aberrant crypt foci formation are inhibited by dairy glycosphingolipids in 1,2-dimethylhydrazine-treated CF1 mice. J. Nutr., 130, 522–527.[Abstract/Free Full Text]
  16. Schmelz,E.M., Roberts,P.C., Kustin,E.M., Lemonnier,L.A., Sullards,C., Dillehay,D.L. and Merrill,A.H. Jr. (2001) Modulation of intracellular ß-catenin localization and intestinal tumorigenesis in vivo and in vitro by sphingolipids. Cancer Res., 61, 6723–6729.[Abstract/Free Full Text]
  17. Selzner,M., Bielawska,A., Morse,M.A., Rüdiger,H.A., Sindran,D., Hannun,Y.A. and Clavien,P.-A. (2001) Induction of apoptotic cell death and prevention of tumor growth by ceramide analogues in metastatic human colon cancer. Cancer Res., 61, 1233–1240.[Abstract/Free Full Text]
  18. Stenius,U. and Högberg,J. (1995) GST-P-positive hepatocytes isolated from rats bearing enzyme-altered foci show no signs of p53 protein induction and replicate even when their DNA contains strand breaks. Carcinogenesis, 16, 1683–1686.[Abstract]
  19. Martens,U. and Stenius,U. (1999) Immunohistochemical detection of induced expression of wild-type p53 tumor supressor protein in the livers of rats treated with diethylnitrosamine. Histochem. J., 31, 75–79.[CrossRef][ISI][Medline]
  20. Brade,L., Vielhaber,G., Heinz,E. and Brade,H. (2000) In vitro characterization of anti-glucosylceramide rabbit antisera. Glycobiology, 10, 629–636.[Abstract/Free Full Text]
  21. Dobrzyn,A. and Górski,J. (2002) Ceramides and sphingomyelins in skeletal muscles of the rat: content and composition. Effect of prolonged exercise. Am. J. Physiol. Endocrinol. Metab., 281, E277–E285.
  22. Pugh,T.M., Kim,J.H., Koen,H., Nychka,D., Chover,J., Wahba,G., He,Y.H. and Goldfarb,S. (1983) Reliable stereological method for estimating the number of microscopic hepatocellular foci from their transections. Cancer Res., 42, 1261–1268.[ISI]
  23. Lennartsson,P., Stenius,U. and Högberg,J. (1999) p53 expression and TGF{alpha}-induced replication of hepatocytes isolated from rats exposed to the carcinogen diethylnitrosamine. Cell Biol. Toxicol., 15, 31–38.[CrossRef][ISI][Medline]
  24. Silins,I., Finnberg,N., Ståhl,A., Högberg,J. and Stenius,U. (2001) Reduced ATM kinase activity and an attenuated p53 response to DNA damage in carcinogen-induced preneoplastic hepatic lesions in the rat. Carcinogenesis, 22, 2023–2031.[Abstract/Free Full Text]
  25. Ji,L., Zhang,G., Uematsu,S., Akahori,Y. and Hirabayashi,Y. (1995) Induction of DNA fragmentation and cell death by natural ceramide. FEBS Lett., 358, 211–214.[CrossRef][ISI][Medline]
  26. Edsall,L.C., Van Brocklyn,J.R., Cuvillier,O., Kleuser,B. and Spiegel,S. (1998) N,N-Dimethylsphingosine is a potent competitive inhibitor of sphingosine kinase but not of protein kinase C: modulation of cellular levels of sphingosine 1-phosphate and ceramide. Biochemistry, 37, 12892–12898.[CrossRef][ISI][Medline]
  27. Tsugane,K., Tamiya-Koizumi,K., Nagino,M., Nimura,Y. and Yoshida,S.A. (1999) Possible role of nuclear ceramide and sphingosine in hepatocyte apoptosis in rat liver. J. Hepatol., 31, 8–17.[CrossRef][ISI][Medline]
  28. Faubion,W.A., Guicciardi,M.E., Miyoshi,H., Bronk,S.F., Roberts,P.J., Svingen,P.A., Kaufmann,S.H. and Gores,G.J. (1999) Toxic bile salts induce rodent hepatocyte apoptosis via direct activation of Fas. J. Clin. Inv., 103, 137–145.[Abstract/Free Full Text]
  29. Xia,P., Wang,L., Gamble,J.R. and Vadas,M.A. (1999) Activation of sphingosine kinase by tumor necrosis factor-{alpha} inhibits apoptosis in human endothelial cells. J. Biol. Chem., 274, 34499–34505.[Abstract/Free Full Text]
  30. Osawa,Y., Banno,Y., Nagaki,M., Brenner,D.A., Naiki,T., Nozawa,Y., Nakashima,S. and Moriwaki,H. (2001) TNF{alpha}-induced sphingosine 1-phosphate inhibits apoptosis through a phosphatidylinositol 3-kinase/akt pathway in human hepatocytes. J. Immunol., 167, 173–180.[Abstract/Free Full Text]
  31. Liu,Y.-Y., Han,T.-Y., Giuliano,A.E. and Cabot,M.C. (2001) Ceramide glycosylation potentiates cellular multidrug resistance. FASEB J., 15, 719–730.[Abstract/Free Full Text]
  32. Inokuchi,J., Mason,I. and Radin,N.S. (1987) Antitumor activity in mice of an inhibitor of glycosphingolipid biosynthesis. Cancer Lett., 38, 23–30.[CrossRef][ISI][Medline]
  33. Grasl-Kraupp,B., Luebeck,G., Wagner,A., Löw-Baselli,A., Gunst,M., Waldhör,T., Moolgavkar,S. and Schulte-Hermann,R. (2000) Quantitative analysis of tumor initiation in rat liver: roll of cell replication and cell death (apoptosis). Carcinogenesis, 21, 1411–1421.[Abstract/Free Full Text]
  34. Jones,B.E., Lo,C.R., Srinivasan,A., Valentino,K.L. and Czaja,M.J. (1999) Ceramide induces caspase-independent apoptosis in rat hepatocytes sensitized by inhibition of RNA synthesis. Hepatology, 30, 215–222.[ISI][Medline]
  35. Kågedal,K., Zhao,M., Svensson,I. and Brunk,U.T. (2001) Sphingosine-induced apoptosis is dependent on lysosomal proteases. Biochem. J., 359, 335–343.[CrossRef][ISI][Medline]
  36. Cremesti,A., Paris,F., Grassmé,H., Holler,N., Tschopp,J., Fuks,Z., Gulbins,E. and Kolesnick,R. (2001) Ceramide enables fas to cap and kill. J. Biol. Chem., 276, 23954–23961.[Abstract/Free Full Text]
  37. Grassmé,H., Jekle,A., Riehle,A., Schwarz,H., Berger,J., Sandhoff,K., Kolesnick,R. and Gulbins,E. (2001) CD95 signaling via ceramide-rich membrane rafts. J. Biol. Chem., 276, 20589–20596.[Abstract/Free Full Text]
  38. Cuvillier,O., Edsall,L. and Spiegel,S. (2000) Involvement of sphingosine in mitochondria-dependent Fas-induced apoptosis of type II Jurkat T cells. J. Biol. Chem., 275, 15691–15700.[Abstract/Free Full Text]
  39. Vesper,H., Schmelz,E.-M., Nikolova-Karakashian,M.N., Dillehay,D.L., Lynch,D.V. and Merrill,A.H. Jr. (1999) Sphingolipids in food and the emerging inportance of sphingolipids to nutrition. J. Nutr., 129, 1239–1250.[Abstract/Free Full Text]
  40. Schmelz,E.-M., Crall,K.J., Larocque,R., Dillehay,D.L. and Merrill,A.H. (1994) Uptake and metabolism of sphingolipids in isolated intestinal loops in mice. J. Nutr., 124, 702–712.[ISI][Medline]
  41. Nyberg,L., Nilsson,Å., Lundgren,P. and Duan,R.-D. (1997) Localization and capacity of sphingomyelin digestion in the rat intestinal tract. J. Nutr. Biochem., 8, 112–118.[CrossRef][ISI]
  42. Radin,N.S. (2001) Killing cancer cells by poly-drug elevation of ceramide levels. Eur. J. Biochem., 268, 193–204.[Abstract/Free Full Text]
Received November 4, 2002; revised March 12, 2003; accepted March 17, 2003.





This Article
Abstract
FREE Full Text (PDF)
All Versions of this Article:
24/6/1077    most recent
bgg055v1
Alert me when this article is cited
Alert me if a correction is posted
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Add to My Personal Archive
Download to citation manager
Search for citing articles in:
ISI Web of Science (4)
Request Permissions
Google Scholar
Articles by Silins, I.
Articles by Stenius, U.
PubMed
PubMed Citation
Articles by Silins, I.
Articles by Stenius, U.