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*
Institute of Biochemistry, Food Science and Nutrition, Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University of Jerusalem, Rehovot 76100, Israel; and
Institute of Pathology, Sheba Medical Center, Tel-Hashomer, Israel
2To whom correspondence should be addressed. E-mail: bschwart{at}agri.huji.ac.il.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • apoptosis • butyrate • caspases • colon cancer
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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–3
). An inverse relationship between colon tumor mass and fecal butyrate levels was observed in rats, supporting this hypothesis (4
,5
). Caderni et al. (6
) provided evidence that oral administration of butyrate in the form of an enteric-resistant slow-release pellet significantly increases colonocyte apoptosis rate in a rat model of colon cancer, indicating a potential anticancer effect in vivo. We have recently shown that high butyrate levels, obtained after fermentation of soluble dietary fibers, or rectally instilled, inhibits early and late events in colon tumorigenesis by controlling the transcription, expression and activity of key proteins involved in the apoptotic cascade. Various mechanisms have been proposed to explain how this particular SCFA inhibits tumorigenesis, including differentiation, cell cycle arrest and apoptosis (4
,7
–10
). Treatment of mammalian cells in vitro with physiological (millimolar) concentrations of butyrate has pleiotropic effects on cellular physiology, including changes in the cell membrane, cytoskeleton (8
), cell cycle (9
,10
) and transcription of multiple genes (11
). Butyrate has several effects on transcription factors and nuclear proteins that could modify gene expression (10
–12
); these effects differ among the many cell lines studied in vitro (8
,10
–14
). Furthermore, although butyrate is a potent inducer of apoptosis in cancer cells in vitro (1
,15
), the molecular mechanisms involved remain largely unknown.
Major advances have been made in our understanding of the molecular mechanisms triggering apoptosis. Regulation of apoptotic cell death is often altered in transformed cells (16
–21
). A key pathway leading to apoptotic cell death is via activation of the intracellular cascade of cysteine proteases, now referred to as caspases (22
). This cascade is believed to represent a major regulatory step in the apoptotic pathway, because it includes upstream (caspase-1) as well as downstream (caspase-3) events (7
,23
–25
). One key endpoint in this cascade is activation of caspase-3, which cleaves several substrates, such as the DNA repair enzyme poly(ADP-ribose) polymerase (PARP) or DNA-fragmentation factor (DFF), leading to the typical 180-bp DNA-strand breaks observed in the course of apoptosis (15
,26
). The Bcl-2 family seems to act as an integrator of diverse positive and negative survival signals, regulating whether the cell lives or die. The members fall into two major opposing subfamilies. One subfamily, such as Bcl-2, enhances survival, while members of the second subfamily, such as Bax and Bak, can trigger apoptosis. Components from the two subfamilies often form heterodimers and seem to titrate one another’s functions. Thus, tension between opposing members of the two families seems to act as a rheostat to determine whether the insult to the cell is sufficient to justify activation of its suicide program (22
).
The objectives of this investigation were to study the mechanisms of butyrate-induced apoptosis using two human colon cancer cell lines, Caco-2 and RSB. Caco-2 is a well-characterized human colon cancer cell line originating and isolated from a primary grade II colorectal adenocarcinoma tumor and defined as well-differentiated. At confluence, the cells express characteristics of enterocytic differentiation, and when injected into nude mice, they become tumorigenic. RSB originated from an ulcerative colitis patient who developed a colon carcinoma. Colon tumors originating in ulcerative colitis undergo a mutational program that differs from sporadic colonic carcinomas (27
). A case in point is the tumor-suppressor gene p53, which is mutated in sporadic carcinoma at late stages of the adenoma-carcinoma sequences, while in colon cancer originating from ulcerative colitis, p53 mutations take place at early stages of cancer development (27
).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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All chemicals and biochemicals were purchased from Sigma Chemical (St. Louis, MO) unless otherwise specified. The caspase-1 inhibitor VI, Z-YVAD-FMK, and the caspase-3 inhibitor II, Z-DVED-FMK, were purchased from Calbiochem (San Diego, CA). Anti-caspase-1 antibody and anti-caspase-3 antibody were from Upstate (Lake Placid, NY). Anti-Bak antibody and anti-PARP antibody were from Santa Cruz Biotechnology (Santa Cruz, CA), and anti-Bcl-2 antibody was from Zymed Laboratories (San Francisco, CA).
Cell lines and culture.
Two million Caco-2 (ATCC Number HTB-37) and RSB cells (generously provided by Dr. R. S. Bresalier, Detroit, MI) were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Biological Industries, Beit Ha’emek, Israel) supplemented with 10% fetal calf serum, glutamine and antibiotics (100,000 U/L penicillin and 100 mg/L streptomycin sulfate), at 37°C under a humidified atmosphere with 5% CO2; 24 h after seeding in flasks, the medium was changed and sodium butyrate was added for 48 h at 2, 5 or 10 mmol/L. At the end of each experiment, the cells were counted and lysed in the appropriate buffer. Additional experiments were performed in 24-multi-well plates (Nunclon, Roskilde, Denmark) at a density of 8 x 104 cells/well. After a 24-h stabilization period, Caco-2 and RSB cells were cultured in the absence (control) or presence of butyrate (2, 5 or 10 mmol/L). Proliferation was quantitated as described previously (28
), by monitoring cell number using a trypan blue exclusion assay. Results were expressed either as cell number or as the percentage of control cells incubated in the absence of butyrate.
Alternatively, Caco-2 and RSB cells were cultured in 24-multi-well plates to subconfluency and exposed to different butyrate concentrations (2, 5 or 10 mmol/L) and/or Z-YVAD-FMK (100 µmol/L) or Z-DVED-FMK (100 µmol/L) for 48 h. The caspase-1 inhibitor Z-YVAD-FMK is a potent cell-permeable and irreversible inhibitor of caspase-1 (ICE). The caspase-3 inhibitor Z-DVED-FMK is a potent cell-permeable and irreversible inhibitor of CPP-32/apopain, a member of the ICE/CED-3 family of cysteine proteases. This tetrapeptide fits the sequence of the cleavage site of PARP by caspase-3. During proteolysis, Z-DVED-FMK binds irreversibly to caspase-3, thereby inactivating the enzyme. Inhibitors were dissolved in dimethylsulfoxide (DMSO) and, consequently, controls contained 0.25% DMSO.
Transmission electron microscopy.
Cells were fixed in 2.5% glutaraldehyde and 0.1 mol/L sodium cacodylate buffer pH 7.2. Dehydration was performed with graded ethanol solutions before propylene oxide exchange and the cells were embedded in Epon. Ultra-thin sections on grids were stained with uranyl acetate and lead citrate and finally examined in a Jeol 1200EX transmission electron microscope (4
,7
,28
,29
).
Western blot analysis.
For the immunodetection of caspase-1, caspase-3, PARP, Bak and Bcl-2, Caco-2 and RSB cells were processed essentially as previously described (4
,7
,29
) following the different treatment protocols. The primary antibodies used were: the rabbit polyclonal antibody ICE p20 equivalent to the 20-kDa fragment of caspase-1; rabbit polyclonal anti-caspase-3 antibody; goat polyclonal anti-PARP antibody; rabbit polyclonal anti-Bak antibody; and mouse monoclonal anti-Bcl-2 antibody. Detection was performed with horseradish peroxidase-linked anti-rabbit, anti-goat or anti-mouse IgG antibody, depending on the primary antibody. Immunoreactive bands were visualized using the SuperSignal (Pierce, Rockford, IL) enhanced chemiluminescence detection reagents following manufacturer’s recommendations. Molecular weight markers and a positive control provided by the suppliers served to identify the appropriate reactive bands. Equal protein loading and quality of the electrotransfer were verified by Ponceau S staining as previously described (4
,7
,29
).
Densitometric analysis of band intensity.
The total intensity of each band obtained in Western blots was detected and quantified with a PhosphorImager system (Fujix, Tokyo, Japan) and corrected for background. The integration values for each band were taken as arbitrary units (4
,7
,29
).
Caspase-3 proteolytic activity.
The caspase-3 activity ratio was calculated by colorimetric assay (R&D Systems, Minneapolis, MN). Cells harvested from the different treatment groups were lysed and 200 µg protein were tested for protease activity by the addition of a caspase-specific substrate peptide, DVED-pnitroaniline. Caspase-3 cleavage of the peptide releases the chromophore p-nitroanilide (p-NA), which was quantitated spectrophotometrically at 405 nm. The level of caspase-3 enzymatic activity in the cell lysate was directly proportional to the color reaction. The results are expressed as the fold increase of caspase activity in apoptotic cells relative to their respective controls. In background reactions, no DVED-p-NA substrate was added, and the values obtained were subtracted from experimental results before to calculating the fold increase (7
).
Nucleosome ELISA kit.
Nucleosome ELISA (Oncogene Research Products, Cambridge, MA) allows the quantitation of apoptotic cells in vitro by DNA affinity-mediated capture of free cytoplasmic nucleosomes followed by their antihistone-facilitated detection. In this assay, mono- and oligonucleosomes are captured on precoated DNA-binding proteins. Anti-histone (H3) biotin-labeled antibody then binds to the histone component of the captured nucleosomes and is detected after incubation with strepavidin-linked horseradish peroxidase (SA-HRP) conjugate. HRP catalyzes the conversion of colorless tetramethylbenzidine to blue, the intensity of the color being proportional to the number of nucleosomes in the sample (30
).
Statistical analysis.
All experiments were performed in duplicate and were repeated at least four times. All values are expressed as mean ± SEM. Data were analyzed by two-way ANOVA, and then differences among means were analyzed using Duncan’s Multiple Range Test. Differences were considered significant at P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Representative electron micrographs of RSB cells cultured under different treatment conditions are shown in Figure 1
. RSB cells incubated in control medium (DMEM) show a high nucleus:cytoplasm ratio - characteristic of high mitotic activity and sparse nonoriented microvilli (Fig. 1
A). Ultrastructural micrographs obtained after incubation with different butyrate concentrations are depicted in Figure 1
, B, C, and D (2, 5 and 10 mmol/L butyrate, respectively). A typical apoptotic cell in which the nucleus contains highly condensed chromatin is show in Figure 1
B. Figure 1
C shows a cell in which membrane blabbing is evident and in Figure 1
D, the cell contains a condensed nucleus, and an apoptotic body.
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Butyrate in the range of 2 to 10 mmol/L inhibited Caco-2 and RSB cell proliferation in a dose-dependent manner (Fig. 2A
). RSB cells were relatively more resistant to butyrate treatment than Caco-2 cells (the same butyrate concentrations led to fewer cells relative to the control in Caco-2 cells than in RSB cells, P < 0.05). Butyrate significantly up-regulated caspase-3 activity in Caco-2 cells in a dose-dependent manner (Fig. 2
B), whereas RSB cells were relatively less responsive. The maximal response was observed with 10 mmol/L butyrate (2131 ± 640% and 890 ± 274% greater than control values, P < 0.05). Butyrate induced apoptosis in a dose-dependent manner (as detected by quantitation of free cytoplasmic nucleosome concentration) in both Caco-2 and RSB cancer cells. However, Caco-2 cells responded more than did RSB cells (P < 0.05, Fig. 2
C).
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Caspase-1 expression.
Expression of the 20-kDa cleaved caspase-1 fragment, indicative of active enzyme, was evident only in Caco-2 cells treated with sodium butyrate (2, 5, 10 mmol/L); in RSB cells this caspase-1 band was not detected. Densitometric analyses of p20 subunit expression in Caco-2 cells under different treatment conditions are summarized in Table 1
.
|
. In contrast, in Caco-2 cells, only the p17 subunit was detected (Table 1)
.
PARP expression.
PARP cleavage is an accepted marker for downstream apoptotic processes (26
,31
–34
). PARP expression was determined by Western immunoblot analysis. The antibody recognized both the full-length (115 kDa) and cleaved (89 kDa) PARP fragment (Table 1)
. Control RSB cells showed the highest relative expression of full-length uncleaved 115-kDa PARP, whereas at the higher butyrate concentrations its expression was lower. In Caco-2 cells the full-length uncleaved 115-kDa PARP was not detectable. Expression of the cleaved 89-kDa fragment generated during active apoptosis was higher at the increasing butyrate concentrations and only faintly expressed in controls or at the low butyrate concentration (2 mmol/L) in both cell lines.
Bcl-2 expression.
The strongest Bcl-2 expression was observed in control (no butyrate) Caco-2 cells (Table 1)
. Increasing butyrate concentrations caused a concomitant decrease in Bcl-2 expression. Bcl-2 was very weakly expressed in RSB colon cancer cells, and no clear response to butyrate was observed (Table 1)
.
Bak expression.
Increasing butyrate concentrations induced in Caco-2 cells increased Bak expression (Table 1)
. RSB cells responded to butyrate in terms of Bak expression only at 5 and 10 mmol/L (Table 1)
.
The effect of caspase inhibitors on butyrate-induced apoptosis
Caspase-1 inhibitor.
Z-YVAD-FMK significantly down-regulated the growth inhibition induced by butyrate in Caco-2 cells. Complete abrogation of butyrate’s effect occurred at an inhibitor concentration of 100 µmol/L, indicating that caspase-1 inhibitor significantly alters the caspase cascade, diminishing the butyrate-induced apoptotic effect (Fig. 3A
). Interestingly, in RSB cells a different pattern was observed: caspase-1 inhibitor did not interact with the antiproliferative effect of butyrate (Fig. 4A
).
|
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B). Exposure to caspase-1 inhibitor did not affect caspase-3 activity in RSB cells (Fig. 4
B).
The use of caspase-1 inhibitor in Caco-2 cells significantly inhibited the concentration of cytoplasmic free nucleosomes at all butyrate concentrations compared with the control (Fig. 3
C). In contrast, in RSB cells there was no response to caspase-1 inhibitor in terms of cytoplasmic free nucleosome concentration (Fig. 4
C).
Caspase-3 inhibitor.
As shown in Figure 3
A, 100 µmol/L Z-DVED-FMK did not suppress the growth-inhibitory effect of butyrate in Caco-2 cells because the cell number did not differ from cells not exposed to caspase-3 inhibitor at any butyrate concentration. In contrast, caspase-3 inhibitor markedly suppressed the effect of butyrate in RSB cells because it nullified the inhibitory effect of butyrate at all butyrate concentrations tested (Fig. 4
A).
Caspase-3 inhibitor was partially effective in blocking caspase-3 activity of Caco-2 cells but only at the highest butyrate concentration (10 mmol/L, Fig. 3
B). In RSB cells, caspase-3 inhibitor markedly decreased caspase-3 activity at all butyrate concentrations (Fig. 4
B).
Caspase-3 inhibitor did not affect accumulation of cytosolic nucleosomes in either control or butyrate-treated Caco-2 cells (Fig. 3
C). However, in RSB cells, caspase-3 inhibitor significantly decreased cytoplasmic free nucleosomes at the highest butyrate concentrations (5 and 10 mmol/L; Fig. 4
C).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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,7
). In this study we investigated whether the reduction in cell proliferation exerted by butyrate in two differently stratified colon cancer cell lines (RSB and Caco-2 cells) is related to apoptosis and whether similar or different molecular events are caused by butyrate in these cell lines.
Caspase-1 expression differed in the two cell lines. Caspase-1 was not detected in either control or in butyrate-treated RSB cells, whereas in Caco-2 cells, at all butyrate concentrations tested, enhanced protein expression of the active caspase-1 p20 subunit was found. Our previous in vivo studies showed similar results to those obtained with Caco-2 cells, i.e., apoptotic death directly correlated with the extent of caspase-1 expression and activity (4
,7
,29
). Supporting these findings, MacCorkle et al. (35
) and Psmantur et al. (16
) demonstrated that caspase-1 and caspase-3 overexpression are both associated with enhanced apoptotic death.
Expression of the downstream apoptosis-related enzyme caspase-3, indicative of active apoptosis (20
,32
,36
–39
), was detected by two different methods. The first measured expression of the enzyme’s precursor and the second the actual enzyme activity using a specific synthetic substrate. Caspase-3 expression also differed in the two cell lines: in RSB cells, both the precursor and the subunits of caspase-3 were detected, whereas in Caco-2 cells, only the active p17 subunit was observed.
A 10- to 20-fold increase in caspase-3 activity over controls was observed in Caco-2 cells exposed to 5 and 10 mmol/L butyrate for 48 h. In RSB cells, a gradual increase in caspase-3 activity was also observed, although its magnitude was much less (fivefold to ninefold). The use of both caspase-3 detection methods enabled us to correlate expression of the precursor with actual enzymatic activity. For Caco-2 and RSB cells, when caspase-3 precursor is overexpressed, higher caspase-3 activity is obtained. We could also detect intrinsic differences between the cell lines in terms of caspase-3 cleavage, activation and activity. In RSB cells, caspase-3 activation apparently is a major pathway involved in butyrate-induced apoptosis whereas in Caco-2 cells, this pathway is less important. Differences in caspase-3 activation may be due to differences in upstream events in these cell lines.
Harvey et al. (40
) and Liu et al. (25
) reported that caspase-3 activity is dependent on upstream caspase-1 activity. Because differences in caspase-1 activation were observed between the cell lines, we suggest that the downstream caspase-3 is activated by different upstream caspases (or other molecules) in RSB and Caco-2 cells.
The decrease in the expression of uncleaved PARP protein in butyrate-treated RSB cells, or the increased 89-kDa PARP product expression when both cell lines were treated with butyrate suggest that butyrate plays an important role in inducing PARP cleavage, which is probably mediated via activation of caspases. Because caspases are differentially expressed and activated in each cell line, we assume that PARP is cleaved by different caspases in RSB and Caco-2 cells or alternatively, by similar caspases at different rates of activity.
The expression levels of anti- and pro-apoptotic proteins of the Bcl-2 family are closely associated with the apoptotic sensitivity of the cells (41
,42
). The relative concentrations of these anti- and pro-apoptosis proteins may determine whether a cell will live or die (43
). Bax, as well as its homologous protein Bak, promote cell death by competing with Bcl-2. While Bax-Bax and Bak-Bak homodimers act as apoptosis inducers, Bcl-2-Bax heterodimer formation evokes a survival signal for the cells (43
). Hague et al. (44
) suggested that Bcl-2 and Bak play a pivotal role in sodium-butyrate-induced apoptosis in colonic epithelial cells, and that overexpression of Bcl-2 does not protect against Bak-mediated apoptosis.
We showed that butyrate down-regulation of Bcl-2 expression in Caco-2 cells is probably associated with enhanced apoptotic activity, whereas in RSB cells the apoptotic pathway seems not to depend only on Bcl-2 expression. In addition, we showed that in Caco-2 cells, Bak protein expression is strongly up-regulated by butyrate, concomitant with Bcl-2 down-regulation.
In RSB cells, Bak was significantly up-regulated by butyrate, while Bcl-2 expression was not affected; the calculated Bcl-2:Bak ratio decreased with butyrate concentration. Thus, butyrate induced a strong shift in the ratio of anti- to pro-apoptotic Bcl-2 family proteins, in favor of an apoptotic response. Similar results have been previously reported for HT29 and Caco-2 colon cancer cells, where butyrate increased Bak expression with no effect on Bcl-2 levels (15
,45
).
The concentration of free cytoplasmic nucleosomes detected by histone antibodies directly correlated with caspase-3 activity and with different butyrate concentrations, in both cell lines. Apoptotic features detected by transmission electron microscopy in RSB cells exposed to different butyrate concentrations directly correlated with all aforementioned apoptotic criteria.
Specific caspase inhibitors were used to further demonstrate the role of different caspases in butyrate-induced apoptosis of Caco-2 and RSB cells. Butyrate-induced apoptosis required the activation of caspase-3 and was abolished by specific caspase-3 inhibition in Caco-2 cells. Down-regulation of caspase-3 activity by caspase-1 inhibitor in Caco-2 cells suggests that butyrate first activates caspase-1 (initiator), and this enzyme may then stimulate downstream enzymes, namely caspase-3.
In RSB cells, the situation was different: caspase-1 inhibitor exhibited strong antagonism to butyrate-induced apoptosis. Caspase-1 inhibitor induced only a slight decrease in caspase-3 activity in these cells, suggesting that butyrate does not activate the apoptotic cascade through caspase-1. These findings suggest that in RSB cells, additional members of the caspase family (such as caspase-8 or caspase-9) may activate caspase-3 (46
). Overall, Caco-2 cells seemed to be more sensitive to butyrate-induced apoptosis than did RSB cells. This suggests that the apoptotic pathway activated by butyrate in Caco-2 cells seems to be more effective than the one activated in RSB cells. The principal similarities and differences between the apoptotic responses to butyrate are summarized in Table 2
.
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FOOTNOTES |
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3 Abbreviations used: DFF, DNA-fragmentation factor; DMSO, dimethylsulfoxide; HRP, horseradish peroxidase; ICE, inhibitor of caspase-1; PARP, poly(ADP-ribose) polymerase; p-NA, p-nitroanilide; SA-HRP, strepavidin-linked horseradish peroxidase; SCFA, short-chain fatty acid.
Manuscript received 17 December 2001. Initial review completed 28 January 2002. Revision accepted 20 March 2002.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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