Effects of colonic lumenal components on AP-1-dependent gene transcription in cultured human colon carcinoma cells

Bjorn Glinghammar1, Kristina Holmberg and Joseph Rafter

Department of Medical Nutrition, Karolinska Institute, Novum,
S-14186 Huddinge, Sweden


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We recently suggested that prolonged deregulated expression of AP-1 activity in colonic cells by bile acids may contribute to tumour promotion in the colon. In the present study, using two human colon carcinoma cell lines, HT-29 and HCT 116, transiently transfected with the AP-1-luciferase reporter construct, we showed that the bile acids, deoxycholate, chenodeoxycholate, ursodeoxycholate and lithocholate, induced AP-1-dependent gene transcription in a dose-dependent manner, whereas cholate was without effect. The greatest effect was observed with deoxycholate, and the ability of this bile acid to induce reporter gene activity was significantly correlated with its ability to induce cell proliferation (r = 0.91, P = 0.01). Cholesterol and the long chain fatty acids, myristate, palmitate and stearate, had no effect on AP-1-dependent gene transcription, whereas the short chain fatty acid, butyrate, exhibited a marked effect. Mindful of the fact that the concentrations of lumenal components that are actually in or entering the epithelial cells in the colon are presumably lower than lumenal values, we considered it of interest to determine the effect of dilution on the capacity of human faecal water to induce AP-1 activity and also cell proliferation. We demonstrated that diluted lipid extracts, from all of the faecal water samples examined, significantly induced AP-1-dependent gene transcription in the colonic cells, and that this effect differed markedly between the extracts. We confirmed that the faecal water lipid extracts, at the same dilution at which they increased AP-1 activity, significantly induced proliferation in the same cell line. These data suggest that lipid components of human faecal water, which is in direct contact with the colon epithelium and may be physiologically more active than the solid phase, can activate AP-1, a transcription factor whose activation has been associated with the promotion of neoplastic transformation.

Abbreviations: AP, activator protein; CA, cholic acid; CDCA, chenodeoxycholic acid; DCA, deoxycholic acid; DMEM, Dulbecco's modified Eagle's medium; DMSO, dimethyl sulphoxide; FBS, fetal bovine serum; LCA, lithocholic acid; MTT, 3-(4,5,dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; OAG, sn-1,2-dioctanoylglycerol; PBS, phosphate-buffered saline; PKC, protein kinase C; PMSF, phenylmethylsulphonyl fluoride; RLU, relative light units; TPA, 12-O-tetradecanoylphorbol-13-acetate; UDCA, ursodeoxycholic acid.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In recent years, there has been considerable interest in the role of the aqueous phase of human faeces (faecal water) in studies examining the mechanisms that underlie the dietary aetiology of colon cancer. The motivation being that components of this faecal fraction are more likely to be able to exert untoward effects on the cells of the colonic epithelium than components bound to food residues and the bacterial mass. The lipid component of faecal water (e.g. bile acids and fatty acids) has received particular attention, in view of the established role of dietary fat as a tumour promoter in colon cancer. Several studies have shown that it is possible to alter the bile acid concentrations in human faecal water through dietary intervention, with diets associated with increased risk for the disease increasing their levels (1,2). Interestingly, in some instances, it appears to be possible to increase levels of faecal water bile acids by dietary changes while leaving total faecal bile acid levels unaffected (2). It has also been demonstrated that cytotoxicity of faecal water is dependent on dietary factors and correlates with the bile acid component of this faecal fraction (2). This cytotoxicity can cause epithelial cell loss in the large bowel, which leads to a compensatory crypt cell proliferation (3). It has been shown that an increased cell proliferation has been linked to a higher risk for the development of colonic cancer (4,5). Indeed, measuring the cytotoxicity of human faecal water is increasingly being used as a risk marker for this disease in dietary intervention studies (6,7). Also, the recent finding of significant amounts of genotoxic activity in human faecal water, as measured by the COMET assay (8), supports the contention that the biochemistry of this faecal fraction may be important in mediating the effects of dietary components on malignant transformation in the colon.

While it is still believed that lumenal bile acids mediate the adverse effect of high dietary fat on colon cancer development, the underlying mechanisms continue to elude us. There are many studies that attest to the adverse effects of bile acids originating from (i) tissue culture, microbial mutagenesis and animal studies of mutagenesis/carcinogenesis, (ii) studies of human populations and high risk patient groups and (iii) studies in humans of the toxicity of bile acids on the intestinal mucosa (9). Thus, bile acids have been shown to act as tumour promoters in colon carcinogenesis in vivo and to enhance cell transformation in vitro, and they appear to be endogenous colon tumour promoters (9,10). There is also a body of evidence that indicates that bile acids, at subtoxic concentrations, are directly mitogenic to colonic cells in culture and that this effect is mediated through the activation of protein kinase C (PKC), a calcium-activated, phospholipid-dependent enzyme that plays a key role in growth-signalling pathways (11,12). Thus, while there is some evidence that bile acids may induce DNA damage (8,13) the general consensus now is that bile acids contribute to colon carcinogenesis by disturbing the fine balance between proliferation, differentiation and apoptosis in the cells of the colonic epithelium (14,15).

It is well established that the mechanisms of tumour promotion are closely related to signal transduction and the action of specific oncogenes (16). Tumour promoting phorbol esters also activate PKC, and it has been shown that the potent phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) can substitute for diacylglycerol, the physiological effector of the enzyme, by interacting with the same binding sites (16). It is well documented that treatment of cells with TPA activates and down-regulates PKC and transiently induces the early-responsive c-fos and c-jun proto-oncogenes in association with the transition of cells into S phase (17,18).

Induction of the expression of c-fos and c-jun also results in the activation of the activator protein-1 (AP-1) transcription factor, a heterodimeric protein complex composed of the products of members of the fos and jun gene family, to enhance its affinity for target DNA sequences and alter the expression of various genes (17,18). Many of the genes in this family are expressed in transformed, rapidly growing cells (17,18). Of considerable interest in this regard, was recent evidence that demonstrated that bile acids could induce AP-1 DNA-binding activity and augment AP-1-responsive gene expression in human cultured colon adenocarcinoma cells, while leaving the function of other transcription factors, such as NF{kappa}B, Sp1 and ATF/CREB, unaffected (19,20). This led to the novel hypothesis that prolonged deregulated expression of AP-1 activity in colonic cells by lumenal components, such as bile acids, may contribute to tumour promotion in the colon (20).

Thus, in view of the increasing interest in human faecal water, referred to above, and the fact that bile acids appear to be an important component of this faecal fraction, we considered it of interest to extend the above studies and determine whether human faecal water/lipid extracts had the capacity to induce AP-1-responsive gene expression in cultured colon carcinoma cells from humans. The ability of faecal water/lipid extracts to induce proliferation in colon cells was also addressed.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Chemicals
All chemicals were purchased from Sigma Chemical Co. (St Louis, MO) unless otherwise specified. Sodium salts of cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), lithocholic acid (LCA) and ursodeoxycholic acid (UDCA), were prepared as stock solutions (0.1 mol/l) in ethanol. Myristic acid, stearic acid, palmitic acid and sn-1,2-dioctanoylglycerol (OAG) were also prepared as stock solutions (0.1 mol/l) in ethanol. TPA was dissolved in dimethyl sulphoxide (DMSO) and used as a positive control in all transfection experiments. Ionomycin was dissolved in DMSO. Cholesterol and 1,2-dimyristan were dissolved in ethylacetate to give stock solutions of 0.1 mol/l. Butyric acid was dissolved in water to give a stock solution of 1 mol/l.

Faecal water preparation
Faecal water was prepared as previously described (7) from 15 healthy individuals on a mixed Western diet. Since the procedure involves a centrifugation step, it is believed to reflect the composition of free water in the stool. Fractionation of the faecal water sample to yield a lipid extract was performed as follows: faecal water (1 ml) was diluted with 9 ml phosphate-buffered saline (PBS; 10 mmol/l, pH 7.2) and applied to a Sep-pak C18 cartridge (Millipore, MA). The cartridge was washed with 10 ml PBS and lipids eluted with 5 ml methanol (Labassco, Partille, Sweden). The methanol eluate was evaporated to dryness at 37°C and the residue was resuspended in Dulbecco's modified Eagle's medium (DMEM) (Gibco BRL, Paisley, UK) with 0.1% fetal bovine serum (FBS) to give the required dilutions. All faecal water samples were sterile filtered (Acrodisc 0.45, Gelman Science, Ann Arbor, MI).

Cells and culture
Human colon carcinoma cells (HCT 116), human colon adenocarcinoma cells (HT-29) and human fetal normal colonic mucosal cells (FHC) were purchased from ATCC (Rockville, MD). To avoid changes in cell characteristics produced by extended cell culture periods, HCT 116 and HT-29 cells were used between passages 3 and 25 and FHC cells were used in passage 9. HCT 116 and HT-29 cells were maintained in DMEM with 10% FBS, 2 mmol/l L-glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin, in a humidified atmosphere of 95% air, 5% CO2 at 37°C. FHC cells were maintained in 50% Ham F12 and 50% DMEM with 10% FBS, 25 mmol/l HEPES, 5 µg/ml transferrin, 100 ng/ml hydrocortisone, 10 ng/ml cholera toxin, 5 µg/ml insulin, 100 U/ml penicillin and 100 µg/ml streptomycin. Cultures were serum starved (0.1% FBS) for 24 h, to obtain quiescent cells, prior to AP-1 induction and proliferation experiments.

Plasmid preparation
The luciferase reporter plasmid (TRE)2tkLuc was a gift from Dr Sam Okret, Karolinska Institute, Sweden (21). The inserted 2x TRE contained the AP-1 consensus sequence TGACTCA (22). The plasmid DNA was purified from bacteria cultures. Briefly, bacteria were grown in Luria broth medium overnight with ampicillin (50 µg/ml, ASTRA, Sodertalje, Sweden). After centrifugation, the pellet was resuspended in buffer that contained 50 mmol/l glucose, 25 mmol/l TRIS and 10 mmol/l EDTA. After addition of 0.2 mol/l NaOH + 1% SDS on ice, the solutions were mixed gently and left on ice for 10 min. After addition of ammonium acetate, the solution was mixed vigorously and left on ice for 5 min. After centrifugation, the supernatant was filtered and precipitation of DNA took place by addition of ethanol. Following centrifugation, the pellet was dissolved in TE buffer and 0.5 vol of ammonium acetate was added and left on ice for 10 min and centrifuged with collection of the supernatant. Further purification was achieved by precipitation with ethanol. The samples were redissolved in TE buffer and ultracentrifuged (76 000 r.p.m., overnight) with caesium chloride and ethidium bromide (10 mg/ml). The gradient band was carefully removed and additional purification took place with ethanol precipitations and TE-saturated butanol. The DNA was visualized on an agarose gel and the DNA purity and concentration was measured spectrophotometrically at 260 and 280 nm.

Transfection and luciferase assay
HCT 116 cells, 35x103 cells (HT-29 cells, 40x103 cells) were placed in each well of a 24-well plate and allowed to grow for 24 h to 50% confluency. The medium was removed and the cells were washed with PBS. OPTIMEM (200 µl) (Gibco), 2 µg/ml plasmid DNA and 10 µg/ml Lipofectin reagent (Gibco) were added to each well. After 6 h of transfection, the OPTIMEM mixture was removed and replaced by DMEM (0.1% FBS) for an additional 24 h. The cells were further incubated with the various test agents (bile acids, bile acid mixtures, fatty acids, diluted faecal water lipid extracts, etc.) in DMEM (0.1% FBS) for an additional 20 h. The medium was removed and the cells were washed with PBS. Lysis buffer [100 µl, 25 mmol/l triphosphate pH 7.8, 15% glycerol, 2% CHAPS, 1% lecithin, 1% bovine serum albumin, 0.1% EGTA pH 8.0, 8 mmol/l MgCl2, 1 mmol/l dithiothreitol and 0.4 mmol/l phenylmethylsulphonyl fluoride (PMSF)] was added to each well and the cells lysed during 30 min. Aliquots of 50 µl of the cell lysates were transferred to a non-transparent 96-well plate and 100 µl luciferin mix (GenGlow, Bioorbit, Turku, Finland) was added to each well. The assay for luciferase activity was performed in the automatic luminometer Lucy 1 (Anthos Labtec Instruments, Salzburg, Austria) which, after addition of the ATP solution, measures the relative light emitted (RLU). The RLU of the untreated control cells in each experiment was set to 100% and the resulting RLU for the test agents was calculated in relation (%) to the control cells.

Proliferation and cytotoxicity assay
Cell proliferation, using the HT-29 and FHC cells, was measured using the Cell-titer 96 proliferation kit (Promega, Madison, WI). Aliquots of 5x103 cells (HT-29) and 10x103 cells (FHC) were plated out in each well of a 96-well plate and covered with 100 µl DMEM (0.1% FBS) and incubated for 24 h at 37°C, in a humidified atmosphere of 95% air and 5% CO2. The medium was then replaced by 100 µl DMEM (0.1% FBS) containing the various test agents, diluted faecal waters/lipid extracts and DCA, and further incubated for 24 h. On each plate, DMEM (0.1% FBS) was used as a negative control and 50 µmol/l DCA as a positive control. Every test agent was performed in octuple. Dye solution (15 µl) containing 3-(4,5,dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) (Promega, Madison, WI) was then added to the wells and incubated for 4 h. The dye/medium in each well was carefully removed and solubilization solution (100 µl, Promega) was placed in each well for a further 1 h. The plates were read at 570 nm in a spectrophotometer (Titertek Multiscan, Eflab Oy, Helsinki). The mean absorbance of each octuple was calculated and cell proliferation was expressed as percentage absorbance of the maximal absorbance in wells incubated with DMEM (0.1% FBS) alone. Cytotoxicity was assayed as previously described (7). Briefly, HT-29 cells (15x103 cells per well) were plated out on a 96-well plate and allowed to grow in full growth medium (DMEM, 10% FBS) for 48 h. Medium was removed and replaced by the faecal water samples/lipid extracts and incubated for 1 h. Thereafter, the faecal waters/lipid extracts were removed and the cells washed with DMEM and allowed to grow for an additional 48 h. An assay for living cells was performed as described above and cell survival was expressed as the percentage absorbance of the maximal absorbance in wells incubated with the negative control (i.e. PBS).

Statistical analysis
In order to analyse changes in proliferation and luciferase induction, Student's t-test (two-tailed) was used. Descriptive and graphical methods were used to characterize the data. All tests were performed with the software package STATISTICA 5.0 (Statsoft, Tulsa, OK). Statistically significant differences are represented as follows: *P < 0.05; **P < 0.01; ***P < 0.001.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Effects of bile acids on transcriptional activation of AP-1-inducible reporter gene
Firstly, using the proliferation assay described in Materials and methods, we confirmed previous results from this laboratory (20), which indicated that the secondary bile acid, DCA, induced proliferation in HT-29 cells in a dose-dependent manner with the maximum effect being observed at 60 µM (Figure 1AGo). Subsequently, in order to address the question as to whether DCA possessed the capacity to induce AP-1-dependent gene transcription, we exposed the HT-29 cells, transiently transfected with the AP-1 luciferase reporter construct, to this bile acid as described under Materials and methods. We considered it of more value to look at gene transcription, exploiting the reporter construct, rather than simply measuring AP-1 endogenous activation. DCA caused a significant increase in reporter gene activity with 60 µM giving the maximum effect (Figure 1BGo). The reduced activity, observed at 80 µM, was presumably because DCA began to exhibit cytotoxicity at this concentration. In addition, the correlation (r = 0.91, P = 0.01) between DCA-induced proliferation and AP-1-dependent gene transcription in the HT-29 cells was significant over the concentration range 0–80 µM. Thus, in this cell system, the concentration of DCA at which maximum induction of AP-1-dependent gene transcription occurs (60 µM) is also the concentration at which the maximum proliferative effect is observed.



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Fig. 1. Effects of DCA on (A) proliferative and (B) AP-1 activity in HT-29 cells. Diagram bars represent mean values (SD) (n = 8) in relation to the control cells, which were set to 100%.

 
Because of the consistent practical difficulties encountered in transiently transfecting the HT-29 cells and the fact that both cell lines appeared to respond in a similar manner to the different `pure substances' studied, much of the further screening was performed using HCT 116 cells, which were found to be significantly easier to transfect. Thus, Figure 2Go shows the fold induction of the reporter gene in HCT 116 cells for the other colonic bile acids. The other secondary bile acid present in the colonic lumen, LCA, induced reporter gene activity in a dose-dependent manner over the concentration range 30–100 µM. The primary bile acid, CDCA, also induced significant effects on reporter gene activity, with the maximum effect observed at 100 µM. Exposure of the cells to UDCA resulted in a lower induction of reporter gene activity, with a significant effect only observed at 25 µM. In general, cytotoxic effects were observed when the cells were exposed to bile acid concentrations >100 µM (data not shown). Interestingly, the primary bile acid, CA, had no effect on reporter gene activity in the HCT 116 cells.



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Fig. 2. Effects of bile acids on AP-1 activity in HCT 116 cells. Diagram bars represent mean values (SD) (n = 4) in relation to the control cells, which were set to 100%.

 
Effects of additional components, which are of relevance to colon carcinogenesis, on transcriptional activation of AP-1-inducible reporter gene
In an attempt to ascertain whether additional compounds, which are of possible relevance to tumorigenesis in the large bowel, had the capacity to modulate AP-1-dependent gene transcription, the experiments outlined below were performed. In Figure 3Go, the effects of the long chain fatty acids, myristic, palmitic and stearic acids, on reporter gene activity in HCT 116 cells are shown. Over the range 50–1000 µM (data shown for 100 and 500 µM), there was no effect on reporter gene activity. Neither did cholesterol have an effect on the reporter gene over the concentration range 25–100 µM (Figure 3Go). However, the short chain fatty acid, butyric acid, at concentrations of 0.5, 1, 2 and 4 mM induced a marked and significant dose–response in reporter gene activity (Figure 3Go).



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Fig. 3. Effects of fatty acids and cholesterol on AP-1 activity in HCT 116 cells. Diagram bars represent mean values (SD) (n = 4) in relation to the control cells, which were set to 100%.

 
When the effects of known activators of PKC: the phorbol ester, TPA, and the diacylglycerols, OAG and 1,2-dimyristan, on reporter gene activity were examined in HCT 116 cells, a significant induction of activity was observed (Figure 4Go). Also of some interest was the observation that exposure of the HCT 116 cells to calcium chloride (10 mM) or the calcium ionophore, ionomycin (1 mM) resulted in a significant induction of AP-1-dependent gene transcription (Figure 4Go).



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Fig. 4. Effects of TPA, OAG, 1,2-dimyristan, CaCl2 and ionomycin on AP-1 activity in HCT 116 cells. Diagram bars represent mean values (SD) (n = 4) in relation to the control cells, which were set to 100%.

 
Effects of human faecal water components on cell proliferation and transcriptional activation of AP-1-inducible reporter gene
Having thus established, with the experiments described above, that effects on AP-1-dependent gene transcription could be effectively studied in our assay system employing both cell lines, we turned to the more interesting question as to whether human faecal water or components thereof possessed the capacity to modulate reporter gene activity.

We firstly addressed the question as to whether faecal water fractions from human faeces were able to induce proliferation, using the proliferation assay described under Materials and methods. When the HT-29 cells were exposed to faecal water fractions, marked cytotoxic effects were consistently observed. Mindful of the fact that the concentrations of lumenal components actually in or entering the epithelial cells in the colon are presumably lower than lumenal values (see Discussion below), we considered it of interest to determine the effect of dilution on the faecal water's capacity to induce cell proliferation. For this purpose, five fecal water samples were randomly chosen from our material of 15. At low dilutions (1 in 5) the faecal water exhibited cytotoxicity, at higher dilutions all five of the faecal waters studied exhibited a capacity to induce cell proliferation in HT-29 cells (Figure 5Go). However, the degree of dilution needed to obtain the maximum proliferative effect (significantly different from control cells for each sample) differed between the different faecal water samples, with the more cytotoxic faecal waters requiring a higher dilution. `Lipid extracts' of a water sample exhibited no effect on reporter gene activity.



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Fig. 5. Effects of five diluted faecal water samples (15) on proliferative activity in HT-29 cells. Values are expressed as mean values (n = 8) in relation to the control cells, which were set to 100%. {blacksquare}, faecal water sample 1; {blacklozenge}, sample 2; x, sample 3; {blacktriangleup}, sample 4; •, sample 5.

 
We then investigated whether the diluted faecal waters also had the ability to induce AP-1-dependent gene transcription. Initially, in order to simplify these experiments, we prepared a lipid extract from the faecal waters as described in the Materials and methods section. In Figure 6Go, the effects of diluted lipid extracts from faecal water samples 6 and 7 on reporter gene activity in HCT 116 and HT-29 cells are shown. Undiluted lipid extracts of faecal water exhibited cytotoxicity in the assays. As is evident from Figure 6Go, faecal water 7, which exhibited 95% cytotoxicity in the cytotoxicity assay, induced maximum and significant reporter gene activity in HT-29 cells at a 1:8 dilution, whereas faecal water 6, which exhibited 15% cytotoxicity, induced maximum and significant reporter gene activity at a 1:4 dilution. The levels of reporter gene activity induced, by the faecal water lipid extracts, in the HCT 116 cells were lower (though significant) than in the HT-29 cells.



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Fig. 6. Effects of diluted lipid extracts from two faecal water samples (6 and 7) on AP-1 activity in HT-29 (broken line) and HCT 116 (solid line) cells. Values are expressed as mean values (n = 8) in relation to the control cells, which were set to 100%. {blacksquare}, faecal water sample 6; x, sample 7. Lipid extracts were not made from faecal water samples 1–5, as there was not sufficient material remaining after the experiment outlined in Figure 5Go.

 
Figure 7Go shows the ability of the lipid extracts from faecal water samples 6–15, at a 1:8 dilution, to induce reporter gene activity in HT-29 cells. HT-29 cells were utilised in these experiments, despite the fact that they were somewhat more difficult to transfect than HCT 116 cells, as they appeared to be more sensitive to the induction of reporter gene activity by faecal water lipid extracts than the HCT 116 cells (Figure 6Go). A dilution of 1:8 was used, as most of the faecal water samples exhibited a cytotoxicity closer to that of faecal water 7 rather than faecal water 6 (data not shown). As is evident from Figure 7Go, the lipid extracts from all the faecal water samples significantly induced reporter gene activity, with values that ranged from 1.3- to 2.9-fold induction compared with the control. We were also able to demonstrate that the lipid extracts from faecal water samples 6–15, at a 1:8 dilution, had the ability to significantly induce cell proliferation in the proliferation assay using HT-29 cells, with values that ranged from 1.3- to 2.0-fold induction compared with the control (Figure 8Go). However, no significant correlation could be established between ability to induce proliferation and ability to induce reporter gene activity for the lipid extracts of the faecal water samples. When we compared the results (% proliferative activities) obtained in the HT-29 cells, which were induced by diluted lipid extracts of two faecal water samples, with those obtained in normal colonic cells (FHC), they were similar: sample 1, 1:8 dilution, 157% (HT-29), 177% (FHC), 1:10 dilution, 145% (HT-29), 168% (FHC); sample 2, 1:12 dilution, 123% (HT-29), 131% (FHC).



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Fig. 7. Effects of diluted (1:8) lipid extracts from faecal water samples 6–15 on AP-1 activity in HT-29 cells. Diagram bars represent mean values (SD) (n = 8) in relation to the control cells, which were set to 100%.

 


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Fig. 8. Effects of diluted (1:8) lipid extracts from faecal water samples 6–15 on proliferative activity in HT-29 cells. Diagram bars represent mean values (SD) (n = 8) in relation to the control cells, which were set to 100%.

 
Effects of bile acid mixtures which reflect profiles in faecal water from individuals with and without colonic polyps or cancer, on transcriptional activation of AP-1-inducible reporter gene
It has been reported that the concentrations of DCA and LCA are significantly higher in faecal water from patients with colonic polyps or cancer than in faecal water from individuals with normal colons (5). We reconstituted two bile acid mixtures to reflect the levels in faecal water from patients with (DCA, 76 µM; LCA, 30 µM) and without (DCA, 17 µM; LCA, 7 µM) polyps or cancer and examined their ability to induce AP-1-dependent gene transcription. Both reconstituted mixtures induced reporter gene activity. However, the mixture that was similar to the faecal water of the polyp/cancer group induced significantly higher activity (330% AP-1 induction) than the mixture that was similar to the faecal water of the group with healthy colons (220% induction).


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The genesis of colorectal cancer is now widely accepted to involve the deregulation of specific genes that control cell division, apoptosis and DNA repair (23). While such deregulation, in many cases, involves mutations in DNA, it is becoming increasingly evident that luminal components, possibly including those of dietary origin, can also alter, either directly or indirectly, the expression of some of the above genes. Meanwhile, research continues to attempt to elucidate the mechanisms by which colonic bile acids contribute to tumour promotion in the colon. While a plethora of biological responses have been attributed to bile acids, it is now generally believed that these acidic lipids contribute to malignant transformation by disturbing the balance between proliferation, differentiation and apoptosis in the colonocytes. Therefore, it becomes of interest to determine whether the activity of transcription factors regulating these processes can be altered by bile acids.

Recently, we demonstrated that the secondary bile acid, DCA, induced AP-1 DNA-binding activity in the human adenocarcinoma cell line HT-29 (20), and Hirano et al. have shown that bile acids also augment AP-1-responsive gene expression, without affecting the function of the transcription factors NF{kappa}B, Sp1 and ATF/CREB, in the colon adenocarcinoma cells, LoVo and DLD-1 (19). It could also be demonstrated that the majority of protein components in the induced AP-1 DNA-binding activity were the products of oncogenes c-fos and c-jun (19,20). It also appeared that the effect of the bile acids on AP-1 activity was relatively selective in colon adenocarcinoma cells, since AP-1 activity was not influenced by bile acids in the neuroendocrine-derived colon cancer cell line COLO320DM (19). In the present study, we have extended this work to include additional components of possible relevance in colorectal carcinogenesis, including the matrix to which the cells are exposed in the human colon and additional cell lines that are widely used in this field. Thus, we showed that DCA induced AP-1-dependent gene transcription in HT-29 cells over a concentration range at which it occurs in the aqueous phase of human faeces (1,2). We were also able to demonstrate, for the first time, that the induction of AP-1 activity by DCA was significantly correlated with its ability to induce proliferation in these cells over this concentration range. This would support the suggestion that the effect on AP-1 activity may mediate the effect of bile acid on cell proliferation. When we screened the other colonic bile acids for their ability to augment AP-1-dependent gene transcription in HCT 116 cells, bile acid-specific effects were obvious, with LCA and CDCA having stronger effects than UDCA, and the primary bile acid CA having no effect. These effects may reflect differences in the hydrophobicity of these lipids and may contribute to their varying potencies as tumour promoters in the colon.

We were also able to demonstrate that the long chain fatty acids, myristate, palmitate and stearate, also components of human faecal water, were unable to induce AP-1-dependent gene transcription in the HCT 116 cells at physiological concentrations. Thus, if these lipids contribute to tumour promotion in the colon, it is not through effects on the AP-1 transcription factor complex as appears to be the case with bile acids. The fact that cholesterol also had no effect on AP-1-dependent gene transcription in the colonic cells gives further support to the idea that it is only specific lipid components of the faecal water that can exert this effect. Interestingly, the most marked effect on gene transcription seen in the present study was with the short chain fatty acid, butyrate, which gave a significant dose–response effect at physiological concentrations. Butyrate has been reported to inhibit cell growth and induce differentiation in colorectal cancer cells (24), and it induces apoptosis in HCT 116 cells at the concentrations studied (unpublished data). There is also evidence that AP-1 activity is increased during apoptosis in several cell lines (25,26). In an effort to further characterize our assay system, prior to the experiments discussed below, we were able to confirm that known activators of PKC (TPA and the diacylglycerols, OAG and 1,2-dimyristan) significantly induced AP-1-dependent gene transcription in HCT 116 cells. We also demonstrated that exposure of the cells to calcium chloride or the calcium ionophore, ionomycin, significantly induced reporter gene activity. It has been reported that increased calcium concentrations in the cell can activate PKC directly, which results in phosphorylation of substrates and leads to activation of c-fos and c-jun, with formation of the AP-1 complex (27).

We then wished to address the question as to whether components of the human faecal fraction in direct contact with the colonic epithelium (i.e. the faecal water) could influence AP-1-dependent gene transcription in colonic cells. But first it was of interest to determine whether this faecal fraction exhibited proliferative effects on the colonic cells. When HT-29 cells were exposed to intact faecal water fractions in the proliferation assay, cytotoxic effects were consistently observed. However, it must be pointed out here that while it is difficult to determine the true concentration of lumenal components (e.g. bile acids) either in or entering the epithelial cells in the colon, it is reasonable to assume that the levels are lower than lumenal values for the following reasons. Intracellular concentrations will be lower than lumenal values because of the concentration gradient established (decreasing in the lumen to cell direction) as a result of the combined effects of the excretion or exocytosis of compounds, such as bile acids, from epithelial cells into the enterohepatic circulation, and the diffusion of compounds across the mucin layer that coats the epithelial cells. In addition, intracellular modification of bile acids by processes such as glucuronidation might also play a role in lowering intracellular concentrations (28). This hypothesis is supported by observations that, in order to reproduce effects seen with TPA and DCA on colonocytes in culture in vivo, it is necessary to perfuse concentrations 10- to 100-fold higher than those used in cell culture through the colon (11, 2931). With this background, we considered it justifiable and relevant to examine the effect of diluting the faecal water fractions on their ability to induce proliferation of HT-29 cells in culture. Interestingly, on dilution, all of the human faecal water samples examined exhibited a capacity to increase the proliferative activity of the cells, with the degree of dilution required to observe the maximum effect dependent on the cytotoxicity of the undiluted sample, i.e. the more cytotoxic faecal waters required a higher dilution. In view of the above discussion, this may indicate that components of human faecal water can influence the proliferative activity of cells of the colonic epithelium in vivo. To our knowledge, this is the first time this observation has been made, and in view of the fact that several studies have clearly demonstrated that the biochemical composition of faecal water is influenced by diet (1,2,6,32), it may well have consequences for the role of dietary factors in tumour promotion in the large bowel.

Next, we examined the capacity of diluted lipid extracts from two representative human faecal water samples (one exhibiting high cytotoxicity and one low cytotoxicity) to increase AP-1-dependent gene transcription in the colonic cell lines. The use of lipid extracts in these initial experiments was justified by the fact that, in accordance with earlier results from our laboratory (20) and from others (19) in combination with the results outlined above, the lipid component of this faecal fraction would appear to be particularly relevant with regard to potential effects on AP-1-dependent gene transcription. Both lipid extracts, on dilution, exhibited a significant induction of reporter gene activity in both HCT 116 and HT-29 cells, with a more marked effect evident in the HT-29 cells. Once again, the extract from the faecal water with the higher cytotoxicity required a slightly higher dilution than that from the faecal water with the lower cytotoxicity in order to induce maximal activity, particularly in the HT-29 cells. When we subsequently screened lipid extracts from additional human faecal water samples to determine whether they could influence AP-1-dependent gene transcription, it was evident that all extracts significantly increased reporter gene activity, although this effect differed markedly between the extracts. We could also confirm that the faecal water lipid extracts significantly induced proliferation in the same cell line at the same dilution at which they increased AP-1 activity. The observation that there was not a significant correlation between the ability of the faecal water lipid extracts to induce AP-1 activity and cell proliferation probably reflects the fact that induction of AP-1 activity invokes cellular responses other than proliferation as alluded to above. Our observation that the proliferative response to faecal water lipid extracts in the normal colonic cells (FHC) was similar to that in the HT-29 cells demonstrates that the effects we are studying are not specific for colon cancer cell lines.

Thus, we have been able to show for the first time that lipid components of human faecal water, the faecal fraction in direct contact with the colonic epithelium, can increase AP-1-dependent gene transcription in colonic cells. The identity of these components remains to be elucidated. However, mindful of the results in the present paper and the concentrations of faecal water bile acids in the literature, and even taking into consideration the fact that intracellular concentrations may be lower than lumenal values, bile acids are candidate components. However, additional components such as short chain fatty acids may also be important for AP-1 induction. This suggestion is supported by our observation that induction of AP-1 activity and induction of cell proliferation by the faecal water lipid extracts were not correlated, which presumably would have been the case if bile acids were solely responsible. Also, of interest was our finding that the bile acid mixture, reconstituted to reflect the profile in faecal water from patients with colonic polyps or cancer, was significantly more efficient in inducing AP-1-dependent gene transcription than the corresponding mixture, reflecting the situation in the normal colon. This may suggest that bile acids in the patient's faecal water may be contributing to induction of AP-1 activity. Of some relevance here is the report that certain bile acids up-regulate the expression of cyclooxygenase (COX)-2, important in colonic tumour formation, in human esophageal adenocarcinoma cells and the suggestion that this effect may be mediated by AP-1 acting on a cyclic AMP response element in the COX-2 promoter (33). However, the determination of the precise role of AP-1 induction in colonic tumour development requires further work. If, as alluded to above, induction of this transcription factor results in a cellular response such as increased cell proliferation, it may well be mediating a tumour promoting effect. If, on the other hand, it results in a response such as apoptosis, it may even be mediating a protective effect. A characterisation of the protein components of the AP-1 complex, induced by the faecal water components, and a comparison with the protein components of the different AP-1 complexes giving rise to different cellular responses should clarify this issue. Finally, the results in the present paper are from faecal water samples from healthy individuals, and may be considered to represent `baseline' values, it will be interesting to determine whether the ability of faecal water to induce gene transcription is sensitive to dietary alterations and differs between patient groups.


    Acknowledgments
 
This study was supported by grants from the Swedish Cancer Society and the Swedish Dairies Association.


    Notes
 
1 To whom correspondence should be addressed Email: bjorn.glinghammar{at}mednut.ki.se Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received October 14, 1998; revised January 29, 1999; accepted February 5, 1999.