Butyrate induces glutathione S-transferase in human colon cells and protects from genetic damage by 4-hydroxy-2-nonenal

Butyrate, one of the major products of gut fermentation, is known to inhibit proliferation, induce apoptosis and differentiation, and increase phase II enzyme activities in tumor cells, whereas little information is available on protective effects in less-transformed colon cells. The aim of this study was to investigate whether the chemoprotective mechanism of glutathione S-transferase (GST) induction by butyrate could also play a role in earlier stages of colon carcinogenesis and whether chemoresistance of cells toward the endogenous genotoxic risk factor 4-hydroxy-2-nonenal (HNE) could be a consequence of butyrate treatment. As cell models, we used the human tumor cell lines HT29 and HT29 clone 19A, a differentiated subclone with properties resembling primary colon cells. We determined the expression of GSTP1 protein (enzyme-linked immunosorbent assay), the major GST in HT29, GSTP1 mRNA (Northern blotting), GST activity, intracellular glutathione, and total protein. The genotoxic impact of HNE (100-200 microM) was compared in butyrate-treated and nontreated cells using single-cell microgel electrophoresis. Our results show that GSTP1 mRNA, GSTP1 protein, GST activity, and total protein were increased (1.2- to 2.5-fold) and glutathione levels were maintained after 24-72 h of incubation with 4 mM butyrate. Moreover, a marked reduction of HNE-induced genotoxicity was caused by preincubation with butyrate. Butyrate also induced the phosphorylation of extracellular signal-regulated kinases (ERK1/2, Western blotting) after 5-30 min, which indicates a regulation of GST expression by this signal pathway. Most effects were greater in HT29 parent cells than in clone cells. In conclusion, butyrate enhances expression of GST and other proteins in both cell lines, which leads to an enhanced chemoprotection, reducing the impact of HNE genotoxicity. Thus butyrate could play a role in early and later stages of cancer prevention by reducing exposure to relevant risk factors.     

Butyrate is only one of several growth inhibitors produced during gut flora-mediated fermentation of dietary fibre sources

Dietary fibre sources are fermented by the gut flora to yield short-chain fatty acids (SCFA) together with degraded phytochemicals and plant nutrients. Butyrate, a major SCFA, is potentially chemoprotective by suppressing the growth of tumour cells and enhancing their differentiation. Conversely, it could lead to a positive selection pressure for transformed cells by inducing glutathione S-transferases (GST) and enhancing chemoresistance. Virtually nothing is known about how butyrate's activities are affected by other fermentation products. To investigate such interactions, a variety of dietary fibre sources was fermented with human faecal slurries in vitro, analysed for SCFA, and corresponding SCFA mixtures were prepared. HT29 colon tumour cells were treated for 72 h with individual SCFA or complex samples. The growth of cells, GST activity, and chemoresistance towards 4-hydroxynonenal were determined. Fermentation products inhibited cell growth more than the corresponding SCFA mixtures, and the SCFA mixtures were more active than butyrate, probably due to phytoprotectants and to propionate, respectively, which also inhibit cell growth. Only butyrate induced GST, whereas chemoresistance was caused by selected SCFA mixtures, but not by all corresponding fermentation samples. In summary, fermentation supernatant fractions contain compounds that: (1) enhance the anti-proliferative properties of butyrate (propionate, phytochemical fraction); (2) do not alter its capacity to induce GST; (3) prevent chemoresistance in tumour cells. It can be concluded that fermented dietary fibre sources are more potent inhibitors of tumour cell growth than butyrate alone, and also contain ingredients which counteract the undesired positive selection pressures that higher concentrations of butyrate induce in tumour cells.     

Intervention with cloudy apple juice results in altered biological activities of ileostomy samples collected from individual volunteers

BACKGROUND: Apple juice is considered to be an important component of the healthy diet, with anticancer activities in colon cancer animal models and key ingredients have numerous chemoprotective activities in human colon cells in vitro. AIM OF THE STUDY: Since only little is known on comparable activities in the human colon in vivo, here a pilot study was performed to assess related mechanisms caused by ileostomy samples from volunteers that had consumed apple juice. METHODS: Ileostomy samples were collected after intervention (0-8 h) with cloudy apple juice (1 l). They were characterized analytically for major apple polyphenols and biologically in HT29 colon cells for their potential to cause genotoxic damage, protect from the genotoxic insult by hydrogen peroxide (H(2)O(2)) and modulate the expression of GSTT2, an enzyme related to antioxidative defence against different peroxides. RESULTS: The analytical determination of polyphenols in the ileostomy samples revealed that the majority of the compounds were recovered in the samples collected 2 h after intervention. The comparison of genotoxic effects of samples before intervention and 2 h after intervention revealed a considerable variation of genotoxic response, but there was a trend for reduced genotoxicity in three of eight persons (P) after intervention. Samples collected at 2 h protected HT29 cells from genotoxic damage by H(2)O(2) (for 4 of 8 persons), resulted in an increased GSTT2 expression (for 2 of 6 persons) and of GSTT2 promotor activity (2 of 6 persons). CONCLUSIONS: The intervention with apple juice results in bioavailable concentrations of related polyphenols in the gut lumen, which could contribute to reduced genotoxicity, enhanced antigenotoxicity and favorable modulation of GSTT2 gene expression in some individuals. The pilot study for the first time used this combination of faecal biomarkers which in larger cohorts may either reveal overall significant alterations of chemoprotection or may be used to identify individuals which could particularly benefit from a personalized nutrition.     

Obituary

Curcumin, quercetin, and eicosapentaenoic acid (EPA) are 3 natural compounds with the capacity to reduce adenoma burden in patients with familial adenomatous polyposis (FAP). The mechanistic basis of this anticarcinogenic capacity is largely unknown, but it was suggested that induction of detoxification enzymes is involved. Therefore, the effects of low-dose curcumin, quercetin, and EPA on phase II detoxification enzymes UDP-glucuronosyltransferase (UGT), glutathione S-transferase (GST), as well as on glutathione (GSH) content were analyzed in 4 cell line models of intestinal carcinogenesis. HT-29, HuTu 80, and Caco-2 intestinal cancer cells and LT97 colon adenoma cells from a patient with FAP were treated with low-dose noncytotoxic concentrations of curcumin, quercetin, and EPA. GST enzyme activity was measured by spectrophotometry, and expression of GSTA1, GSTM1, GSTP1, GSTT1, and UGT1 by Western blotting. Cytosolic GSH levels were determined by high performance liquid chromatography. An inducing effect of curcumin and quercetin on GST or UGT was seen in Caco-2, LT97, and HuTu 80 cells. GSH levels were reduced by quercetin and EPA in HT-29 cells and induced by curcumin in Caco-2 cells. In LT97 cells, GST activity and expression was reduced, but UGT1 expression was induced by curcumin and quercetin; whereas EPA only decreased GST or UGT levels. In summary, enhancement of the detoxification capacity by low dose of the potential anticarcinogens curcumin, quercetin, or EPA seems only a minor factor in explaining their anticarcinogenic properties.     

The Influence of Curcumin, Quercetin, and Eicosapentaenoic Acid on the Expression of Phase II Detoxification Enzymes in the Intestinal Cell Lines HT-29, Caco-2, HuTu 80, and LT97

Curcumin, quercetin, and eicosapentaenoic acid (EPA) are 3 natural compounds with the capacity to reduce adenoma burden in patients with familial adenomatous polyposis (FAP). The mechanistic basis of this anticarcinogenic capacity is largely unknown, but it was suggested that induction of detoxification enzymes is involved. Therefore, the effects of low-dose curcumin, quercetin, and EPA on phase II detoxification enzymes UDP-glucuronosyltransferase (UGT), glutathione S-transferase (GST), as well as on glutathione (GSH) content were analyzed in 4 cell line models of intestinal carcinogenesis. HT-29, HuTu 80, and Caco-2 intestinal cancer cells and LT97 colon adenoma cells from a patient with FAP were treated with low-dose noncytotoxic concentrations of curcumin, quercetin, and EPA. GST enzyme activity was measured by spectrophotometry, and expression of GSTA1, GSTM1, GSTP1, GSTT1, and UGT1 by Western blotting. Cytosolic GSH levels were determined by high performance liquid chromatography. An inducing effect of curcumin and quercetin on GST or UGT was seen in Caco-2, LT97, and HuTu 80 cells. GSH levels were reduced by quercetin and EPA in HT-29 cells and induced by curcumin in Caco-2 cells. In LT97 cells, GST activity and expression was reduced, but UGT1 expression was induced by curcumin and quercetin; whereas EPA only decreased GST or UGT levels. In summary, enhancement of the detoxification capacity by low dose of the potential anticarcinogens curcumin, quercetin, or EPA seems only a minor factor in explaining their anticarcinogenic properties.     

Prolonged butyrate treatment inhibits the migration and invasion potential of HT1080 tumor cells

Butyrate, a normal constituent of the colonic luminal contents, is produced by the bacterial fermentation of dietary fibers and resistant starches. It has been hypothesized that butyrate may inhibit the invasion of tumor cells. The purpose of the present study was to investigate the effects of butyrate treatment on the growth, migration, and invasion characteristics of tumor HT1080 cells. HT1080 cells cultured in the presence of 0.5 and 1 mmol/L butyrate for 14 d exhibited an increase in the G(1) and G(2) fractions with a concomitant drop in the S-phase, thus showing slower cell growth. Interestingly, 0.5 and 1 mmol/L butyrate inhibited the migration and invasion rate of the tumor cells compared with the untreated (control) cells. The protein and mRNA levels of the tissue inhibitors of metalloproteinase-1 (TIMP-1) and TIMP-2 were significantly increased in HT1080 cells cultured with 0.5 and 1 mmol/L butyrate. Enzymatic activities and the mRNA level of the latent forms of matrix metalloproteinase (MMP), pro-MMP-2 and pro-MMP-9, were also increased in HT1080 cells cultured with 0.5 and 1 mmol/L butyrate. In contrast, the active form of MMP-2 was detectable by zymographic analysis in control but not butyrate-conditioned media. Collectively, these results demonstrate that prolonged and low-dose butyrate treatment increases both prometastasis MMP-2, -9 and antimetastasis TIMP-1, -2 expression, and the net effect of these increases is the inhibition of pro-MMP-2 activation and of tumor cell migration/invasion potential.     

The effects of short-chain fatty acids on human colon cancer cell phenotype are associated with histone hyperacetylation

The short-chain fatty acid (SCFA) butyrate is produced via anaerobic bacterial fermentation within the colon and is thought to be protective in regard to colon carcinogenesis. Although butyrate (C4) is considered the most potent of the SCFA, a variety of other SCFA also exist in the colonic lumen. Butyrate is thought to exert its cellular effects through the induction of histone hyperacetylation. We sought to determine the effects of a variety of the SCFA on colon carcinoma cell growth, differentiation and apoptosis. HT-29 or HCT-116 (wild-type and p21-deleted) cells were treated with physiologically relevant concentrations of various SCFA, and histone acetylation state was assayed by acid-urea-triton-X gel electrophoresis and immunoblotting. Growth and apoptotic effects were studied by flow cytometry, and differentiation effects were assessed using transient transfections and Northern blotting. Propionate (C3) and valerate (C5) caused growth arrest and differentiation in human colon carcinoma cells. The magnitude of their effects was associated with a lesser degree of histone hyperacetylation compared with butyrate. Acetate (C2) and caproate (C6), in contrast, did not cause histone hyperacetylation and also had no appreciable effects on cell growth or differentiation. SCFA-induced transactivation of the differentiation marker gene, intestinal alkaline phosphatase (IAP), was blocked by histone deacetylase (HDAC), further supporting the critical link between SCFA and histones. Butyrate also significantly increased apoptosis, whereas the other SCFA studied did not. The growth arrest induced by the SCFA was characterized by an increase in the expression of the p21 cell-cycle inhibitor and down-regulation of cyclin B1 (CB1). In p21-deleted HCT-116 colon cancer cells, the SCFA did not alter the rate of proliferation. These data suggest that the antiproliferative, apoptotic and differentiating properties of the various SCFA are linked to the degree of induced histone hyperacetylation. Furthermore, SCFA-mediated growth arrest in colon carcinoma cells requires the p21 gene.     

Short-chain fatty acids modulate gene expression for vascular endothelial cell adhesion molecules

OBJECTIVE: Leukocyte infiltration into the intestinal wall is central to the pathogenesis of tissue injury that occurs in patients with a variety of inflammatory bowel diseases. Migration of leukocytes from the intestinal circulation into bowel tissues is mediated by chemotactic substances and adhesion molecules (i.e., intercellular adhesion molecule-1 [ICAM-1] and E-selectin) on the surface of endothelial cells lining blood vessels. Short-chain fatty acids (SCFAs) derived from dietary fiber decrease inflammatory responses in colon cells. However, the effect of SCFAs on vascular adhesion molecules is unknown. We investigated the effects of SCFAs on vascular endothelial cell adhesion molecule expression. METHODS: We assessed the effect of physiologically relevant concentrations of butyrate on expression of ICAM-1 protein and mRNA in cultures of human umbilical vein endothelial cells. We also assessed the effect of butyrate on levels of HLA-DR, E-selectin, vascular cell adhesion molecule-1, and endoglin. In additional experiments, we evaluated the effect of butyrate on ICAM-1 mRNA stability and the effect of valerate, isobutyrate, and propionate on ICAM-1 expression. The effect of butyrate on ICAM-1 expression was compared with that of trichostatin A, a specific inhibitor of histone deacetylase. Data were evaluated with Student's t tests or Tukey's multiple comparison tests, with P < 0.05 considered statistically significant. RESULTS: Butyrate concentrations of 2.5 to 5 mM significantly increased endothelial expressions of ICAM-1 protein and mRNA. The effect of butyrate (5 mM) on ICAM-1 expression was time dependent, with significant increases in levels occurring after 16 h of incubation. Butyrate (5 mM) also increased expression of E-selectin but not of HLA-DR, vascular cell adhesion molecule-1, or endoglin. Isobutyrate had little effect on ICAM-1 expression, whereas valerate and propionate significantly increased expression of ICAM-1 but were weaker stimulants compared with butyrate. Butyrate (5 mM) did not alter stability of ICAM-1 mRNA. The effect of butyrate (5 mM) was comparable to that of trichostatin A. The stimulatory effect of butyrate on ICAM-1 expression was reversed after 48 h of butyrate withdrawal. CONCLUSIONS: Butyrate increases vascular endothelial expressions of ICAM-1 and E-selectin. We speculate that butyrate-induced effects on vascular adhesion molecules modulate gut inflammation. The role of SCFAs and fiber in the pathogenesis and modulation of gut inflammation in vivo requires further study.     

Resveratrol enhances the differentiation induced by butyrate in caco-2 colon cancer cells

Butyrate, a short-chain fatty acid produced in the colon by microbial fermentation of fiber, inhibits growth of colonic carcinoma cells while inducing differentiation. Resveratrol, a plant polyphenol found in red wine and peanuts, has been shown to exert chemopreventive properties on colon cancer cells. The aim of this study was to determine whether resveratrol modulates the effects of butyrate on Caco-2, a colonic adenocarcinoma cell line. The growth inhibitory effect of resveratrol (50 micromol/L) was more powerful than that of butyrate (2 mmol/L). Butyrate did not intensify the inhibition of proliferation exerted by resveratrol. Although the polyphenol enhanced the differentiation-inducing effect of butyrate, it did not elevate alkaline phosphatase activity or E-cadherin protein expression, markers of epithelial differentiation, when applied alone. Butyrate-induced transforming growth factor-beta1 secretion was inhibited by resveratrol. Treatment with the combination of resveratrol and butyrate attenuated levels of p27(Kip1), whereas resveratrol enhanced butyrate's effect on the induction of p21(Waf1/Cip1) expression. These data demonstrate a possible combined chemopreventive effect of two substances naturally occurring in the colonic lumen after ingestion of fibers and resveratrol-containing food.     

Butyrate activates the cAMP-protein kinase A-cAMP response element-binding protein signaling pathway in Caco-2 cells

Butyrate is a major SCFA produced by microbial fermentation of dietary fiber in the gastrointestinal tract. Butyrate is widely thought to mediate the benefits of fiber and resistant starch consumption to colon health in humans. Besides serving as a substrate for energy production, butyrate has many regulatory effects in animals. Little is known about the signaling mechanisms underlying the regulatory effects of butyrate and other SCFA. In this study, we determined whether butyrate can activate cAMP-protein kinase A (PKA)- cAMP response element (CRE)-binding protein (CREB) signaling in Caco-2 cells, a model of intestinal epithelial cells. Butyrate promoted luciferase expression from a CRE-reporter construct, induced phosphorylation of CREB, increased the activity of PKA, and elevated the levels of cAMP in Caco-2 cells. These data suggest that butyrate activates cAMP-PKA-CREB signaling in Caco-2 cells. Butyrate, however, had no effect on the activities of adenylyl cyclase (AC) and phosphodiesterase (PDE), two enzymes that determine the production and degradation of intracellular cAMP, respectively. Because the activities of AC and PDE are primarily regulated by G protein-coupled receptor (GPR)-mediated intracellular signaling, lack of an effect of butyrate on these two enzymes suggests that butyrate does not activate cAMP-PKA-CREB signaling through GPR. Butyrate-treated Caco-2 cells had greater concentrations of ATP than untreated cells. Because ATP is the substrate for cAMP production, this difference suggests that butyrate may activate cAMP-PKA-CREB signaling in Caco-2 cells through increased ATP production. Overall, this study raises the possibility that some of the regulatory effects of butyrate in animals, including those on the colonocytes, may be mediated by the cAMP-PKA-CREB signaling pathway at the cellular level.     

Fecal water genotoxicity is predictive of tumor-preventive activities by inulin-like oligofructoses, probiotics (Lactobacillus rhamnosus and Bifidobacterium lactis), and their synbiotic combination

The measurement of fecal water genotoxicity in human colon cells could be a useful biomarker to study effects of diet in the colon. Here we assessed aqueous fecal extracts of samples from a chronic study with rats fed prebiotics, probiotics, and their combination. Treatments were maltodextrins (controls), inulin/oligofructoses (prebiotic), Lactobacillus rhamnosus, and Bifidobacterium lactis (probiotics) or both (synbiotic). Azoxymethane (AOM) was administered to initiate tumors. Rat feces were collected at 0 and 10 days and 2, 4, and 8 mo, and cecal contents were collected at 8 mo. Aqueous phases were prepared and tested for genotoxicity in HT29 colon cells using the comet assay. The studied types of intervention reduced fecal and cecal genotoxicity. DNA damage by samples from AOM-treated, tumor-free rats was significantly lower than from tumor-bearing animals, especially after 4 mo of synbiotic and prebiotic interventions. Inulin-based diets reduced exposure to genotoxins in the feces, directly reflecting the reported reduction of tumor incidence in these animals. Evidence is provided for the validity of this measurement as a biomarker of chemoprevention because 1) fecal water genotoxicity reflected genotoxic exposure in the cecum, 2) tumor incidence and fecal genotoxicity were directly related, and 3) these interventions reduced tumor risks by reducing exposure to genotoxins in the gut.     

Dietary intervention with the probiotics Lactobacillus acidophilus 145 and Bifidobacterium longum 913 modulates the potential of human faecal water to induce damage in HT29clone19A cells

Probiotics reduce the risk of colon cancer by inhibiting carcinogen-induced DNA damage in animals, but there are no analogous data in human subjects. To enhance knowledge of the effects of probiotics in human subjects, we have investigated the genotoxicity of faecal water after dietary intervention with standard yoghurt or with probiotic yoghurt, which included the strains Lactobacillus acidophilus 145 and Bifidobacterium longum 913. Faeces were collected from nine healthy volunteers after intervention with probiotic yoghurt or standard yoghurt. Faecal water was isolated and incubated with human colon tumour cells HT29clone19A. DNA strand breaks, oxidised DNA bases and damage after challenge with H2O2 were determined by micro-gel-electrophoresis. Faecal water was genotoxic in comparison with NaCl, but protected against H2O2-induced DNA strand breaks. The intervention with probiotic yoghurt significantly lowered faecal water genotoxicity compared with standard yoghurt. However, probiotic intervention also increased oxidative damage; this either reflected prooxidative activity or stimulation of endogenous defence systems. Altogether, the balance of effects favoured protection, since faecal water from the probiotic group reduced overall genetic damage. Thus, there was a reduction of strand break-inducing compounds in human faeces after dietary intervention with probiotic bacteria. This protection reflected results from previous studies in carcinogen-exposed animals where probiotics reduced DNA damage in colon cells.     

In vitro studies on the inhibition of colon cancer by butyrate and carnitine

ObjectiveEpidemiologic studies support an association between diet and the incidence of colorectal cancer. Butyrate, a short-chain fatty acid present in dietary fiber and dairy products, is a potential anticarcinogenic compound. We previously showed that carnitine can enhance the bioavailability of butyrate in vivo. In the present study, we evaluated the effects of butyrate alone and in combination with carnitine on colon cancer cells in vitro, examining proliferation and apoptosis and the molecular mechanisms by which these nutrients may inhibit colon cancer.MethodsCaco-2 cells, a well-established cell model, were incubated with butyrate (2.5–20 mM) with or without carnitine (10 mM) for various incubation periods. Proliferation was measured by incorporation of ³H-thymidine, and apoptosis was detected using flow cytometry, and then confirmed by analyzing the presence of single-strand DNA breaks typical of apoptotic cells. Prostaglandin E₂ production was assayed and Bcl-2 and cyclo-oxygenase-2 expressions were examined by western blotting.ResultsButyrate and carnitine inhibited Caco-2 cell proliferation (P < 0.05) and induced apoptosis (P < 0.05). Prostaglandin E₂ production was decreased in treated Caco-2 cells. At the molecular level, the expression of proapoptotic Bax and Bak proteins were increased in cells incubated with butyrate and carnitine, whereas expression of antiapoptotic Bcl-x_L was decreased. Cyclo-oxygenase-2 expression was decreased in cells incubated with butyrate and carnitine.ConclusionsButyrate and carnitine inhibit human colon carcinoma cell proliferation and induce apoptosis in human colon carcinoma cells. This is accompanied by an appreciable alteration of the Bax-to-Bcl-x_L and Bak-to-Bcl-x_L ratios in favor of apoptosis. This study provides a scientific rationale to study the effects of carnitine and butyrate in colon cancer in vivo.     

Differential Antineoplastic Effects of Butyrate in Cells With and Without a Functioning DNA Mismatch Repair

The aim of this study was to investigate the differential antineoplastic effects of butyrate in cells with and without a functional mismatch repair and to determine the molecular mechanisms underlying these effects. SW48 colon cancer cells in which the MLH1 gene is silenced by promoter hypermethylation and demethylated SW48 cells in which the MLH1 gene is reexpressed were treated with butyrate (0-5mM) for 8 days and the effects on cell number, MLH1 gene promoter methylation, and expression of two cell cycle regulatory genes, CDK4 and GADD45A, were assessed. Butyrate suppressed viable cell number ( P < 0.001) and reduced MLH1 promoter methylation ( P < 0.05) in SW48 cells. However, in demethylated SW48 cells, butyrate caused an increase in viable cells ( P < 0.05) and promoter methylation ( P < 0.05). CDK4 expression was downregulated by butyrate exposure, but the effect was significantly greater for demethylated SW48 cells ( P = 0.025). Butyrate treatment caused upregulation of GADD45A expression in SW48 cells but downregulation of GADD45A expression in demethylated SW48 cells ( P = 0.045). This study supports the hypothesis that butyrate has more potent antineoplastic effects on colon cancer cells with MLH1 dysfunction. Differential expression of key cell cycle regulatory genes may explain some of the molecular mechanisms underlying these effects.     

Dietary compounds that induce cancer preventive phase 2 enzymes activate apoptosis at comparable doses in HT29 colon carcinoma cells.

Dietary agents that induce glutathione S-transferases and related detoxification systems (Phase 2 enzyme inducers) are thought to prevent cancer by enhancing elimination of chemical carcinogens. The present study shows that compounds of this group (benzyl isothiocyanate, allyl sulfide, dimethyl fumarate, butylated hydroxyanisole) activated apoptosis in human colon carcinoma (HT29) cells in culture over the same concentration ranges that elicited increases in enzyme activity (5-25, 25-100, 10-100, 15-60 micromol/L, respectively). Pretreatment of cells with sodium butyrate, an agent that induces HT29 cell differentiation, resulted in parallel increases in Phase 2 enzyme activities and induction of apoptosis in response to the inducers. Cell death characteristics included apoptotic morphological changes, appearance of cells at sub-G1 phase on flow cytometry, caspase activation, DNA fragmentation and TUNEL-positive staining. The results suggest that dietary Phase 2 inducers may protect against cancer by a mechanism distinct from and in addition to that associated with enhanced elimination of carcinogens. If this occurs in vivo, diets high in such compounds could eliminate precancerous cells by apoptosis at time points well after initial exposure to chemical mutagens and carcinogens.     

Histone-deacetylase inhibition and butyrate formation: Fecal slurry incubations with apple pectin and apple juice extracts

OBJECTIVE: Butyrate plays a major role among the short-chained fatty acids formed by the microbial flora of the colon. It is considered to be an important nutrient of the colon mucosa and has been shown to trigger differentiation and apoptosis of colon-derived cells in culture. Inhibition of histone deacetylase (HDAC) seems to play a central role in these effects. Butyrate was thus suggested to act as a chemopreventive metabolite that can prevent the occurrence of colorectal cancer, one of the most abundant types of cancer in Western industrialized countries. Some polymeric carbohydrates such as pectin, resistant to digestion in the small intestine, have been shown to serve as substrates for butyrate formation by the microflora of the colon. METHODS: In this study we investigated fermentation supernatants (FSs) from incubations of human fecal slurry with apple pectin and with polyphenol-rich apple juice extracts (AJEs). RESULTS: We found that FSs from fermentations with pectin were rich in butyrate and very active in HDAC inhibition in nuclear extracts prepared from the colon tumor cell lines HT-29 and Caco-2 and in intact HeLa Mad 38 cells bearing a reporter gene driven by HDAC inhibition. The butyrate levels explained most of the HDAC-inhibitory potency in FSs from pectin-rich fermentations. FSs from fermentations with AJEs showed lower butyrate yields but comparable HDAC inhibition. Combined incubations of pectin with AJEs led to effects similar to those with FSs from incubations with pectin as the only substrate added. These effects could not be explained by a direct HDAC-inhibitory potency of AJEs. Furthermore, the FSs were not cytotoxic at the HDAC-inhibitory concentrations. CONCLUSION: These findings suggest that butyrate is the most relevant HDAC inhibitor formed in fermentations of human fecal slurry with apple pectin, whereas addition of AJEs leads to the formation of butyrate and other, yet unknown, HDAC inhibitors.     

Fermented wheat aleurone enriched with probiotic strains LGG and Bb12 modulates markers of tumor progression in human colon cells

Fermentation of dietary fiber by the microflora enhances the levels of effective metabolites, which are potentially protective against colon cancer. The specific addition of probiotics may enhance the efficiency of fermentation of wheat aleurone, a source of dietary fiber. We investigated the effects of aleurone, fermented with fecal slurries with the addition of the probiotics LGG and Bb12 (aleurone(+)), on cell growth, apoptosis, and differentiation, as well as expression of genes related to growth and apoptosis using two different human colon cell lines (HT29: adenocarcinoma cells; LT97: adenoma cells). The efficiency of fermentation of aleurone was only slightly enhanced by the addition of LGG/Bb12, resulting in an increased concentration of butyrate. In LT97 cells, the growth inhibition of aleurone(+) was stronger than in HT29 cells. In HT29 cells, a cell cycle arrest in G(0)/G(1) and the alkaline phosphatase activity, a marker of differentiation, were enhanced by the fs aleurone(+). Treatment with all fermentation supernatants resulted in a significant increase in apoptosis and an upregulation of genes involved in cell growth and apoptosis (p21 and WNT2B). In conclusion, fs aleurone(+) modulated markers of cancer prevention, namely inhibition of cell growth and promotion of apoptosis as well as differentiation.