Mixtures of SCFA, composed according to physiologically available concentrations in the gut lumen, modulate histone acetylation in human HT29 colon cancer cells.

Intake of fibre has beneficial properties on gut health. Butyrate, a product of bacterial gut fermentation, is thought to contribute to positive effects by retarding growth and enhancing apoptosis of tumour cells. One mechanism is seen in its capacity to modulate histone acetylation and thereby transcriptional activity of genes. Next to butyrate, propionate and acetate are also major products of gut fermentation and together they may exert different potencies of cellular effects than butyrate alone. Since virtually nothing is known on combination effects by SCFA mixtures, here we had the aim to assess how physiological relevant concentrations and mixtures of SCFA modulate histone acetylation in human colon cells. HT29 colon cancer cells were incubated with mixtures of butyrate, acetate and propionate and with the individual compounds as controls. Histone acetylation was determined with acid-urea gel electrophoresis and immunoblotting. Acetylated histones slowly increased over 24 h and persisted up to 72 h in butyrate-treated HT29 cells. Butyrate (5-40 mM) and propionate (20-40 mM) enhanced histone acetylation significantly after 24 h incubation, whereas acetate (2.5-80 mM) was ineffective. Mixtures of these SCFA also modulated histone acetylation, mainly due to additive effects of butyrate and propionate, but not due to acetate. In conclusion, physiological concentrations of propionate together with butyrate could have more profound biological activities than generally assumed. Together, these SCFA could possibly mediate important processes related to an altered transcriptional gene activation and thus contribute to biological effects possibly related to cancer progression or prevention.

The gut fermentation product butyrate, a chemopreventive agent, suppresses glutathione S-transferase theta (hGSTT1) and cell growth more in human colon adenoma (LT97) than tumor (HT29) cells.

PURPOSE: The gut fermentation product of dietary fiber, butyrate, inhibits growth of HT29, an established tumor cell line. It also induces detoxifying enzymes belonging to the glutathione S-transferase family (GSTs), namely hGSTM2, hGSTP1, hGSTA4, but not of hGSTT1 . Here we investigated kinetics of effects in HT29 and compared sensitivities with preneoplastic LT97 colon adenoma cells, to assess mechanisms of colon cancer chemoprevention in two stages of cell transformation. METHODS: We determined cell growth after butyrate treatment by quantifying DNA, GST expression by Northern/Western Blotting or biochemical analysis and butyrate consumption by measuring the residual concentrations in the cell culture supernatants. Stability of GST-theta (hGSTT1) mRNA was assessed in HT29 cells after inhibition of transcription with actinomycin D. RESULTS: LT97 adenoma cells consumed twofold more butyrate and were more sensitive to growth inhibition than HT29 (EC(50)1.9 mM and 4.0 mM, respectively). Butyrate did not induce GSTs, but instead reduced hGSTT1 in LT97 and HT29. CONCLUSIONS: Butyrate has suppressing-agent activities in human colon cells by inhibiting two survival factors, namely hGSTT1 and cell growth, with LT97 more sensitive than HT29. These findings indicate that butyrate formation in the gut lumen of humans could be protective by reducing survival of transformed colon cells.

Analysis of the growth phase-associated transcriptome of Streptococcus pyogenes.

Streptococcus pyogenes (group A streptococci, GAS) is a human pathogen which probably varies its multiplication rate and thus, growth phases in association with the type of infection caused in its host. To create a basis for future determinations of such associations, the genome-wide growth phase-related GAS transcriptome was assessed in the present study. Therefore, the published serotype M1 S. pyogenes genome sequence as well as the partially sequenced serotype M18 and M49 GAS genomes were used to produce DNA microarrays that carried 2256 oligonucleotide probes matching 3662 open reading frames (ORFs). With these microarrays, the transcriptome of the serotype M49 GAS strain 591 grown to the exponential, transition, and early stationary growth phases was assessed in seven independent experiments. The gained data were compared to real-time RT-PCR assays. Data analysis was refined by a novel approach, i.e. grouping of expressed genes to four classes according to relative transcript abundance and gene functions. At the different growth phases, 86.7%, 79.5% and 55.7% of the at least 1883 ORFs contained in the serotype M49 genome were expressed above the defined detection level. Contrary to the general trend, transcript amounts of genes in the functional groups of transport and membrane proteins as well as stress response factors peaked at the transition phase. The most prominent changes in the transcript abundances were predominantly observed for sugar compound transport and turnover-related ORFs. The majority of known virulence genes had their maximum expression during the transition phase, consistent with the proposed associated change in virulence behavior of the bacteria. With these results, it will now be feasible to assess the in situ growth phase of a given GAS strain during any type of infection by measuring the expression of selected marker genes.

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.

Human adenoma cells are highly susceptible to the genotoxic action of 4-hydroxy-2-nonenal.

Oxidative stress and resulting lipid peroxidation are important risk factors for dietary-associated colon cancer. To get a better understanding of the underlying molecular mechanisms, we need to characterise the risk potential of the key compounds, which cause DNA damage in cancer-relevant genes and especially in human target cells. Here, we investigated the genotoxic effects of 4-hydroxy-2-nonenal (HNE) and hydrogen peroxide (H(2)O(2)) in human colon cells (LT97). LT97 is a recently established cell line from a differentiated microadenoma and represents cells from frequent preneoplastic lesions of the colon. The genomic characterisation of LT97 was performed with 24-colour FISH. Genotoxicity was determined with single cell microgelelectrophoresis (Comet assay). Comet FISH was used to study the sensitivity of TP53-a crucial target gene for the transition of adenoma to carcinoma-towards HNE. Expression of glutathione S-transferases (GST), which deactivates HNE, was determined as GST activity and GSTP1 protein levels. LT97 cells were compared to primary human colon cells and to a differentiated clone of HT29. Karyotyping revealed that the LT97 cell line had a stable karyotype with only two clones, each containing a translocation t(7;17) and one aberrant chromosome 1. The Comet assay experiments showed that both HNE and H(2)O(2) were clearly genotoxic in the different human colon cells. HNE was more genotoxic in LT97 than in HT29clone19A and primary human colon cells. After HNE incubation, TP53 migrated more efficiently into the comet tail than the global DNA, which suggests a higher susceptibility of the TP53 gene to HNE. GST expression was significantly lower in LT97 than in HT29clone19A cells, which could explain the higher genotoxicity of HNE in the colon adenoma cells. In conclusion, the LT97 is a relevant model for studying genotoxicity of colon cancer risk factors since colon adenoma are common preneoplastic lesions occurring in advanced age.