Short Chain Fatty Acids (SCFA) Reprogram Gene Expression in Human Malignant Epithelial and Lymphoid Cells

<div><p>The effect of short chain fatty acids (SCFAs) on gene expression in human, malignant cell lines was investigated, with a focus on signaling pathways. The commensal microbial flora produce high levels of SCFAs with established physiologic effects in humans. The most abundant SCFA metabolite in the human microflora is n-butyric acid. It is well known to activate endogenous latent Epstein-Barr virus (EBV), that was used as a reference read out system and extended to EBV+ epithelial cancer cell lines. N-butyric acid and its salt induced inflammatory and apoptotic responses in tumor cells of epithelial and lymphoid origin. Epithelial cell migration was inhibited. The n-butyric gene activation was reduced by knock-down of the cell membrane transporters MCT-1 and -4 by siRNA. N-butyric acid show biologically significant effects on several important cellular functions, also with relevance for tumor cell phenotype.</p></div

Microflora associated characteristics (MACs) of the CD patients (n = 4) and controls (n = 5). Concentrations of relevant short chain fatty acids (SCFAs) in faeces (mmol/kg) (mean and range).

<p>Microflora associated characteristics (MACs) of the CD patients (n = 4) and controls (n = 5). Concentrations of relevant short chain fatty acids (SCFAs) in faeces (mmol/kg) (mean and range).</p

N-butyric acid induces PARP cleavage.

<p>Western blot analysis of cells treated with butyrate or TPA for 24hr, A-C) Raji cells, D-F) C666-1 cells. A,D) PonceauS staining. C,E) Actin. B,F) anti PARP Ab.</p

Increase of Faecal Tryptic Activity Relates to Changes in the Intestinal Microbiome: Analysis of Crohn's Disease with a Multidisciplinary Platform

<div><p>Objective</p><p>To investigate—by molecular, classical and functional methods—the microbiota in biopsies and faeces from patients with active Crohn's disease (CD) and controls.</p><p>Design</p><p>The microbiota in biopsies was investigated utilizing a novel molecular method and classical cultivation technology. Faecal samples were investigated by classical technology and four functional methods, reflecting alterations in short chain fatty acids pattern, conversion of cholesterol and bilirubin and inactivation of trypsin.</p><p>Results</p><p>By molecular methods we found more than 92% similarity in the microbiota on the biopsies from the two groups. However, 4.6% of microbes found in controls were lacking in CD patients. Furthermore, NotI representation libraries demonstrate two different clusters representing CD patients and controls, respectively. Utilizing conventional technology, Bacteroides (alt. Parabacteroides) was less frequently detected in the biopsies from CD patients than from controls. A similar reduction in the number of Bacteroides was found in faecal samples. Bacteroides is the only group of bacteria known to be able to inactivate pancreatic trypsin. Faecal tryptic activity was high in CD patients, and inversely correlated to the levels of Bacteroides.</p><p>Conclusions</p><p>CD patients have compositional and functional alterations in their intestinal microbiota, in line with the global description hypothesis rather than the candidate microorganism theory. The most striking functional difference was high amount of faecal tryptic activity in CD patients, inversely correlated to the levels of <i>Bacteroides</i> in faeces.</p></div

Analysis of short chain fatty acids induced BZLF1 gene expression in C666-1 NPC cells.

<p>RT-PCR analysis upon overnight treatment with 10mM of short chain fatty acids as indicated. A. BZLF1 expression. B. BART A expression. C. GAPDH expression. Cells were treated with 10mM of SCFAs as indicated or were left untreated. D. Digital analysis of data from panels A-C, demonstrating BZLF1 expression in relation to GAPDH.</p

N-butyric acid induces proinflammatory cytokine expression.

<p>A. qRT-PCR analysis of Il-6 and IL-8 expression after overnight exposure to 10mM n-butyric acid. Values are adjusted to the EF1α gene expression. B. ELISA analysis of IL-8 secretion from C666-1 cells after 48hr of exposure to 10mM n-butyric acid.</p

N-butyric acid inhibits migration of C666-1 NPC cells.

<p>Evaluation of the gap distance in migration assay of C666-1 NPC cells upon treatment as indicated.</p

N-butyric acid affects cellular gene expression in LMP2A expressing cells.

<p>Network enrichment analysis of differentially expressed genes in HONE1 and HONE1 LMP2A cells upon n-butyric acid exposure.</p

Analysis of n-butyric acid induced BZLF1 gene expression in C666-1 NPC cells.

<p>RT-PCR analysis upon treatment with 10mM of n-butyric acid or HCl. A. BZLF1 expression. B. GAPDH expression. C. BART A expression. D. Digital analysis of relative BZLF1 expression calibrated to GAPDH, calculated from data in panels A-C.</p

A comparison of the differentially activated signaling pathways in LMP2A positive cells.

<p>A comparison of the differentially activated signaling pathways in LMP2A positive cells.</p