The transition from normality to malignancy in colorectal cancer is characterized by alterations in the expression of genes associated with the maintenance of tissue homoeostasis. Butyrate, a product of microbial fermentation of dietary fibre in the colon, is known to regulate a number of genes associated with the processes of proliferation, differentiation and apoptosis of colonic epithelial cells, and, hence, homoeostasis of colonic tissue. We have shown previously that the transport of butyrate into colonocytes is of fundamental importance to butyrate's regulatory ability, and therefore sought to assess the expression profile of butyrate-responsive genes in colon cancer tissue, where the expression of the colonic luminal-membrane butyrate transporter, MCT1 (monocarboxylate transporter 1), is significantly down-regulated. In the present paper, we first employed microarray analysis to assess global changes in butyrate-responsive genes using HT29 human colon carcinoma cells treated with butyrate. There was consistency in the butyrate response of selected genes in two other human colonic cell lines (HCT116 and AA/C1) using quantitative real-time PCR. Furthermore, we report that expression levels of selected butyrate-responsive genes involved in the processes of proliferation, differentiation and apoptosis, are deregulated in colon cancer tissue, correlating with decreased expression of MCT1. These findings support our hypothesis that a reduction in MCT1 expression, and hence butyrate transport, can lead to a reduction in the intracellular butyrate levels required to regulate gene expression. Collectively, our results highlight the important contribution of butyrate transport to the maintenance of tissue homoeostasis and disease prevention.
- colon cancer
- gene expression
- real-time PCR
Butyrate is a naturally occurring short-chain fatty acid that is produced in the lumen of the colon by bacterial fermentation of dietary fibre and resistant starch . As well as serving as the principal energy source for colonic epithelial cells , butyrate exhibits a range of anti-tumorigenic effects on many cancer cell lines and plays a significant role in the regulation of a number of genes associated with the key processes of apoptosis, proliferation and differentiation [3–7].
We have demonstrated previously that the expression of the colonic luminal-membrane butyrate transporter, MCT1 (monocarboxylate transporter 1), is significantly down-regulated during the transition from normality to malignancy in human colon , and that siRNA (small interfering RNA)-silencing of MCT1 expression in colonic epithelial cell lines abrogates the butyrate-induced response of several key genes [9,10]. These results have led us to propose that butyrate transport into colonocytes is of fundamental importance for the regulation of processes involved in tissue homoeostasis.
To assess the significance of these findings to butyrate-induced gene regulation in vivo and so examine our proposal that butyrate transport is important to its regulatory function, we sought to investigate the expression of butyrate-responsive genes in colon carcinoma where expression of MCT1 is significantly down-regulated. We first identified genes altered in expression by exposure to butyrate using in vitro colon cancer model systems. HT29 colon carcinoma cells treated with butyrate were compared with untreated controls using microarray analysis to assess simultaneously the expression levels of 19400 human genes. Using the results obtained, we identified butyrate-responsive genes that are involved specifically in the processes of apoptosis, proliferation and differentiation of colonic epithelial cells. To verify the array analysis, we quantitatively assessed the butyrate response of a number of key genes by real-time PCR. The results were consistent in two other colonic epithelial cells, HCT116 and AA/C1. Having identified butyrate-responsive genes in vitro, we report that, in colon cancer tissue, in which expression of MCT1 is significantly down-regulated, the levels of mRNA encoding selected genes involved in the processes of proliferation, differentiation and apoptosis are also deregulated. This supports our proposition that the transport of butyrate across the colonic luminal membrane, via MCT1, is of fundamental importance to the regulation of butyrate-responsive genes and appears directly relevant to the maintenance of tissue homoeostasis in vivo.
Microarray analysis of butyrate response in HT29 colon carcinoma cells
HT29 colon carcinoma cells treated with 5 mM sodium butyrate for 24 h were compared with untreated control cells using microarray analysis to assess simultaneously the expression levels of 19400 human genes. Results indicated that a total of 1983 genes (10.2%) were differentially expressed, being ≥2-fold up- or down-regulated in response to butyrate exposure; 796 genes were found to be up-regulated (4.1%), and 1187 genes were down-regulated (6.1%). Of these, we identified 221 genes (1.1%) as being specifically associated with the processes of apoptosis, proliferation and differentiation. Of these genes, 59 were up-regulated (0.3%) and 162 genes were down-regulated (0.8%). Notable genes are listed in Table 1.
Quantitative assessment of butyrate response by real-time PCR
The butyrate response of nine genes associated with the processes of apoptosis, proliferation and differentiation, were quantitatively assessed using real-time PCR. To demonstrate that the responses are not cell-line-specific, the assay was performed on two other colonic cell lines, HCT116, a colon carcinoma-derived cell line, and AA/C1, derived from a non-tumorigenic colonic adenoma . Cells were treated with 5 mM sodium butyrate for up to 72 h. The nine selected genes, and the changes in the corresponding mRNA levels in response to butyrate treatment, are shown in Table 2. The results of quantitative real-time-PCR analysis confirmed the butyrate-responsiveness of these genes and demonstrated that the responses shown were not cell-line specific; butyrate-induced regulation could be demonstrated in all three cell lines tested, albeit with some variations in degree and speed of response. In accordance with butyrate's ability to induce cell cycle arrest, differentiation and apoptosis, CDKN1A (cyclin-dependent kinase inhibitor 1A) (p21WAF1/CIP1) and GADD45A (growth-arrest and DNA-damage-inducible protein 45A), both cell cycle inhibitors, ALPI (intestinal alkaline phosphatase), a marker of differentiation, and BAK1, a pro-apoptotic gene, were all up-regulated in response to butyrate treatment. BIRC5 (survivin), CFLAR (cFLIPL) and Bcl-XL, all anti-apoptotic genes, CCND1 (cell cycle regulatory protein cyclin D1), a cell cycle progression gene, and CCT5, also involved in cell cycle progression, were down-regulated in response to butyrate.
Importance of butyrate transport to maintenance of tissue homoeostasis
We have shown previously that the expression of MCT1 is significantly down-regulated during the transition from normality to malignancy in the human colon  and have proposed that this decline in transporter expression results in a reduction in the intracellular concentration of butyrate and therefore deregulation in butyrate-induced gene expression. We have previously tested this proposal directly, by inhibiting the expression of MCT1 in vitro, and found a profound inhibitory effect on butyrate's ability to regulate the expression of a number of key target genes, including CDKN1A, ALPI and CCND1 . Conversely, inhibition of MCT1 expression did not affect butyrate-induced regulation of the apoptotic genes BAK1 and Bcl-XL, suggesting that the regulation of these genes is independent of MCT1 function.
To assess the significance of these findings in vivo, and so examine our hypothesis that a reduction in intracellular butyrate may lead to deregulation of butyrate-responsive genes, we have assessed the expression of the nine previously selected genes in colon cancer tissues, in which MCT1 expression was demonstrated to be significantly down-regulated. In agreement with our in vitro data, we have found that five of these key genes are deregulated in cancer tissue. ALPI and CDKN1A, both up-regulated by butyrate in vitro, are down-regulated (3–6-fold) in colon carcinoma compared with normal tissue. BIRC5, CCND1 and CCT5, all down-regulated by butyrate in vitro, are significantly up-regulated (<5-fold) in colon carcinoma (Table 2). These results highlight the important contribution of butyrate transport to the regulation of key colonic genes and their associated processes of cellular apoptosis, proliferation and differentiation.
Also in agreement with our in vitro data, the apoptotic genes BAK1 and Bcl-XL remained unchanged in colon carcinoma compared with normal tissue, supporting the proposal that regulation of these genes is independent of MCT1 expression.
In conclusion, we have identified a large number of butyrate-responsive genes that are involved in the processes of apoptosis, proliferation and differentiation, and demonstrated that many of these genes are deregulated in colon carcinoma tissue, correlating with decreased expression of MCT1. Accordingly, these findings support our hypothesis that a reduction in the transport of butyrate across the luminal membrane of colonic epithelial cells may contribute to the genetic changes that characterize the adenoma–carcinoma sequence, and highlight the fundamental importance of butyrate transport to the maintenance of tissue homoeostasis and the prevention of colorectal cancer.
Financial support of Tenovus, The Cancer Charity, the Biotechnology and Biological Sciences Research Council, and the Wellcome Trust is gratefully acknowledged. We thank Professor C. Paraskeva, University of Bristol, for providing the AA/C1 cells. For work using human colonic tissue samples, permission of the Liverpool (Adult) Research Ethics Committee (Ref. M01/074) was obtained.
The Molecular Biology of Colorectal Cancer: Focused Meeting held at the UBHT Education Centre, Bristol, U.K., 10–11 March 2005. Organized and edited by T. Corfield (Bristol, U.K.), C. Paraskeva (Bristol, U.K.) and H. Wallace (Aberdeen, U.K.).
Abbreviations: ALPI, intestinal alkaline phosphatase; CCND1, cell cycle regulatory protein cyclin D1; CDKN1A, cyclin-dependent kinase inhibitor 1A; GADD45A, growth-arrest and DNA-damage-inducible protein 45A; MCT, monocarboxylate transporter
- © 2005 The Biochemical Society