Abstract
BackgroundWe have determined that butyrate, which is derived from the fermentation of dietary fiber in the colonic lumen, hyperactivates Wnt activity in colorectal (CRC) cells, and that this upregulation of Wnt signaling is causatively related to the induction of apoptosis. To better understand the genetic program regulated by butyrate-mediated Wnt hyperactivation, we performed total human genome microarray analyses on HCT-116 CRC cells in the presence or absence of a physiologically relevant concentration of butyrate. To evaluate changes in Wnt-specific gene expression, Wnt activity was suppressed with inducible dominant negative Tcf4 (DN-Tcf4). Six biological replicates of a full human genome microarray were performed, and the data deposited into the Gene Expression Omnibus database, according to Minimum Information About A Microarray Experiment standards.ResultsReporter assay and western blot data confirm that DN-Tcf4 is expressed at high levels in stably transfected HCT-116 cells upon cotreatment with doxycycline and butyrate, and that these cells exhibit a marked repression of butyrate-mediated Wnt hyperactivation. Analysis of six biological replicates of microarray analyses indicated that 1008 genes are modulated by butyrate (>two-fold, P < 0.01) in a Wnt signaling-specific manner, while 1587 genes are similarly modulated at P < 0.05. The modulated genes include members of a variety of gene families; including the Biological Process category, such as regulation of development, regulation of metabolism, cytokine and chemokine mediated signaling pathways, and DNA replication; the Cellular Component category such as cytoskeleton and organelle factors, and intermediate filaments; and the Molecular Function category, such as GTPase activator activity.ConclusionsWe have identified, for the first time, in CRC cells, the total array of direct and indirect Wnt-target genes whose expression is modulated by butyrate. Knowledge of the molecular mechanisms determining the response of CRC cells to butyrate in vitro may assist in determining more effective preventive and therapeutic strategies against CRC.
Highlights
We have determined that butyrate, which is derived from the fermentation of dietary fiber in the colonic lumen, hyperactivates Wnt activity in colorectal (CRC) cells, and that this upregulation of Wnt signaling is causatively related to the induction of apoptosis
Characterization of system We have established that physiologically relevant concentrations of butyrate induce apoptosis, and repress clonal growth, in CRC cells dependent upon the hyperactivation of Wnt activity [12,13,14]
To better evaluate differences in gene expression that contribute to the butyrate response, we determined the pattern of gene expression changes due to butyrate treatment, focusing on genes which are direct or indirect Wnt targets
Summary
We have determined that butyrate, which is derived from the fermentation of dietary fiber in the colonic lumen, hyperactivates Wnt activity in colorectal (CRC) cells, and that this upregulation of Wnt signaling is causatively related to the induction of apoptosis. Active Wnt signaling, caused by mutations in the APC and beta-catenin genes [8,9,10] promotes colonic cell proliferation and tumorigenesis; both relatively high and relatively low levels of Wnt transcriptional activity lead to CRC cell apoptosis [12,13,14,15,16]. The ability of butyrate, and other HDACis, to promote CRC apoptosis and repress cell growth, is casually related to the degree of Wnt hyperactivation induced by these agents [12,13,14]
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