Effect of Colonic Luminal Components on Induction of Apoptosis in Human Colonic Cell Lines

Abstract

Apoptosis is central to cell number regulation in the colonic epithelium, and interest in its role in colon carcinogenesis has been growing rapidly. It thus becomes of interest to characterize luminal components, possibly of dietary origin, that may influence this process. We have investigated the sensitivity of two human colonic cell lines, the human adenocarcinoma cell line (HT-29) and the human fetal colonic mucosa cell line (FHC), to induction of apoptosis by sodium butyrate, bile acids, and human fecal water fractions. The apoptotic effect has been studied by 1) morphological changes in cells examined by fluorescence microscopy, 2) DNA fragmentation analysis by gel electrophoresis, 3) flow cytometry analysis of DNA strand breaks assessed by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay (TUNEL), and 4) poly(ADP-ribose) polymerase cleavage by Western blot. Sodium butyrate and bile acids induced a time- and concentration-dependent apoptosis in both cell lines. Quantitation of this effect, by use of the TUNEL assay, indicated that deoxycholic acid was most effective in inducing this effect at lower concentrations and at shorter times. Apoptotic effects were also observed, in both cell lines, when the cells were exposed to intact human fecal waters (the fecal fraction in direct contact with the epithelium) and their lipid extracts, with the intact samples being more effective. Although all fecal waters examined induced apoptosis, quantitation of the effect by the TUNEL assay indicated that the ability to induce apoptosis differed markedly between samples. Induction of apoptosis by the fecal waters was not correlated to cytotoxicity but was negatively correlated to the pH of the samples. Interestingly, the cells derived from the fetal mucosa (FHC) were consistently less sensitive to apoptotic effects of the luminal components than the tumor-derived cells (HT-29). Thus human fecal water fractions induce apoptosis in colonic cells, and this effect is not due to lipid components alone.