Abstract
Bile acids are steroid detergents that are toxic to mammalian cells at high concentrations; increased exposure to these steroids is pertinent in the pathogenesis of cholestatic disease and colon cancer. Understanding the mechanisms of bile acid toxicity and apoptosis, which could include nonspecific detergent effects and/or specific receptor activation, has potential therapeutic significance. In this report we investigate the ability of synthetic enantiomers of lithocholic acid (ent-LCA), chenodeoxycholic acid (ent-CDCA), and deoxycholic acid (ent-DCA) to induce toxicity and apoptosis in HT-29 and HCT-116 cells. Natural bile acids were found to induce more apoptotic nuclear morphology, cause increased cellular detachment, and lead to greater capase-3 and -9 cleavage compared with enantiomeric bile acids in both cell lines. In contrast, natural and enantiomeric bile acids showed similar effects on cellular proliferation. These data show that bile acid-induced apoptosis in HT-29 and HCT-116 cells is enantiospecific, hence correlated with the absolute configuration of the bile steroid rather than its detergent properties. The mechanism of LCA- and ent-LCA-induced apoptosis was also investigated in HT-29 and HCT-116 cells. These bile acids differentially activate initiator caspases-2 and -8 and induce cleavage of full-length Bid. LCA and ent-LCA mediated apoptosis was inhibited by both pan-caspase and selective caspase-8 inhibitors, whereas a selective caspase-2 inhibitor provided no protection. LCA also induced increased CD95 localization to the plasma membrane and generated increased reactive oxygen species compared with ent-LCA. This suggests that LCA/ent-LCA induce apoptosis enantioselectively through CD95 activation, likely because of increased reactive oxygen species generation, with resulting procaspase-8 cleavage.
Highlights
High fat western diets induce extensive recirculation of the bile acid pool, resulting in increased exposure of the colonic epithelial cells to these toxic steroids [12, 13]
In this study we explore the enantioselectivity of LCA, CDCA, and DCA-mediated toxicity and apoptosis in two human colon adenocarcinoma cell lines, HT-29 and HCT-116
Bile acids are unique among naturally occurring apoptotic agents because they have the potential to induce apoptosis through both nonspecific detergent effects and through receptor-mediated interactions
Summary
Chemicals and Reagents—LCA was obtained from Sigma, CDCA was obtained from Acros Organics (Morris Planes, NJ), and DCA was obtained from Steraloids (Newport, RI). entLCA, ent-CDCA, and ent-DCA were synthesized as previously described [34, 35]. For staining of attached cells, the medium was removed after 1 h, and the cells were washed with PBS. The cells were allowed to attach overnight, resulting in nearly 30% confluency, after which they were treated with 200 l of medium containing bile acids or vehicle (EtOH). After the desired treatment time, the medium was removed, and the cells in each well were washed with 200 l of PBS. Cell proliferation was normalized to vehicle (EtOH)-treated control cells, and all of the means were calculated from a sample size of n Ն 3. Protein Detection by Western Blotting—HT-29 and HCT-116 cells were plated in 6-well plates and treated with bile acid or vehicle (EtOH) at about 80% confluency. Ent-Bile Acid Apoptosis removed after 30 min, and the cells were washed with PBS and fixed for 30 min with 4% formalin/. 1:100 rabbit anti-CD95 antibody (sc-714; Santa Cruz Biotechnology) in PBS overnight at 4 °C, washed with 3ϫ PBS and incubated at room temperature with 1:2000 AlexaFluor-488 conjugated goat anti-rabbit antibody (A11008; Invitrogen) in PBS for
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