Possible mechanisms involved in apoptosis of colon tumor cell lines induced by deoxycholic acid, short-chain fatty acids, and their mixtures.

Apoptosis of tumor cells is an important growth-regulating event in tumor masses. In this study we have confirmed that deoxycholic acid (DCA) and the short-chain fatty acids (SCFA) butyrate and propionate induce a time- and concentration-dependent apoptosis in two human colon tumor cell lines: HT-29 and CaCO2. DCA is more potent, inducing effects at low concentration (50 microM) and after 24 hours of incubation, whereas SCFA (4 mM) requires 72-96 hours of treatment. Combining low concentrations of DCA (12.5-25 microM) with butyrate and propionate (4 mM) produces an additive effect on the percentage of apoptotic cells, as demonstrated by flow cytometry and DNA fragmentation. Protein kinase C, protein tyrosine kinase, and gene transcription/translation inhibitors do not significantly modify the rate of apoptosis, whereas the intracellular Ca2+ chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA-AM) completely abolishes the DCA-induced effect without affecting the SCFA-induced apoptosis. Measurement of intracellular Ca2+ by inverted fluorescence microscopy reveals that DCA induces a rapid increase of cytosolic Ca2+ that is abolished when the cells are preincubated with BAPTA-AM, whereas ethyleneglycolbis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid has a minimal effect. In contrast, SCFA does not modify the intracellular Ca2+ concentration. Thus the DCA-induced apoptosis is a Ca(2+)-dependent process, whereas the intracellular signals responsible for the SCFA-induced effect remain unknown. The ionophore activity of DCA could be responsible for the increased intracellular Ca2+, but other mechanisms, such as activation of phospholipase C and phosphoinositide hydrolysis, have to be considered.