Hydrophobic bile acids induce apoptosis in both colon cancer cells and hepatocytes. The mechanism by which colon cancer cells respond to bile acids is thought to be different from that of hepatocytes. Therefore, we investigated the characteristics of apoptosis in colon cancer cell line HCT116. Hydrophobic bile acids, i.e., deoxycholic acid (DCA), and chenodeoxycholic acid, induced apoptosis in HCT116 cells. Apoptotic indications were detectable at as early as 30 min and the extent increased in time- and concentration-dependent manners. SDS and a hydrophilic bile acid, cholic acid, did not induce apoptosis even at cytotoxic concentrations. Pretreatment with cycloheximide failed to inhibit apoptosis, suggesting that protein synthesis is not involved in the apoptotic response. Release of cytochrome c from mitochondria and activation of caspase-9 were detectable after 5 and 10 min, respectively, whereas remarkable activation of Bid was not detected. Ursodeoxycholic acid (UDCA) protected HCT116 cells from DCA-induced apoptosis but a preincubation period of > or =5 h was required. Nevertheless, UDCA did not inhibit cytochrome c release from mitochondria. Our results indicate that hydrophobic bile acids induce apoptosis in HCT116 cells by releasing cytochrome c from mitochondria via an undefined but specific mechanism, and that UDCA protects HCT116 cells by acting downstream of cytochrome c release.

A secondary bile acid, namely, deoxycholic acid (DCA), has been known to promote colon tumors; on the other hand, it also induces apoptosis in several human colon cancer cell lines. A hydrophobic primary bile acid, namely, chenodeoxycholic acid (CDCA), exhibits a similar property of apoptosis induction; DCA and CDCA also trigger some specific intracellular signal pathways in the human colon cancer cell line HCT116. In this article, we report that hydrophobic bile acids induce different cellular responses depending on their concentration, that is, a sublethal concentration of hydrophobic bile acids can suppress the apoptosis induced by a higher concentration of DCA. Pretreatment with DCA or CDCA at a concentration of < or = 200 microM for 8 h suppressed the apoptosis induced by 500 microM DCA in HCT116 cells. Under this condition, the association of caspase-9 and Apaf-1 and subsequent activation of caspase-9 were inhibited, but the release of cytochrome c from the mitochondria was not. At 200 microM, DCA and CDCA induced the phosphorylation of Akt and ERK1/2, although these phosphorylations do not appear to be indispensable for the cytoprotection. It is interpreted that prolonged exposure to sublethal concentrations of hydrophobic bile acids induces resistance to apoptosis, leading to promotion of colorectal tumorigenesis.

In the human colon cancer cells HCT116, deoxycholic acid (DCA) induces apoptosis via the mitochondrial pathway by triggering the release of mitochondrial factors such as cytochrome c. To elucidate if Bax, a proapoptotic member of the Bcl-2 family known to trigger cytochrome c release in response to various types of apoptotic stimuli, is involved in DCA-induced apoptosis in HCT116 cells, we analyzed DCA-induced apoptosis in Bax-knockout (Bax(-/-)) HCT116 cells. Cytochrome c release and caspase-9 activation were detectable after 5 min in both Bax(-/-) and Bax(+/-) HCT116 cells. Caspase-3 and caspase-8 activation was observed after 15 and 30 min, respectively. Bax(-/-) cells were protected from apoptosis by treating them with ursodeoxycholic acid for 12 h prior to DCA treatment. These results are consistent with our previous observations that were obtained by using wild-type HCT116 cells and suggest that Bax is not indispensable for DCA-induced apoptosis in HCT116 cells.

We previously demonstrated that ursodeoxycholic acid (UDC) requires prolonged (≥5 h) preincubation to exhibit effective protection of colon cancer HCT116 cells from deoxycholic acid (DC)-induced apoptosis. Although UDC diminished DC-mediated caspase-9 activation, cytochrome c release from the mitochondria was not inhibited, indicating that UDC acts on the steps of caspase-9 activation. In the present study, therefore, we investigated the effects of UDC on the factors involved in caspase-9 activation. We found that UDC had no significant effect on the expression of antiapoptotic XIAP. Furthermore, UDC did not affect the expression or release of proapoptotic Smac/DIABLO, or the association of XIAP and Smac/DIABLO. In contrast, association of Apaf-1 and caspase-9 stimulated by 500 μM DC was inhibited by UDC pretreatment. Although UDC caused remarkable activation of Akt/PKB, phosphatidylinositol-3-kinase (PI3K) inhibitor did not significantly reduce UDC-mediated cytoprotection. Furthermore, phosphorylation of threonine residues on caspase-9 after UDC pretreatment could not be detected. UDC-mediated cytoprotection was independent of the MAPK pathway, and cyclic AMP (cAMP) analogue did not inhibit DC-induced apoptosis. Our results indicate that UDC protects colon cancer cells from apoptosis induced by hydrophobic bile acids, by inhibiting apoptosome formation independently of the survival signals mediated by the PI3K, MAPK, or cAMP pathways.

Hydrophobic bile acids but not hydrophilic bile acids induce apoptosis in HCT116 cells. We expressed sodium-dependent bile acid transporters in HCT116 cells, and the intracellular concentration of hydrophilic bile acids increased to that of the hydrophobic bile acids. But no sign of apoptosis was observed, which suggests a hydrophobic-bile acid-specific mechanism for the induction of apoptosis in HCT116 cells.