Ceramide induces release of mitochondrial proapoptotic proteins in caspase-dependent and -independent manner in HT-29 cells

In this study, the release of mitochondrial proapoptotic intermembrane space proteins induced by exogenous C-2-ceramide in human colon carcinoma (HT-29) cell line was investigated. HT-29 cells were treated with 12.5, 25 and 50 mu mol/L C-2-ceramide in vitro. Flow cytometer was used to detect the mitochondrial membrane potential (Delta Psi(m)). Subcellular fractions were extracted by Mitochondrial/Cytosol Fractionation Kit after C-2-ceramide treatment for 24 h. SDS-PAGE was used to determine the level of cytochrome c (Cyt c), high temperature requirement A2 (HtrA2) and second mitochondrial-derived activator of caspases (Smac) released from mitochondria, the expression of X-linked inhibitor of apoptosis protein (XIAP) and caspase-3 for 24 h. The results showed that Delta Psi(m) began to decrease from 6 h after 25 and 50 mu mol/L C-2-ceramide treatment (P < 0.05) and cyclosporin A (CsA) could inhibit the collapse of Delta Psi(m) through regulating mitochondrial membrane permeability transition pore. There was no effect of C-2-ceramide on the expression of Cyt c, HtrA2 and Smac in the total levels. 12.5, 25 and 50 mu mol/L C-2-ceramide could induce Cyt c, HtrA2 and Smac to release from mitochondria to cytosol and down-regulate the expression of XIAP (P < 0.05). Also there was expression of cleaved caspase-3 with C-2-ceramide treatment. After the treatment with caspase inhibitor, C-2-ceramide still induced the release of Cyt c and HtrA2, but Smac did not. Therefore, C-2-ceramide could induce apoptosis of HT-29 cells through the mitochondria pathway. The release of Cyt c, HtrA2 and Smac from mitochondria did not occur via the same mechanism, the release of Cyt c and HtrA2 was caspase-independent and the release of Smac was caspase-dependent.