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

摘要

In this study,the release of mitochondrial proapoptotic intermembrane space proteins induced by exogenous C2-ceramide in human colon carcinoma (HT-29) cell line was investigated.HT-29 cells were treated with 12.5,25 and 50 umol/L C2-ceramide in vitro.Flow cytometer was used to detect the mitochondrial membrane potential (DELTA PAI_m).Subcellular fractions were extracted by Mitochondrial/Cytosol Fractionation Kit after C2-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 apop-tosis 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 umol/L C2-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 C2-ceramide on the expression of Cyt c,HtrA2 and Smac in the total levels.12.5,25 and 50 umol/L C2-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 C2-ceramide treatment.After the treatment with caspase inhibitor,C2-ceramide still induced the release of Cyt c and HtrA2,but Smac did not.Therefore,C2-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.