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 micromol/L C(2)-ceramide in vitro. Flow cytometer was used to detect the mitochondrial membrane potential (DeltaPhi(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 DeltaPhi(m) began to decrease from 6 h after 25 and 50 micromol/L C(2)-ceramide treatment (P<0.05) and cyclosporin A (CsA) could inhibit the collapse of DeltaPhi(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 micromol/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.
Zhang
XiaoFeng
X
Public Health College, Harbin Medical University, Harbin 150081, China.
Li
BaiXiang
B
Zhang
Yang
Y
Liu
JiaRen
J
D051017:Apoptosis Regulatory Proteins / Q000378:metabolism*; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism*; D002518:Ceramides / Q000494:pharmacology*; D019073:HT29 Cells; D006801:Humans; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D051336:Mitochondrial Membranes / Q000187:drug effects / Q000378:metabolism; D024101:Mitochondrial Proteins / Q000378:metabolism*; D011480:Protease Inhibitors / Q000494:pharmacology
D051017:Apoptosis Regulatory Proteins; D061945:Caspase Inhibitors; D002518:Ceramides; D024101:Mitochondrial Proteins; D011480:Protease Inhibitors; D020169:Caspases