Potential mechanisms involved in ceramide-induced apoptosis in human colon cancer HT29 cells
Jing Wang1, Xiao-Wen Lv, Yu-Guo Du
1State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China. avaecn@gmail.com
Objective:
To investigate the potential mechanisms of cell death after the treatment with ceramide.
Methods:
MTT assay, DNA ladder, reporter assay, FACS and Western blot assay were employed to investigate the potential mechanisms of cell death after the treatment with C2-ceramide.
Results:
A short-time treatment with C2-ceramide induced cell death, which was associated with p38 MAP kinase activation, but had no links with typical caspase activation or PARP degradation. Rather than caspase inhibitor, Inhibitor of p38 MAP kinase blocked cell death induced by a short-time treatment with ceramide (<12 h). However, inhibition of p38 MAP kinase could not block cell death induced by a prolonged treatment with ceramide (>12 h). Moreover, incubation of cells with ceramide for a long time (>12 h) increased subG1, but reduced S phase accompanied by caspase-dependent and caspase-independent changes including NFkappaB activation.
Conclusion:
Ceramide-induced cell apoptosis involves both caspase-dependent and -independent signaling pathway. Caspase-independent cell death occurring in a relatively early stage, which is mediated via p38 MAP kinase, can progress into a stage involving both caspase-dependent and -independent mechanisms accompanied by cell signaling of MAPKs and NFkappaB.
Insights
Ceramide treatment triggers cell death through p38 MAP kinase activation in early stages. Prolonged exposure involves both caspase-dependent and -independent pathways, including NFkappaB signaling.
Area of Science:
- Cell biology
- Biochemistry
Background:
- Ceramides are bioactive lipids involved in various cellular processes.
- Understanding ceramide-induced cell death mechanisms is crucial for therapeutic development.
Purpose of the Study:
- To elucidate the molecular mechanisms underlying ceramide-induced cell death.
- To differentiate the roles of various signaling pathways in early and late stages of ceramide treatment.
Main Methods:
- Cell viability assessed using MTT assay.
- DNA fragmentation analyzed by DNA ladder assay.
- Apoptosis and cell cycle analysis performed via Flow Cytometry (FACS).
- Protein expression and activation studied using Western blot and reporter assays.
Main Results:
- Short-term ceramide treatment (<12h) induced cell death via p38 MAP kinase (MAPK) activation, independent of caspases.
- Inhibition of p38 MAPK blocked early ceramide-induced cell death.
- Prolonged ceramide treatment (>12h) resulted in cell death involving both caspase-dependent and -independent pathways, with increased subG1 population and NFkappaB activation.
Conclusions:
- Ceramide-induced cell death involves complex signaling crosstalk.
- Early-stage cell death is mediated by p38 MAPK (caspase-independent).
- Later stages involve both caspase-dependent and -independent pathways, with MAPK and NFkappaB signaling playing key roles.
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