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F. Rizvi, T. Heimann, W. J. O'Brien; Ceramide Induced Apoptosis in Human Corneal Stromal Cells Mediated by Harakiri (HRK) Gene. Invest. Ophthalmol. Vis. Sci. 2009;50(13):5550.
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These studies were conducted to understand the molecular mechanism of ceramide induced cell death in human corneal stromal fibroblasts (HCSF).
HCSF were treated with 40µM C6 ceramide (minimum concentration that brought the optimal cell death). Cells were harvested at 3,6,12 and 24 hours. Caspase 3 and 9 activities were measured at all time points. DNA laddering was studied 24 hrs post ceramide treatment. TUNEL assays were performed at 12 and 24 hrs. MitoSOX Red was used to evaluate the production of reactive oxygen molecules. Real time PCR arrays were used to screen for the differential expression among 84 genes known to be involved in apoptosis. Significant differences among caspase activities and TUNEL assays were determined by ANOVA.
Low but measurable caspase 3 activity was significantly higher in the cells undergoing death at 6 and 12 hours post-ceramide treatment than in controls (dihydroceramide) but dropped to non-significant levels in 24 hrs. No significant caspase 9 activity was measureable. TUNEL assays demonstrated a greater number apoptotic cells in cultures treated with C6 ceramide for 24 hrs than for 12 hrs. DNA ladders were detected at 24 hrs post-ceramide treatment. Real-time PCR array data from ceramide treated cells showed no change or under expression in the genes such as Caspase 2, 3, 4, 5, 6,7,8,9. Only two apoptotic genes; tumor necrosis factor (TNF) and HRK showed more than 3-fold over expression compared to 26 over expressed apoptotic genes in staurosporin treated HSCFs. Increased superoxide production assessed by MitoSOX Red oxidation was observed in the cells treated by ceramide than in cells treated with dihydroceramide.
These findings suggest that ceramide induced apoptosis by a novel pathway independent of caspases. Ceramides play the role in releasing the proapoptotic molecules from mitochondria. HRK is a pro-apoptotic BH3-only protein belonging to the Bcl-2 family. It exerts its function by altering the physical properties of the membrane by associating with the mitochondrial outer. HRK induced by ceramide may also play an important role in the production of ROS leading to cell death.
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