Abstract: OBJECTIVE:To investigate the effect of acid sphingomyelinase (ASMase) and ceramide (Cer) on the death receptor pathway and on oxidative stress in vitamin E succinate (VES)-induced apoptosis in gastric cancer cells. METHODS:Human gastric cancer cells in cultures were treated with medium,desipramine (DES,12.5 μmol/L),VES (20 μg/mL) or VES+DES (cells were pretreated with DES at 12.5 μmol/L for 2 h,then with VES at 20 μg/mL). ASMase activity was assessed using the ELISA assay and cellular expression of Cer was detected by immunostaining at different time points. Apoptosis was evaluated using DAPI staining at 24 h. Western blotting was used to detect the expression of proteins in the death receptor pathway,including Fas,death receptor 5 (DR5),caspase-8,caspase-9 and PARP. 2',7'-dichlorofluorescein-diacetate was used to quantitate the levels of reactive oxygen species (ROS). RESULTS:VES increased the activity of ASMase and accumulation of Cer in cellular membrane at 1.5 h and reaching the peak at 3 h. The ratio of apoptosis was (41.00±1.00)% at 24 h. The inhibition of ASMase activities was significantly associated with blockage of VES-induced apoptosis (20.00±2.00)%,via perturbing the levels of Fas,DR5,caspase-8/9 and PARP cleavage,and oxidative stress (all P < 0.01). CONCLUSION:In the VES-induced apoptosis in the SGC-7901 human gastric cancer cells,our results demonstrate that ASMase and Cer were upstream events which were followed via the death receptor pathway and expression of oxidative stress.

Key words: vitamin E succinate, acid sphingomyelinase, ceramide, death receptor, oxidative stress