Roles of Fas signaling pathway in vitamin E succinate-induced apoptosis in human gastric cancer SGC-7901 cells

Abstract

Aim: To investigate the roles of Fas signaling pathway in vitamin E succinate-induced apoptosis in human gastric cancer SGC-7901 cells.

Methods: Human gastric cancer SGC-7901 cells were treated with VES at 5, 10, 20 mg x L(-1), succinic acid and vitamin E as vehicle control and condition media only as untreated (UT) control. Apoptotic morphology was observed by DAPI staining. Western blot analysis was applied to measure the expression of Fas, FADD and caspase-8 proteins. After the cells were transiently transfected with Fas and FADD antisense oligonucleotides, respectively, caspase-8 activity was determined by flurometric method.

Results: The morphologically apoptotic changes were observed after VES treatment by DAPI staining. 23.7 % and 89.6 % apoptosis occurred after 24 h and 48 h of 20 mg x L(-1) VES treatment, respectively. The protein levels of Fas, FADD and caspase-8 were evidently increased in a dose-dependent manner after 24 h of VES treatment. The blockage of Fas by transfection with Fas antisense oligonucleotides obviously inhibited the expression of FADD protein. After SGC-7901 cells were transfected with Fas and FADD antisense oligonucleotides, caspase-8 activity was obviously decreased (P<0.01), whereas Fas blocked more than FADD.

Conclusion: VES-induced apoptosis in human gastric cancer SGC-7901 cells involves Fas signaling pathway including the interaction of Fas, FADD and caspase-8.

Figure 1

SGC-7901 cells stained with DAPI. A: UT control; B: VES at 20 mg·L^-1

Figure 2

The expression of Fas protein in SGC-7901 cells follow-ing treatment of VES for 24 h. Lane1: Molecular weight marker; Lane2: UT control; Lane3: succinate; Lane4: vitamin E; Lane5: VES at 5 mg·L^-1; Lane6: VES at 10 mg·L^-1; Lane7: VES at 20 mg·L^-1.

Figure 3

The expression of FADD protein in SGC-7901 cells fol-lowing treatment of VES for 24 h. Lane1: Molecular weight marker; Lane2: VES at 20 mg·L^-1; Lane3: VES at 10 mg·L^-1; Lane4: VES at 5 mg·L^-1; Lane5: vitamin E; Lane6: succinate; Lane7: UT control.

Figure 4

The expression of caspase-8 protein in SGC-7901 cells fol-lowing treatment of VES for 24 h. Lane1: Molecular weight marker; Lane2: UT control; Lane3: succinate; Lane4: vitamin E; Lane5: VES at 5 mg·L^-1; Lane6: VES at 10 mg·L^-1; Lane7: VES at 20 mg·L^-1.

Figure 5

The expression of FADD protein when Fas antisense was transfected into SGC-7901 cells following treatment of VES for 24 h.