A novel selenium-containing glutathione transferase zeta1-1, the activity of which surpasses the level of some native glutathione peroxidases.

Glutathione peroxidase (GPX) is a critical antioxidant selenoenzyme in organisms that protects cells against oxidative damage by catalyzing the reduction of hydroperoxides by glutathione (GSH). Thus, some GPX mimics have been generated because of their potential therapeutic value. The generation of a semisynthetic selenoenzyme with peroxidase activity, which matches the catalytic efficiencies of naturally evolved GPX, has been a great challenge. Previously, we semisynthesized a GPX mimetic with high catalytic efficiency using a rat theta class glutathione transferase (rGST T2-2) as a scaffold, in which the highly specific GSH-binding site is adjacent to an active site serine residue that can be chemically modified to selenocysteine (Sec). In this study, we have taken advantage of a new scaffold, hGSTZ1-1, in which there are two serine residues in the active site, to achieve both high thiol selectivity and highly catalytic efficiency. The GPX activity of Se-hGSTZ1-1 is about 1.5 times that of rabbit liver GPX, indicating that the selenium content at the active site plays an important role in enhancement of catalytic performance. Kinetic studies revealed that the catalytic mechanism of Se-hGSTZ1-1 belong in a ping-pong mechanism similar to that of the natural GPX.

Apoptotic effects of ginsenoside Rh2 on human malignant melanoma A375-S2 cells.

AIM: To study the mechanism of ginsenoside-Rh2 (G-Rh2)-induced growth inhibition of A375-S2 cells. METHODS: A375-S2 cell viability and the effect of caspase inhibitors on G-Rh2-induced apoptosis were measured by crystal violet assay. Changes in cellular morphology were observed by phase-contrast microscopy. Apoptosis-specific nucleosomal DNA fragmentation was assayed by agarose gel electrophoresis. Cell cycle distribution was measured by flow cytometry. RESULTS: G-Rh2 inhibited the A375-S2 cell growth in concentration- and time-dependent manners. Caspase family inhibitor, z-Val-Ala-Asp-fluoromethylketone (z-VAD-fmk), caspase-3 inhibitor, z-Asp-Glu-Val-Asp-fluoromethylketone (z-DEVD-fmk), and caspase-8 inhibitor, z-Ile-Glu-Asp-fluoromethylketone (z-IETD-fmk), partially inhibited G-Rh2-induced apoptosis. But caspase-1 inhibitor, Ac-Tyr-Val-Ala-Asp-chloromethyl-ketone (Ac-YVAD-cmk), did not antagonize G-Rh2 induced-cell death. CONCLUSION: G-Rh2 suppresses the growth of A375-S2 cells in vitro by inducing apoptosis. G-Rh2-induced apoptosis is partially dependent on caspase-8 and caspase-3 pathway in A375-S2 cells. Other apoptotic pathways might be also related to the induction of apoptosis by G-Rh2.

[Studies on evodiamine induced HeLa cell apoptosis].

AIM: To study the mechanism of evodiamine-induced growth inhibition of HeLa cells. METHODS: HeLa cells viability and the effect of caspase inhibitors on evodiamine-induced apoptosis were measured by crystal violet assay. Changes in cellular morphology were observed by phase-contrast microscopy. Apoptosis-specific nucleosomal DNA fragmentation was assayed by agarose gel electrophoresis. RESULTS: Evodiamine was found to inhibit HeLa cell growth in dose- and time-dependent manners. Caspase-3 inhibitor, z-Asp-Glu-Val-Asp-fmk (z-DEVD-fmk) was shown to partially inhibit evodiamine-induced apoptosis. However, caspase-1 inhibitor, Ac-Tyr-Val-Ala-Asp-chloromethyl-ketone (Ac-YVAD-cmk), did not antagonize evodiamine induced cell death. CONCLUSION: Evodiamine suppresses the growth of HeLa cells in vitro by apoptosis. Evodiamine-induced apoptosis is partially dependent on caspase-3 pathway in HeLa cells. Other apoptotic pathways might be also related to the induction of apoptosis by evodiamine.