| Objective:To determine the glutathione (GSH) content in a human hepatoma cell line (SMMC-7221) treated with xylitol/selenit, and explore the relationship between GSH content and the concentration and treatment time of the drug, providing a part of an investigation of its anti-cancer mechanisms.Methods:Firstly, in order to explore the effect of xylitol/selenite, Hoechst 33258 was used to lable cell nucleus, and then we can vertify the previous reaearch that xylitol/selenite can induce apoptosis, according to the changes of cells and cell nucleus in morphology compared with normal cells under an inverted fluorescence microscope. Secondly, Capillary electrophoresis (CE) was applied to determine the GSH content in SMMC-7221 cells treated with different concentrations (0,0.5 mg/L,1 mg/L) and treatment time (12 h,36 h,60 h) of xylitol/selenite. Finally, the relationship between apoptosis and GSH content could be built, according to compare and analyse the different data in three group cells. Then the anti-cancer mechanisms of xylitol selenite can be speculated.Results:0.5 mg/L xylitol selenite was used to do the apoptosis assay, morphology significantly changed from circular to crescent shape compared with mormal SMMC-7221 cells and apoptotic body was also observed.Single factor experiments and orthogonal experiments are used to optimize the experimental conditions. The selected conditions:the derivatization was 2 h, the running buffer was 10 mM pH 11.4 Na2HPC>4, the concentration of 5-IAF was 0.4 mM, the separation voltage was 18 kV, the cartridge temperature was 25℃ and the injection time was 5 s. A calibration curve was obtained by plotting the GSH/NAC peak-area ratios against the corresponding GSH concentrations from5 to 70 μM. The regression equation obtained was y= 0.06 x+0.02 (R2=0.99).Finally, uncoated CE was applied to determine the GSH content in three group cells (0,0.5 mg/L and 1 mg/L). The levels of GSH markedly decreased after treated with 0.5 and 1mg xylitol/selenite L-1 for 12,36 and 60 h (12 h:from 95.57±19.57 to 29.09±7.74 and 24.27±11.15; 36 h:from 70.73±11.35 to 19.54±6.39 and 9.35±6.69; 60 h:from 72.63±16.94 to 7.43±3.84 and 0. The data were from control to 0.5 mg/L group and 1 mg/L group). (**p< 0.01). lmg xylitol/selenite L-1 caused a slight decrease in the GSH content at every time point compared with 0.5 mg xylitol/selenite L-1, but this was not significant (p> 0.05). The depletion rate of GSH was more related to the concentration of xylitol/selenite than the treatment time (depletion rate was from 69.95± .87% to 100% vs 0.22±0.2% to 100%)Conclusions:1. Xylitol/selenite is a promising anti-cancer drug to induce apoptosis in SMMC-7221 cells.2. The apoptosis and GSH depletion occurred simultaneously, and they were more related to the concentration of xylitol/selenite than the treatment time.3.Xylitol/selenite may regulate the apoptosis through the co-action of mechanisms related to GSH depletion. |
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