| Objective:To study the inhibition effect of dioscin to human gastric carcinoma SGC-7901 cells and then to investigate the mechanism.Methods:In this study, different concentrations of dioscin (0,0.65,1.3 and 2.6μg/ml, final concentration) under different treatment times (12,24,48 and 72 h) were tested to inhibit the growth of human gastric cancer SGC-7901 cells by MTT assay, and 10-hydroxycamptothecin (10-HCPT) was used as the positive control. Acridine orange/Ethidium bromide (AO/EB) staining was used to evaluate cell apoptosis. The rates of morning apoptosis and advanced stage apoptosis as well as necrosis were evaluated by flow cytometry asssy (FCA). The concentrations of TNF-αin the supernatants were determined by radio-immunity assay (RIA), and the activities of Caspase-3 and -8 were detected. The expressions of the proteins and/or mRNA of Fas, FasL, TNF-α, TNF Receptor 1 (TNFR1), TNF receptor- associated factor 1 (TRAF-1) and Fas-associated protein with death domain (FADD), Bcl-2, Bax, cytochrome c, Bcl-x1, BAK, Bid, procaspase-3, p53 and vimentin by western blotting and reverse transcription polymerase chain reaction (RT-PCR).Result:The growth of human gastric SGC-7901 cells was strikingly inhibited by dioscin in a dose-dependent manner. Compared with the positive drug, the inhibition effect produced by dioscin at low concentration was stronger than the action produced by 10-HCPT at high concentration for 48 h treatment. The adherence cells were markedly decreased and large amount of cells with stained jacinth fluorescent were observed based on AO/EB double fluorescent staining. The rates of morning apoptosis, advanced stage apoptosis and necrosis of the cancer cells caused by dioscin were increased in a dose-dependent manner based on FCA. Meanwhile, the concentration of TNF-αin the supernatant was also markedly increased. Deeply mechanism study indicated that 2.6μg/ml of dioscin can up-regulate the protein and mRNA expressions of Fas/FasL with 2.78/22.5 times,3.75/2.11 times, respectively. The mRNA expressions of TNF-α/TNFR1 were up-regulated with 1.13/2.56 times, and the protein expression of TRAF-1 were up-regulated with 1.99 and 1.89 times by dioscin at the concentrations of 1.3 and 2.6μg/ml. Compared with the un-treated cells, the protein and mRNA expressions of FADD were up-regulated. The above results showed that the death receptor pathway was activated. Furthermore, compared with the control group,2.6μg/ml of dioscin down-regulated the protein and mRNA expressions of bcl-2, Bcl-xl and up-regulated the protein and mRNA expressions of Bax, BAK with 2.4,1.4,14.1, 1.36 and 1.28 times, respectively. The protein and mRNA expression of Bid were down-regulated with 9 and 2 times. Cytochrome c was released from mitochondrion to cytosol, which manifested that the mitochondrial pathway was activated by dioscin. The protein expression of procaspase-3 was down-regulated and the activations of Caspase-3 and -8 were also observed. In addition, we also found that the mRNA expression of p53 was up-regulated with p<0.01 and the protein expression of vimentin was also markedly down-regulated with p<0.001 by dioscin compared with the un-treated cells.Conclusion:The present study is the first time to discover the significant anti-tumor activity of dioscin against human gastric cancer SGC-7901 cells, and the action was related to activate death receptor and mitochondrial pathways. These findings provide useful experimental data for further investigation of dioscin. |
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