| Objective: The study was to investigat the inhibiting effectiveness of Salinomycin,17-AAG and the combination of them against human gastric cancer SGC-7901 cells in vitro, and examine whether the two drugs have synergistic inhibitory effect on the gastric cancer and to explore the underlying synergistic mechaniam.Methods : In this study, we used MTT to investigat the inhibition ratio of Salinomycin(in single and combined with 17-AAG) on the proliferation of human gastric cancer SGC-7901 cell line. The morphological changes of cells were observed by inverted phase-contrast microscope. Morphological changes of cells apoptosis observed by PI staining,AO staining and under the Fluorescence microscope. Cell cycle and Cells apoptosis assay ware detected by flow cytometry analysis.The expression of Caspase-3 was evaluated by Western-blotting. The expression of NF-κBp65 and FAS-L were evaluated by immunocytochemistry. All statistical analysis was performed using SPSS software(version, 22.0). The results are presented as the mean values ± standard deviation. Comparisons among groups were achieved using one-way analysis of variance.Multiple Comparisons between multiple average numbers using LDS-t test. P-values < 0.05 were statistically significant.Results :1. The results measured by MTT showed that gastric cancer SGC-7901 cells treated with a certain concentration range(salinomycin: 1, 2, 4, 8, 16, 32μmol/L)for a long time(24,48,72h) inhibited the growth of SGC-7901 cells effectively(P<0.05 or P<0.01). The results indicated that salinomycin could inhibited the growth of human gastric cancer SGC-7901 cells in a time- and dose-dependent manner. The combination of salinomycin(4,8,16μmol/L) and 17-AAG(0.625μmol/L) could inhibited the growth of gastric cancer SGC-7901 cells effectively(P<0.05 or P<0.01). It showed that the combination of salinomycin and 17-AAG have a synergistic inhibitory effect on the proliferation of gastric cancer SGC-7901 cell(P < 0.05). Gastric cancer SGC-7901 cells were treated with the combination of salinomycin and 17-AAG could inhibited the growth of them higher than that were treated with them alone(P < 0.05 or P < 0.01).The results indicated that the combination of salinomycin and 17-AAG could inhibited the growth of human gastric cancer SGC-7901 cells in a time- dependent manner.2. Microscopic and Fluorescence microscope observation revealed morphological changes and typical apoptotic morphological changes.3. Flow cytometric analysis of cell cycle showed that Salinomycin(4,8,16μmol/L) blocked SGC-7901 cells in S phase,The proportion of S phase in cell cycle were 26.90%,34.57%,41.83%, which were significantly higher than that in control group(P<0.05).The combination of salinomycin(8μmol/L) and 17-AAG(0.625μmol/L) could blocked SGC-7901 cells in S phase too. The proportion of S phase in cell cycle were 34.57%,26.92%,40.10% in SGC-7901 cells treated with 8μmol/L Salinomycin, 0.625μmol/L 17-AAG and the combination of them. The combination group were significantly higher than that in control group(23.52%) and the group of monotreatment(P<0.05).4. Flow cytometric analysis of cell apoptosis showed that apoptosis rates were 10.52%,14.69% and 20.46% in human gastric cancer SGC-7901 cells treated with Salinomycin(4,8,16μmol/L)for 48 h, which were significantly higher than that in control group 3.24%(P<0.05). The apoptosis rate of Salinomycin 8μmol/L,17-AAG 0.625μmol/L and the combination of them were 14.69%,7.96% and 26.87%, which were greater than that in the control groupt(P<0.05). The combination group were significantly higher than that in the group of monotreatment(P<0.05).5. Western-blotting analysis of the expression levels of cell protein showed that Salinomycin and17-AAG could up-regulated the expression levels of protein Caspase-3 of human gastric cancer SGC-7901 cells,obviously,and their combination up-regulated it more significantly than either of the drugs did.6. Immunocytochemistry analysis of the expression levels of cell protein showed that Salinomycin and 17-AAG could down-regulated the expression levels of protein NF-κBp65 and up-regulated the expression levels of protein FAS-L of human gastric cancer SGC-7901 cells,obviously,and the combination of Salinomycin and 17-AAG regulated it more significantly than either of the drugs did.Conclusion:1. Salinomycin could inhibited cell growth of gastric cancer SGC-7901 cells in a time- and dose-dependent manner.2. The combination of Salinomycin and 17-AAG could inhibited cell growth of gastric cancer SGC-7901 cells in a time- and dose-dependent manner.3. Salinomycin,17-AAG and the combination of them could induce cell apoptosis of human gastric cancer SGC-7901 cells,Maybe we could attributed the change to the increase in Caspase-3,FAS-L and the decrease in NF-κBp65 expression. |
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