| ObjectiveTo investigate the effect of melatonin on proliferation and apoptosis of human gastric carcinoma cell SGC-7901, to explore some probable mechanisms of melatonin-mediated of tumor cell's apoptosis by analyzing the expression change of survivin and livin.Methods①Mode of human gastric carcinoma cell SGC-7901 treated with melatonin was established : human gastric carcinoma cell SGC-7901 was subcultured; The cell in the exponential phase were divided into 5 groups according to the different concentration of melatonin , 1mM group,2mM group,3mM group,4mM group,5mM group. The control group without melatonin intervention was established at the same time. All the index of each group were detected after melatonin intervention of 24 hours.②CCK-8 assay was used to detect the growth inhibition of cell SGC-7901 in each group.③Flow cytometry was used to detect the apoptosis rate of cell SGC-7901 in each group.④The apoptosis of cell SGC-7901 in each group was detected by TUNEL method.⑤Immunocytochemistry was used to detect the expression change of survivin and livin in each group.⑥Western blot was used to detect the expression change of survivin and livin protein in each group. ⑦Real time fluorescence quantitive PCR was used to detect the expression change of survivin and livin mRNA in each group.Results①CCK-8 assay results demonstrated that the cell growth in each group with melatonin intervention was inhibited compared with the control group.②Flow cytometry analysis showed that the apoptosis rate of tumor cell increased with the melatonin concentration increased; Compared with control group, the apotosis rate of 3,4 and 5mM group results were statistically significant.③TUNEL results showed that the apoptosis rate of tumor cell increased after melatonin intervention, which consisted with the flow cytometry results.④The results of immunohistochemistry showed that the average optical density of survivin-positive tumor cells reduced in each melatonin treatment group; Compared with the control group, the result of each group showed significant differences except the 1 mM group. The average optical density of livin-positive tumor cells significantly reduced in each melatonin treatment group except the 1mM group, compared with the control group.⑤Western bolt results showed that survivin protein expression of each melatonin treatment group decreased compared with the control group. Each group showed significant differences compared with the control group except the 1mM group. The livin protein of each melatonin treatment group significantly decreased compared with the control group.⑥Real time fluorescence quantitive PCR analyses demonstrated that survivin mRNA expression of each melatonin treatment group decreased compare with the control group. Each group showed significant differences compared with the control group except the 1mM group, which consisted with the western blots results. The livin mRNA expression of each melatonin treatment also significantly decreased compared with the control group. ConclusionAfter melatonin intervention of 24 hours, growth inhibition and elevated apoptosis of cell SGC-7901 were detected, which showed in a dose-dependent manner. At the same time, the transcription, translation and synthesis level of survivin and livin gene declined in varying degrees, which also showed in a probable dose-dependent manner. This indicted that melatonin could down-regulate the expression of survivin and livin, which might be one of the mechanisms of melatonin anti-cancer effect. |
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