| Sterigmatocystin (ST) is the fungal secondary metabolite produced by many Aspergillus species such as A. versicolor, A. chevalieri, etc. ST is one of the most common contaminant in different foodstuffs and environment, and it was regularly detected in grains, corn, bread, cheese and animal feed. Structurally, ST is related to a?atoxins and is generally recognized as a potential carcinogen, mutagen and teratogen. ST was found to have different toxicological, mutagenic and carcinogenic effects in animals and has been recognized as a 2B carcinogen (possible human carcinogen) by International Agency for Research on Cancer. ST could induce hepatocellular carcinoma after oral administration or intraperitoneal injection and squamous cell carcinoma after repeated application to the skin in rats, and could induce lung adenocarcinoma after oral administration in NIH mice. In short-term tests, ST, although considered less potent, is known to cause DNA damage and form DNA adducts, lead to DNA breaks, chromosome aberration, and sister-chromatid exchange in animal experiments. Our previous studies showed that ST could induce malignant transformation and p53 mutation in human fetal gastric mucosa cells in vitro. Oral administration of ST for long period of time could induce intestinal metaplasia and atypical hyperplasia of glandular epithelium in mice. In China, ST has been one of the most commonly detected mycotoxins in the maize from a high incidence area of gastric carcinoma, such as Taihang Mountains in North China. Many epidemiological studies of tumor etiology showed that diet exposure of ST may have some relationship with high incidence of gastric cancer in these areas. As gastric epithelium would be directly exposured to oral intake of ST, evaluation of the cytotoxic effects of ST on human gastric epithelial cells can be of further usage for the elucidation of the possible carcinogenic mechanism of ST.The imbalance between cell proliferation and death is regularly considered to be an important event in carcinogenesis. At key transitions during eukaryotic cell cycle progression, signaling pathways monitor the successful completion of upstream events prior to proceeding to the next phase and these regulatory pathways are commonly referred to as cell cycle checkpoints. In mammalian cells, proliferation is controlled by factors that regulate the transition at cell cycle checkpoints, which are responsible for the initiation and completion of DNA replication and control of cell division respectively. Cell cycle progression is regulated by the checkpoint controls that function to allow time for the DNA repair and result in activation of pathways leading to apoptosis if cellular damage cannot be properly repaired. Defects in cell cycle checkpoints can result in cell genomic instability, gene mutations, and aneuploidy, all of which can contribute to carcinogenesis. Many studies have shown apoptosis, proliferation inhibition and cell cycle arrest are among the early effects of carcinogenic toxins on animal cells. Xie, et al. found that ST could cause G2/M arrest and overexpression of p53 protein in murine fibroblasts. Our primary study confirmed that ST treatment for 24 hours could induce the G2 arrest by affecting the MAPK signaling pathway in GES-1 cells in vitro.DNA damage checkpoint signal pathway is a highly conserved signal induction process. ATM/ATR is a early sensor of protein kinase to DNA damage signal and the increase of its activity constitutes the first step in the whole activation pathway. p53 is a substrate of ATM, which can either activate cell cycle checkpoints and induce cycle arrest to repair damaged DNA, or activate cells into the apoptosis pathway, according to severity of injury. To further evaluate the effect of ST treatment on cell cycle and apoptosis and to explore their corresponding mechanism, in this study, we used a human gastric epithelial cell line (GES-1) and carried out the following studies. First, we observed the effect of ST treatment on GES-1 cell cycle distribution, proliferation and apoptosis. And then, we investigated the effect of ST treatment on DNA damage of GES-1 cells and the changes in DNA damage downstream signaling pathway. Finally, we chose p53-p21WAF1/CIP1 signaling pathway, which were closely related with DNA damage and cell cycle arrest, and further explored the molecular mechanism of ST-induced G2 arrest. The aim of this study is to lay an experimental foundation for revealing the possible relationship between diet exposure of ST and gastric mucosa damage as well as the possible effects on gastric carcinogenesis.. The following three parts are included in the studyPartâ… The effect of ST on the distribution of cell cycle, proliferation and apoptosis in GES-1 cells in vitroObjective: To further confirm the effect of ST on the regulation of the cell cycle distribution, proliferation and apoptosis of human gastric epithelium cells (GES-1) in vitro.Methods: The effect of ST treatment at different concentrations (0.0348μM) for 24, 48 and 72 h on GES-1 cells survival rates was tested by MTT. The distribution of cell cycle after ST treatment was analyzed by FCM. Western Blot and RT-PCR were used for the determination of expression of cell cycle key factors (Cdc25C, Cdc2 and CyclinB1) at protein and mRNA level respectively. The effect of ST on the Cdc2-CyclinB1 complex was evaluated with immunoprecipitation. The effect of ST on apoptosis was measured by flow cytometry after Annexin V-PI staining and PI staining and detected morphologically by Hoechst 33258 staining. Western Blot was used for the expression of apoptosis related factor Bcl-2, Bax, NF-κB, and Caspase-3.Results:1.1 Effects of ST on survival rates in GES-1 cellsMTT assay showed that ST had a potent inhibitory effect on the growth of GES-1 cells. When GES-1 cells were treated by ST for 24 hours, the cell survival rates in ST treated groups ranging from 3 to 48μM displayed a significant decrease (P<0.05) in a dose-dependent manner (r24h=-0.961, P<0.01) and treated for 48 and 72 hours, the cell survival rates in ST treated groups ranging from 1.5 to 48μM respectively displayed a significant decrease (P<0.05) in a dose-dependent manner (r48h=-0.955, r72h=-0.913, P<0.01). When treated at 24μM and 48μM, the cell survival rates in ST treated groups significantly decreased in a time-dependent manner (r24μM=-0.998, r48μM=-0.998, P<0.05).1.2 Effects of ST on the distribution of cell cycle in GES-1 cells1.2.1 Effects of ST on cell cycle and G2 phase key regulatory factor in GES-1 cells at various time points1.2.1.1 Effects of ST on cell cycle in GES-1 cells at various time points Flow Cytometric results showed that when the GES-1 cells were treated by 3μM ST for 2, 4, 8 and 12d, the proportion of cells in G2/M phase in all ST treatment groups was significantly increased as compared with that in control group (P<0.05). The peak value of the proportion of cells in G2/M phase was reached appeared at the 4th day after exposure to 3μM ST. Subsequently, this G2 delay was sustained till day 8 of ST treatment (P<0.05).1.2.1.2 Effects of ST on G2 phase key regulatory factor in GES-1 cells at various time pointsWestern Blot analysis showed that the expression of the key G2 phase regulatory proteins Cdc25C was significantly decreased after 2d, 4d, 8d and 12d in 3μM ST treatment cells (P<0.05) in a time-dependent manner (r=-0.814, P<0.01). At the same time, the phosphorylation level of Cdc25C was increased in ST treatment groups (P<0.05) time-dependently(r=0.807, P<0.01). RT-PCR analysis showed that the expression of Cdc25C mRNA in ST treatment groups was significantly decreased as compared with that in control group (P<0.05).3μM ST treatment significantly decreased the key G2 phase regulatory proteins Cdc2 expression from 2 to 4d after treatment, When the treatment continued for 8 and 12days, the expression of Cdc2 was gradually recovered to level of the control group In the time range from 2 to 8 days, the phosphorylation levels of Cdc2 were increased at all ST treatment groups in time-dependent manner (r= 0.603,P<0.01). Different from the expression at protein level, RT-PCR analysis showed that the expression of Cdc2 mRNA in ST treatment groups was significantly decreased as compared with that in control group (P<0.05).Western Blot analysis and RT-PCR analysis showed that the expression of Cyclin B1 at protein and mRNA level were all significantly increased in all ST treatment groups as compared with that in control group (P<0.05).1.2.2 Effects of ST treatment with different concentrations on cell cycle and G2 phase key regulatory factor in GES-1 cells1.2.2.1 Effects of ST treatment with different concentrations for 4 days on cell cycle and G2 phase key regulatory factor in GES-1 cells1.2.2.1.1 Effects of ST treatment with different concentrations for 4 days on cell cycle in GES-1 cells Flow Cytometric results showed that when GES-1 cells were treated by ST for 4 days, the proportions of cells in G2/M phase in 0.3μM, 1.5μM, 3μM ST treatment groups were all significantly increased as compared with that in solvent control group in a dose-dependent manner (r=0.927, P<0.01).1.2.2.1.2 Effects of ST treatment with different concentrations for 4 days on G2 phase key regulatory factor in GES-1 cellsWestern Blot analysis results showed that 4 days after ST treatment at 0.3μM, 1.5μM, 3μM, the expressions of Cdc25C and Cdc2 were all significantly decreased as compared with that in solvent control group (P<0.05) and a dose effects could be seen (r=-0.800, r=-0.876, P<0.01). At the same time, the phosphorylation level of Cdc25C and Cdc2 was increased at all ST treatment groups dose-dependently (r=0.714, r=0.767, P<0.01). Similar to the expression at protein level, RT-PCR analysis showed that the expression of Cdc25C and Cdc2 mRNA in ST treatment groups was also significantly decreased as compared with that in control group (P<0.05).Four days after ST treatment, the expressions of Cyclin B1 at protein and mRNA level were all significantly increased in 0.3μM, 1.5μM, 3μM ST treatment groups as compared with that in solvent control group (P<0.05) in a dose-dependent manner (r=0.929, P<0.01).1.2.2.1.3 Effects of ST treatment with different concentrations for 4 days on Cdc2-CyclinB1 complex in GES-1 cells Immunoprecipitation results suggested that when treated by ST for 4 days, ST could induce the decrease of Cdc2-CyclinB1 complex of GES-1 cells in 0.075μM, 0.3μM, 1.5μM and 3μM ST treatment groups.1.2.2.2 Effects of ST treatment with different concentrations for 8 days on cell cycle and G2 phase key regulatory factor in GES-1 cells1.2.2.1.1 Effects of ST treatment with different concentrations for 8 days on cell cycle in GES-1 cellsFlow Cytometry showed that when GES-1 cells were treated by ST for 8 days, the proportion of cells in G2/M phase in 0.3μM to 3μM ST treatment groups for 8 days was significantly increased (P<0.05) as compared with that in solvent control group in a dose-dependent manner (r=0.910, P<0.01).1.2.2.2.2 Effects of ST treatment with different concentrations for 8 days on G2 phase key regulatory factor in GES-1 cellsWestern Blot analysis showed that when GES-1 cells were treated by ST for 8 days, the expression of Cdc25C was significantly decreased in 1.5μM, 3μM ST treatment groups as compared with that in solvent control group (P<0.05) in a dose-dependent manner ranging from 0.3 to 3μM (r=-0.830, P<0.01). Conversely, the phosphorylation level of Cdc25C was increased in 1.5μM, 3μM ST treatment groups in dose-dependent manner (r=0.714, r=0.767, P<0.01). RT-PCR analysis showed that the expression of Cdc25C mRNA in ST treatment groups was significantly decreased as compared with that in control group (P<0.05).Western Blot analysis showed that when GES-1 cells were treated by ST for 8 days, the expression of Cdc2 was significantly decreased in only 3μM ST treatment groups as compared with that in solvent control group (P<0.05). Conversely, the phosphorylation level of Cdc2 was increased at all ST treatment groups in dose-dependent manner (r=0.781,P<0.01). RT-PCR analysis showed that the expression of Cdc2 mRNA in ST treatment groups was significantly decreased as compared with that in control group (P<0.05).Western Blot analysis showed that when GES-1 cells were treated by ST for 8 days, the expression of Cyclin B1 was significantly increased in 0.3μM, 1.5μM, 3μM ST treatment groups as compared with that in solvent control group (P<0.05) in a dose-dependent manner(r=0.753, P<0.01). RT-PCR analysis showed that the expression of Cyclin B1 mRNA in ST treatment groups was significantly increased as compared with that in control group (P<0.05).1.3 Effects of ST on apoptosis in GES-1 cells1.3.1 Effects of ST on apoptosis in GES-1 cells at various time points1.3.1.1 Effects of ST on apoptosis rate in GES-1 cellsThe PI staining result showed that when the GES-1 cells were treated by 3μM ST for 4, 8 and 12d, the apoptosis rate in all ST treatment groups (4.07±0.71%,7.96±1.17% and 3.89±0.28%) was significantly increased compared with that in control group (2.19±0.08%, P<0.05). Exposure to 3μM ST, the peak value of apoptosis rate appeared at the 8th day and subsequently decreased after ST treatment for 12 days(P<0.05). It is worth noting that the peak value of apoptosis rate was later four days than that of G2 arrest.1.3.1.2 Apoptosis was detected in GES-1 cells with ST treatment at various time points by stainingCells treated with 3μM ST were stained by Hoechst 33258 and observed through fluorescence microscope. The typical morphological changes, such as condensation of chromatin and nuclear fragmentations appeared in GES-1 cells treated with ST at different time points. Apoptotic index of 2d, 4d and 8d ST treatment groups was significantly increased as compared with that in control group (r=0.827, P<0.01) and the peak value appeared at the 8th day, subsequently decreased (P<0.05) .1.3.1.3 Effects of ST on apoptosis related proteins in GES-1 cells at various time points1.3.1.3.1 Effects of ST on expression of Bcl-2 in GES-1 cells at various time pointsWestern Blot analysis showed that when the GES-1 cells were treated by 3μM ST for 2, 4, 8 and 12d, the expression of Bcl-2 was significantly decreased in all ST treatment groups(P<0.05) as compared with that in control group in time-dependent manner (r=-0.557, P<0.05).1.3.1.3.2 Effects of ST on expression of Bax in GES-1 cells at various time pointsWestern Blot analysis showed that when the GES-1 cells were treated by 3μM ST for 2, 4, 8 and 12d, the expression of Bax was significantly increased in all ST treatment groups as compared with that in control group(P<0.05). Protein expression peaked at 8th day and decreased at 12th day (P<0.05), which consistented with the change of cell cycle arrest.1.3.1.3.3 Effects of ST on expression of NF-κB in GES-1 cells at various time pointsWestern Blot analysis showed that when the GES-1 cells were treated by 3μM ST for 2, 4, 8 and 12d, the expression of NF-κB was significantly decreased in all ST treatment groups as compared with that in control group(P<0.05) in time-dependent manner(r=-0.825,P<0.01).1.3.1.3.4 Effects of ST on activation of caspase-3 in GES-1 cells at various time pointsWestern Blot analysis showed that ST could activate caspase-3 (P<0.05). When the GES-1 cells were treated by 3μM ST for 2, 4, 8 and 12d, the peak value of the cleaved (activated) form of Caspase-3 could be seen in 8 day ST treated groups and decreased in 12 day ST treated groups (P<0.05), which consistented with the changes of apoptosis rate.1.3.2 Effects of ST treatment with different concentrations on apoptosis in GES-1 cells1.3.2.1 Effects of ST treatment with different concentrations for 4 days on apoptosis in GES-1 cells1.3.2.1.1 Effects of ST treatment with different concentrations for 4 days on apoptosis rate in GES-1 cells The PI staining result showed that after ST treatment for 4 days the apoptosis rate in 0.075μM, 0.3μM, 1.5μM and 3μM ST treatment groups was significantly increased as compared with that in solvent control group(P<0.05) in dose-dependent manner (r=0.854, P<0.01).Annexin V-PI staining showed that the apoptosis percentage of cells including early and late apoptotic cells was significantly increased in different concentrations ST treatment groups as compared with that in solvent control group (P<0.05), which consistented with the result of PI staining.Based on the above two methods shows that ST treatment for 4 days could induce apoptosis in GES-1 cells in dose-dependent manner.1.3.2.1.2 Effects of ST treatment with different concentrations for 4 days on apoptosis related proteins in GES-1 cells1.3.2.1.2.1 Effects of ST treatment with different concentrations for 4 days on Bcl-2 in GES-1 cellsWestern Blot analysis showed that after ST treatment for 4 days, the expression of Bcl-2 was significantly decreased in 0.075μM, 0.3μM, 1.5μM, 3μM ST treatment groups as compared with that in solvent control group (P<0.05) in a dose-dependent manner (r=-0.857, P<0.05).1.3.2.1.2.2 Effects of ST treatment with different concentrations for 4 days on Bax in GES-1 cellsWestern Blot analysis showed that after ST treatment for 4 days, the expression of Bax was significantly increased in 0.075μM3μM ST treatment groups as compared with that in solvent control group (P<0.05) in a dose-dependent manner(r=0.907, P<0.01).1.3.2.1.2.3 Effects of ST treatment with different concentrations for 4 days on NF-κB in GES-1 cellsWestern Blot analysis showed that after ST treatment for 4 days, the expression of NF-κB was significantly decreased in 0.3μM3μM ST treatment groups as compared with that in solvent control group (P<0.05) in a dose-dependent manner (r=-0.828, P<0.01).1.3.2.1.2.4 Effects of ST treatment with different concentrations for 4 days on Caspase-3 in GES-1 cellsWestern Blot analysis showed that after ST treatment for 4 days, the expression of the cleaved fragment of Caspase-3 was significantly decreased in 0.075μM3μM ST treatment groups as compared with that in solvent control group (P<0.05) in a dose-dependent manner (r=0.905, P<0.05).1.3.2.2 Effects of ST treatment with different concentrations for 8 days on apoptosis in GES-1 cells1.3.2.2.1 Effects of ST treatment with different concentrations for 8 days on apoptosis rate in GES-1 cellsThe PI staining result showed that after ST treatment for 8 days the apoptosis rate in 0.3μM, 1.5μM and 3μM ST treatment groups was significantly increased as compared with that in solvent control group(P<0.05) in dose-dependent manner (r=0.933, P<0.01).1.3.2.2.2 Effects of ST treatment with different concentrations for 8 days on apoptosis related proteins in GES-1 cells1.3.2.1.2.1 Effects of ST treatment with different concentrations for 8 days on Bcl-2 in GES-1 cellsWestern Blot analysis showed that after ST treatment for 8 days, the expression of Bcl-2 was significantly decreased in 0.3μM, 1.5μM, 3μM ST treatment groups as compared with that in solvent control group (P<0.05) in a dose-dependent manner (r=-0.912, P<0.05).1.3.2.2.2.2 Effects of ST treatment with different concentrations for 8 days on Bax in GES-1 cellsWestern Blot analysis showed that after ST treatment for 8 days, the expression of Bax was significantly increased in 0.3μM3μM ST treatment groups as compared with that in solvent control group (P<0.05) in a dose-dependent manner(r=0.901, P<0.01).1.3.2.1.2.3 Effects of ST treatment with different concentrations for 8 days on NF-κB in GES-1 cellsWestern Blot analysis showed that after ST treatment for 8 days, the expression of NF-κB was significantly decreased in 0.3μM3μM ST treatment groups as compared with that in solvent control group (P<0.05) in a dose-dependent manner (r=-0.803,P<0.01).1.3.2.1.2.4 Effects of ST treatment with different concentrations for 8 days on Caspase-3 in GES-1 cellsWestern Blot analysis showed that after ST treatment for 8 days, the expression of the cleaved fragment of Caspase-3 was significantly decreased in 0.075μM3μM ST treatment groups as compared with that in solvent control group (P<0.05) in a dose-dependent manner (r=0.850, P<0.01).Partâ…¡The study on the effect of DNA damage and the activation of damage pathway in GES-1 cells induced by ST in vitroObjective: To explore the effect of ST on DNA damage and the activation of damage pathway in human gastric epithelium cells (GES-1). And to reveal that the possible reason for ST-induced G2 phase arrest in GES-1 cells.Methods: The situation of DNA damage was measured by single cell gel electrophoresis assay after different time (2, 4, 8 and 12d) of ST treatment. Western Blot detected the expression of cell cycle key factors (Cdc25C, Cdc2 and CyclinB1). The distribution of cell cycle after ST treatment was detected by FCM.Results:2.1 Effects of ST on DNA damage in GES-1 cellsSingle cell gel electrophoresis assay result showed that 3μM ST treatment could induce DNA damage. Damage Index of different time ST treatment groups were significantly higher than that of corresponding solvent control groups (P<0.001) in time-dependent manner (r=0.903, p<0.001).The analysis of DNA content of the comet tail, tail length and tail moment by the software after ST treatment at different time in GES-1 cells after electrophoresis were significantly increased as compared with the corresponding solvent control groups(P<0.01) in a time-dependent manner.The result suggested that ST could induce GES-1 cells DNA damage, which was possible reason on ST-induced G2 phase arrest.2.2 Effects of ST on G2 arrest and cell cycle related proteins in GES-1 cells, with caffeine pretreatment2.2.1 Effects of ST on cell cycle distribution in GES-1 cells, with caffeine pretreatmentFlow Cytometry showed that the proportion of cells in G2/M phase in caffeine pretreatment group was reduced as compared with that in 3μM ST treatment group (P<0.05). And caffeine could inhibit ST-induced G2 phase arrest in GES-1 cells. The results indicated that ST induced G2 phase arrest in GES-1 cells involving the activation of ATM/ATR and its related signaling pathway.2.2.2 Effects of ST on cell cycle related proteins in GES-1 cells, with caffeine pretreatment2.2.2.1 Effects of ST on Cdc25C in GES-1 cells, with caffeine pretreatmentThe results of Western Blot showed that the expression of Cdc25C in caffeine pretreatment group were significantly increased as compared with that in ST (1.5μM, 3μM) treatment groups (P<0.05) and the phosphorylation level of Cdc25C was opposite (P<0.05). The results suggested that ATM/ATR signaling pathway may be involved in the changes of Cdc25C and p-Cdc25C ST-induced.2.2.2.2 Effects of ST on Cdc2 in GES-1 cells, with caffeine pretreatmentThe results of Western Blot showed that the expression of Cdc2 in caffeine pretreatment group were significantly increased as compared with that in ST (1.5μM, 3μM) treatment groups (P<0.05) and the phosphorylation level of Cdc2 was opposite (P<0.05). The results suggested that ATM/ATR signaling pathway may be involved in the changes of Cdc2 and p-Cdc2 ST-induced.2.2.2.3 Effects of ST on CyclinB1 in GES-1 cells, with caffeine pretreatmentThe results of Western Blot showed that no significant difference was found between caffeine + ST treatment groups and ST treatment groups (P>0.05).2.3 Effects of ST on p-p53 in GES-1 cells, with caffeine pretreatmentThe results of Western Blot showed that the expression of p-p53 in caffeine pretreatment group was significantly decreased and in 1.5μM and 3μM ST treatment groups were significantly increased as compared with that in solvent control groups (P<0.05). The expression of p-p53 in caffeine pretreatment group were significantly decreased as compared with that in ST (1.5μM, 3μM) treatment groups (P<0.05). The results suggested that after ST treatment for 2 days, ST could activate p53; p53 is a downstream protein of ATM/ATR signaling pathway and ATM/ATR signaling pathway may be involved in the changes of p-p53 ST-induced.Partâ…¢The study on the molecular mechanisms of G2 Arrest in GES-1 cells induced by ST in vitroObjective: To explore the possible molecular mechanisms on ST induced G2 phase arrest in GES-1cells in vitro.Methods: The expression of p53-p21WAF1/CIP1 signaling pathway after ST treatment was analysized with Western Blot and real-time PCR. GES-1 cells were transfected with p53 siRNA, Western Blot detected the expression of signaling pathway related molecule and G2/M phase key regulatory factors.The cell cycle distribution of GES-1cells after p53 siRNA transfection was detected with FCM. Results:3.1 Effects of ST on p53-p21WAF1/CIP1 signaling pathway in GES-1 cells3.1.1 Effects of ST on p53-p21WAF1/CIP1 in GES-1 cells at various time points3.1.1.1 Effects of ST on p53 in GES-1 cells at various time pointsWestern Blot analysis showed that when the GES-1 cells were treated by 3μM ST for 2, 4, 8 and 12d, the expression of p53 and p-p53 was significantly decreased in all ST treatment groups (P<0.05). The peak value of p-p53 appeared at 8th day, subsequently decreased at 12th day(P<0.05). The results suggested that ST treatment could activate p53 and the peak value of p-p53 appeared at 8th day.Real time-PCR analysis showed that the expression of p53 mRNA was significantly increased after different time ST treatment(P<0.01), while the peak value appeared at 8th day which was consistent with expression of p53 at protein level.Based on the above, the activation of p53 was basically consistent in time with G2 arrest and apoptotsis.3.1.1.2 Effects of ST on p21WAF1/CIP1 in GES-1 cells at various time points Western Blot analysis showed that when cells were treated by 3μM ST from 2 to 12d, the expression of p21 was significantly decreased in all ST treatment groups (P<0.05). The peak value of p21 appeared at 8th day, subsequently decreased at 12th day(P<0.05). The results suggested that ST treatment could activate p53 and the peak value of p-p53 appeared at 8th day. The activation of p21 was basically consistent in time with G2 arrest and apoptotsis.3.1.2 Effects of ST treatment with different concentrations on p53-p21WAF1/CIP1 signaling pathway in GES-1 cells3.1.2.1 Effects of ST treatment with different concentrations for 4 days on p53-p21WAF1/CIP1 signaling pathway in GES-1 cells3.1.2.1.1 Effects of ST treatment with different concentrations for 4 days on p53 in GES-1 cellsWestern Blot analysis showed that after ST treatment for 4 days, the expression of p-p53 was significantly increased in 0.075μM3μM ST treatment groups as compared with that in solvent control group (P<0.05) in a dose-dependent manner(r=0.893, P<0.01). The expression of p53 was significantly increased in 0.3μM3μM ST treatment groups as compared with that in solvent control group (P<0.05) in a dose-dependent manner(r=0.866, P<0.01).Real time-PCR analysis showed that the expression of p53 mRNA was significantly increased after different concentrations ST treatment(P<0.01),3.1.2.1.2 Effects of ST treatment with different concentrations for 4 days on p21WAF1/CIP1 in GES-1 cellsWestern Blot analysis showed that after ST treatment for 4 days, the expression of p21 was significantly increased in 0.075μM3μM ST treatment groups as compared with that in solvent control group (P<0.05) in a dose-dependent manner(r=0.910, P<0.01). 3.1.2.2 Effects of ST treatment with different concentrations for 8 days on p53-p21WAF1/CIP1 signaling pathway in GES-1 cells3.1.2.1.1 Effects of ST treatment with different concentrations for 8 days on p53 in GES-1 cellsWestern Blot analysis showed that after ST treatment for 8 days, the expression of p-p53 was significantly increased in 0.075μM3μM ST treatment groups as compared with that in solvent control group (P<0.05) in a dose-dependent manner(r=0.836, P<0.01). The expression of p53 was significantly increased in 0.3μM3μM ST treatment groups as compared with that in solvent control group (P<0.05) in a dose-dependent manner(r=0.879, P<0.01).Real time-PCR analysis showed that the expression of p53 mRNA was significantly increased after different concentrations ST treatment(P<0.01),3.1.2.1.2 Effects of ST treatment with different concentrations for 8 days on p21WAF1/CIP1 in GES-1 cellsWestern Blot analysis showed that after ST treatment for 8 days, the expression of p21 was significantly increased in 0.075μM3μM ST treatment groups as compared with that in solvent control group (P<0.05) in a dose-dependent manner(r=0.926, P<0.01).3.2 Effects of ST on GES-1 cells with p53 siRNA transfection3.2.1 Effects of ST on p53-p21WAF1/CIP1 signaling pathway in GES-1 cells with p53 siRNA transfection3.2.1.1 Effects of ST on p53 in GES-1 cells with p53 siRNA transfectionReal time-PCR analysis showed that the expression of p53 mRNA was significantly decreased after p53 siRNA transfection for 2 days (P<0.01,data not given). Western Blot analysis showed that the expression of p53 and p-p53 was significantly increased in p53 siRNA transfection group as compared with that in solvent control group (P<0.05). The inhibition rate of p53 at mRNA and protein level reached 70%80% which proved p53 siRNA transfection was successful in interfering with the expression of p53.Western Blot analysis showed that after ST treatment for 2 days, the expression of p53 and p-p53 was significantly decreased in p53 siRNA + ST (3μM) treatment group as compared with that in ST treatment group (P<0.05), while increased in p53 siRNA treatment group (P<0.05). The results suggested that p53 siRNA transfection could inhibit ST-induced the activition of p53.3.2.1.2 Effects of ST on p21WAF1/CIP1 in GES-1 cells with p53 siRNA transfectionWestern Blot analysis showed that after ST treatment for 2 days, the expression of p53 and p-p53 was significantly decreased in p53 siRNA + ST (3μM) treatment group as compared with that in ST treatment group (P<0.05), while increased in p53 siRNA treatment group (P<0.05). The results suggested that p53 siRNA transfection could inhibit ST-induced the activition of p21WAF1/CIP1.3.2.2 Effects of ST on the key regulatory factors of G2 phase in GES-1 cells with p53 siRNA transfection3.2.2.1 Effects of ST on Cdc25C in GES-1 cells with p53 siRNA transfectionWestern Blot analysis showed that the expression of Cdc25C was significantly increased in p53 siRNA + ST (3μM) treatment group as compared with that in ST treatment group (P<0.05), while that of p-Cdc25C decreased in ST treatment group (P<0.05). The results suggested that p53 was involved in the ST-induced decreasement of Cdc25C and increasement of p-Cdc25C.3.2.2.2 Effects of ST on Cdc2 in GES-1 cells with p53 siRNA transfection Western Blot analysis showed that the expression of Cdc2 was increased in p53 siRNA + ST (3μM) treatment group as compared with that in ST treatment group (P<0.05), while that of p-Cdc2 significantly decreased in ST treatment group (P<0.05). The results suggested that p53 was involved in ST-induced the decreasement of Cdc2 and increasement of p-Cdc2.3.2.2.3 Effects of ST on CyclinB1 in GES-1 cells with p53 siRNA transfectionWestern Blot analysis showed that the expression of CyclinB1 was significantly decreased in p53 siRNA + ST (3μM) treatment group as compared with that in ST treatment group (P<0.05), while significantly increased as compared with that in p53 siRNA treatment group (P<0.05). The results suggested that p53 was involved in the ST-induced increasement of CyclinB1.3.2.3 Effects of ST on apoptosis related proteins in GES-1 cells with p53 siRNA transfection3.2.3.1 Effects of ST on Bcl-2 in GES-1 cells with p53 siRNA transfectionWestern Blot analysis showed that the expression of Bcl-2 was significantly decreased in ST treatment group as compared with that in solvent control group (P<0.05), but no significant difference was found between p53 siRNA + ST (3μM) treatment group and ST treatment group (P>0.05). The results suggested that p53 could not reverse the ST-induced decreasement o... |
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