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Objective: Sterigmatocystin （ST） is a mycotoxin,mainly produced by some Aspergillus species fungi, such as A versicolor, A nidulans,etc. It is quite commonly found in the environment as one of the contaminants in feedstuff and foodstuff all over the world. ST have carcinogenic potency, it could induce a variety of cancer. Our previous studies showed that ST could induce malignant transformation in human fetal gastric mucosa cells in vitro, and induce G2 phase arrest in human gastric GES-1 cells in vitro. In addition, we found that ST treatment could cause DNA damage in GES-1 cells. All these findings revealed the pupative carcinogenesis of ST on human gastric mucosa.When the DNA damage caused by the drugs was happened, the ATM-Chk2 signal pathway would be activated rapidly to inhibit the tyrosine phosphatase Cdc25C. Consequently, the cells were arrested in G2 phase due to the reduction of Cdc2-CyclinB1 complex formation. It have reported that the activation of ATM-Chk2 signal pathway and the changes of Cdc25C, Cdc2, CyclinB1 induced by it played an important role in OTA induced G2 arrest in GES-1 cells in vitro. However, whether the ATM-Chk2 signal pathway play a role in ST induced G2 arrest in GES-1 cells has not been reported.Therefore, in the present study, the effects of ST on ATM-Chk2 signal pathway and the related factors of G2 phase arrest were detected in a human gastric epithelial cell line （GES-1） by western blot and co-immunoprecipitation assays, and the correlation between these factors were analyzed further. Our results may explore the putative role of ATM-Chk2 signal pathway in the related factors of ST induced G2 arrest in GES-1 cells in vitro. Methods:1 GES-1 cells in logarithmic growth phase were randomly divided into 6 groups: solvent control group, 0.075μM, 0.3μM, 1.5μM, 3μM and 6μM ST treatment group. Cells were harvested after 48h. In addition, we randomly divided the GES-1 cells in logarithmic growth phase into 6 groups: control group, 12h, 24h, 48h, 72h and 96h ST treatment group, with 3μM ST treatment. Cells were collected at each time respectively. 2 Western Blot assay was used to detecte the expression level of ATM/p-ATM, Chk2/p-Chk2, Cdc25C/p-Cdc25C, Cdc2/p-Cdc2 and CyclinB1. 3 The expression of Cdc2-CyclinB1 complex was detected by co-immunoprecipitation.Results:1 Effects of ST treatment on ATM-Chk2 signal pathway in GES-1 cellsThe results of western blot showed that, compared with solvent control group, the level of ATM and p-ATM were both significantly increased in 0.075μM, 0.3μM, 1.5μM, 3μM and 6μM groups （P<0.05）, and the expression of p-ATM was increased in a dose-dependent manner （r=0.908, P<0.01）. In addition, the change of Chk2 and p-Chk2 after ST treatment showed the same way as ATM or p-ATM （P<0.05）, and the p-Chk2 was also increased in a dose-dependent manner in the 0.075μM～6μM dose range （r=0.826,P<0.01）.Compared with control group, the level of ATM and p-ATM were significantly increased in 24h, 48h, 72h and 96h groups （P<0.05）, and the expression level of p-ATM was increased in a time-dependent manner （r=0.826, P<0.01）. Meanwhile, the Chk2 and p-Chk2 showed the same change as ATM or p-ATM （P<0.05）, the p-Chk2 was also increased in a time-dependent manner in the 24h96h time range（r=0.675, P<0.01）.All these results suggested that the ATM-Chk2 signal pathway could be activated by ST treatment in dose-dependent and time-dependent manners in GES-1cells.2 Effects of ST treatment on the related factors of G2 phase arrest in GES-1 cellsCompared with solvent control group, the expression of Cdc25C was significantly decreased after 0.3μM, 1.5μM, 3μM and 6μM ST treatment（P<0.05）, but the p-Cdc25C level was increased after 1.5μM～6μM ST treatment（P<0.05）. Meanwhile, the expression of Cdc2 was also reduced （P<0.05）, and it decreased in a dose-dependent manner in the 0.075μM～6μM dose range（r=-0.933,P<0.01）.while the p-Cdc2 level was increased in 1.5μM～6μM ST treatment groups（P<0.05）. At the same time, the expression of CyclinB1 was significantly increased after 3μM and 6μM ST treatment （P<0.05）.Immunoprecipitation results showed that the expression of Cdc2-CyclinB1 complex was also significantly decreased after 0.075μM, 0.3μM, 1.5μM, 3μM and 6μM ST treatment as compared with that in solvent control group （P<0.05）. In addition, the expression of Cdc2 protein decreased in a dose-dependent manner （r=-0.977, P<0.01）.Compared with control group, the expression of Cdc25C was significantly decreased after 12h96h ST treatment（P<0.05）, but the p-Cdc25C level was increased in 24h, 48h, 72h and 96h groups（P<0.05）. The expression of Cdc2 was also decreased in a time-dependent manner in the 12h96h time range（r=-0.889, P<0.01）. On the contrary, the p-Cdc2 was increased after 24h96h ST treatment（P<0.05） .Meanwhile, the CyclinB1 increased in 48h, 72h and 96h ST treatment groups（P<0.05）.These results suggested that ST could effect significantly on the related factors of G2 arrest, for example, the expression of Cdc25C and Cdc2 was decreased, while the level of p-Cdc25C, p-Cdc2 and CyclinB1 was increased, and the Cdc2-CyclinB1 complex was reduced in a dose-dependent manner.3 The correlation analysis of effects of ST treatment on ATM-Chk2 signal pathway and the related factors of G2 arrestThe results of different dose experiment showed that the expression of p-Chk2 increased with the increasing of p-ATM in the 0.075μM～6μM dose range（r=0.675, P<0.01）.At the same time,the expression of Cdc2 decreased with the increasing of p-ATM （r=-0.983, P<0.01）.And the Cdc2-CyclinB1 complex also decreased （r=-0.969, P<0.01）.The results of different time experiment showed that the expression of p-Chk2 increased with the increasing of p-ATM in the 24h96h time range （r=0.885, P<0.01）.At the same time,the expression of Cdc2 decreased （r=-0.921, P<0.01）.So we can see that with the increasing of p-ATM, the expression of the p-Chk2 was also increased, and the Cdc2 and the Cdc2-CyclinB1 complex were decreased.These results revealed that the p-ATM activated p-Chk2, and the activation of ATM-Chk2 signal pathway involved in the regulation of the related factors of G2 arrest.Conclusions:1 ST treatment could activate the ATM-Chk2 signal pathway in dose-dependent and time-dependent manners in GES-1 cells in vitro. At the same time, the expression of related factors of G2 arrest such as Cdc25C and Cdc2 were reduced, but the level of p-Cdc25C, p-Cdc2, CyclinB1 were increased, and the complex of Cdc2-CyclinB1 was decreased.2 The activation of ATM-Chk2 signal pathway by ST treatment may be one of the mechanisms in ST induced G2 arrest in GES-1 cells in vitro.
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