Study On The Effects Of ST On The Expression Of CyclinB1, Cdc2 And Cdc25C In GES-1 In Vitro

Objective: Sterigmatocystin (ST), the secondary metabolite of Aspergillus versicolor and Aspergillus nidulans etc, is a carcinogenic mycotoxin. It is quite commonly found in the environment as a contaminant in grains and animal feeds. The frequency of contamination of ST in foodstuff samples and gastric juice of patients with chronic gastritis was very high in areas of high mortality rates of gastric cancer in our country. Several studies indicated that ST could induce adenocarcinoma in lung and dysplasia of glandular stomach in mice, and unscheduled DNA synthesis of human gastric epithelial cells in vitro. ST-DNA adduct was detected in gastrointestinal cancers.One defining feature of cancer is the rapid creation of abnormal cells that grow beyond their usual boundaries. Cancer was thought to be the result of dysregulations in cell proliferation, differentiation and apoptosis. It is now accepted that cancer is more accurately described as being the product of malfunctions within the regulation of the cell cycle. In previous studies, we observed that ST could induce and promote apoptosis of cerebral cells, renal cells and hepatocytes in mice, and inhibit proliferation of hepatocytes to some extent. ST could also induce apoptosis in human peripheral blood lymphocytes in vitro. The Proliferation Index of the ST-treated gastric mucosa increased dramatically. We recently demonstrated that ST could induce G2 arrest in human gastric GES-1 cells in vitro, but the mechanism is still not well established.The progression of eukaryotic cells from the G2 to M phase of the cell cycle depends on the activity of a functional complex formed between cyclin B1 and Cdc2 (CyclinB1/Cdc2). The Cdc25C phosphatase is a key activator of CyclinB1/Cdc2 that controls M-phase entry. Cdc25C activation is essential to promote cell cycle progression whereas the inhibition of Cdc25C is associated to the initiation of cell cycle checkpoints.The aim of the present study, therefore, was to examine the effectiveness of ST in modulating the expression of CyclinB1, Cdc2, Cdc25C in human gastric GES-1 cells in vitro.Methods:1 Cell culture and treatmentGES-1 cells were grown in DMEM medium supplemented with 10% new-born calf serum (NBCS), streptomycin (100μg/ml), penicillin (100U/ml) in an atmosphere of 5% CO2/95% air at 37℃. GES-1 cells in logarithmic growth phase were randomly divided into 6 groups: control group, solvent control group, ST treatment groups. ST final concentration of ST treatment groups were 100, 500, 1000 and 2000μg/L respectively. The cells in control group were treated with NS and in solvent control group were treated with DMSO(all in same volume).2 Western blottingThe expression of CyclinB1, Cdc2, Phospho-Cdc2 and Cdc25C, Phospho-Cdc25C at protein level in GES-1 cells in vitro in different groups was determined by the method of Western blotting.3 Abstraction and quantitation of the total RNAThe total RNA was abstracted by single-step method with guanidinium isothiocyanate. The integrity of the total RNA was identfied at 90V on 1% agarose gels. The UV Spectro- photometer was used for the quantitation of the total RNA.4 The expression of mRNA detected by RT-PCRThe effects of ST on the expression of CyclinB1, Cdc2 and Cdc25C at mRNA level in GES-1 cells in vitro in different groups were determined using RT–PCR method. The relative expression was calculated as the ratio between the density of target gene and that of GAPDH by BIO-LD densitometric image analyzer. 5 StatisticsData from these studies were analyzed by one-way analysis of variance (ANOVA) and bivariate correlation. The SPSS 13.0 was employed for all calculations. Data were expressed as means±SD.Results1 The effect of ST on the expression of cell cycle related protein in GES-1 in vitro1.1 The expression of CyclinB1 at protein levelThe molecular weight of CyclinB1 is 48kD. After SDS-PAGE electrophoresis and Western blotting, 48kD positive bands were found in lanes of both the control and ST treatment groups. The relative expression of CyclinB1 in all treatment groups were higher compared with the solvent control. The result of image analysis revealed that ST could significantly increase the expression of CyclinB1 in GES-1 cells in vitro.1.2 The dephosphorylation of Cdc2 detected by Western blottingThe molecular weight of Cdc2 is 34kD. After SDS-PAGE electrophoresis and Western blotting, 34kD positive bands were found in lanes of both the control and ST treatment groups. Image analysis showed that the dephosphorylation of Cdc2 was reduced in GES-1 cells treated with ST for 24 h in vitro. 1.3 The phosphorylation of Cdc2 detected by Western blottingThe molecular weight of Phospho-Cdc2 is 34kD. After SDS-PAGE electrophoresis and Western blotting, 34kD positive bands were found in lanes of both the control and ST treatment groups. The result of Western blotting suggested that ST could significantly increase the phosphorylation of Cdc2 in GES-1 cells in vitro.1.4 The expression of Cdc25C at protein levelThe molecular weight of Cdc25C is 60kD. After SDS-PAGE electrophoresis and Western blotting, 60kD positive bands were found in lanes of both the control and ST treatment groups. In comparison with the solvent control, the level of Cdc25C was reduced in ST-treated GES-1 cells. The result showed that ST could dramatically decrease the expression of Cdc25C in GES-1 cells in vitro.1.5 The phosphorylation of Cdc25C detected by Western blottingThe molecular weight of Phospho-Cdc25C is 60kD. After SDS-PAGE electrophoresis and Western blotting, 60kD positive bands were found in lanes of both the control and ST treatment groups. The result of Western blotting showed that ST could markedly increase the phosphorylation of Cdc25C in GES-1 cells in vitro. 2 The effects of ST on the expression of CyclinB1, Cdc2 and Cdc25C mRNA in the GES-1 cells in vitro2.1 The expression of CyclinB1 mRNA detected by semi-quantitative RT-PCRRT-PCR products were detected by 1.5% agarose gel electrophoresis and analyzed by BIO-LD. The result showed that there was no significant difference in the expression of CyclinB1 at mRNA level between the control group and the solvent control group. The relative contents of CyclinB1 mRNA in all treatment groups were higher compared with the solvent control, and the expression of CyclinB1 mRNA increased in a concentration-dependent manner(r=0.818,n=3,P<0.01). The result indicated that ST could significantly increase the expression of CyclinB1 mRNA in GES-1 cells in vitro.2.2 The expression of Cdc2 mRNA detected by semi-quantitative RT-PCRRT-PCR products were detected by 1.5% agarose gel electrophoresis and analyzed by BIO-LD. The result showed that there was no significant difference in the expression of Cdc2 at mRNA level between the control group and the solvent control group. Results from concentration-dependent studies indicated that ST up-regulated the expression of Cdc2 mRNA in a concentration-dependent manner(r=0.962,n=3,P<0.01). These results suggested that ST could dramaticaly increase the expression of Cdc2 mRNA in GES-1 cells in vitro.2.3 The expression of Cdc25C mRNA detected by semi-quantitative RT-PCRRT-PCR products were detected by 1.5% agarose gel electrophoresis and analyzed by BIO-LD. There was no significant difference in the expression of Cdc25C at mRNA level between the control group and the solvent control group. In comparison with the solvent control, the level of Cdc25C mRNA was reduced in ST-treated GES-1 cells. Results from concentration-dependent studies revealed that ST could down-regulate Cdc25C mRNA in GES-1 cells in vitro in a concentration-dependent manner(r=-0.881,n=3,P<0.01).Conclusion:1 ST could increase the expression of CyclinB1 and the phosphorylation of Cdc2 and Cdc25C in concentration -dependent manners with decreasing the dephosphorylation of Cdc2 and Cdc25C in GES-1 cells in vitro.2 ST could up-regulate the expression of CyclinB1 and Cdc2 and down-regulate the expression of Cdc25C in GES-1 cells in vitro at mRNA level.3 All results in this study showed that ST could influence the expression of CyclinB1, Cdc2 and Cdc25C in GES-1 cells at both transcriptional and translational levels. These effects may be involved in the ST-induced G2 arrest.