| Sterigmatocystin (ST) is a toxic fungal metabolite with low molecular weight. It is produced by some Aspergillus species fungi, such as, A versicolor, A nidulans,etc. ST is one of the common contaminants of the ingredients of animal feed and human food all over the world. ST has been proved to have carcinogenic potency in experimental animal models. It could induce lung adenocarcinoma, hepatoma and mesotheliomas in different experimental animals. ST is one of genotoxic agents and could form DNA adducts with target cell DNA and result in direct DNA damages. Our previous studies with human tissue 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.As we know that the control of cell cycle is one of the most imortant biological processes. Disregulation of cell cycle may result in carcinogenesis. It has be showed that ST could affect cell cycle of target cells. Xie et al found that ST could cause G2/M arrest in murine fibroblasts. Our primary study with human gastric epithelial cell line also confirmed that ST could induce G2 phase arrest in human gastric GES-1 cells in vitro. But the putative mechanisms of G2 arrest induced by ST was still not clear enough.Cyclins are key components of the cell cycle progression machinery. They activate their partner cyclin-dependent kinases (CDKs) and possibly target them to respective substrate proteins within the cell. CDK-mediated phosphorylation of specific sets of proteins drives the cell through particular phases or checkpoints of the cell cycle. It has been established that the G2 checkpoint initiation is primarily regulated through the control of Cdc2 (Cdk1) activity, which is regulated at multiple levels, including periodic association with the B-type cyclins, reversible phosphorylation, and intracellular compartimentalization. CyclinB1 is the cylcin at stage of G2, and the compound formed with its kinase Cdc2 is the key factor for mitoschisis and play key role to precipitate cell cycle from stage of G2 to stage of M. Cyclin B1 reaches maximum levels in G2, when it enters into the nucleus to form a complex with Cdk1 in a phosphorylation-dependent manner. Protein phosphatase Cdc25C could induce dephosphorylation of Cdc2 and promote the progression of cell cycle. Phosphorylation of Cdc25C could result in the loss of enzyme activity, which in turn increase the phosphorylation activity of Cdc2 and the cell cycle may be arrested at G2 phase as a result. eIF4E is the key regulatory factor in the process of protein synthesis. In association with its binding protein- 4E-BP1, eIF4E modulates the synthesis of cap-dependent protein, such as CyclinD1. The elevated level of phosphorylation could enhance the protein synthesis of eIF4E. Studies showed that eIF4E and 4E-BP1 are associated with the disruption of cell cycle, and expressed abnormally in malignant tumor cell. Our recent experiment studies indicated that ST could inhibit the proliferation and induce human gastric GES-1 cells arrested at stage of G2 in vitro. And ST was found to have impact on the expression of CyclinB1 as well as the activity of Cdc2 kinase and Cdc25 phosphatase, which are all the key factors for the G2 regulatory point. At the same time, we found that ST could inhibit the initiated translation function of eIF4E.JNK, ERK and p38 are the important members of MAPK signal transduction pathway. Studies showed that environmental carcinogens(such as mycotoxins)and chemotherapeutic agents could affect the expressions of the related genes and biological behaviors of the target cells by their effects on JNK, ERK and p38 signal transduction pathways. PI3K is one of the important signal transduction pathways in Her2 family. It played important roles in the control of cell proliferation, protein synthesis, etc through the regulation of the downstream nuclear factor-mTOR. In malignant tumors, PI3K was associated with the invasion, metastasis and prognosis and chemotherapy tolerance of tumor.To further explore the putative mechanisms of G2 arrest induced by ST, the present study was carried out based on our previous studies. The effects of ST on the the activities of JNK, ERK, p38 and PI3K signal transduction pathway in human gastric epithelial cell line-GES-1 were studied. And the roles of JNK, ERK and PI3K activation on G2 arrest of GES-1 induced by ST in vitro was studied.The following three parts are included in the study:1 Effects of Sterigmatocystin on MAPK and PI3K signal transduction pathway in GES-1 cells in vitroObjective: To explore the effects of different concentration of ST on JNK, ERK, p38 and PI3K/mTOR signal transduction pathway in GES-1 cells.Methods: After initial culture for 24h, GES-1 cells were harvested, centrifuged and resuspended in fresh DMEM medium supplemented with 10% FCS at the concentration of (1~2)×104 cells/L in culture flasks (8 ml). The cells were randomly divided into 6 groups. The cells in ST groups were treated with ST at 100μg/L, ST 500μg/L, ST 1000μg/L and ST 2000μg/L, respectively, while DMSO and saline were used in solvent control and control group. Cells were harvested 24 h after ST treatment. The expression of ERK/p-ERK, p38/p-p38 and PI3K/mTOR/p-mTOR at protein level in GES-1 cells treated with ST was determined by Western blot. The expression of ERK, p38 and PI3K/mTOR mRNA in GES-1 cells was detected by RT-PCR.GES-1 cells seeded at the concentration of (1~2)×104 cells/L in culture flasks were randomly divided into 5 groups: control, solvent control, ST 1000μg/L, blocking agent and blocking agent +ST 1000μg/L. The medium of GES-1 cells was replaced by new DMEM medium supplemented with 2% FCS 24h later. The cells of blocking agent groups were pretreated for 30 min with 1μM SP600125 (JNK inhibitor), 50μM PD98059 (ERK inhibitor ), 0.5μM SB203580 (p38 inhibitor) and 1μM LY-294002 (PI3K inhibitor) respectively. Then the cells in blocking agent +ST 1000μg/L group were treated with ST 1000μg/L, while these in solvent control and control groups were incubated with DMSO and saline respectively. Cells were harvested 24 h after ST treatment. The JNK, ERK, p38 and PI3K/mTOR activation of GES-1 cells treated with ST were determined with Western blot.Results:1.1 Effects of ST on JNK signal transduction pathway1.1.1 Effects of ST with different concentrations on JNK signal transduction pathwayThe results of Western blot showed that there was no significant difference in JNK protein expression in GES-1 cells between all the ST treatment groups and solvent control group (DMSO) (P>0.05), while the phosphorylation of JNK in ST 500μg/L, 1000μg/L and 2000μg/L treatment groups was significantly increased as compared with that in corresponding DMSO group (P<0.05). And there is a significant dose-dependent correlation between ST concentration and the phosphorylation of JNK (r=0.925, P<0.01, n=3).The results of RT-PCR confirmed that no significant difference in JNK mRNA expression in GES-1 cells between all the ST treatment groups and solvent control group (DMSO) (P>0.05).1.1.2 Effects of ST on JNK signal transduction pathway, with SP600125 pretreatmentThe results of Western blot showed that the phosphorylation of JNK in GRS-1 cells in SP600125 treatment group was significantly decreased than that in corresponding DMSO group (P<0.05). And the phosphorylation of JNK in SP600125+ST treatment group was dramatically decreased compared with that in ST treatment alone, while still higher than that in solvent control (P<0.05). The result indicated that the SP600125 could specifically inhibit the activation of JNK MAP kinase, but the effect of activation of JNK in ST-treated cells was partially inhibited by the presence of SP600125.1.2 Effects of ST on ERK signal transduction pathway1.2.1 Effects of ST with different concentrations on ERK signal transduction pathway The results of Western blot suggested that there was no significant difference in ERK protein expression in GES-1 cells between ST treatment groups and solvent control group (DMSO) (P>0.05). In comparison with solvent control group, the phosphorylation of ERK was significantly increased in ST treatment groups (P<0.05). Results from concentration-dependent studies indicated that ST up-regulated the phosphorylation of ERK in a dose-dependent correlation (r=0.887, P<0.01, n=3).The results of RT-PCR showed that ST had no effect on the expression of ERK mRNA.1.2.2 Effects of ST on ERK signal transduction pathway, with PD98059 pretreatmentThe results of Western blot revealed that the phosphorylation of ERK in GRS-1 cells of PD98059 treatment group was significantly decreased compared with that in solvent control group (P<0.05). And the phosphorylation of ERK in PD98059+ST treatment group was decreased significantly compared with that in ST 1000μg/L treatment, while still higher than that in solvent control (P<0.05). The result revealed that the PD98059 could inhibit the activation of ERK MAP kinase, but the effect of activation of ERK in ST-treated cells was partially inhibited by the presence of PD98059.1.3 Effects of ST on p38 signal transduction pathway1.3.1 Effects of ST with different concentrations on p38 signal transduction pathwayNo significant difference in p38 protein expression of GES-1 cells between all the ST treatment groups and solvent control group was found by Western blot (P>0.05), while the phosphorylation of p38 in ST treatment groups was lower compared with that in corresponding solvent control group (P<0.05). And the phosphorylation of p38 decreased with ST treatment in a dose-dependent manner (r=-0.843, P<0.01, n=3).RT-PCR results confirmed that there was no significant difference in p38 mRNA expression in GES-1 cells between all the ST treatment groups and solvent control group (P>0.05). 1.3.2 Effects of ST on p38 signal transduction pathway, with SB203580 pretreatmentWestern blot results showed that SB203580 could significantly decreased the level of phosphorylation of p38 in GES-1 cells (P<0.05). And the phosphorylation of p38 in SB203580+ST treatment group was markedly decreased compared with that in ST treatment (P<0.05). The result indicated that there might be a synergistic effect of the inhibitor of p38, SB203580 and ST on the phosphorylation of p38.1.4 Effects of ST on PI3K/mTOR signal transduction pathway1.4.1 Effects of ST with different concentrations on PI3K/mTOR signal transduction pathwayThe results of Western blot showed that the expression of PI3K protein in GES-1 cells in both the ST treatment groups and solvent control group showed no significant differences (P>0.05), while the expression of mTOR protein in ST 1000μg/L and 2000μg/L treatment groups was higher compared with that in solvent control group (P<0.05). And there is a significant dose-dependent correlation between ST concentration and the expression of mTOR protein (r=0.831, P<0.01, n=3). Meanwhile, the phosphorylation of mTOR was significantly increased by ST in a dose-dependent way (r=0.868, n=3, P<0.01).RT-PCR analysis suggested that there was no difference in PI3K mRNA expression in GES-1 cells between all the ST treatment groups and solvent control group (P>0.05). But the expression of mTOR mRNA was increased by ST in a dose-dependent way (r=0.831, n=3, P<0.01).1.4.2 Effects of ST on PI3K/mTOR signal transduction pathway, with LY-294005 pretreatmentThe results of Western blot showed that the expression of mTOR protein and phosphorylation in GES-1 cells in LY-294002 treatment group showed no significant difference compared with that in solvent control group (P<0.05). Compared with the group with ST only treatment, the phosphorylation of mTOR in LY-294002+ST treatment group was decreased significantly (P<0.05), while there was no difference as compared with that of solvent control (P>0.05). But the exptession of mTOR protein in LY-294002+ST treatment group was higher than that in solvent control (P<0.05), and it showed no difference between LY-294002+ST treatment group and ST treatment only (P>0.05).The results of RT-PCR displayed that there was no difference in mTOR mRNA expression in GES-1 cells between ST treatment groups and solvent control group (P>0.05). But in comparison with solvent control group, mTOR mRNA expression in LY-294002+ST treatment group was higher (P<0.05), but was not different from that in ST treatment only groups (P>0.05).The results indicated that the LY-294002 could inhibit the activity of PI3K/mTOR signal pathway, but it had no effect on the expression of mTOR at both protein and mRNA levels in GES-1 cells.2 Effects of JNK, ERK and PI3K signal transduction pathway activation on the G2 arrest induced by Sterigmatocystin in GES-1 in vitroObjective: To explore the effects of inhibitors of JNK, ERK and PI3K on the ST-induced proliferation inhibition and G2 arrest.Methods:GES-1 cells were harvested, centrifuged and resuspended in fresh DMEM medium supplemented with 10% FCS at the concentration of (1~2)×104 cells/L in 96-well culture plates. The cells were randomly divided into 5 groups: control, solvent control, ST 1000μg/L, blocking agent and blocking agent +ST 1000μg/L. The cells of blocking agent groups were pretreated for 30 min with 1μM SP600125 (JNK inhibitor), 50μM PD98059 (ERK inhibitor), 0.5μM SB203580 (p38 inhibitor) and 1μM LY-294002 (PI3K inhibitor) respectively. Then the cells in blocking agent +ST 1000μg/L group were treated with ST 1000μg/L, while these in solvent control and control groups were incubated with DMSO and saline respectively. Cell viability for GES-1 cells was determined by MTT assay.For the blocking studies, GES-1 cells seeded at the concentration of (1~2)×104 cells/L in culture flasks were randomly divided into 5 groups: control, solvent control, ST 1000μg/L, blocking agent and blocking agent +ST 1000μg/L (the treatment was same as above). Cells were harvested 24 h after ST treatment. The changes of cell cycle in response to ST was measured using FCM. And the expression of CyclinB1, the activity of Cdc2 kinase and Cdc25C phosphatase in GES-1 cells were determined with Western blot and RT-PCR.Results:2.1 Effects of JNK signal pathway on the G2 arrest induced by ST2.1.1 Effect of SP600125 pretreatment on the changes of survival rate in GES-1 cells induced by STThe result of MTT showed that the cell viability in SP600125+ST treatment group was much higher than that in ST group, but it still lower than that of controls (P<0.05). The result indicated that SP600125 could partly block the inhibition of proliferation induced by ST in GES-1 cell.2.1.2 Effect of SP600125 pretreatment on G2 arrest of GES-1 cells induced by STFCM analysis revealed that the G0/G1 fraction was increased and the G2/M fraction was decreased in SP600125+ST treatment group compared with ST 1000μg/L group. But the G2/M faction was still increased compared with control group. Thus, addition of SP600125 to the cells could partly decrease the ability of ST to increase the percentage of G2/M faction.2.1.3 Effect of SP600125 pretreatment on the ST-induced changes of the key factors of G2 phase in GES-1 cells2.1.3.1 Effect of SP600125 pretreatment on the expression of CyclinB1The results of Western blot showed that the expression of CyclinB1 protein in SP600125+ST treatment group was significantly decreased compared with that in solvant control group and ST group (P<0.05).The results of RT-PCR suggested that the expression of CyclinB1 mRNA in SP600125+ST treatment group was significantly decreased as compared with that in ST group (P<0.05) and there was no difference of that compared with solvent control group. Thus, the results in this study revealed that the increase in the expression of CyclinB1 at both protein and mRNA level was blocked by the presence of SP600125 in ST-treated GES-1 cells.2.1.3.2 Effect of SP600125 pretreatment on Cdc2 kinaseWestern blot results showed that in comparison with ST treatment group, the dephosphorylation level of Cdc2 in SP600125+ST treatment group was significantly increased, meanwhile the phosphorylation level of Cdc2 was decreased (P<0.05). And no significant difference was found between SP600125+ST treatment group and solvent control group.The results of RT-PCR displayed that the expression of Cdc2 mRNA had no difference between SP600125+ST treatment group and ST treatment group.Thus, the results in this study suggested that alteration in dephosphorylation and phosphorylation of Cdc2 caused by ST were blocked by the JNK inhibitor SP600125, which had no effect on the expression of Cdc2 mRNA.2.1.3.3 Effect of SP600125 pretreatment on Cdc25C phosphataseThe results of Western blot showed that the presence of the JNK inhibitor simultaneously with ST induced a significant increase in the expression of Cdc25C, as compared with that in solvent control group and ST treatment group (P<0.05). The level of phosphorylation of Cdc25C was reduced as compared with ST treatment group (P<0.05), and had no difference compared with solvent control group.RT-PCR results confirmed that there was no difference in the expression of Cdc25C mRNA between SP600125+ST treatment group and ST treatment group.Thus, the results in this study indicated that SP600125 could inhibit the alterations in protein expression and phosphorylation level of Cdc25C induced by ST, but had no influence on the expression of Cdc25C mRNA.2.2 Effects of ERK signal pathway on the G2 arrest induced by ST2.2.1 Effect of PD98059 pretreatment on the changes of GES-1 cells survival rate induced by ST The result of MTT showed that the cell viability in PD98059+ST treatment group was much higher than that of ST group, and there was no difference of that as compared with solvant control group (P<0.05). The result indicated that the PD98059 could partly block the ST-induced antiproliferative action in GES-1 cells.2.2.2 Effect of PD98059 pretreatment on the G2 arrest of GES-1 cells induced by STFCM analysis showed that the number of S faction was increased and that of G2/M faction was decreased in PD98059+ST treatment group compared with ST 1000μg/L groups. But the G2/M faction was still increased compared with solvant control group. Thus, addition of PD98059 to the cells could partly decrease the ability of ST to increase the percentage of G2/M faction.2.2.3 Effect of PD98059 pretreatment on the changes of the key factors expression of G2 stage induced by ST in GES-1 cells2.2.3.1 Effect of PD98059 pretreatment on the expression of CyclinB1Western blot results showed that the expression of CyclinB1 protein in PD98059+ST treatment group was significantly decreased as compared with that in ST group, but it was still higher than that in solvant control group (P<0.05).The results of RT-PCR showed that the expression of CyclinB1 mRNA in GES-1 cells had no difference between PD98059+ST treatment group and ST treatment group (P>0.05).Thus, the results in this study suggested that the alterations in protein expression of CyclinB1 caused by ST were blocked by the presence of ERK inhibitor PD98059, which had no effect on the expression of CyclinB1 mRNA.2.2.3.2 Effect of PD98059 pretreatment on Cdc2 kinaseThe results of Western blot showed that no difference in Cdc2 dephosphorylation in PD98059+ST treatment group and ST group could be found. But the phosphorylation of Cdc2 in PD98059+ST treatment group was decreased as compared with that in ST group (P<0.05), and there was no difference of that compared with solvent control group (P>0.05).The results of RT-PCR confirmed that the expression of Cdc2 mRNA in PD98059+ST treatment group was decreased as compared with that in ST treatment group, but it was still higher than that in solvent control group (P<0.05).Thus, the results in this study revealed that the increase in the level of phosphorylation of Cdc2 was blocked by the addition of PD98059 which could partly inhibit the expression of Cdc2 mRNA in ST-treated GES-1 cells.2.2.3.2 Effect of PD98059 pretreatment on Cdc25C phosphataseThe results of Western blot showed that, the expression of Cdc25C protein had no difference in PD98059+ST treatment group and ST group. The phosphorylation of Cdc25C in PD98059+ST treatment group was significantly decreased as compared with ST group, but it was still higher than that in solvent control group (P<0.05).The results of RT-PCR revealed that there was no difference of the expression of Cdc25C mRNA between PD98059+ST treatment group and ST group (P>0.05).Thus, the results in this study indicated that PD98059 could inhibit the alterations in phosphorylation of Cdc25C induced by ST, but had no effect on the expression of Cdc25C mRNA.2.3 Effects of PI3K signal pathway on the G2 arrest induced by ST2.3.1 Effect of LY-294002 pretreatment on the changes of GES-1 cells survival rate induced by STThe result of MTT showed that there was no difference in the cell viability between LY-294002+ST treatment group and ST group (P>0.05), and it was much lower than that of controls (P<0.05). The result indicated that LY-294002 had no effect on the cell viability.2.3.2 Effect of LY-294002 pretreatment on the G2 arrest of GES-1 cells induced by STFCM analysis showed that the number of G0/G1 and S faction were all decreased and that of G2/M faction was increased in LY-294002+ST treatment group compared with ST 1000μg/L groups (P<0.05). The result indicated that there might be a synergistic effect of LY-294002 and ST on the increase of G2/M faction of GES-1 cells.2.3.3 Effect of LY-294002 pretreatment on the changes of the key factors expression of G2 stage induced by ST in GES-1 cells2.3.3.1 Effect of LY-294002 pretreatment on the expression of CyclinB1.Western blot results showed that addition of the PI3K inhibitor combined with ST induced a significant decrease of CyclinB1 protein expression, as compared with that in solvant control group and ST group (P<0.05).The results of RT-PCR confirmed that the expression of CyclinB1 mRNA in LY-294002+ST treatment group was significantly decreased as compared with ST group and solvent control group (P<0.05).Thus, the results in this study revealed that the increase in the expression of CyclinB1 at protein and mRNA level was blocked by the addition of LY-294002 in ST-treated GES-1 cells.2.3.3.2 Effect of LY-294002 pretreatment on Cdc2 kinaseThe results of Western blot showed that there was no difference in the level of dephosphorylation of Cdc2 in LY-294002+ST treatment group (P>0.05), meanwhile the level of phosphorylation of Cdc2 was increased as compared with that in ST group and solvent control group (P<0.05).RT-PCR results confirmed that the expression of Cdc2 mRNA was increased in LY-294002+ST treatment group as compared with solvant control group and ST group (P<0.05).The results in this study suggested that a synergistic effect might exist between LY-294002 and ST on the phosphorylation and mRNA expression of Cdc2.2.3.3.2 Effect of LY-294002 pretreatment on Cdc25C phosphataseThe results of Western blot showed that the expression of Cdc25C protein in LY-294002+ST treatment group had no difference compared with ST group (P>0.05). But the level of phosphorylation of Cdc25C was higher compared with ST group and solvent control group (P<0.05).The results of RT-PCR confirmed that there was no difference in the expression of Cdc25C mRNA in LY-294002+ST treatment group compared with ST group (P>0.05).Thus, the results in this study suggested that LY-294002 might have synergistic effect with ST on the phosphorylation of Cdc25C, but had no effects on the expression of Cdc25C at both protein and mRNA level.3 Effects of JNK, ERK and PI3K signal transduction pathway activation on the protein dyssynthesis of GES-1 cells induced by Sterigmatocystin in vitroObjective: To explore the effects of inhibitors of JNK, ERK and PI3K on the ST-induced protein dyssynthesis.Methods: GES-1 cells seeded at (1~2)×104 cells/L in culture flasks were randomly divided into 5 groups: control, solvent control, ST 1000μg/L, blocking agent and blocking agent +ST 1000μg/L (the treatment was the same as above). The expression of CyclinD1, the activity of eIF4E and 4E-BP1 in GES-1 cells were determined with Western blot and RT-PCR.Results:3.1 Effects of JNK signal pathway on the protein dyssynthesis induced by ST 3.1.1 Effect of SP600125 pretreatment on the expression of eIF4E The results of Western blot showed that the expression of eIF4E protein in SP600125+ST treatment group was significantly decreased as compared with that in solvant control group and ST group (P<0.05), meanwhile the phosphorylation of eIF4E in SP600125+ST treatment group was significantly increased (P<0.05).The results of RT-PCR confirmed that the expression of eIF4E mRNA in SP600125+ST treatment group was significantly decreased as compared with that in ST group, but it was still higher than that in solvent control group (P<0.05).Thus, the results in this study revealed that the decrease in the phosphorylation of eIF4E was blocked by the addition of SP600125, which could partly inhibit the expression of eIF4E mRNA in ST-treated GES-1 cells.3.1.2 Effect of SP600125 pretreatment on the expression of 4E-BP1The results of Western blot showed that in comparison with ST group, the expression of 4E-BP1 protein in SP600125+ST treatment group was significantly decreased (P<0.05), but had no difference compared with solvent control group. And the phosphorylation of 4E-BP1 in SP600125+ST treatment group was significantly increased as compared with that both in ST group and solvant control group (P<0.05).The results of RT-PCR confirmed that the expression of 4E-BP1 mRNA in SP600125+ST treatment group was reduced compared with ST group, but it was still higher than that in solvant control group (P<0.05).The results in this study suggested that SP600125 could inhibit the alteration of 4E-BP1 activity induced by ST. And it could partly decreased the ability of ST to increase the expression of 4E-BP1 mRNA.3.1.3 Effect of SP600125 pretreatment on the exprssion of CyclinD1 proteinThe results of Western blot showed that addition of the JNK inhibitor combined with ST induced a significant increase of CyclinD1 expression, as compared with that in ST group, but the expression of CyclinD1 was still lower than that in solvent control group (P<0.05). And the results suggested that the increase in the expression of CyclinD1 at protein level was blocked by the addition of SP600125 in ST-treated GES-1 cells.3.2 Effects of ERK signal pathway on the protein dyssynthesis induced by ST3.2.1 Effect of PD98059 pretreatment on the expression of eIF4EThe results of Western blot showed that there was no difference in the expression of eIF4E protein in PD98059+ST treatment group compared with ST group (P>0.05). But the phosphorylation of eIF4E in PD98059+ST treatment group was significantly increased as compared with that in solvant control and ST group (P<0.05). The results of RT-PCR revealed that the expression of eIF4E mRNA in PD98059+ST treatment group was significantly decreased as compared with that in ST group, and had no difference as compared with that in solvent control group (P<0.05).Thus, the results in this study suggested PD98059 could block both of the decrease in phosphorylation of eIF4E and the increase in eIF4E expression at mRNA level induced by ST.3.2.2 Effect of PD98059 pretreatment on the expression of 4E-BP1The results of Western blot showed that the expression of 4E-BP1 protein in PD98059+ST treatment group was significantly decreased as compared with that in ST group (P<0.05), and had no difference compared with solvent control group. And addition of the ERK inhibitor combined with ST induced a significant increase of 4E-BP1 phosphorylation, as compared with that both in ST group, and had no difference compared with solvant control group (P<0.05).The results of RT-PCR confirmed that the expression of 4E-BP1 mRNA in PD98059+ST treatment group was decreased compared with ST group and solvant control group (P<0.05).The results in this study suggested that the additon of PD98059 could block the ST-induced increase in 4E-BP1 protein expression and decrease in the phosphorylation of 4E-BP1, and also partly block the increase in the expression of 4E-BP1 mRNA induced by ST.3.2.3 Effect of PD98059 pretreatment on the exprssion of CyclinD1 proteinThe results of Western blot showed that addition of PD98059 combined with ST induced a significant increase of CyclinD1 expression, as compared with solvent control group and ST group (P<0.05). And the results suggested that the decrease in the expression of CyclinD1 at protein level in ST-treated GES-1 cells was blocked by the addition of PD98059. 3.3 Effects of PI3K signal pathway on the protein dyssynthesis induced by ST 3.3.1 Effect of LY-294002 pretreatment on the expression of eIF4EThe results of Western blot showed that the expression of eIF4E protein in LY-294002+ST treatment group was significantly increased as compared with that in solvant control group and ST group (P<0.05). Meanwhile the phosphorylation of eIF4E in LY-294002+ST treatment group was significantly decreased as compared with that in ST group and solvant control group (P<0.05).The results of RT-PCR confirmed that the expression of eIF4E mRNA in LY-294002+ST treatment group was significantly increased as compared with that in ST group and solvent control group (P<0.05).Thus, the results in this study suggested that there might be a synergistic effect between LY-294002 and ST on the expression of eIF4E at both protein and mRNA level. And there might be a synergistic effect between LY-294002 and ST on the phosphorylation of eIF4E.3.3.2 Effect of LY-294002 pretreatment on the expression of 4E-BP1The results of Western blot showed that in comparison with ST group, the expression of 4E-BP1 protein in LY-294002+ST treatment group was significantly decreased (P<0.05), but had no difference compared with solvent control group. And the phosphorylation of 4E-BP1 in LY-294002+ST treatment group was significantly decreased as compared with ST group and solvant control group (P<0.05).The results of RT-PCR confirmed that the expression of 4E-BP1 mRNA in LY-294002+ST treatment group was decreased compared with ST group, but it was still higher than that in solvant control group (P<0.05). The results in this study suggested that the increase in 4E-BP1 expression at both protein and mRNA level induced by ST could be blocked by LY-294002. But LY-294002 could produce a synergistic effect with ST on the phosphorylation of 4E-BP1.3.3.3 Effect of LY-294002 pretreatment on the exprssion of CyclinD1 proteinThe results of Western blot showed that the addition of LY-294002 simultaneously with ST induced a significant increase of CyclinD1 expression, as compared with solvent control group and ST group (P<0.05). And the results in this study suggested that there might be a synergistic effect between LY-294002 and ST on the protein expression of CyclinD1.Conclusions:1. ST could activate JNK, ERK and PI3K signal trusduction pathway, and block p38 signal trusduction pathway at the same time.2. ST induced G2 arrest of GES-1 cells may be through activion of JNK, ERK signal pathways. But the activation of PI3K signal pathway may play negative regulating role on G2 arrest of GES-1 cells induced by ST.3. The changes in the expression and function of Cdc25C-Cdc2/CyclinB1 by the activation of JNK, ERK and PI3K signal transduction pathways may be the putative mechanisms of G2 arrest of GES-1 cells induced by ST in vitro.4. The activation of JNK, ERK and PI3K signal transduction pathways were all involved in the effects of ST on GES-1 cells in vitro. But the activation of PI3K signal pathway may mainly play the negative regulating role.
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