Study Of Synergistic Effect And Mechanism Of MTOR Inhibitor Rapamycin Combined With MEK Inhibitor PD98059 On Gastric Cancer Cell

Objectives:1.This thesis aims to compare the expression differences of PI3K,AKT,mTOR,MEK,ERK and other genes in gastric cancer cell SGC-7901 and normal gastric epithelial cell GES-1 and to verify the activation of PI3K/AKT/mTOR and MAPK/ERK signaling pathways in gastric cancer.2.To investigate the possible molecular mechanism of PI3K/AKT/mTOR signaling pathway in the regulation of gastric cancer cell growth,we observed the effect of mTOR inhibitor rapamycin on gastric cancer MGC-803 cells,and thus provide more experimental evidence for clinical use of this pathway inhibitor.3.To provide more experimental basis for the basic research and clinical treatment of gastric cancer,we observed and discussed the effect of MEK inhibitor PD98059 on the signal transduction of gastric cancer SGC-7901 cells and its mechanism.4.To investigate the interaction of PI3K/AKT/mTOR and MAPK/ERK signaling pathways,we investigated whether rapamycin and PD98059 have synergistic effect on gastric cancer SGC-7901 cells,thus revealing the regulatory mechanism of two signaling pathways.In this way,we can control the prevention and treatment of gastric cancer gene levels and provide academic basis for the clinical study of new targeted therapy drugs and a new combination of treatment program.Methods:1.In vitro culture of human poorly low-differentiated gastric cancer cell line SGC-7901 and normal gastric epithelial cells GES-1.Real-time fluorescence quantitative PCR was used to detect the mRNA expression of key signal molecules such as PI3K,AKT,mTOR,MEK and ERK.The expression of total protein AKT,mTOR,MEK1/2,ERK1/2 and phosphorylated protein p-AKT,p-mTOR,p-MEK1/2 and p-ERK1/2 were detected by Western Blotting.2.mTOR targeting inhibitor rapamycin was applied to intervene the gastric cancer cell line MGC-803.MTT assay was used to detect the effect of rapamycin on cell proliferation.RT-qPCR was used to detect the mRNA expression of PI3K,AKT,mTOR,4EBP and P70S6K.Western Blotting was used to detect the quantitative expression of mTOR(Ser2448),P70S6K and phosphorylated protein p-mTOR(Ser2448),p-P70S6K(Thr389).Annexin V-FITC/PI staining and flow cytometry were used to detect the cell cycle distribution and apoptosis rate.3.Intervene on gastric cancer cell line SGC-7901 with MEK-targeted inhibitor PD98059.CCK-8 array was used to detect the effect of PD98059 on cell proliferation.RT-qPCR was used to detect the mRNA expression of MEK and ERK.Western blot was used to detect the quantitative expression of MEK 1/2,ERK 1/2 and phosphorylated protein p-MEK 1/2,p-ERK1/2.Effects of PD98059 on cell cycle distribution and apoptosis rate of SGC-7901 cells were detected by flow cytometry.4.According to the results of rapamycin and PD98059 inhibition of gastric cancer cell proliferation test,we selected a concentration point of drug and designed therapy groups:control group,rapamycin group,PD98059 group and combined group.RT-qPCR was used to detect the mRNA expression of the key signal molecules of PI3K/AKT/mTOR signal pathway such as PI3K,AKT,mTOR and MAPK/ERK signaling pathway such as MEK snd ERK.The quantitative expression of AKT,mTOR,MEK1/2,ERK1/2,p-AKT,p-mTOR,p-MEK 1/2 and p-ERK1/2 were detected by Western Blot.Annexin V-FITC/PI staining combined with flow cytometry was used to detect the effect of single treat and combination therapy on cell cycle distribution and apoptosis rate of SGC-7901 cells.Results:1.Expression of key signal molecules of PI3K/AKT/mTOR and MAPK/ERK pathways in SGC-7901 and GES-1 cellsWe used RT-qPCR to determine the expression of PI3K,AKT,mTOR,MEK and ERK.The results showed that their amplification curves were good and the dissolution curve showed good specificity.We found that the mRNA expression level in SGC-7901 cells was significantly higher than that in GES-1 cells(P<0.05).There was no significant difference in the expression levels of total protein mTOR,AKT,MEK and ERK between SGC-7901 cells and normal gastric epithelial cells(P>0.05).The expression of phosphorylated protein p-mTOR,p-AKT,p-MEK and p-ERK protein in SGC-7901 cells was significantly higher than that in GES-1 cells(P<0.05).2.Effects of mTOR inhibitor Rapamycin on the biological function of gastric cancer MGC-803 cellsCompared with control group,different concentrations of rapamycin had inhibitory effect on MGC-803 cells.With the increase of rapamycin concentration,the proliferation of MGC-803 cells decreased gradually and the inhibitory effect was dose-dependent(P<0.01).The expression of PI3K,AKT,mTOR,4EBP and P70S6K mRNA in the experimental group was significantly lower than that in the control group(P<0.01).There was no significant difference in the expression of mTOR(Ser2448)and P70S6K in the experimental group.The phosphorylated protein p-P70S6K(Thr389)and p-mTOR(Ser2448)decreased gradually with the inhibitor concentration increases(P<0.05).The proportion of G0/G1 phase cells in the experimental group was higher than that in the control group(P<0.01),and the proportion of S phase cells was lower than that of the control group(P<0.01).The apoptotic rate of the experimental group was higher than that of the control group(P<0.01)and increased correspondingly with the increase of the concentration.3.The effect of PD98059 targeted inhibiting MAPK/ERK signaling pathway on gastric cancer SGC-7901 cellsThe proliferation rate of SGC-7901 cells in the experimental group was lower than that in the control group(P<0.01).In a certain range,with the increase of PD98059 concentration,SGC-7901 cells proliferation gradually decreased,and the inhibitory effect was dose-dependent.When the concentration of inhibitor reached 200?mol/L,the inhibitory effect was slowed down.There was no significant difference between 300?mol/L group and 400?mol/L group(P>0.05).The mRNA expression of MEK and ERK in the experimental group was lower than that in the control group(P<0.01).With the increase of PD98059 concentration,the mRNA expression of ERK gene decreased gradually and appeared dose-dependent(P<0.01).The mRNA expression of MEK gene in 50?mol/L group was higher than that in 25?mol/L and 100?mol/L group,which was lower than that in control group.Compared with the control group,the protein expression of MEK1/2 in 25?mol/L group had no change(P>0.05),while the expression of 50?mol/L and 100?mol/L group was higher than that of the control group(P<0.05).The expression of p-MEK1/2 was significantly lower than that of the control group(P>0.05).There was no significant difference in total ERK1/2 expression(P>0.05),while the phosphorylated protein p-ERK1/2 decreased gradually with the increase of inhibitor concentration(P<0.05).Compared with the control group,the proportion of G0/G1 phase cells in the experimental group increased gradually,which was higher than that in the control group(P<0.01),and the proportion of S phase cells was gradually higher than that of the control group(P<0.01),and the proportion of cells decreased gradually(P<0.01),but there was no significant difference between the 50?mol/L and 100?mol/L groups(P>0.05).The apoptotic rate of 25?mol/L and 50?mol/L concentration group increased with the increase of concentration.4.Synergistic effect of mTOR inhibitor Rapamycin combined with MEK inhibitor PD98059 on gastric cancer cells and its mechanismThe gene expression level of AKT,mTOR,MEK and ERK in rapamycin group and PD98059 group was lower than those in control group(P<0.05).The mRNA expression of AKT,mTOR,MEK and ERK genes in the combined group were lower than those in the control group(P<0.05).There was no significant difference in total protein expression of AKT,mTOR,MEK1/2 and ERK1/2 between the two groups(P>0.05).The expression levels of p-AKT,p-mTOR,p-MEK1/2 and p-ERK1/2 in the phosphorylated protein of rapamycin group and PD98059 group were lower than the control group(P<0.05).The expression levels of p-AKT,p-mTOR,p-MEKl/2 and p-ERK1/2 in the combination group were lower than rapamycin group and PD98059 group(P<0.05).The ratio of G0/G1 phase in rapamycin group and PD98059 group was higher than that in control group(P<0.01).The proportion of G0/G1 phase cells in rapamycin group and PD98059 group was higher than rapamycin group and PD98059 group(P<0.01).The proportion of S phase cells in rapamycin group and PD98059 group was lower than control group(P<0.01).The proportion of S phase cells in rapamycin group and PD98059 group was lower than rapamycin group and PD98059 group(P<0.01).The proportion of G2/M phase cells in PD98059 group was not significantly different from control group(P>0.05).The proportion of G2/M phase in combination group was lower than control group(P<0.05).The apoptotic rates of rapamycin group and PD98059 group were higher than control group(P<0.01).The apoptotic rate of the combined group was higher than the rapamycin group and PD98059 group(P<0.01).Conclusions:1.PI3K/AKT/mTOR and MAPK/ERK signaling pathway are active in gastric cancer SGC-7901 cells.The abnormal changes of these two signaling pathways are closely related to the occurrence and development of tumor.2.The mTOR targeting inhibitor rapamycin inhibits the transcription and translation of key genes of PI3K/AKT/mTOR signaling pathway and the proliferation of gastric cancer cells.It blocks the cell cycle in G0/G1 phase,causing apoptosis.Therefore,the mTOR targeting inhibitor can inhibit the growth of gastric cancer cells,revealing the relevance of PI3K/AKT/mTOR signaling pathway and gastric cancer chemotherapy effect.3.The MEK-targeted inhibitor PD98059 can inhibit the proliferation of gastric cancer cells by inhibiting the biological activity of the cells by inhibiting the MAPK/ERK signaling pathway,causing G0/G1 arrest,promoting cell apoptosis,and finally inhibiting the growth of gastric cancer cells.It indicated that MAPK/ERK signaling pathway inhibitor PD98059 can be used as an effective anticancer drug in clinical practice.Deepening the study of MAPK/ERK signaling pathway in the development of tumor abnormalities in the role of abnormal activation,both to deepen the understanding of the occurrence and development of cancer,but also for the clinical treatment of gastric cancer to provide new targets and new ideas for the clinical treament of gastric cancer.4.The combination of mTOR inhibitor rapamycin and MEK inhibitor PD98059 could significantly inhibit the transcription and translation of key cell genes,to promote cell apoptosis and cell cycle arrest,which could inhibit cell growth.Combined use of multi-target inhibitors may be a new direction for the treatment of resistant gastric cancer patients.This experiment provides experimental basis for the clinical study of new targeted therapy drugs and new combination therapy regimen.