| Objectives:1.Select PI3 K inhibitor LY294002,mTOR inhibitor AZD8055 and MEK inhibitor AZD6244,observe the effect of the three inhibitors individually on the proliferation,apoptosis,and cell cycle of gastric cancer cell line MGC-803.2.By observing the changes of the m RNA and protein levels of key signal molecules in PI3K/Akt/mTOR and Ras/Raf/MEK/ERK signaling pathways in MGC-803 cells after treated with LY294002,AZD8055,AZD6244 alone,explore the possible molecular mechanisms of the effect of three inhibitors on the biological function of MGC-803 cells through the PI3K/Akt/mTOR and Ras/Raf/MEK/ERK signaling pathways,illuminate the existence of compensatory activation,mutual regulation mechanism and feedback loop between the two signaling pathways.3.Combined the PI3 K inhibitor LY294002 and MEK inhibitor AZD6244,mTOR inhibitor AZD8055 and MEK inhibitor AZD6244 to treat gastric cancer MGC-803 cells.Observing the effects of combined LY294002+AZD6244 and AZD8055+AZD6244 on the cell proliferation,apoptosis,cycle and the m RNA and protein expression levels of Akt,ERK,P70S6 K.By comparing the effects of the three inhibitors alone with the combination of LY294002+AZD6244 and AZD8055+AZD6244,the combination of LY294002+AZD6244 with AZD8055+AZD6244,explore the optimal combination of three different inhibitors,and explain the synergistic effect PI3K/Akt/mTOR and Ras/ Raf/MEK/ ERK signaling pathways,providing an academic basis for clinical research of new targeted therapeutic drugs and the search for new combined treatment options.Methods:1.The gastric cancer cell line MGC-803 were treated with PI3 K inhibitor LY294002,the mTOR inhibitor AZD8055 and the MEK inhibitor AZD6244 respectively.CCK-8 was used to detect the effects of LY294002,AZD8055 and AZD6244 on the proliferation of gastric cancer cells;Annexin V-FITC/PI staining and flow cytometry was used to detect the changes in cell cycle and apoptotic;RT-q PCR was used to detect the m RNA expression of key signal molecules PI3 K,Akt,TSC2,mTOR,P70S6 K,RAS,ERK in PI3K/Akt/mTOR and Ras/Raf/MEK/ERK signal pathway.Western blot was used to detect the expression of total proteins PI3 K,Akt,TSC2,mTOR,P70S6 K,RAS,ERK and phosphorylated proteins of PI3 K,Akt,TSC2,mTOR,P70S6 K and ERK.2.MGC-803 cells were treated with LY294002+AZD6244,AZD8055+AZD6244,respectively.CCK-8 was used to detect the proliferation of MGC-803 cells;Annexin V-FITC/PI staining and flow cytometry was used to detect the cell cycle and apoptosis of LY294002+AZD6244,AZD8055+AZD6244 combination group;RT-q PCR was used to detect the m RNA expression of the key signal molecules Akt,P70S6 K,ERK in LY294002+AZD6244,AZD8055+AZD6244 combined group;Western blot was used to detect the expression of total proteins Akt,P70S6 K,ERK and phosphorylated proteins Akt,P70S6 K,ERK.Results:1.The results of CCK-8 showed that different concentrations of LY294002 inhibitedthe proliferation of MGC-803 cells in a dose-dependent manner(P <0.01).Annexin V-FITC/PI staining was used to detect the apoptosis rate of LY294002 group and control group at different concentrations.The apoptosis rate of the LY294002 groups showed a dose-dependent relationship with the concentration(P <0.01).The flow cytometry was used to detect the effect of LY294002 on the cell cycle.The proportion of cells in G1 phase increased significantly after treated with different concentrations of LY294002(P <0.05).2.The results of CCK-8 showed that compared with control group,different concentrations of AZD8055 inhibited the proliferation of MGC-803 cells in a dose-dependent style(P <0.01).Annexin V-FITC / PI staining flow cytometry was used to detect apoptosis,the apoptosis rate of MGC-803 cells showed no significant change(P> 0.05).Flow cytometry results showed that the proportion of G1,S,G2 cells treated with different concentrations of AZD8055 was not significantly different(P> 0.05).3.The results of CCK-8 showed that different concentrations of AZD6244 inhibited the proliferation of MGC-803 cells in a dose-dependent manner(P <0.01).Annexin V-FITC/PI staining flow cytometry was used to detect apoptosis.The apoptosis rate was higher in the 25,125,and 175 μmol/L concentration groups than in the control group(P <0.05).Flow cytometry results showed that the proportion of cells in the G1 phase increased significantly(P <0.05),and the cells in the S phase decreased after treated with different concentrations of AZD6244(P <0.05).4.The results of RT-q PCR showed that after treated with LY294002 for48 h,the m RNA expression of PI3 K,TSC2,and P70S6 K decreased in a dose-dependent manner(P <0.05).The m RNA expression of mTOR was higher than that in the control group but gradually decreased(P <0.05),the change in ERK expression was no significant(P>0.05).Additionally,Western blot results showed that the protein expression of p-PI3 K,p-Akt,p-mTOR,and p-P70S6 K decreased in a dose-dependent manner,and the expression of P-TSC2 fluctuated with the increase of LY294002 concentrations.p-ERK protein expression gradually increased with the increase of inhibitor concentration(P<0.05)after treated with LY294002 for 24 h.The expression of total proteins of PI3 K,Akt,mTOR,TSC2,P70S6 K,and RAS in the pathway decreased(P <0.05),and the protein expression of ERK had no significant change(P> 0.05).5.The results of RT-q PCR showed that the m RNA expression of mTOR,P70S6 K and RAS in the experimental groups decreased and mTOR decreased in a dose-dependent manner(P <0.05)after 48 h of AZD8055 treatment.m RNA expression of Akt and TSC2 increased in a dose-dependent manner(P <0.05),while the expression of ERK did not change significantly(P> 0.05).Western blot results showed that the protein levels of p-mTOR,p-TSC2 and p-P70S6 K decreased in a dose-dependent manner(P <0.05).With the increase of inhibitor concentration,the protein of p-PI3 K,p-Akt gradually increased(P <0.05),the p-ERK fluctuated with the inhibitor concentration(P <0.05).The expressions of total proteins PI3 K,Akt,mTOR,TSC2,P70S6 K,RAS,and ERK had no significant change(P> 0.05).6.RT-q PCR results showed that the m RNA expression levels of PI3 K,TSC2,and P70S6 K in the experimental group increased in a concentration-dependent manner(P<0.05),the m RNA levels of RAS and ERK showed a downward trend after AZD6244 was applied to MGC-803 cells for 48 h.The results of Western blot showed that the levels of p-PI3 K,p-Akt,p-mTOR,and p-P70S6 K protein increased in a dose-dependent manner(P <0.05),the protein level of p-TSC2 had no obvious trend(P<0.05).p-ERK protein expression level decreased in a concentration-dependent manner(P <0.05)after treated with AZD6244 for 24 h.The expression of total proteins PI3 K,Akt,mTOR,P70S6 K and ERK did not change significantly(P> 0.05),the protein expression of TSC2 gradually decreased while RAS gradually increased with the increase of inhibitor concentration(P <0.05).7.Treat MGC-803 cells with single LY294002,AZD8055 and AZD6244 or combined LY294002 with AZD6244,AZD8055 with AZD6244.The results showed that the combination of LY294002 and AZD6244 at different concentrations had inhibitory effects on the proliferation of MGC-803 cells in a dose-dependent manner(P<0.01).Similarly,the combination of AZD8055 and AZD6244 at different concentrations inhibited the cell proliferationin a dose-dependent style(P <0.01).Further analysis showed that the proliferation rate of MGC-803 cells in the LY294002+ AZD6244 combined group was lower than that in the single use of LY294002 and AZD6244 groups.Similarly,the proliferation rate of AZD8055 + AZD6244 combined groups was lower than that of single use of AZD8055 and AZD6244 groups.8.Treat MGC-803 cells with different concentrations of LY294002,AZD8055,AZD6244,and combined LY294002+ AZD6244,AZD8055+ AZD6244.Flow cytometry was used to detect the apoptosis rate of each group.The results showed that the combination of LY294002 and AZD6244 at different concentrations induced cells apoptosisin a dose-dependent manner(P <0.01).The same situation existed in the group of combind AZD8055 and AZD6244(P <0.01).Further analysis showed that the cell apoptosis of combined LY294002 with AZD6244 group was higher than single use of LY294002 and AZD6244 groups.Similarly,the cell apoptosis of combined AZD8055 with AZD6244 group was higher than single use of AZD8055 and AZD6244 sgroups.In addition,the cell apoptosis induced by LY294002 +AZD6244 group was higher than AZD8055 +AZD6244 group.9.Flow cytometry was used to detect the cell cycle of each group.The results showed that the group of LY294002+AZD6244 significantly increased the number of cells in the G1 phase and decreased the number of cells in the S phase compared with the control group(P <0.05).The effect of G1 phase arrest in group of LY294002+AZD6244 was significantly stronger than that of single use LY294002 and AZD6244 groups.AZD8055 group had no significant effect on the cell cycle.AZD6244 group induced G0 / G1 phase cell cycle arrest,the G1 phase significantly increased,and the S phase decreased.After the combination of AZD8055 and AZD6244,the number of cells in the G1 phase increased significantly(P <0.05)and the number of cells in the S phase decreased(P <0.05),.But the combined group did not show a stronger effect of cycle arrest than that in the groups of single use.10.The results of RT-q PCR showed that the m RNA expression of Akt,P70S6 Kand ERK in the group of LY294002+AZD6244 significantly changed,the m RNAs expression of Akt,P70S6 K and ERK showed a concentration-dependent decreasing trend(P <0.05).After treated with combined AZD8055 with AZD6244 at different concentrations,the m RNA expression of Akt,P70S65 K and ERK in AZD6244 combined group gradually decreased in a dose-dependent manner(P < 0.05).The results of Western blot showed that no significant changes in the total protein expression of Akt,P70S6 K and ERK(P> 0.05)in the groups of single use LY294002,AZD8055 and AZD6244,and the groups of LY294002+AZD6244,AZD8055+AZD6244.The phosphorylation protein expression of Akt,P70S6 K,ERK in groups of LY294002 + AZD6244,AZD8055 + AZD6244 were lower than the LY294002,AZD8055,AZD6244 alone group(P <0.05)),the inhibitory effect of the combined group on the phosphorylation protein is stronger than that of the single use LY294002,AZD8055,AZD6244 groups.In addition,the decrease of p-AKT,p-P70S6 K and p-ERK in the group of LY294002+AZD6244 was more significantly than that in the group of AZD8055+AZD6244.Conclusions:1.The inhibitors LY294002,AZD6244 inhibit the MGC-803 cells proliferation,induce apoptosis,and blocking the cell cycle;,however,AZD8055 inhibits the cell proliferation significantly,but has no significant effect on the apoptosis and cell cycle.2.The PI3 K inhibitor LY294002 inhibits the key signal molecules of PI3 K Akt/mTOR signaling pathway to reduce the proliferation rate,change the cell cycle,and increase the apoptosis rate of gastric cancer cells.mTOR inhibitor AZD8055 has an inhibitory effect on the transcription and translation of key genes in PI3K/Akt/mTOR signaling pathway,thereby inhibiting the proliferation of gastric cancer cells.Furthermore,the MEK inhibitor AZD6244 inhibits the transcription and translation of key genes in the Ras/Raf/MEK/ERK signaling pathway,and the biological function of gastric cancer cells can be regulated through the changes of MEK,ERK and downstream key factors.In conclusion,LY294002,AZD8055 and AZD6244 can affect the biological activity of gastric cancer cells by the two signaling pathways.3.After treated MGC-803 cells with LY294002,the resultsdemonstrates that there is compensatory activation between the PI3K/AKT/MTOR and Ras/Raf/MEK/ERK signaling pathways.It is speculated that mTOR activity was inhibited in MGC-803 cells and then regulate the activates Raf/MEK/ERK signal pathway through P70S6K-PI3K-RAS feedback.AZD8055 inhibits the PI3K/Akt/mTOR signaling pathway.The activation of the Ras/Raf/MEK/ERK signaling pathway shows that inhibition of one signaling pathway may lead to activation of another the compensatory signaling pathway.It also shows that the inhibition of downstream signaling moleculars leads to the feedback activation of upstream signaling molecules like PI3 K and Akt,The study results confirms the existence of PI3K-Akt-TSC2-mTOR pathway in MGC-803 cells and also demonstrates that the Ras-ERK pathway cross-activates the signaling pathway of PI3K/AKT/mTOR through regulating the PI3 K,TSC2 and mTOR.Besides,it is showed that AZD6244 inhibits the downstream moleculars of the Ras/Raf/MEK /ERK signaling pathway and cross-activates the PI3K/Akt/mTOR signaling pathway through regulating PI3 K,TSC2 and mTOR.The inhibition of ERK leads to TSC2 dephosphorylation,and inhibition of TSC2 further activates mTOR.In conclusion,there are compensatory activation and the specific feedback mechanism between PI3K/AKT/mTOR and Ras/Raf/MEK/ERK signaling pathways.4.The combination of LY294002+AZD6244,AZD8055+AZD6244 inhibits cell proliferation,promotes apoptosis,induces cell cycle arrest and significantly inhibits the transcription and translation processes of Akt,P70S6 K and ERK.The combination of LY294002+AZD6244 showed stronger inhibition of cell proliferation,promotion of apoptosis,and induction of cell cycle arrest compared with the single treatment of the two inhibitors.Combined AZD8055 with AZD6244 showed a strong inhibitory effect on cell proliferation,but has no significant differences on the apoptosis and cell cycle in MGC-803 cells compared with the use two inhibitors alone.Futher study indicates that group of LY294002+AZD6244 has better effect on the inhibition of cell proliferationand promotion of cell apoptosis than group of AZD8055+AZD6244.The two groups of the combined inhibitors have stronger effect on the phosphorylation of key protein than that of the individual drug group,and the decline of p-AKT,p-P70S6 K and p-ERK in the LY294002 + AZD6244 group is more obvious than that of the AZD8055 + AZD6244 group.The findings provid an experimental basis for clinical research of new targeted therapeutic drugs and the search for new combined treatment plans. |
