The Effects Of Akt Inhibitor MK-2206 On Proliferation And Invasion Of Colon Cancer SW480 Cells Under Hypoxia Conditions

Objective:1.To evaluate the effect of Akt inhibitor MK-2206 on the proliferation and invasion of colorectal cancer SW480 cells under different oxygen conditions.2.To compare the differences of Akt inhibitor MK-2206 on the mRNA and protein expression of Akt,mTOR and HIF-1?in SW480 cells under different oxygen conditions.3.To discuss the role of PI3K/Akt/mTOR signal pathway and HIF-1?in regulating the proliferation and invasion of colorectal cancer SW480 cells combined with the previous experimental results.Methods:1.The SW480 cell lines were routinely cultured,when the cell lines adherenting,the cell lines were treated with 0?100?200?250 and 300?mol/L CoCl₂ to induce hypoxia,and then they were divided into different concentration gradient hypoxia model groups to observe cell growth.The experiment needs to select the appropriate concentration of CoCl₂ by reference,and the proliferation activity of the cells was detected by CCK-8 test,then we got the Hypoxia model group.2.The cell lines were treated with 0,5,10,15 and 20?mol/L Akt inhibitor MK-2206for 24 h,48 h and 72 h,the proliferation activity of the cells was detected by CCK-8test.3.The concentration of MK-2206 which can inhibit growth and has little effect on cell activity was selected,and then the experiment was divided into four groups:control group,MK-2206 group,hypoxia?CoCl₂-induced hypoxia?experimental group,MK-2206 hypoxia group;Transwell chamber was used to detect the cell migration and invasion ability of the four groups.4.The mRNA expression levels of Akt,mTOR and HIF-1?in each cell group were detected by RT-PCR technique,then comparing the data.5.The differences of Akt,p-Akt,mTOR,p-mTOR and HIF-1?protein expression in each cell group were detected by Western-blot technique,and the data of each group were need to be analyzed.Results:1.CoCl₂-induced anoxic environment can not only promote the invasion and migration of SW480 cells,but also promote the expression of HIF-1?protein and mRNA?P<0.05?.However,CoCl₂ at low concentration had no significant effect on the proliferation of SW480 cells?P>0.05?,and the higher the concentration,the more obvious the inhibitory effect on cell proliferation.2.MK-2206 can inhibit the proliferation,invasion and migration of SW480 cells both normoxic and anoxic environment?P<0.05?,however,hypoxia may weaken the inhibitory effect of MK-2206 on the migration of SW480 cells?P<0.05?.MK-2206could inhibit the mRNA expression of Akt,mTOR and HIF-1?in SW480 cells that cultured in normoxic environment?P<0.05?,and MK-2206 could significantly reduce the protein levels of pAkt,pmTOR and HIF-1?in normoxic and hypoxic environment?P<0.05?.Conclusion:1.Akt inhibitor MK-2206 can significantly inhibit the proliferation and invasion of SW480 cells under normoxic and hypoxic conditions in vitro.2.HIF-1?is located in the downstream of Akt/mTOR signal pathway,and the change of HIF-1?expression can affect the invasiveness of SW480 cells,but has no significant effect on cell proliferation.PI3K/Akt/mTOR signal pathway may affect the invasive ability of colon cancer SW480 cells through HIF-1?.