The Mechanism Of DRAM1-induced Autophagy And Suppressed Cell Proliferation

Aim:To investigate if DRAM1 induces autophagy via Akt-p70S6K-S6 and inhibits cell proliferation.Methods:(1)To confirm that DRAM1 can induce autophagy in cells transfected with FLAG-DRAM 1 plasmids using Western blot method.(2)To detect whether DRAM1 can downregulate phospho-S6 protein levels in HEK293T,SW480 and HCT116 cells.(3)To examine the impact of DRAM1 on Akt-mTOR-p70S6K pathway.(4)Considering the important role of mTOR in regulating Akt activity,we evaluate whether DRAM1 influences the assembly of mTORC1 and mTORC2 with the methods of IP-mTOR.(5)Using CCK-8 and clone formation analysis to observe if DRAM1 inhibits cell proliferation.Results:We have successfully established overexpressing-DRAM 1 cells by transfection with a FLAG-DRAM1 plasmid.Data showed overexpression of DRAM1 induced autophagy,enhancing the turnover of LC3-? to LC3-? and attributing to the degradation of p62.Meanwhile,DRAM1 downregulated phospho-S6 protein levels.We also detected that DRAM1 played as a negative regulator in the Akt-mTOR-p70S6K pathway.Phospho-p70S6K acted as a negative feedback to IRS-1-PI3K pathway,and DRAM1 dramatically upregulated p55 regulatory subunit of class ? PI3K.As the important role of mTOR in regulating Akt activity,we found DRAM 1 made no difference in the assembly of mTORCl and mTORC2.DRAM1 inhibiting the Akt-p70S6K-S6 pathway which plays an important role in cell proliferation,we detected that DRAM1 reduced cell viability under the condition of serum-starvation and inhibited cell proliferation.Conclusion:DRAM1 induced autophagy via inhibiting Akt-p70S6K-S6 pathway;DRAM1 upregulated the protein level of phospho-p55 regulatory subunit of class ? PI3K;DRAM1 had no influence on the assembly of mTORCl and mTORC2.DRAM1 inhibited cell viability and cell proliferation to a certain extent.