AMPK Inhibits Epithelial To Mesenchymal Transition,Invasion And Metastasis Of Gastric Cancer

Background and Aim:Invasion and metastasis are critical biological characteristics of gastric carcinoma.Most patients die of metastatic cancer instead of primary cancer.Therefore,it is of significance for treatment and prognosis of gastric cancer to discuss the mechanism of invasion and metastasis in depth.Epithelial-mesenchymal transition(EMT)is a basic phenomenon in embryo development.Not only does EMT participate in the embryogenesis and development but also play an important role in tumor invasion and metastasis.AMPK(AMP-activated Protein Kinase)is an intracellular energy sensor which regulates the energy balance the whole organism.However,its role in EMT and the regulation of tumor invasion and metastasis has not been a a systematic research.our previous study has shown that AMPK is capable of inhibiting TGF-β/smads signal pathway although the effect of AMPK on downstream targets pf TGF-β has not been verified.Therefore the present study aims to investigate the regulation of AMPK on EMT induced by TGF-β and cell invasion and metastasis.Methods:1.The expression of AMPK activity and EMT markers in clinical gastric carcinoma samples:The correlation between EMT markers and AMPK activity is analyzed with immunohistochemistry technology.16 samples are selected from all samples that are cryopreserved(-86℃)in tissue specimen storage of the hospital.AMPK activity and EMT markers are inspected with western blot.The correlation between them as well as the correlation between both and clinical pathological features are analyzed.2.The expression of AMPK activity and EMT markers in gastric cells:The expression of AMPK activity and EMT markers of gastric cells are inspected with western blot.The capacity of cell metastasis is inspected with scratch experiments.3.TGF-β induces EMT:The expression of EMT related protein are inspected with western blot in SGC-7901 and MKN-28 cells.The cell morphology and migration ability at different time points of TGF-β acting on gastric carcinoma cells are inspected with scratch experiment.4.AMPK inhibits induction of EMT by TGF-β:The expression of AMPK phosphorylated protein is inspected with western blot at different time points of metformin acting on cells to figure out whether the activation of metformin on AMPK is time-dependent.TGF-β and metformin are combined to act on cells and EMT related proteins are inspected with western blot to further investigate whether the activation of AMPK is capable of inhibiting EMT.Then cell migration ability is inspected with scratch experiment and Transwell experiments to observe the effect of AMPK on cell ethology.Results:1.The expression of AMPK activity and EMT markers in clinical gastric carcinoma samples:The expression of p-AMPK,E-cadherin decline with clinical pathological stages development,the expresssion of P-smad2,Vimentin,N-cadherin rise with clinical pathological stages development.It is indicated that p-AMPK is negatively correlated with P-smad2,Vimentin,N-cadherin and positively correlated with E-cadherin.The expression of P-smad2,Vimentin,N-cadherin in gastric carcinoma tissue is higher than those in para-carcinoma tissue while the expression of p-AMPK,E-cadherin in gastric carcinoma tissue is higher than those in para-carcinoma tissue.2.The expression of AMPK activity and EMT markers in gastric cells:The expression of Vimentin,N-cadherin in gastric carcinoma cell lines(MKN28,SGC-7901,823)is relatively lower which can be selected as cells to be induced.3.TGF-β induces EMT:Along with the TGF-β acting time,cells gradually become fusiform and mesenchyme phenotype appears.The expression of mesenchyme proteins including Vimentin、N-cadherin、β-catenin in SGC-7901 and MKN-28 is increasing and the expression of epithelium protein E-cadherin is decreasing.4.AMPK inhibits induction of EMT by TGF-β:The expression of p-AMPK,p-ACC increases with the processing time of metformin.After treated with combined TGF-β and metformin,the expression of EMT mesenchyme protein in this group is significantly lower than that in TGF-β group.The cell migration distance is longer in TGF-β group.After combined treatment of metformin plus TGF-β the cell migration distance is obviously decreased.In Transwell experiment,the quantity of invasion cells increases after induction by TGF-β.After the combined treatment of metformin and TGF-β,the quantity of invasion cells notably decreases.Conclusions:1.The AMPK activity declining in gastric carcinoma slices is together with the EMT markers increasing and closely correlated with the extent of gastric carcinoma metastasis which indicates that the EMT markers increasing is related to AMPK activity2.The activation of AMPK will result in the dramatically decrease of EMT markers and the inhibition of cell invasion and metastasis.