The Mechanisms Of Cordycepin Affecting Biological Behavior Of Gastric Cancer Cells Via Regulating Akt And MAPK Signaling Pathway

Objective:To investigate the the regulation mechanisms about cordycepin on biological behavior of gastric cancer cell line MGC-803.Methods:The effects of different concentrations of cordycepin on the activity of normal gastric epithelial mucous membrane GES-1 cells and gastric cancer MGC-803 cells were detected by MTT;Cell colony forming ability was detected by colony formation assay;Hoechst33342 staining was used to observe the apoptotic cells;Annexin V-FITC/PI double staining flow cytometry was used to detect cell apoptosis;PI single flow cytometrywas used to detect cell cycle;Wound healing experiment and transwell compartment migration experiment were used to detect cell lateral and vertical migration ability;Western blot assay detected the protein of apoptosis,cell cycle,EMT,meanwhile the protein of Akt signaling pathway and MAPK signal pathway.Verifying the expression level of C type lectin like receptor 2(CLEC2)gene in MGC-803,SGC-7901,HGC-27 and GES-1 by RT-PCR and Western blot,as well as the expression level of CLEC2 gene in MGC-803 cells after treatment with cordycepin;The CLEC2 gene was silenced by siRNA transfection,and the transfection efficiency was verified by Western blot;MTT method and wound healing experiment verified the effect of CLEC2 gene silencing on cell proliferation and migration ability;The effect of CLEC2 gene silencing on Akt signaling pathway was detected by Western blot assay.Results:1.MTT results showed the inhibition ability of cordycepin on GES-1 cells was much weaker than that of MGC-803 cells,and compared with the control group,cordycepin with different concentrations(25μM,50μM,100μM)all could inhibit the viability of MGC-803 cells both 24h,48h and 72h(P<0.01);2.colone formation test showed that the colony formation ability of the cells in the drug group decreased significantly(P<0.01);3.Compared with the control group,the number of apoptotic cells increased gradually by Hoechst33342 staining;4.Annexin V-FITC/PI double dye flow cytometry showed compared with the control group,the apoptotic rate was significantly higher in the 50 μM and 100 μM groups.(P<0.05 or P<0.01);5.PI single dye flow cytometry showed G2/M phase block in the drug group(P<0.05 or P<0.01);6.Wound healing experiment and transwell compartment migration experimen showed that the lateral and longitudinal migration ability of the cells in the drug group wassignificantly lower than that of the control group(P<0.01);7.Western blot experiment showed that cordycepin could up tegulation the protein of apoptosis related protein cleaved-caspase3 and cleaved-caspase9,the apoptotic substrate cleaved-PARP,the epithelial cell marker E-cadherin and MAPK signaling pathway protein:p-JNK and p-P38(P<0.05 or P<0.01);down regulation the protein of apoptosis suppressor protein survivin,cell cycle related proteins CDK1,CyclinB1,CDC25C,interstitial cell markers Vimentin,Snail,Slug,members of MMPS MMP2 and MMP9,Akt signaling pathway protein:p-Akt and p-4EBP1(P<0.05 or P<0.01);8.RT-PCR and Western blot results showed that mRNA and protein expression levels of CLEC2 in MGC-803,SGC-7901 and HGC-27 cells were significantly lower than GES-1 cells;9.The results of RT-PCR and Western blot showed that the expression level of mRNA and protein of CLEC2 in MGC-803 cells were increased with the increasing concentration of cordycepin;10.After silencing of CLEC2 gene,the effect of cordycepin on proliferation and migration of gastric cancer cells was weakened(P<0.01).The Western blot results showed that the expression level of p-Akt protein in the silent group was up(P<0.05)than that of the control group.Conclusion:1.Cordycepin inhibited the proliferation and migration of gastric cancer MGC-803 cells,and the mechanism was related to the downregulation of Akt signaling pathway;2.Cordycepin blocked MGC-803 cells in G2/M phase,and its mechanism was related to up regulation of JNK and P38 phosphorylation leve;3.Cordycepin inhibits proliferation and migration of gastric cancer cells by regulating Akt signaling pathway through up regulation of CLEC2.