A Study Of The Effect Of Curcumin Isothiouracil Derivative 1g Modulate EMMPRIN On Inducing Autophagy In Colon Cancer Cells And Its Mechanism

Objectives: This paper observed the effect of a new isothiourea modified curcumin pyrimidine derivative 1g on the proliferation,autophagy,migration and invasion of human colon cancer cells HCT116 and HT29 cells,and explored possible molecular mechanisms.Methods: The CCK8 method was used to observe the inhibitory effect of 1g on colon cancer cells HCT116 and HT29;The transwell method was used to observe the effect of 1g(IC50)on the migration and invasion of colon cancer cells HCT116 and HT29;Electron microscopy was used to observe the formation of autophagic vesicles after 1g(IC50)treatment on HCT116 and HT29 cells;Western blot was used to observe the expression of LC3,MMP-2 and MMP-9 proteins after 1g(IC50)treatment on HCT116 and HT29 cells.Western blot was used to detect the expression of EMMPRIN and PI3K/ AKT/mTOR protein after 1g(IC50)treatment on HCT116 and HT29 cells;HCT116 and HT29 cells were transfected with EMMPRIN-siRNA and pCMV-EMMPRIN-FLAG and real-time fluorescence quantitative PCR(Real-time PCR)was used to detect the expression of EMMPRIN in HCT116 and HT29 cells;Western blot was used to detect the expression of LC3,EMMPRIN,PI3K/AKT/m TOR pathway protein in HCT116 and HT29 cells,in control group,1g group,1g+EMMPRIN-siRNA group,1g+Negativesi RNA group;1g+pCMV-EMMPRIN-FLAG group,1g+pCMV-FLAG group,1g+pCMV-EMMPRINFLAG+ PI3 K inhibitor20μM(LY294002)group.Results: CCK8 test results showed that 1g can inhibit the proliferation of human colon cancer cell lines HCT116 and HT29 cells,and the survival rate was reduced in a dose-dependent manner(P<0.05);Compared with the control group,transwell experiment results showed that in 1g(IC50)group the number of migrating and invasive cells of HCT116 and HT29 cells decreased(P<0.01);TEM results showed that no obvious autophagosomes were found in the cytoplasm of HCT116 and HT29 in the normal group,but autophagosomes were clearly visible in the cells of the 1g(IC50)group;Western blot results showed that 1g(IC50)the expression of LC3Ⅱ increased,but the expression of MMP-2 and MMP-9 decreased(P<0.05).Western blot results showed that the expression of EMPPRIN protein in HCT116 and HT29 cells under the effect of 1g(IC50)was reduced(P<0.05),and the expression of phosphorylated protein in PI3 K / AKT / mTOR pathway decreased(P<0.05);qRT-PCR results confirmed that EMMPRIN was lowly expressed in EMMPRIN-siRNA ang highly expressed in pCMV-EMMPRIN-FLAG(P<0.05);Compared with the control group,Western blot experiments showed that the expression of EMMPRIN,P-PI3K/PI3 K,P-AKT/ AKT,P-mTOR/mTOR protein in the 1g group was significantly reduced(P <0.05);Compared with the 1g group,the expression of EMMPRIN,P-PI3K/PI3 K,P-AKT/AKT,P-m TOR/mTOR protein in 1g+pCMV-EMMPRIN-FLAG group was increased prominently(P<0.05),but LC3II/LC3 I protein expression decreased significantly(P<0.05),while the 1g+EMMPRIN-siRNA group showed an opposite trend;Compared with the EMMPRIN overexpression group,the 1g+pCMV-EMMPRIN-FLAG,P-PI3K/PI3 K,P-AKT/AKT,P-mTOR/mTOR protein was reduced notably after the treatment of PI3 K inhibitor(P<0.05),but the expression of LC3 II / LC3 I protein increased dramatically(P <0.05).Conclusions:Curcumin isothiouracil derivative 1g can inhibit the proliferation ability of HCT116 and HT29 cells.and inhibition of EMMPRIN and PI3K/AKT/mTOR pathway may be one of the molecular mechanisms of the1 g inducing the autophagy of HCT116 and HT29 cells.