Effects Of Curcumin On The Epithelial Mesenchymal Transition Of HCT116 Cell Line And The Related Mechanisims

Objective: To investigate the effect of curcumin on the growth,proliferation and migration of human colon cancer cell lines HCT116,and to explore the effect of curcumin on TNF-?-induced colon epithelial-mesenchymal transition(EMT)and migration and the possible mechanism,providing a theoretical basis of the Anti-tumor therapy with curcumin.Method: 1.Using MTT assay to detect the toxic effect of human colon cancer cell line HCT116 at different times and at different concentrations:Respectively,selecting curcumin with 0 ?mol/L,5 ?mol/L,10 ?mol/L,15 ?mol/L,20 ?mol/L,25 ?mol/L and 30 ?mol/L deal with the cells.After 12 h,24h,36 h and 48 h,taking advantage of MTT colorimetric detection to detect the survival rate of human colon cancer cells HCT116.2.To explore the effect of curcumin on human colon cancer cell line HCT116 of epithelial mesenchymal transition,the impact of migration and its possible mechanism: According to the results of MTT assay,selecting the appropriate drug concentration of curcumin for the next intervention experiments.The human colorectal cancer cell HCT116 was divided into control group,TNF-? group and curcumin + TNF-? group.Using inverted microscope to observe the morphological changes of the cells at each group after the curcumin effect.Detecting the expression levels of Epithelial phenotype protein E-cadherin and Interstitial phenotype protein Vimentin in the EMT-related markers by Western blot,as well as the expression of p65 protein on the NF-?B signal pathway.Through the cell scratch test,we examined the migration ability of the human colorectal cancer cell HCT116 after treatment.Results: 1.When the concentration of curcumin is less than 20 ?mol/L,the cell’s survival rates were not statistically significant both in drug concentrations and in acting times(P>0.05).If the concentration of curcumin is more than 20?mol/L,it significantly inhibited human colon cancer cell line HCT116’s survival rate after 12 h,and the inhibition gradually increased with the increase of drug concentration and acting time and the difference of cell’s survival rates were statistically significant,no matter in drug concentrations or in acting times(P<0.05).2.The relative expression levels of E-cadherin in control group and TNF-? group were(0.1278 + 0.0082)and(0.0921 + 0.0076),respectively(P<0.05).The relative expression levels of Vimentin in control group and TNF-? group were(0.1353 + 0.0140)and(0.2864 + 0.0096),respectively(P<0.05),and the relative expression levels of NF-?Bp65 in control group and TNF-? group were(0.6516 + 0.0268)and(0.8407 + 0.0307),respectively(P<0.05).The results showed that after treated with TNF-?,the expression of epithelial phenotype protein E-cadherin was down-regulated,the Interstitial phenotype protein Vimentin was up-regulated,at the same time,NF-?Bp65increased expression.In addition,most of HCT116 cells in TNF-? group were long spindle shaped cells,and most of them grew slender filopodia.Compared with the control group,the arrangement between the cells was significantlylooser.The intercellular gap was widened and the cells appeared morphological change of mesenchymal cells.The cell scratch test showed that average migration distance of HCT116 cells in control group and TNF-? group after 48 h were(144.07 + 11.31)?m and(319.84 + 20.93)?m,respectively(P<0.05),indicated that TNF-? can induce epithelial mesenchymal transition and enhance the ability of migration in HCT 116 cells by regulating NF-?Bp65.3.Treated the epithelial mesenchymal transition cell model induced by TNF-? with curcumin after 48 h,using Western Blot to detecte the expression levels of Epithelial phenotype protein E-cadherin and Interstitial phenotype protein Vimentin in the TNF-? group and curcumin + TNF-? group,as well as the expression of NF-?Bp65.The relative expression levels of E-cadherin in control group and TNF-? group were(0.0921 + 0.0076)and(0.1185 + 0.0033),respectively(P<0.05).The relative expression levels of Vimentin in control group and TNF-? group were(0.2864 + 0.0096)and(0.2043 + 0.0238),respectively(P<0.05),and the relative expression levels of NF-?Bp65 in control group and TNF-? group were(0.8407 + 0.0307)and(0.5406 + 0.0224),respectively(P<0.05).Results showed that compare with the TNF-? group,the expression of E-cadherin in the curcumin + TNF-? group was up-regulated,and the expression of Vimentin in the curcumin + TNF-? group was down-regulated,at the same time,NF-?Bp65 decreased expression.Moreover,there were long spindle cells and circular cells of apoptosis coexist in the curcumin +TNF-? group.The slender filopodia in the curcumin + TNF-? group was significantly reduced,and the arrangement of cells in the curcumin +TNF-? group is tighter than in the TNF-? group.The cell scratch test showed that average migration distance of HCT116 cells in TNF-? group and curcumin+ TNF-? group after 48 h were(319.84 + 20.93)?m and(90.70 + 7.25)?m,respectively(P<0.05),indicated that curcumin inhibited epithelial mesenchymal transition in HCT 116 cells induced by TNF-? and metastasis of the epithelial mesenchymal transition cell model through depressing the expression of NF-?Bp65.Conclusion: Curcumin inhibited epithelial mesenchymal transition and metastasis in HCT 116 cells induced by TNF-? through depressing the expression of NF-?Bp65.