| Objective:The malignant degree of gastric cancer is very high nowadays,and malignant distant metastasis can easily occur at the very early stage,which is one of the chief reason of the treatment failure of the vast majority of the gastric cancer patients.Studies have clearly shown that epithelial mesenchymal transition is very closely related to tumor metastasis and the invasion.This experiment mainly studied the effects of ZuJinFang and its main active components-berberine and evodiamine on the migration and invasion of human gastric cancer cells(SGC7901)and EMT markers to provide experimental basis in clinical treatment for gastric cancer metastasis.Methods:(1)The Effect and mechanism of berberine on the inhibition of epithelial mesenchymal transition in SGC7901 cells.1.The effect of berberine on the growth of SGC7901 cells was detected by MTT method to determine the berberine concentration used in the study of subsequent experiments.2.The function of berberine on the migration and the invasion of SGC7901 cells can be tested by scratch assay and transwell assay.3.Real-Time qPCR was used to detect the function of berberine on the gene transcription of N-cadherin and E-cadherin in SGC7901 cells.4.Immunofluorescence or Western blot were adopted to test the changes of EMT-related proteins for instance E-cadherin,Vimentin,Z0-1,N-cadherin,Snail,MMP9 and also AKT,pAKT pathway protein.5.The effects of PI3 K / AKT inhibitor(LY294002)and agonist(IGF-1)alone or combined with berberine on the expression of E-cadherin,N-cadherin ? pAKT were sequentially inspected by Western blot.(2)The function of Evodiamine on the invasion and the migration of SGC7901 cells and the effect of EMT.1.The effect of evodiamine on the viability of SGC7901 cells was detected by MTT assay to determine the evodiamine concentration for subsequent experiments.2.Scratch assay ? transwell assay were used for examining evodiamine on SGC7901 cells migration and invasion ability.3.The effect of evodiamine on the protein expression of N-cadherin and E-cadherin in SGC7901 cells ware tested by Western blot.(3)Effect of Zuojinfang and its main component berberine on epithelial-mesenchymal transition of SGC7901 cells1.Making Zuojinfang freeze-dried powder and using high performance liquid chromatography(HPLC)to clear the purity and content of freeze-dried powder.Comparison of standards,detecting single drug content of freeze-dried powder.2.Effect of Zuojinfang on the Growth of SGC7901 Cells by MTT Method.3.The function of Zuojinfang and berberine on migration and the invasion of SGC7901 cells were compared by scratch and Transwell test.4.Western blot test compared the effect of Zuojinfang and berberine on the expression of E-cadherin and N-cadherin protein in SGC7901 cells.Results:(1)The Effect and mechanism of berberine on the inhibition of epithelial mesenchymal transition in SGC7901 cells.1.The results of MTT showed that berberine had a significant effect on the growth inhibition of SGC7901,and the IC50 of berberine-treated SGC7901 cells at 24 h was 127?M.10,20,40?M berberine had inhibitory effect on SGC7901 cell activity,but the killing effect was weak,so this concentration range was determined for following experiments.2.Scratch and Transwell experiments showed that berberine could significantly inhibit the migration and invasion of SGC7901 cells,and had a concentration dependence.3.Berberine obviously increased the expression of E-cadherin mRNA,compared with the control group was significantly different(P <0.01),and which is in a concentration-dependent manner.Berberine at 10?M and 20?M reduced the transcription level of N-cadherin(P <0.05).4.Immunofluorescence and Western bolt detection of berberine in SGC7901 cells showed that berberine could significantly increase E-cadherin and Zo-1expression in SGC7901 cells,and contraily down regulate MMP9,Snail,Vimentin,and N-cadherin protein expression in SGC7901 cells.The PI3K/AKT pathway protein showed that with the increase of concentration,berberine could significantly reduce the level of pAKT expression.5.Western bolt results showed that berberine 40 uM was added to SGC7901 cells treated with activator IGF-1(100 ng/mL)for 24 h,compared with IGF-1alone,E-cadherin was sharply increased,N-cadherin and pAKT decreased clearly.The inhibitor LY294002(10 uM)alone or berberine combined with SGC7901 cells showed significant difference in the expression of E-cadherin,N-cadherin,pAKT(P < 0.05).(2)The function of Evodiamine on invasion and the migration of SGC7901 cells and the effect of EMT.1.The results of MTT showed that evodiamine had a strong lethality on SGC7901 cells,and there was a clearly relationship between the aging and dose-dependent effects.The killing effect of 0.1,0.2,0.4 uM evodiamine on SGC7901 cells is weak,so this concentration range is selected for subsequent experiments.2.The results of scratch test and Transwell performed 0.2 and 0.4 uM evodiamine could distinctnessly prevent the migration and the invasion of SGC7901 cells.3.The results of Western bolt showed that the expression of E-cadherin and N-cadherin had no obvious change after the effect of 0.1,0.2,0.4 uM evodiamine on SGC7901 cell.(3)Effect of Zuojinfang and its main component berberine on epithelial-mesenchymal transition of SGC7901 cells.1.HPLC showed that Zuojinfang Freeze-dried powder was with 9.85%berberine.2.The results of MTT expressed that Zuojinfang had a sharp effect on the growth suppression of SGC7901.Compared with 40?M(16.3 mg / L)berberine and 165 mg / L(16.3 mg / L berberine)Zuojinfang,the prohibitive function of Zuojinfang on SGC7901 cells was stronger.3.Scratches and transwell experiments showed that Zujinfang and berberine could significantly inhibit the migration and invasion ability of SGC7901 cells,while there was no especial otherness between Zuojinfang group and berberine group.4.Western bolt results showed that 165 mg / L Zujinfang group and 16.3mg / L berberine group E-cadherin,N-cadherin expression was not statistically different.Zuojinfang and berberine could obviously inhibit the EMT of SGC7901 cells,and berberine might play a major role in Zuojinfang.Conclusion:1.Berberine can inhibit the invasion and metastasis of gastric cancer cell SGC7901 and inhibit the epithelial-mesenchymal transition of SGC7901 cells by inhibiting PI3 K / AKT pathway.2.Evodiamine significantly inhibited the migration and invasion of SGC7901 cells,but had no obvious inhibitory effect on EMT.3.Zuojinfang can control SGC7901 cell growth,invasion and migration,prevent the SGC7901 cell epithelial-mesenchymal transition,in addition to berberine evodiamine other components also have synergistic inhibition of gastric cancer cells,but in inhibiting EMT,berberine is main valid components in compound. |
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