Diallyl Disulfide Inhibit Migration And Invasion In Human Colon Cancer SW480 Cells Of Overexpression Of UPRA

Objective: diallyl disulfide(DADS),the main component in garlic, is great potential for development of cancer drugs. Our previous studies reported that diallyl disulfide(DADS) plays different roles in different tumors, such as inhibiting proliferation,migration and invasion in human lung cancer SW480 cells. Urokinase-type plasminogen activator receptor(urokinase plasminogen activator, uPAR) can reverse tumor cell EMT, inhibit tumor cell invasion and metastasis, through blocking uPAR signaling pathway. So far, the effect of DADS to inhibit tumor cell invasion,metastasis and EMT was rarely reported. The issue on the basis of former studies, constructed an overexpression uPAR SW480 cell lines, researching DADS downregulated uPAR by signaling pathway to mediate inhibition of human colon cell proliferation, EMT, invasion and migration, confirming that uPAR plays a role of key molecule that DADS inhibited tumor EMT, invasion and metastasis.Which provides experimental evidence for the clinical development and application of DADS.Methods: pIRES2-EGFP-uPAR eukaryotic expression vector was transfected into SW480 cells to establish stable cell lines. Transwell invasion assay and wound healing assay detected the effect of DADS on migration and ivasion in SW480 cells of overexpression of uPAR. RT-PCR and Western blot detected the effect of DADS on EMT and uPAR related signaling molecules in SW480 cells of overexpression of uPAR. nude mice xenograft experiments detected the effect of DADS on the growth of tumor in SW480 cells of overexpression of uPAR.Results:1. The successful construction of uPAR/SW480 cells with stably overexpressed uPAR.The pIRES2-EGFP and pIRES2-EGFP-uPAR eukaryotic expression vector was transfected into SW480 cells. Green fluorescence was visible under the fluorescence microscopy. Which shows that the pIRES2-EGFP and pIRES2-EGFP / uPARtransfected into SW480 cells. PCR and Western blot results showed that uPAR mRNA and protein expression in uPAR/SW480 cells increased.A stable uPAR / SW480 cell line was constructed.2.Effect of DADS on the migration in SW480 cells of overexpression of uPAR.Wound healing test showed that, the migration distance of uPAR / SW480 cells is significantly longer than SW480 cells(P<0.05).After DADS treatment, the migration distance of vector/SW480 cells is significantly shorter than SW480 cells and uPAR/SW480 cells(P<0.05), the migration distance of uPAR/SW480 cells is significantly shorter than it before DADS treatment(P<0.05).3. Effect of DADS on the invasion in SW480 cells of overexpression of uPAR.Invasion test showed that, the invasion cells number of uPAR / SW480 cells is significantly more than SW480 cells(P<0.05).After DADS treatment, the invasion cells nunmer of vector/SW480 cells is significantly fewer than SW480 cells and uPAR/SW480 cells(P<0.05), the invasion cells nunmer of uPAR/SW480 cells is significantly fewer than it before DADS treatment(P<0.05).4. Effect of DADS on the EMT in SW480 cells of overexpression of uPAR.RT-PCR and Western blot showed that,the expression level of vimentin in uPAR/SW480 cell is significantly higher than SW480 cells,and the expression level of E-cadherin is lower than SW480 cells(P<0.05).After DADS treatment, the expression level of vimentin in vector/SW480 cells is significantly lower than SW480 cells and uPAR/SW480 cells,and the expression level of E-cadherin is higher than SW480 cells and uPAR/SW480 cells(P<0.05).5. Effect of DADS and uPAR on the expression of,the uPAR uPA, MMP-9 in SW480 cells.According to the results of Western blot and RT-PCR, the expression level of uPAR,uPA,and MMP-9 in uPAR/SW480 cell is significantly higher than SW480 cells(P<0.05).After DADS treatment, the expression level of uPAR,uPA,and MMP-9 in vector/SW480 cells is significantly lower than SW480 cells and uPAR/SW480cells(P<0.05), the expression level of uPAR,uPA,and MMP-9 in uPAR/SW480 cells is significantly lower than it before treatment(P<0.05).6.Effect of DADS and uPAR on SW480 cells in nude mice xenograft experiments.nude mice xenograft experiments results showed that tumor volume and weight in DADS treated group were significantly different,compared with the related untreated groups(P <0.05),.Which shows that DADS inhibit tumorigenicity of SW480 cells. There were significant differences between uPAR / SW480 and SW480(P <0.05), confirming that overexpression of uPAR increase SW480 cells tumorigenic capacity.Conclusion:1. Overexpression of u PAR can promote EMT,migration,invasion and tumorigenicity in SW480 cell.2.. DADS can inhibit EMT,migration,invasion and tumorigenicity in SW480 cells of overexpression of uPAR.