| Objective: To investigate whether upregulation of ROR? expression by DADS can enhance apoptosis and sensitivity to 5-FU in SGC-7901,SGC-7901/VCR and SGC-7901/T0901317 cells,respectively.Methods: SGC-7901 cells were treated with ROR? inhibitor T0901317 and treated with DADS for three groups of SGC-7901,SGC-7901/VCR and SGC-7901/T0901317,respectively.Under the different concentration of 5-FU,the change of proliferation inhibition rate of SGC-7901 cells in each group was detected by MTT assay.The change of apoptosis rate of SGC-7901 cells in each group was detected by flow cytometry.The expression of Bcl-2,XIAP,Caspase-3 and P-gp in each group of SGC-7901 cells was detected by immunofluorescence and Western blot.Results:1.ROR? low expression of SGC-7901 cellsSGC-7901 cells were treated by using the ROR? inhibitor T0901317.The expression of ROR? in SGC-7901 cells was significantly decreased(P<0.05).However,the expression of ROR? in SGC-7901 cells was significantly decreased after treatment with SGC-7901/T0901317 increased(P<0.05).2.Effect of DADS and downregulation of ROR? on the proliferation in SGC-7901 cellsThe results of MTT showed that the proliferation inhibition rates of SGC-7901 were 1.95%,5.09% respectively after being treated with 5-Fu of 1.25mg/L,2.5mg/L,5mg/L,10mg/L and 20mg/L,13.43%,22.87% and 46.63%,respectively.The inhibition rate of SGC-7901 group after DADS treatment was 4.33%,9.85%,22.5%,48.17% and 57.44%,respectively(P<0.05).The proliferation inhibition rates of SGC-7901/VCR were 0.18%,0.59%,1.01%,2.82% and 4.69%,respectively(P<0.05).The inhibition rates of SGC-7901/VCR treated with DADS were 2.23%,3.31%,7.3%,19.43% and 27.91%,respectively(P<0.05).The proliferation inhibition rate of SGC-7901/T0901317 group was 1.24%,4.3%,11.83%,18.34% and 40.89%,respectively.Compared with SGC-7901 group,the proliferation inhibition rate was significantly decreased(P<0.05).The inhibitory rates of SGC-7901/T0901317 group after DADS treatment were 2.24%,8.7%,19.54%,40.83% and 52.34%,respectively(P<0.05).The results suggest that down-regulation of ROR? expression can enhance cell proliferation and increase the resistance of cells to 5-FU to a certain extent.DADS can inhibit the cell proliferation of SGC-7901,SGC-7901/VCR and SGC-7901/T0901317,and reduce the cell resistance to 5-Fu to a certain extent.3.The effect of DADS and downregulation of ROR? on the apoptosis of SGC-7901 cellsFlow cytometry showed that the apoptosis rates of SGC-7901,SGC-7901/VCR and SGC-7901/T0901317 group were 6.8%,1.7% and 2.91%,respectively.The apoptosis rate of SGC-7901/T0901317 group was significantly lower than that of SGC-7901 group(P<0.05).The apoptotic rates of SGC-7901,SGC-7901/VCR and SGC-7901/T0901317 group were 12.66%,7.88% and 9.54%,respectively after DADS treatment,which were significantly higher than those of untreated group(P<0.05).It indicated that low ROR? expression could reduce the apoptosis rate of SGC-7901 cells and DADS could increase the apoptosis rate of SGC-7901 cells.4.Effect of DADS and down-regulation of ROR? in SGC-7901 cell-related protein expressionThe results of Western blot showed that the expression of Bcl-2,P-gp and XIAP in SGC-7901 group and the untreated SGC-7901 group were significantly increased(P<0.05)and Casepase3 protein(P<0.05).After DADS treatment,the expressions of Bcl-2,P-gp and XIAP in SGC-7901,SGC-7901/VCR and SGC-7901/T0901317 group were significantly decreased(P<0.05).The results of immunofluorescence showed that the fluorescence of Bcl-2,P-gp and XIAP in T0901317-treated SGC-7901 group was enhanced and that of Casepase3 was decreased in untreated SGC-7901 group.After DADS treatment,the fluorescence of Bcl-2,P-gp and XIAP in SGC-7901,SGC-7901/VCR and SGC-7901/T0901317 group was weakened and the fluorescence of Casepase3 was enhanced.Immunofluorescence and Western blot results suggest that the resistance-related proteins and apoptosis-inhibitory protein can be upregulated by ROR? low expression,and pro-apoptotic protein by low ROR? expression down.After treated with DADS,the expression of drug-resistance-related protein and apoptosis protein were down-regulated and the expression of pro-apoptotic protein was up-regulated.Conclusions:1.Low-expression of ROR? can up-regulate the expression of Bcl-2,P-gp and XIAP,and down-regulate the expression of Casepase3,which inhibit the apoptosis and decrease the chemosensitivity to 5-FU in SGC-7901 cells.2.DADS could down-regulate the expression of Bcl-2,P-gp and XIAP,and upregulate the expression of Casepase3 through upregulating the expression of ROR? increase apoptosis and increase chemosensitivity of SGC-7901 cells to 5-FU. |
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