| Objective:Our previous experiments have confirmed that DADS treatment can inhibit the migration and invasion of gastric cancer cells with high expression of DJ-1.There is no related study on DADS and down-regulation of DJ-1 and apoptosis and drug resistance of gastric cancer cells.In this study,human gastric cancer SGC7901 cells,SGC7901 / VCR and SGC7901 / DJ-1 were used to study the effect of DADS and down-regulation of DJ-1 on gastric cancer cells,to investigate whether it could enhance the apoptosis of SGC7901 cells and also enhance the sensitivity to 5-Fu,and to further explore that DJ-1 was one of the targets of DADS to enhance the chemosensitivity of gastric cancer.To provide theoretical basis for molecular targeted therapy of gastric cancer.Methods: Stable SGC7901 cells line with high expression of DJ-1 was constructed.The DJ-1 protein level in SGC7901 cells was detected by Western blot.MTT detect the cell proliferation inhibition in SGC7901,SGC 7901 + DADS,SGC7901/VCR,SGC 7901/VCR + DADS,SGC7901/DJ-1 and SGC7901/DJ-1+DADS.The rate of cell apoptosis was detected by flow cytometry.Western blot,q RT-PCR and the immunofluorescence prescription were used to detect the expression of protein and mRNA of XIAP,Caspase-3,Bcl-2 and P-gp in SGC7901 cell lines treated with 30 mg / L DADS.Results: 1.Stable SGC7901 cells line with high expression of DJ-1 was established successfully.The high expression lentivirus vector of DJ-1 was constructed by the purchasing company.The high expression lentivirus vector of DJ-1 was transfected into SGC7901 cell line of human gastric cancer,and the lentivirus transfection was promoted by adding polybrene adjuvant.After 24 hours of transfection,0.25 ugrml puromycin medium was added.The transfected cells were observed under fluorescence microscope,and SGC-7901 cell lines with moderate expression of DJ-1 were selected.The cells were frozen after amplification and were verified by Western blot.It was found that the expression of DJ-1 in SGC7901/DJ-1 was significantly higher than that in SGC7901 cells(P< 0.05),and DJ-1 down-regulation in SGC7901/DJ-1 DADS group was the most obvious(P< 0.05).This indicated that the high expression of DJ-1 in SGC7901 cell line with high DJ-1 expression could be further used in further experiments and DADS could down-regulate DJ-1 expression.2.Effect of DADS and DJ-1 overexpression on proliferation of human gastric cancer SGC7901 cells.MTT results showed that in different concentrations of 5-Fu,the cells of SGC7901 cell lines were treated by DADS as compared to the untreated inhibition rate was significantly increased(P<0.05),cell proliferation was significantly decreased,but the high expression of DJ-1 in group DADS after treatment compared with the untreated group SGC7901 proliferation the rising rate of SGC7901 in combination with no resistant strains of SGC7901/VCR combination significantly.When the concentration of 5-Fu was 1.25mg/L,2.5 mg/L,5 mg/L,10 mg/L and 20 mg/L,SGC7901 and SGC7901/VCR were 2.2%,4.9%,16.1%,23.2% and 44.6% and 0.2%,0.8%,1.5%,3.1% and 4.4%.Compared with DADS treatment,SGC7901 and SGC7901/VCR inhibition rates were 4.1%,10.3%,25.1%,47.4% and 58.6% and 1.9%,3.5%,7.4%,20.3% and 29.4% increased significantly(P<0.05).In the same concentration of 5-Fu,high expression of SGC7901 DJ-1 cell proliferation inhibition rates were 1.1%,3.1%,12.1%,17.8% and 35.8%,treated with DADS over expression of DJ-1 in SGC7901 cells after inhibition rates were 2.3%,7.4%,17.3%,30.9% and 51%,the inhibition rate of the former was significantly increased(P<0.05).This indicates that DADS can inhibit SGC7901 proliferation of SGC7901/VCR cells,while DADS can also be enhanced through down-regulation of DJ-1 on the expression of SGC7901/DJ-1 inhibited the proliferation of DJ-1 cells,and enhance the drug sensitivity of 5-Fu.3.The effect of high expression of DADS and DJ-1 on apoptosis of human gastric cancer SGC7901 cell lines.The results of flow cytometry showed that the apoptotic rates of SGC7901 / VCR and SGC7901/DJ-1 in the control group were 6.5% and 3.2%.Respectively,The apoptosis rate of SGC7901/VCR was the lowest and the highest was SGC7901.After treated with DADS,the apoptotic rates of SGC7901 / VCR and SGC7901/DJ-1 were 13.991% and 10.26%,respectively,which were significantly higher than those of the control grouP(P< 0.05).4.The effect of high expression of DADS and DJ-1 on the expression of SGC7901 cell protein in each group.Western blot showed that the protein expression of protein P-gp,Bcl-2 and XIAP in SGC-7901 / DJ-1 cells was significantly up-regulated than that in SGC7901 and SGC7901/VCR cells(P< 0.05).After DADS treatment,the expression of P-gp,Bcl-2 and XIAP in SGC7901 ? SGC7901/VCR cells and SGC7901/DJ-1 cells was significantly down-regulated(P< 0.05).Before DADS treatment,the expression of Caspase-3 protein in SGC7901/DJ-1 with stable overexpression of DJ-1 was lower than that in SGC7901 and SGC7901/VCR cells(P< 0.05).Caspase-3 protein was up-regulated in SGC7901,SGC7901 / VCR and SGC-7901/DJ-1 cells after DADS treatment(P< 0.05).QRT-PCR results showed that compared with SGC7901,the expression of mRNA in the high expression group of P-gp,Bcl-2 and XIAP are significantly up-regulated(P< 0.05),while the expression of Caspase-3 mRNA are significantly down-regulated(P< 0.05).After treatment with 30 mg / L DADS for 24 h,the expression of P-gp,Bcl-2 and XIAP mRNA in the experimental group are significantly down-regulated(P< 0.05),while the expression of Caspase-3 mRNA was significantly up-regulated(P< 0.05).Immunofluorescence showed that in the control group,the fluorescence intensity of the cells transfected with DJ-1 protein of P-gp,Bcl-2 and XIAP were significantly higher than that of SGC7901 and SGC7901/VCR cells,but it was treated with DADS 30 mg / L for 24 h,compared with the untreated group,the staining of P-gp,Bcl-2 and XIAP protein in the three groups was decreased.The fluorescence expression of Caspase-3 in SGC7901/DJ-1 was lower than that in SGC7901 and SGC7901/VCR cells,and the fluorescence intensity in SGC7901/VCR group was lower than that in SGC7901 group.After DADS treatment,Caspase-3 fluorescence expression in all groups was significantly increased.The results were consistent with those of qRT-PCR-Western Blot.Conclusion: 1.The high expression of DJ-1 could up-regulate the expression of P-gp,Bcl-2 and XIAP and down-regulate the expression of Caspase-3 and inhibit the apoptosis of SGC7901 cells,and also could down-regulate the sensitivity to 5-Fu.2.The down-regulation of DJ-1 by DADS could enhance the apoptosis of SGC7901 cells and its sensitivity to 5-Fu was related to the down-regulation of P-gp,Bcl-2 and XIAP and up-regulation of Caspase-3. |
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