Effect Of Suppression Of Mcl-1 Gene Expression Via SiRNA On TRAIL-Induced Apoptosis Of Human Gastric Cancer SGC7901/ADR Cells

Objective To study the effect of suppression of myeloid cell leukemia-1 (Mcl-1) gene expression via siRNA on apoptosis of adriamycin (ADR)-resistant human gastric cancer SGC7901/ADR cells induced by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), and to investigate the possible mechanism of TRAIL resistance of SGC7901/ADR cellsMethods 1. Human gastric cancer drug-resistant cell line SGC7901/ADR cells were treated with final concentrations of 10, 50 and 100 ng/mL TRAIL for 48 h, respectively, and the control group(SGC7901/ADR cells untreated with TRAIL) and bank group (culture solution without cells)were set up at the same time. The cell viability of SGC7901/ADR cells in treatment groups were detected by MTT.2. SGC7901/ADR cells were treated with final concentrations of 10,50 and 100 ng/mL TRAIL for 24 h, respectively.and the control group were set up at the same time. Annexin V-FITC/PI double staining and the flow cytometry were used to detect the apoptosis rate of SGC7901/ADR cells3. SGC7901/ADR cells were treated with final concentrations of 10,50 and 100 ng/mL TRAIL for 48 h, respectively. The expression levels of Mcl-1 mRNA and protein were determined by real-time fluorescent quantitative PCR and Western blotting, respectively.4. SGC7901/ADR cells were transfected with Mcl-1-siRNA, NC-siRNA(negative control-siRNA) using Lipofectamine(?) 3000, respectively, meanwhile, the bank group(only Lipofectamine(?) 3000) should be set up. Real-time PCR and Western blotting were used to measure the expression levels of Mcl-1 mRNA and protein,in order to identify gene silencing effiacy.5. After treatment with TRAIL(50ng/mL) alone or transfected with Mcl-1-siRNA in combination with TRAIL(50ng/mL) treatment for 48 h, the cell viability of SGC7901/ADR cells in treatment groups were determined by MTT.6. After treatment with TRAIL(50ng/mL) alone or transfected with Mcl-1-siRNA in combination with TRAIL(50ng/mL) treatment for 24 h, the apoptosis rates of SGC7901/ADR cells in treatment groups were measured by flow cytometry.7. After treatment with TRAIL(50ng/mL) alone or transfected with Mcl-1-siRNA in combination with TRAIL(50ng/mL) treatment, the expression levels of active-caspase 9, active-caspase 3 and Cytochrome C protein were determined by Western blotting. respectively.Results 1. Compared wiith the control group, TRAIL can decrease the survival rate of SGC7901/ADR cells, but with the increasing of TRAIL concentration, the degree of decrease for the survival rate of SGC7901/ADR cells were not obvious.2.The result of flow cytometry showed that TRAIL could induce apoptosis of SGC7901/ADR cells. With the increasing of TRAIL concentration, the apoptosis rate of SGC7901/ADR cells were not significantly increased, and there was no significant differences between 50 ng/mL TRAIL group and 100ng/mL TRAIL group.3. The results of real-time fluorescent quantitative PCR and Western blotting showed that the expression levels of Mcl-1 mRNA and protein were up-regulated after treatment with TRAIL.4.After transfected with Mcl-1-siRNA, the expression levels of Mcl-1 mRNA and protein in SGC7901/ADR cells were significantly down-regulated.5. Compared with TRAIL alone, combination of TRAIL and Mcl-1-siRNA more significantly inhibited the proliferation of SGC7901/ADR cells.6. Compared with TRAIL alone, Mcl-1 gene suppress by Mcl-1-siRNA can further enhance TRAIL-induced apoptosis of SGC7901/ADR cells.7. Compared with TRAIL alone, Down-regulation of Mcl-1 gene by Mcl-1-siRNA can further increase the expression levels of active-caspase 9, active-caspase 3 and Cytochrome C protein induced by TRAIL. Conclusion 1. SGC7901/ADR cells were not very senstive to TRAIL, and TRAIL could significantly up-reguate the expression levels of Mcl-1 mRNA and protein in SGC7901/ADR cells.2. Down-regulation of Mcl-1 gene by by siRNA interference enhances human gastric cancer cell line SGC7901/ADR cells to TRAIL-Induced Apoptosis., which indicate gastric cancer drug resistance cell line SGC7901/ADR cells are not sensitive to TRAIL may be associated with high expression of Mcl-1.