Establishment Of Cisplatin-resistant Gastric Cancer Cell SGC7901/DDP And Study On Its Drug Resistant Mechanism And Reversal Of Tamoxifen

Objective:1. To establish cisplatin-resistant gastic cancer SGC7901/DDP and provide the cell model for multidrug resistance study;2. To explore the resistance reversal mechanisam of cisplatin-resistant gastic cancer SGC7901/DDP with estrogen receptor antagonist tamoxifenMethods:A cisplatin-resistant gastic cancer SGC7901/DDP was established by increasing cisplatin concentration step by step and continuing induction. Then we observed the morphological changes of SGC7901/DDP through an inverted microscope, draw the cell growth curve and calculate the population doubling time of SGC7901/DDP by cell counting. The resistance index and cross-resistance characteristics of SGC7901/DDP cells was detected by CCK-8assay. The drug resistance associated genes expression of SGC7901/DDP cells and SGC7901cells were detected by Real time Quantitative PCR.The Rhodaminel23test and the cell cycle of drug-resistant cells interference with TAM was detected by flow cytometry(FCM). The influences of TAM and DDP on drug-resistant cells apoptosis was detected by double fluorescent labeling with Annexin V-PE.7-AAD. Multidrug resistance associated proteins were detected by western blotting.Results:We took6months to raise the cisplatin concentration8times and successfully established the drug-resistant gastric cancer cell SGC7901/DDP which could tolerate lug/ml DDP by gradually increasing cisplatin concentration and continuing exposure. Compared with SGC7901under inverted microscope, SGC7901/DDP cells showed heterogeneity for the size, irregular shape, unclear boundaries, cell volume slightly increased, irregular nuclear and giant cells. Compared with SGC7901cells, the proliferation rate of SGC7901/DDP cell slowed down and the population doubling time extended approximately5hours (P<0.05) from the cell growth curve. The resistance index of SGC7901/DDP to DDP,5-FU, ADM were9.86,5.69and2.51respectively, indicating SGC7901/DDP cross-resistance characteristics. The multidrug-resistance associated genesMDR-1、MRP1、MRP2、MRP3、MRP5、Survivin and ERCC1of SGC7901/DDP cells expressed higher than those of SGC7901cells and the expressionof apoptosis associated genes Bax and Caspase-3were increased and Bcl-2was decreased by real time Quantitative PCR. The expression of resistance associated proteins P-gp and survivin of SGC7901/DDP were higher than SGC7901by western blotting.After SGC7901/DDP cells were interference with TAM the expression of multidrug-resistance associated gene MDR1、MRP1、MRP2、MRP3、MRP5,ERCC1and apoptosis gene Bcl-2, Survivin decreased and the expression of the apoptosis gene Bax and Caspase-3enhanced. After SGC7901/DDP cells were interferenced with TAM the cells of G0/G1phase and G2/M phase increased, the cells of S phase decreased, and apoptosis peak appeared before G1phase. Resistance associated protteins P-gp,Survivin significantly lower with TAM than before.Conclusion:1. The cisplatin-resistance gastric cancer cell line SGC7901/DDP was established by increasing the concentration step by step and continuing induction.2. SGC7901/DDP cells had multidrug resistant characteristics. The possible multidrug resistant mechanism of SGC7901/DDP correlate with the population doubling time extending and multidrug resistance gene MDR1、MRP1、MRP2、MRP3、MRP5Survivin and ERCC1upregulation and apoptosis associated proteins P-gp,Survivin have effect on multidrug resistance.3. The resistance reversal by TAM maybe related to the upgratulation of Bax、Caspase-3and downregulation of MDR1、MRP1、MRP2、MRP3、MRP5、 ERCC1、Bcl-2, Survivin and resistance associated proteins P-gp and Survivin...