Study On The Mechanisms And Intervention Of Human Esophageal Cancer Multidrug Resistance Cells

Part I. Establishment of human esophageal cancer cells mult id rug resistant cell line YES-2/DDP and its biological characteristicsObjective To establish human esophageal cancer cells multidrug resistant cell line YES-2/DDP and to analyze its biological characteristics.Methods In this study, increasing concentrations and intermittent of cisplatin was applied to establish YES-2/DDP resistant strains. The half of inhibition concentrations (IC50) and resistance indexs (RI) of different anticancer drugs were detected by CCK-8in wild-type and drug resistance human esophageal cancer cells. Further the cell adherence rate, growth curve, doubling time differences, and double-layer soft-agar colony formation rate were observed in the YES-2/WT and YES-2/DDP cells. Cell cycle, rhodamine efflux, apoptosis were detected by flow cytometry, respectively. The MDR-1, MRP-1and LRP protein expression were detected by Western blotting.Results During the nine months, human esophageal cancer YES-2/DDP resistance cells line was induced and its resistance index to cisplatin was16.4, and YES-2/DDP cells for other types of anticancer drugs are different degrees of resistance, Freezing and withdraw of drug had not affect to the RI of YES-2/DDP. Compared to YES-2/WT Cell line, YES-2/DDP showed a slow growth curve, increased cell population doubling time and cell colony formation, decreased the cell cycle in G1and S phase and increased the proportion of G2phase. Western blot result showed that in the resistant cell line YES-2/DDP, MRP1protein expression was significantly increased, while the expression of MDR1and LRP was no significant changed. The concentration of rhodamine was markedly decreased in the YES-2/DDP cells compared with YES-2/WT cells. Flow cytometry and fluorescence microscopy analysis showed that72h after treatment with cisplatin (10μg/ml), the number of cell apoptosis was significantly in YES-2/DDP cells compared with YES-2/WT cells.Conclusion We successfully established a human esophageal cancer multidrug resistance cell line YES-2/DDP. The resistance may be related to increased MRP1expression, resulting in reducing intake and increasing efflux of anti-cancer drugs, which inhibit the effect of anticancer drugs on tumor cell apoptosis. Part Ⅱ Study on the mechanisms of multidrug resistance in human esophageal cancer cellsObjective To explore the relationship of COX-2, PPAR-β and MRP-1in the YES-2/DDP cells and reveal the molecular mechanism of multidrug resistance in human esophageal cancer cells.Methods1. The expression of COX-2, PPAR-β protein in the YES-2/WT and YES-2/DDP cells were determined by western blotting, and further the production of PGE2and PPRE report gene activity were assayed by enzyme-linked immunosorbent assay and luciferase assay, respectively.2. YES-2/DDP cells were administrated with NS398(25μM), or PGE2(μM) for72h, respectively, the expression of MRP-1and PPAR-β, PPRE luciferase reporter gene activity, the concentration of cellular rhodamine, the IC50and RI of cisplatin at48h were determined, respectively. Further the concentration of intracellular cisplatin at1h and the rate of cell apoptosis at72h after treatment with10μg/ml cisplatin were assayed.3. YES-2/DDP cells were administrated with GSK0660(2μM) or transfer5μg of PPAR-(3expression plasmid for72hours, respectively, the expression of MRP-1and COX-2, PGE2production, the concentration of cellular rhodamine, the IC50and RI of cisplatin at48h were determined, respectively. Further the concentration of intracellular cisplatin at1h and the rate of cell apoptosis at72h after treatment with10μg/ml cisplatin were assayed.Results1. Compared with YES-2/WT cells, COX-2protein expression and PGE2production were significantly increased in the YES-2/DDP cells, further PPAR-β expression and PPRE activity were also increased.2. Compared with YES-2/DDP cells, in the25μM NS398-treated YES-2/DDP cells, the expressions of MRP-1and PPAR-β protein, the PPRE activity, IC50and RI of cisplatin were significantly reduced, while the concentrations of intracellular Rhodamine were significantly increased. Further the concentration of intracellular cisplatin at1h and the rate of cell apoptosis at72h after treatment with10μg/ml cisplatin were significantly increased. Compared with the YES-2/DDP cells, in the1μM PGE2-treated YES-2/DDP cells, the expressions of MRP-1and PPAR-β protein, the PPRE activity, IC50and RI of cisplatin were significantly increased, while the concentrations of intracellular Rhodamine were significantly reduced. Further the concentration of intracellular cisplatin at1h and the rate of cell apoptosis at72h after treatment with10μg/ml cisplatin were significantly reduced.3. Compared with YES-2/DDP cells, in the2μM GSK0660-treated YES-2/DDP cells, the expression of COX-2protein and PGE2production had no changes, but the MRP-1protein expression, IC50and RI of cisplatin at48h were markedly reduced; the concentrations of intracellular Rhodamine were significantly increased. Further the concentration of intracellular cisplatin at1h and the rate of cell apoptosis at72h after treatment with10μg/ml cisplatin were significantly increased.4. Compared with the YES-2/DDP cells, in the5μg of PPAR-β expression plasmid-transfected YES-2/DDP cells, the expression of COX-2protein and PGE2production had no changes, but the MRP-1protein expression, IC50and RI of cisplatin at48h were markedly increased; the concentrations of intracellular Rhodamine were significantly reduced. Further the concentration of intracellular cisplatin at lh and the rate of cell apoptosis at72h after treatment with10μg/ml cisplatin were significantly reduced.Conclusion1.The COX-2protein expression and PGE2production might mediate the expression of MRP1protein, which reduced the concentration of intracellular cisplatin, resulting in reducing the toxicity and apoptosis of the antitumor drugs in human esophageal cancer cells.2. The PPAR-β protein expression and activation might mediate expression of MRP1protein, which reduced the concentration of intracellular cisplatin, resulting in reducing the toxicity and apoptosis of the antitumor drugs in human esophageal cancer cells.3. In conclusion, the mechanism of multidrug resistance in human esophageal cancer cells YES-2/DDP may be through the promotion of COX-2/PGE2system, increasing of PPAR-β protein expression and activity, which mediated MDR1expression, and promoted the efflux of antitumor drugs, lead to reducing the concentrations of anti-tumor drugs, thereby reducing the cellular toxicity of these drug, resulting in multidrug resistance. Part Ⅲ.Effect and mechanism of tetrandrine on the YES-2/DDP resistant cellsObjectiveTo research the effect of tetrandrine on human esophageal cancer resistant cell lines YES-2/DDP, and further explore its possible mechanism.MethodsHuman esophageal cancer cells were divided into four groups:the control group (no intervention tetrandrine),10μ.M tetrandrine in the intervention group,30μM tetrandrine in the intervention group,100μM tetrandrine in the intervention group. At72hours after tetrandrine administration, the COX-2, PPAR-β and MRP expression were determined by western blotting, PGE2levels were assayed by enzyme-linked immunosorbent assay, PPRE luciferase enzyme activity was detected, intracellular Rhodamine concentration was analyzed by flow cytometry, the IC50and RI of cisplatin at48h were detected by CCK-8. Further the concentrations of intracellular cisplatin were measured by HPLC at1h after treatment with10μg/ml cisplatin.Results1. Compared with YES-2/DDP control cells, tetrandrine dose-dependently inhibited the expression of COX-2protein and the production of PGE2, PPAR-β expression and activation. Further we also showed that tetrandrine inhibited the expression of MRP-1protein and accumulation of cellular rhodamine in a dose-dependent maner.2. When cells were treated with10μg/ml cisplatin in different intervention groups, tetrandrine concentration-dependently increased the concentration of intracellular cisplatin, decreased the IC50and RI of cisplatin.ConclusionTetrandrine could effectively reverse the resistance of YES-2/DDP resistant cells line; the mechanism may be related to inhibition of COX-2expression and production of PGE2, PPAR-β expression and activity, and the expression of MRP-1.