Experimental Study On Reversal Effect Of GBEE On S180MDR Cell Line

Objective:According to the clinical PFC(DDP+FU+CPA) scheme and using the gradually increasing dose to induce S180 ascites tumor mice in vivo to obtain S180 tumor multidrug resistance(MDR) cell line and doing a study of the reversal activity of ginkgo biloba exocarp extracts(GBEE) on it in vitro and in vivo and its related mechanisms.Methods:According to the clinically chemotherapetic PFC scheme and using the gradually increasing dose to induce S180 ascites tumor mice and establish the obtained mouse model of S180MDR.The resistance factor of induced cells by low, medium and high dose of chemotherapeutics drugs was determined by MTT assay, the accumulation of adriamycin(ADR) and functional activity of P-glycoprotein(P-gp) were determined by flow cytometry,which all were used for monitoring the resitant strength of indued cells.The mRNA expression of MDR-1 and multidrug resistance-associated protein 1(MRP-1) of induced cells by different dose were detected by real time quantitative PCR(RT-qPCR).Taking the S180MDR cells induced by high dose to research the reversal effect of GBEE in vitro and in vivo.Studies in vitro:MTT assay was used to detect the reversal index of GBEE on S180MDR cells;Using flow cytometric technology to detect the influence of GBEE on the accumulation of ADR and the functional activity of P-gp on S180MDR cells. Studies in vivo:S180MDR ascites and S180MDR implanted solid tumor mice models were established.The mice were randomly divided into blank group,model group,parent cells cis-dichlorodiamineplatinum(DDP)3mg/kg、ADR3mg/kg drug groups,S180MDR cells DDP3mg/kg、ADR3mg/kg、GBEE50、25、12.5mg/kg、Verapamil(VER)15mg/kg drug groups,S180MDR cells GBEE50、25、12.5mg/kg、VER15mg/kg+DDP3mg/kg combination groups,14 groups in total,10 mice in each group.With the drug intervention 1 week, monitoring the survival time of ascites tumor mice and calculating the tumor inhibition rate of solid tumor mice.The mRNA expression of MDR-1 and MRP-1 in different group cells of ascites tumor mice were detected by RT-qPCR.The level of Interleukin 3(IL-3),Interleukin 18(IL-18)) and Interferon-γ(IFN-γ) in blood serum of solid tumor mice were evaluated by double antibody ELISA.Results:The results of model experiment show that,compared with parent cells,the resistant factors of induced S180 cells induced by high,medium and low three doses to chemotherapeutics drugs were gradually increased with increasing the induced dose, the accumulation of ADR was gradually reduced with increasing the induced dose,the functional activity of P-gp was gradually strengthened with increasing the induced dose.The induced S180 cells not only have a strong resistance to DDP and fluorouracil(FU),but also have a certain degree of cross resistance to ADR,which hasn’t been contacted and has a different function mechanism.The mRNA expression of MDR-1 and MRP-1 of induced cells had a positive correlation to the induced dose.In vitro,the reverse indexes of GBEE30、15、7.5ug/mL to S180MDR cells were 8.48、11.34,9.88.Each dose of GBEE could significantly increase the accumulation of ADR and inhibit the functional activity of P-gp.In vivo,GBEE50、25、 12.5mg/kg could effectively reverse the resistant about S180 MDR of ascites and soild tumor cells to DDP,could significantly extend survival time of ascites tumor mice and inhibit the tumor growth in solid tumor mice. The life-prolonging rate of GBEE25mg/kg combined with DDP group on ascites tumor in mice was 37.41%,the inhibition rate of solid tumor mice was 65.43%(P< 0.01 or P< 0.001).Each dose of GBEE could inhibit the expression of MDR-1mRNA and MRP-1mRNA in S180MDR ascites tumor mice cells and improve the levels of immune factors,such as IL-3、IL-18 and IFN-γ in blood serum of S180MDR solid tumor mice (P<0.05).Conclusion:According to the PFC scheme and using the gradually increasing dose to induce S180 ascites tumor mice to obtain the S180MDR cell model.In a definite dose range, there was a positive correlation between the resistant strength of obtained S180MDR cells and the induced dose.GBEE had the reversal effect of MDR tumor in vivo and in vitro,which may be related to inhibit the expression of MDR-1mRNA,MRP-1mRNA of tumor resistant cells,then reduce the composition of resistant protein and inhibit the functional activity of P-gp,which lead to the concentration of chemotherapetic drugs increased within tumor cells.What’s more,which may be also related to promote the level of immune factors of IL-3, IL-18 and IFN-γ in blood serum of mice.