The Effect Of Bmi-1 On Multidrug Resistance In K562/ADR Cells And Its Mechanisms

Background: Chronic myeloid leukemia(CML)is a clonal myeloproliferative disorder of a pluripotent stem cell;chemotherapy is a primary treatment of CML.However,one of the major reasons for chemotherapy failure and leukemia relapse is multidrug-resistance(MDR).The MDR1 gene/P-glycoprotein(P-gp)is a popular and important subject in the leukemic cells drug-resistance research field.Bmi-1 is a vital member of Polycomb group.Bmi-1 as an oncogene has been linked to oncogenesis and cancer progression in various types of human cancers including leukemia.However,the biological mechanism of Bmi-1 in multidrug resistance of leukemia remains unclear.In our present study,we choose Chronic myelogenous leukemia(CML)MDR cell line K562/ADR to find out whether Bmi-1 gene is involved in the MDR of K562/ADR cells or not,and further to explore its possible molecular mechanisms.Methods: 1.K562/ADR leukemia cells were engineered to be silenced for Bmi-1 expression using RNA interfering method by transient transfecting three already constructed transfectants 48 hours.2.Detected PTEN,p-AKT,NF-?B P65 in nuclear and P65 in cytoplasm,P-gp expressions and the function of MDR1/P-gp after knockdown Bmi-1.3.The expression of P-gp and MDR1/P-gp function were detected after K562/ADR cells treated with NF-?B inhibitor 80?M PDTC 24 h.4.Detected p-AKT,NF-?B P65 in nuclear and P65 in cytoplasm,P-gp expressions after PI3K/AKT inhibitor LY294002(20?M,24h)was used.5.The expression of p-AKT,NF-?B P65 in nuclear and P65 in cytoplasm,P-gp were detected after the Bmi-1-siRNA transfected cells were treated by 80 n M BPV(PTEN inhibitor)4h.The K562/ADR cells transient transfection efficiency was observed by fluorescence inversion microscope system.The expression of Bmi-1 and MDR1 mRNA were analyzed by RT-PCR.Various proteins of different groups were measured by Western Blotting.The drug efflux function of MDR1/P-gp was detected by flow cytometric analysis.Results: 1.Bmi-1 gene knockdown could inhibit NF-?B signaling(NF-?B p65 in nuclear was down regulated and P65 in cytoplasm was up regulated)and P-gp expression.Bmi-1 knockdown increased the doxorubicin intracellular accumulation,suggesting that MDR1/P-gp drug efflux function was reduced.2.The activity of NF-?B and P-gp expression was inhibited and the function of P-gp was reduced by using NF-?B inhibitor(PDTC)in K562/ADR cells.3.Bmi-1 gene knockdown could increase PTEN,inhibit p-AKT.4.Additionally,the expression of Ser473 p-AKT and expression of NF-?B P65 in nuclear as well as P-gp were inhibited significantly by using PI3K/AKT inhibitor LY294002.5.NF-?B activation and P-gp expression were restored when the Bmi-1-siRNA transfected K562/ADR cells were treated with PTEN inhibitor BPV(phen).Conclusions: The effect of Bmi-1 on MDR1 might via activating NF-?B through PTEN/PI3K/AKT pathway in K562/ADR cells.