Experimental Study Of Multidrug-resistance Leukemia Mechanism Reversed By Proteasome Inhibitor Bortezomib

ObjectiveLeukemia cells of multi-drug resistance(MDR)is one of the main reasons of chemotherapy failure and remission relapse.To overcome multidrug resistance(MDR) leukemia effect is to raise an important way.MDR(multidrug resistance,MDR)refers to tumor cells after exposure to a drug,not only for drug resistance,but also to other structures and mechanisms of different drugs have become drug resistant.It is a complex process caused by a combination of factors.Now the main reason of t drug resistance is that the mdr1 gene encoding the P-glycoprotein(P-gp)over-expression of anti-cancer drug efflux increased,lowering effect.The latest research results show that NF-κB can mediate the multi-drug resistance mechanism through up-regulated expression of P-gp/mdr1-mRNA.Anticancer drugs in the anti-rumor cells also activated NF-κB,NF-κB into the nucleus and combined,IκB sites,induced mdr1 gene expression. Protease inhibitor bortezomib by preventing degradation of IκB(NFκB of the negative regulator),could significantly inhibit the activation of NF-κB,down-regulate the expression of mdr1 gene,and induce cell apoptosis.So as to enhance treatment and reverse drug resistance.Recentl preclinical studies have shown that the proteasome inhibitor bortezomib decreases proliferation,induces apoptosis,enhances the activity of chemotherapy and radiation,,and reverses chemoresistance in a variety of hematologic and solid malignancy models in vitro and in vivo.At present on the bortezomib used in the study of tumor cell lines were more sensitive to chemotherapy,and the multi-drug resistant tumor cell lines seldom study.Using the multi-drug resistance of leukemia cell lines, we observed the change of P-gp/mdr1-mRNA,the cell cycle and apoptosis at same time when cells were treated by bortezomib for 24 h.In order to further explore in the relationship between changes of P-gp/mdr1-mRNA and cell apoptosis.We investigated the responsible molecular mechanisms of bortezomib reversed multi-drug resistance mechanism of leukemia.Materials1.K562/S and K562/DNR Cells.2.Protease inhibitor inhibitor Bortezomib and Daunorubicin.3.Reagents for MTT colorimetric assay.4.Multidrug Resistance Gene(MDR1)Fluorescence quantitative PCR Diagnostic kit.5.Anti-P-glycoprotein(P-gp).6.Reagents for FCM levels of Cell Cycle assay.and Annexin V-FITC / PI double labeled assay.Methods1.Cell CultureK562/S and K562/DNR cells were cultured in RPMI 1640 medium with12%fetal bovine serum,100units/ml penicillin,and 100ug/ml streptomycin.They were maintained in a 37℃,5%CO₂,fully humidified incubator.2.Determination of resistance of K562/DNR cells to Daunorubicin.K562/S and K562/DNR cells in logarithm growth stage were transferred in 96-well plates.Cells were cultured for 12 hours.Daunorubicin added at various concentrations after 68 hours,then detected with an MTT assay.Cell growth-inhibitory rate were assayed by a Spectra max miroplate at 540 nm.Complete medium was used as blank control.Repeated three wells were set up for each concentration in each time.3.Determination of the concentration of daunorubicin.K562/S and K562/DNR cells in logarithm growth stage were withdrawn,washed with ice-cold PBS three time,resuspended in fresh medium.Cell suspension were added or not bortezomib(final concentration of 10 nmol/L),together with the work of DNR,the DNR final concentration of 5 umol/L,were incubated 90 min at 37℃,The supernatant was removed,washed with ice-cold PBS three time,by adding fresh medium,flow cytometry of cells DNR fluorescence intensity. 4.Fluorescence Quantitative PCR DetectionK562/DNR cells,treated by 0,5,10,50,100nmol/L bortezomib for 24 hours, were extracted RNA using Trizol,according to a statement kit retrovirus of cDNA, PCR amplification.After the reaction,the computer automatically by the quantitative analysis of the results.5.Determination of P-gpK562/DNR cells,treated by 0,5,10,50,100nmol/L bortezomib for 24 hours, were washed three times with PBS,and incubated with Anti-p-glycoprotein PE in the dark at 20-25℃for 30 minutes.The percentage of P-gp positive cells and the mean fluorescence intensity(MFI)of the samples were determined by flow cytometry.6.Cell Apoptosis AnalysisK562/DNR cells,treated by 0,5,10,50,100nmol/L bortezomib for 24 hours, were incubated with FITC-conjugated Annexin V and counterstained with propidium iodide(PI)in order to allow exclusion of necrotic cells.The cells were subsequently analyzed using a flow cytometer.10000 cells were analyzed in every sample.7.Cell Cycle AnalysisK562/DNR cells,treated by 0,5,10,50,100nmol/L bortezomib for 24 hours, were made permeable by the addition of 70%ethanol for 4 h at 4℃and stained with PI in the presence of 5μg/mL RNAse(Sigma).Ten thousand events were acquired on a FACScalibur flow cytometer(BD Biosciences)and analyzed with the Paint-a-Gate program,the percentage of cells in the apoptotic sub-G1 and the G1 phase,S phase and G2/M phases were calculated using Multicycle software.Results1.DNR intracellular accumulation of K562/DNR was partially reversed only by addition of bortezomib.Bortezomib had no effect on DNR intracellular accumulation in K562/S cells.2.5-100nmol/L bortezomib in vitro remarkably down-regulated expression of P-gp/mdr1-mRNA.They were negatively correlated with the number of apoptosis K562/DNR cells(r=-0.912,P＜0.01).The increase in apoptosis and decrease in P-gp/mdr1-mRNA happened at same time when cells were treated by 0,5,10,50,100nmol/L bortezomib for 24 h. 3.Bortezomib caused an increase in G2/M and a marked decrease in G0/G1 and S phases in a dose-dependent manner.ConclusionProtease inhibitor bortezomib not only can reverse the mdr1 gene encoding the P-gp expression mediated multi-drug resistance mechanism of leukemia.,but also can reverse the anti-apoptosis-mediated multidrug resistance mechanisms of leukemia.