Effects Of Bortezomib On The Expression Of NF-κB,1κB And P-gp Of DNR Resistant K562 Cells

PrefaceNF-κB is a transcriptional modulator existing in eukaryocyte universally.Under normal circumstance,it is combined with inhibitor protein IκB in cytoplasma.When stress occurs,IκB is phosphorylated and then recognized and degraded by proteasome. Subsequently,NF-κB is released into the nucleus,combined with its specific sequence and induces the expression of multi-drug resistance gene(mdr1 gene).The product of this gene is P-glycoprotein(P-gp) which is an ATP-dependent drug pump and can pump out the drug entering into the cell.This leads to the decrease of the intracellular drug concentration and resistance ocuurs.Bortezomib(PS-341) is a proteasome inhibitor with anti-tumor effect which is used in the treatment of recurrence and refractory multipl myeloma in clinic.But its use in leukemia is in theory and exploration stage.This study was carried out in vitro to observe the effect of PS-341 on the expression of NF-κB,IκB and P-gp of K562 cells induced by daunorubicin(K562/DNR) to investigate the molecular mechanism and feature of its action and supply experimental basis for overcoming leukemia multi-drug resistance in clinic.MaterialsCell lineHuman leukemia multi-drug resistant cell line K562/DNR was offered by oncology laboratory of the first affiliated hospital of China Medical University.This cell line was stimulated by 0.5mmol/L DNR and the drug resistant activity was maintained.Main instrument and reagents1.Reagents detecting cell drug resistance by MTT methods2.Rabbit antihuman NF-κB,IκB and P-gp antibody(SANTA CRUZ American) 3.AKP-labeled goat anti-rabbit and horse anti-mice antibody(Zhongshan Beijing)4.Mice anti-β-actin(sigma American)5.NF-κB activity kit(Active Motif American)6.Cell apoptosis detecting kit(Baosai Beijing)7.PS-341(Yangshen Xian)MethodsCell cultureK562/S and K562/DNR cells were cultured in RPMI1640 nutrient liquid containing 12%fetal bovine serum,100U/ml penicillin and 100ug/ml streptomycin with 37℃,5%CO₂ and saturated humidity condition.The experiment was not carried out until the cells experienced high passage and reached exponential growth phase. K562/DNR cell was stimulated by 0.5mmol/L DNR and the drug resistant activity was maintained.Assessment of drug resistant cell line by MTT methodGrouping(1)K562/S;(2)K562/DNR;the quality concentration of the cells was adjusted to 2×10⁵/mL and the cells and DNR were added into the 96 holes of the plate with 5 parallel holes for each quality concentration.After 72h of culture, MTT(5g/L)20μL was added into each hole for another 4 hours.After centrifugalization, the upper serum was discarded and 150μL DMSO was added into the system to end the reaction.Then,after 15 minutes of oscillation,the value of 570nm absorbance density(A) was measured on automatic enzyme-labeled meter.The growth inhibitor rate and the multiple of drug resistance were calculated.Assessment of direct cellular toxicity of PS-341 by MTT methodThe procedure was similar to the above.PS-341 of 0.4 u g/L,4 u g/L and 40 u g/L was added into each hole respectively.The survival rate of the blank group was regarded as 100%.The relative survival rate in each concentration group was calculated according to the following equation.Survival rate(%)= A value of experimental group/ A value of blank group×100%.IC₁₀ that is 90%survival rate was calculated.A concentration that was lower than IC₁₀ was selected as the experimental concentration of PS-341 to act as reversing drug resistance. Western blot100 u g/ml DNR using alone or combined with 4 u g/L PS-341 for 12h,24h and 36h were acted on K562/DNR cells.100 u g/ml DNR using alone or combined with 0.4 u g/L,4 u g/L and 40 u g/L PS-341 were acted on K562/DNR cells for 36 hours.The expression of NF-κB,IκB and P-gp were measured by Western blot.1×10⁷ cells were added with 200 ul cell disruption buffer solution and the total cellular protein was abstracted and quantitated by Larry method.Then,50μg protein sample was added to 15%SDS-PAG for electrophoresis(100V,1.5h).After BPB entered the bottom of the gel,the protein was transferred onto the nitrocellulose filter.NF-κB,IκB and P-gp antibody andβ-actin antibody was incubated,then AP-labeled second antibody was added into the system.Developer was used for coloration and the image was analyzed on gel formatter.Measurement of activity of NF-κB by ELISA methodThe extraction of nuclear protein and the procedure of the manipulation was carried out all according to the description of the kit.Detecting of cell apoptosis rate by FACS methodThe cells in each group were collected and Annexin-â…¤-FITC double staining was introduced.The cell apoptosis rate of each group was detected and calculated by FACS.Statistical analysisGray scale analysis was carried out by gel formatter and the expression value of each index was illustrated by the relative A value to that ofβ-actin.SPSS11.5 software was used for analysis,F test was used for variations comparison and X² test was used for ratio comparison.Results1.The IC₅₀of DNR to K562/S was 1.16μg/mL while 50.43μg/mL for K562/DNR cells and the multiple of drug resistance was 43.47.2.IC₁₀ vale of PS-341 on K562/DNR cell lines was 4 u g/L.That is to say,when the concentration of PS-341 was less than 4 u g/L,nearly 90%cells survived.This concentration of PS-341 could be regarded as free of cellular toxicity. 3.Compared with the control group,the expressions of NF-κB and P-gp in K562/DNR could be induced by DNR.4.When K562/DNR were cultured with bortezomib,the expressions and activity of NF-κB and expression of P-gp induced by DNR were significantly suppressed and this effcet showed the characteristic of concentration and time-dependent pattern.5.Compared with the control group,the apoptosis rate increased significantly in DNR group,40 u g/L PS-341 group and DNR plus different concentrations of PS-341 group.While the apoptosis rate did not change much in 0.4 u g/L PS-341 group and 4 u g/L PS-341 group.Compared with the DNR group,addition of PS-341 could significantly increase the apoptosis rate induced by DNR and this effect showed a concentration and time -dependent pattern.Compared with 40 u g/L PS-341 group, DNR plus 40 u g/L PS-341 could increase the apoptosis rate.ConclusionBortezomib could decrease the expressionsof NF-κB and P-gp in K562/DNR, convert the cellular durg resistance and promote cell apoptosis.