| Objective:To provide powerful experiment basis and find novel therapeutic strategies for reversing MDR in leukemia clinical treatment,the effect of silencing Apollon gene by RNAi combined Timosaponin A-? on reversing multi-drug resistance in human CML K562/ADM cells and its molecular mechanism is investigated.Methods:1.CCK-8 was used to determine the drug-resistant fold of K562/ADM cells.The expression levels of Apollon and MDR1 in K562 and K562/ADM cells were measured by RT-PCR.The pGPHl/GFP/Neo/Apollon shRNA eukaryotic expression vector was stably transfected with K562/ADM cells.Then the effect of silencing Apollon gene by eukaryotic expression vector was determined by RT-PCR and Western blotting analysis.The reversal effect and intracellular ADM accumulation was respectively determined by CCK-8 and FCM.Then,the expression of MDR1 mRNA and P-gp was determined by RT-PCR and Western blotting assay.The pGPH1/GFP/Neo/MDR1 shRNA eukaryotic expression vector was stably transfected with K562/ADM cells.Then the effect of silencing MDR1 gene by eukaryotic expression vector was determined by reverse RT-PCR and Western blotting analysis.The reversal effect and intracellular ADM accumulation was determined by CCK-8 and FCM.Then,the expression of Apollon mRNA and its protein was determined by RT-PCR and Western blotting assay.2,CCK-8 assay was used to determine the none cytotoxic does(cell growth inhibition<5%and<10%)and the reversal effect of TAIII on K562/ADM cells.After K562/ADM cells pretreated with the none cytotoxic does of TAIII(IC<5 and IC<10)for 24 h,the cell-associated MFI of ADM,Rho-123 and CFDA was detected by FCM;the mRNA expression levels of MDR1 and MRP1 were measured by RT-PCR;the protein expression levels of p-Akt,t-Akt,P-gp and MRP1 was determined by Western blotting.After treatment with wortmannin for 24h,the protein expression levels of p-Akt,t-Akt,P-gp and MRP1 in K562/ADM cells was determined by Western blotting.3.The CCK-8 was used to determine the survival rate of cells in blank group(K562/ADM cells),negative control(NC)group(shControl cells),Apollon RNAi group(shApollon cells),TAIII group(K562/ADM cells incubated with 2?M TAIII),TAIII+NC group(shControl cells incubated with 2?M TAIII),TAIII +Apollon RNAi group(shApollon cells incubated with 2?M TAIII).Results:1.The drug-resistant fold of K562/ADM cells was 31.78.The expression of Apollon mRNA in K562 and K562/ADM cells were(31.58±1.954)%and(54.58?3.240)%respectively(P<0.05).The expression of Apollon mRNA and Apollon protein were(9.752±1.161)%and(22.58±3.166)%respectively after transfected with pGPH 1/GFP/N eo/Apollon shRNA eukaryotic expression vector(P<0.05).The reversal fold of Apollon interference group was 1.86(P<0.05).ADM MFI in shApollon cells was(315.33±3.511),there was no significant difference compared with control groups.The expression of MDR1 mRNA and P-gp in shApollon cells were(45.19 ±2.023)%and(78.92 ±3.079)%respectively,there was no significant difference between Apollon RNAi group and the controls.The expression of MDR1 mRNA in K562 and K562/ADM cells were(35.68±2.534)%and(67.30±5.339)%respectively(P<0.05).The expression of MDR1 mRNA and P-gp were(14.91±2.788)%and(16.04±1.837)%respectively after transfected with pGPH1/GFP/Neo/MDR1 shRNA eukaryotic expression vector(P<0.05).The reversal fold of MDR1 RNAi group was 5.64(P<0.05).ADM MFI in shMDR1 cells was(597.0±12.00),significant higher than the controls.The expression of Apollon mRNA and protein in MDR1 RNAi group were(78.94±3.737)%and(76.43±2.788)%respectively,there was no significant difference compared with the controls.2.The cell growth inhibition rate of TAIII 1?M and 2?M were(3.82±0.73)%and(8.74 ± 0.81)%.To minimize TAIII itself on K562/ADM cells growth,1±M(IC<5)and 2±M(IC<10)was selected to reverse MDR of K562/ADM cells.The reversal fold of TAIII IC<5 and TAIII IC<10 were 1.50 and 2.52(P<0.05).ADM MFI,Rho-123 MFI and CFDA MFI in TAIII IC<5 group and TAIII IC<io group were 1605±78.50 and 1888±54.50,1841 ± 130.0 and 2179±98.50,1137±44.50 and 1340±84.50,respectively,all of them significantly higher than the controls(P<0.05).The proteins expression of p-Akt,P-gp and MRP1 in TAIII IC<5 group and TAIII IC<10 group were(43.62±1.258)%and(39.90± 1.880)%,(51.50 ± 1.789)%and(46.29±2.754)%,(46.78± 1.643)%and(42.64± 1.327)%,respectively,significantly decreased compared to the negative control group.However,the protein expression of t-Akt in TAIII IC<5 group and TAIII IC<10 group were(52.64±0.6965)%and(54.88±0.8231)%,did not change markedly compared with the control.Moreover,the proteins expression of p-Akt,P-gp and MRP1 in K562/ADM cells with the specific PI3K/Akt inhibitor wortmannin treatment were(21.84 ± 1.726)%,(26.04±2.257)%and(32.44±2.832)%,all of them significantly decreased compared with the control.Similarly,the expression of t-Akt in wortmannin treatment group was(62.71±0.6994)%,hasnot changed much compared with the control.3.The IC50 of ADM and reversal fold of Apollon RNAi+TAIII group,Apollon RNAi group and TAIII group were(18.883±2.381)mg/L and 3.36,(13.519±1.278)mg/L and 1.78,(9.998±1.204)mg/L and 2.48,respectively.The drug resistance reversal effect of Apollon RNAi+TAIII group is stronger than Apollon RNAi group and TAIII group alone.Conclusions:1.Compared with its parental K562 cells,K562/ADM cells showed clear drug-resistant property.Resistant K562/ADM cells had higher Apollon and MDR1 expression compared with sensitive K562 cells.pGPH1/GFP/Neo/Apollon shRNA eukaryotic expression vector inhibited Apollon mRNA and protein expression effectively thus reverse K562/ADM cells MDR.Apollon RNAi could not inhibit MDR1 mRNA and P-gp expression and function,and could not increasing the intracellular accumulation of ADM.Inhibition of MDR1 by pGPHl/GFP/Neo/MDR1 shRNA eukaryotic expression vector significantly inhibited expression of P-gp,increased ADM intracellular accumulation of K562/ADM cells,thus reverse K562/ADM cells MDR.MDR1 RNAi had no effect on the expression of Apollon.Take together,the reversal mechanism of Apollon and MDR1 silencing does not directly interact with each other.2.TAIII induced a MDR reversal activity through increase the intracellular accumulation of ADM in K562/ADM cells at nontoxic concentrations by down-regulating P-gp and MRP1 expressions,function and transcription which might mainly correlate to the regulation of PI3K/Akt signaling pathway.3.The drug resistance reversal effect of Apollon RNAi combined TA? is stronger than Apollon RNAi and TA? alone. |
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