Study On The Interventional Effect Of Tetrandrine On The Acquired Multidrug Resistance In Leukemia Cells

Objective: 1. We applied doxorubicin (DOX) to induce multidrug resistance (MDR) in human leukemia cell line K562. Based on this model, K562 cells were treated with the traditional Chinese medicine tetrandrine (TTD) and the same dosage of doxorubicin. The expression of mdr1 gene and P-gp (P-glycoprotein),the function of P-gp were investigated to verify the interventional effect of tetrandrine on doxorubicin-induced MDR; 2. Setting about the transcriptional control of mdr1 gene, we try to find out the mechanisms of the interventional effect of tetrandrine on mdr1 overexpression from the mRNA expression level,nuclear protein expression level and the activity of transcription factors. We also investigated the role of apoptosis in the intervention of MDR initially.Methods: 1. MTT assay was applied to investigate the toxic effect of tetrandrine on K562 cells. IC₅,IC₁₀,IC₂₀ were calculated and choosen to carry out the following experiments; 2. K562 cells were treated with 0.6μg/ml doxorubicin only or combind 0.6μg/ml doxorubicin with various concentrations of tetrandrine (IC₅,IC₁₀,IC₂₀) together; 3. Reverse transcription-PCR(RT-PCR) was used to detect the mRNA expression levels of mdr1,NF-κB,c-jun,YB-1 survivin genes; 4. Flow cytometry (FCM) was complied to measure the expression level of P-gp; 5. Intracellular Rhodamine 123 (Rho123) retention assay was applied to test the function of P-gp; 6. Western blotting was used to investigate the expressin levels of transcription factors NF-κB,c-jun and YB-1; 7. EMSA was adopted to detect the binding activity of transcription factors NF-κB and c-jun with DNA; 8. Annexin V was used to test the apoptosis of K562 cells treated with 0.6μg/ml doxorubicin only,2μg/ml tetrandrine only and the two drug used together.Results: 1. After treated with 0.6μg/ml doxorubicin for 24 hours, compared with K562 cells , the expression of mdr1 mRNA increased(P<0.05) . When cells were treated with various concentrations(0.5,1.0,2.0μg/ml) of tetrandrine and 0.6μg/ml doxorubicin, compared with expression of mdr1 mRNA in the cells treated with 0.6μg/ml doxorubicin only, 0.5μg/ml tetrandrine hardly had any effect on the doxorubicin-induced overexpression of mdr1 mRNA(P>0.05). 1μg/ml tetrandrine can inhibit the doxorubicin-induced overexpression of mdr1 mRNA(P<0.05) . 2μg/ml tetrandrine can significantly inhibit the doxorubicin-induced overexpression of mdr1 mRNA(P<0.01). Compared with the expression of mdr1 mRNA in the control, the expression in the cells treated with both 0.6μg/ml doxorubicin and 0.5μg/ml or 1μg/ml tetrandrine was higher(P<0.05), but there were no obvious dfferenerences between the cells treated with 0.6μg/ml doxorubicin and 2μg/ml tetrandrine together and the control(P>0.05); 2. We then chose 2μg/ml tetrandrine for further study. The expression level of P-gp in K562 cells treated with doxorubicin only was much higher than the control (P<0.05). After cells were treated with both 0.6μg/ml doxorubicin and 2μg/ml tetrandrine, the overexpression of P-gp induced by 0.6μg/ml doxorubicin was inhibited by 2μg/ml tetrandrine(P<0.05); 3. The function of P-gp was highly up-regulated after cells were treated with 0.6μg/ml doxorubicin only(P<0.05). Corresponding with the expression of P-gp, compared with 0.6μg/ml doxorubicin treated only the function of P-gp was down-regulated in the cells treated with both 0.6μg/ml doxorubicin and 2μg/ml tetrandrine together(P<0.05) and had no obvious differences with the control(P>0.05); 4. After treated with 0.6μg/ml doxorubicin, the expression levels of NF-κB mRNA and nuclear protein increased(P<0.05). 2μg/ml tetrandrine can repress the elevation of NF-κB expression(P<0.05) induced by 0.6μg/ml doxorubicin. After treated with 0.6μg/ml doxorubicin, the expression level of NF-κB nuclear protein was 2.05 times as the control. In the cells treated with both 0.6μg/ml doxorubicin and 2μg/ml tetrandrine, compared with the cells treated with 0.6μg/ml doxorubicin only the expression level of NF-κB nuclear protein was reduced by 49%. The binding ativity of NF-κB with DNA in the cells treated with 0.6μg/ml doxorubicin decreased, but in the cells treated with both 0.6μg/ml doxorubicin and 2μg/ml tetrandrine, the binding activity increased; 5. After treated with 0.6μg/ml doxorubicin, the expression of c-jun mRNA and nuclear protein decreased (P<0.05). 2μg/ml tetrandrine can repress the doxorubicin-induced down-regulation of c-jun mRNA and nuclear protein expression(P<0.05). After treated with 0.6μg/ml doxorubicin, the expression level of c-jun nuclear protein was reduced by 72% as the control. In the cells treated with both 0.6μg/ml doxorubicin and 2μg/ml tetrandrine, the expression level of c-jun nuclear protein was 3.64 times as the cells treated with 0.6μg/ml doxorubicin only. The binding ativity of c-jun with DNA in the cells treated with 0.6μg/ml doxorubicin only decreased, but in the cells treated with both 0.6μg/ml doxorubicin and 2μg/ml tetrandrine, the binding activity increased; 6. After treated with 0.6μg/ml doxorubicin only, the expression of YB-1 mRNA and nuclear protein was up-regulated(P<0.05). 2μg/ml tetrandrine had no obvious effect on the expression of YB-1 mRNA and nuclear protein(P>0.05) compared with 0.6μg/ml doxorubicin treated only. In cells treated with with 0.6μg/ml doxorubicin or both 0.6μg/ml doxorubicin and 2μg/ml tetrandrine the expression level of YB-1 nuclear protein was respectively 2.72,2.80 times as the control; 7. After treated with 0.6μg/ml doxorubicin, the expression of survivin mRNA had hardly any changes (P>0.05). After treated with 0.6μg/ml doxorubicin and 2μg/ml tetrandrine the expression of survivin mRNA was inhibitted(P<0.05) compared with both the control and 0.6μg/ml doxorubicin treated only. The results of AnnexinV suggested: 0.6μg/ml doxorubicin had little effect on the apoptsis of K562 cells and 2μg/ml tetrandrine had nearly no effect on the apoptsis in K562 cells, but when the two drug were used together, the apoptosis in the cells icreased significantly.Coclusions: 1. After K562 cells were treated with doxorubicin only, the expression levels of mdr1 mRNA,its corresponding protein P-gp increased and the function of P-gp was up-regulated, resulting in the decreased intracellular drug concentration of doxorubicin. The MDR took place . Tetrandrine can interfere with the doxorubicin-induced overexpressions of mdr1 mRNA,P-gp and up-regulation of the function of P-gp in a dose dependent manner. 2μg/ml tetrandrine is the best concentration in this model; 2. The mechanisms of those effects of tetrandrine may be that it can reduce the expression of NF-κB and increase the expression of c-jun in the nuclear, but it had no effect on the expression of YB-1; 3. Tetrandrine can inhibit the expression of survivin mRNA in K562 cells, thus increased doxorubicin-induced apoptsis. This may be another mechanism of the interventional effect of tetrandrine on the doxorubicin-induced MDR. In conclusion, tetrandrine can interfere with MDR by many mechanisms. This study provides a new strategy for clinical chemotherapy.