屈洛昔芬与他莫昔芬对K562/A02耐药性影响的比较

Comparative study on effects of droloxifene and tamoxifen on multidrug resistance in K562/A02 cell line

目的:比较屈洛昔芬 (droloxifene,DRO)和他莫昔芬 (tamoxifen,TAM)对K562/A02耐 药性的逆转作用。方法:应用 MTT法、RT PCR和免疫细胞化学 染色观察细胞毒活性和MDR1、 GSTpi耐药基因表达变化,采用流 式细胞仪测定细胞内多柔比星 (ADR)浓度。结果:在20、10和5 mmol/L浓度时,DRO和TAM均 显著逆转K562/A02的耐药性, DRO和TAM的逆转倍数相比较 差异无统计学意义。MDR1和 GSTpi的mRNA和蛋白表达在 DRO和TAM处理后第2天开始 降低,第5天出现明显降低。20 mmol/L浓度的DRO和TAM分别 孵育K562/A02,可使其细胞内 ADR积累分别增加2.9和3.0 倍。结论:DRO与TAM类似,有 较强的逆转活性,可明显抑制 MDR1和GSTpi基因的表达及增 加细胞内ADR的积累。

OBJECTIVE: To study the reversal effects of droloxifene (DRO) and tamoxifen (TAM) on MDR in K562/A02.METHODS:K562 cell line resistant to adriamycin (ADR) cell line (K562/A02) and K562 cell line sensitive to ADR (K562) were treated with DRO and TAM, respectively. By using tetrazolium dye (MTT) assay, effects of various concentrations of DRO and TAM on MDR in K562/A02 was studied. Before and after the treatment with 10 mmol/L of DRO and TAM, MDR1 and GSTpi genes, expressions were assayed by reverse transcription-polymerase chain reaction (RT-PCR), immunocytochemistry (ICC) assay. By using flow cytometry (FCM), intracellular ADR concentration was measured. RESULTS:DRO and TAM significantly reversed MDR in K562/A02, respectively. After 20, 10, and 5 mmol/L of DRO and TAM treatment, the reversal times had significant differences between DRO and TAM. After 10 mmol/L DRO and TAM treatment, both MDR1 and GSTpi mRNA genes, expressions began to decline on the second day,and significantly decreased on the fifth day,P<0.01. Changes of P-gp and GSTpi protein expressions were similar to those of their mRNA expressions. Two hours after 20 mmol/L DRO and TAM treatment, intracellular ADR concentration increased to 2.9 and 3.0 times respectively. However, both agents did not significantly increase ADR accumulation in K562. CONCLUSIONS:Reversal activity of DRO is similar to that of TAM. Both agents have strong reversal effects. The reverse is via different routes, i.e, down regulating mRNA and protein expression levels of MDR1/P-gp and GSTpi, and increasing intracellular drug concentration. Since DRO has low toxicity, it might have good prospects in clinical application.