槲皮素和山柰酚逆转白血病细胞系K562/A02多药耐药性的机制及相关基因表达谱的研究

Study on Mechamism of Reversing Multidrug Resistance by Quercetin and Keampferol and Their Effects on the Expression of Related Genes in Human Erythroleukemic K562/A02 Cells

目的研究槲皮素(quercetin,Que)及山柰酚(kaempferol,Kae)单用及联合应用时对K562/A02细胞系多药耐药的影响及机制。方法体外培养K562和K562/A02细胞经Que及Kae处理,采用噻唑蓝(MTT)法计算药物单独及联合作用后细胞的生长抑制率及对多柔比星(adriamycin,ADM)的增敏倍数;流式细胞术检测药物作用后细胞内ADM浓度的变化;Annexin V/PI法观察细胞凋亡情况;Real-time PCR基因芯片检测药物转运蛋白及凋亡相关基因表达的变化。结果药物作用48 h后,Que及Kae在5～160μmol.L-1时对K562和K562/A02细胞均有剂量依赖性的生长抑制作用,K562/A02对ADM的敏感性显著增强,二者联用效果有协同性;在ADM为5μmol.L-1时,药物与细胞共培养2 h即可检出Que能增加细胞内ADM的浓度,而Kae则对ADM浓度无明显影响,两药联合作用的效果不及Que单独作用。Que和Kae均可剂量依赖性地诱导K562和K562/A02细胞的凋亡。二者可影响ABC、SLC家族等药物转运蛋白相关基因的表达,并可调节Bcl-2、TNF等凋亡相关基因的表达。结论 Que和Kae可通过多种途径逆转K562/A02的多药耐药性,但二者的作用机制可能不完全相同。

OBJECTIVE To investigate the possibility and mechanism of quercetin and kaempferol, ilavonoid molecules, for reversing multidrug resistance （MDR） in K562/A02 cells. METHODS 562 and K562/A02 cells were cultured in vitro with the flavonoids as single and in combination respectively. Cell growth inhibition and adriamycin （ADM） sensitivity were detected by MTY assay. Intracellular ADM concentration was determined by flow eytometry when K562/A02 cells was cultured with ADM and the flavonoids for 2 h, Cell apoptosis was examined by Annexin V/PI staining method. Moreover, the expression of related genes for drug . transporters and apoptosis was also tested by real time PCR array. RESULTS The MTT assay showed that quercetin and keampferol inhibited the proliferation of K562 and K562/A02 at the concentrnation of 5 -160 mol . L-1. They also increased the sensitivity of K562/A02 cells to ADM at a low toxicity concentration. Flow cytometry showed quercetin increased intracellular ADM in a dose dependent manner, but keampferol showed trivial effect on intracellular ADM accumulation. However, quercetin ＋ keampferol treatment showed a similar effect to that of quercetin alone. Cell apoptosis experiment showed both compounds had a dose dependent effect on I（562 and K562/A02 cells. Furthermore, PCR array showed that some of the genes related to drug transporters such as ATP-binding cassette（ABC） and solute carrier（SLC） and some of the genes related to apoptosis such as Bcl-2, TNF and TNFR families were regulated. CONCLUSION The experiment indicated that quercetin and kaempferol may be used as reveratrol in leukemia chemotherapy, but their interaction and difference should be noticed.