Zhai Xiaofeng, Lu Jianying, Wang Ying, Fang Fanfu, Li Bai, Gu Wei
J Tradit Chin Med. 2014 Dec;34(6):678-83. doi: 10.1016/s0254-6272(15)30082-0.
To probe insights into the reversal effect of bufalin on vincristine-acquired multidrug resistance (MDR) in human leukemia cell line K562/VCR.
Proliferative inhibition rate and the reversal index (RI) of bufalin were determined by Methyl thiazolyl tetrazolium assay. The uptake of Adriamycin (ADM) in K562/VCR cells, cell cycle and apoptosis rate were determined by flow cytometry (FCM). Cell morphologic changes were observed with Wright-Giemsa staining. The expression of P-glycoprotein (P-gp), multidrug-associated protein-1 (MRP1), Bcl-xL and Bax protein were measured by immunocytochemistry.
The human leukemia multidrug resistant K562/VCR cells showed no cross-resistance to bufalin. The RIs of bufalin at concentrations of 0.0002, 0.001 and 0.005 μmol/L were 4.85, 6.94 and 14.77, respectively. Preincubation of 0.001 μmol/L bufalin for 2 h could increase intracellular ADM fluorescence intensity to 28.07% (P < 0.05) and down-regulate MRP1 expression simultaneously, but no remarkable effect was found on P-gp protein. Cell cycle analysis indicated increased apoptosis rate and apparent decreased G2/M phase proportion after treatment with bufalin. When exposed to 0.01 μmol/L bufalin, typical morphological changes of apoptosis could be observed. Down-regulation of Bcl-xL and up-regulation of Bax expression in K562/VCR cells could be detected by immunocytochemistry.
Bufalin could partly reverse the MDR of K562/VCR cells, with a possible mechanism of down-regulating MRP1 expression and activating apoptosis pathway by altering Bcl-xL/Bax ratio.
探讨蟾毒灵对人白血病细胞株K562/VCR长春新碱诱导的多药耐药（MDR）的逆转作用机制。
采用甲基噻唑基四氮唑蓝法（MTT法）检测蟾毒灵对K562/VCR细胞的增殖抑制率及逆转指数（RI）；采用流式细胞术（FCM）检测K562/VCR细胞阿霉素（ADM）摄取、细胞周期及凋亡率；采用瑞氏-吉姆萨染色观察细胞形态学变化；采用免疫细胞化学法检测P-糖蛋白（P-gp）、多药耐药相关蛋白-1（MRP1）、Bcl-xL和Bax蛋白的表达。
人白血病多药耐药K562/VCR细胞对蟾毒灵无交叉耐药。0.0002、0.001和0.005 μmol/L浓度的蟾毒灵逆转指数分别为4.85、6.94和14.77。0.001 μmol/L蟾毒灵预处理2 h可使细胞内ADM荧光强度增加至28.07%（P<0.05），同时下调MRP1表达，但对P-gp蛋白无明显影响。细胞周期分析显示，蟾毒灵处理后凋亡率增加，G2/M期比例明显降低。当细胞暴露于0.01 μmol/L蟾毒灵时，可观察到典型的凋亡形态学变化。免疫细胞化学检测发现，K562/VCR细胞中Bcl-xL表达下调，Bax表达上调。
蟾毒灵可部分逆转K562/VCR细胞的多药耐药，其机制可能与下调MRP1表达、改变Bcl-xL/Bax比例激活凋亡通路有关。
