大黄素对HL-60／ADR耐药细胞多药耐药逆转作用的研究

本研究旨在探讨大黄素（emodin）对人急性白血病HL-60／ADR耐药细胞多药耐药（mulfidrugresistance，MDR）逆转作用及其相应的作用机制。采用MTT法检测HL-60／ADR细胞对大黄素以及8种临床常用化疗药物的耐药性，比较大黄素联合化疗药物后对HL-60／ADR细胞耐药逆转效果，应用DNA倍体和DNALadder分析检测大黄素与阿霉素联合用药后细胞凋亡改变，RT-PCR和Westernblot分别检测耐药相关基因和蛋白表达变化，流式细胞术检测大黄素处理后HL-60／ADR细胞内阿霉素荧光阳性率和柔红霉素平均荧光强度（MFI），激光共聚焦显微镜检测细胞内柔红霉素分布变化。结果表明：大黄素对HL-60／ADR耐药株和相应HL-60细胞敏感株的IC5值接近，分别为24．09±1.72μmol/L和23．18±0．87μmol／L，对阿霉素（ADR）、柔红霉素（DNR）、依托泊苷（VP16）、长春新碱（VCR）、阿糖胞苷（Ara-C）、高三尖杉酯碱（HHT）、米托蒽醌（MTZ）和吡柔比星（THP）呈现不同程度耐药。小剂量大黄素对8种药物耐药逆转倍数介于1．58-4．12之间，其中对ADR的耐药细胞逆转效果最好。大黄素与ADR联合用药组可见明显的亚二倍体凋亡峰和典型的DNA降解梯状带形成。与单药组比较，联合用药组MRP1、TOPOⅡB、GSTπ、BcL-2耐药相关基因mRNA和蛋白表达水平下调明显，细胞内ADR和DNR蓄积水平增加，胞浆和胞核DNR分布增加，该作用与大黄素呈浓度依赖性。结论：大黄素具有逆转HL-60／ADR细胞多药耐药作用，其作用机制可能与下调耐药相关基因表达水平、增加细胞内化疗药物蓄积和促进细胞凋亡作用有关。

Reversing Effects of Emodin on Multidrug Resistance in Resistant HL-60/ ADR Cells

This study was aimed to investigate the reversing effects of emodin on multidrug resistance （MDR） in resistant HL-60/ADR cells, and to explore the underlying mechanisms. The MTT assay was used to assess the chemoresistance of HL-60/ADR cells to emodin and 8 chemotherapeutic agents commonly used in clinic. The reversal effects of emodin on MDR of HL-60/ADR ceils were also evaluated by MTT method. DNA ploid analysis and DNA Ladder assay were used to detect apoptosis-induced effects on HL-60/ADR cells via the adriamycin （ADR） and emodin combination. The expression changes of the drug resistance-associated genes and proteins were detected by RT-PCR and Western Blot respectively. The intracellular accumulation and subcellular distribution of ADR and DNR were measured by flow cytometry and confocal laser scanning microscopy. The results showed that emodin inhibited HL-60/ADR cell proliferation with an average IC50 value of 24. 09 ± 1.72 μmol/L, which was similar to that of the parental HL-60 cells （average IC50 = 23.18 ± 0. 87 μmol/L）. HL-60/ADR cells were resistant to a variety of chemotherapeutic agents, such as ADR, DNR, VP16, VCR,Ara-C, HHT, MTZ and THP. The reversal multiple were between 1.58 and4. 12 after the treatment with low concentration of emodin combined with the above mentioned different agents. The combination of ADR with emodin showed the best reversal effects,and the typical hypodiploid peak （apoptotic peak） and DNA ladder could be detected after the co-treatment. In addition, emodin down-regulated the mRNA and protein expression levels ofMRP1 ,TOPO Ⅱ β, GST π and BCL-2. Furthermore, the addition of emodin enhanced ADR and DNR intracellular accumulation and subcellular distribution in HL-60/ADR cells in dose-dependent manner. It is concluded that the emodin shows reversing effects on the multidrug resistant HL-60/ADR cells, possibly via decreasing the expression levels of drug resistance-associated genes, increasing the intracellular accumulation of chemotherapeutic agents and activating the apoptosis pathway.