Chen Ying-Yu, Li Jing, Hu Jian-Da, Zheng Jing, Zheng Zhi-Hong, Zhu Liang-Fang, Chen Xin-Ji, Lin Zhen-Xing
Fujian Institute of Hematology, Fujian Provincial Key Laboratory of Hematology, Fujian Medical University Union Hospital, Fuzhou 350001, Fujian Province, China.
Fujian Institute of Hematology, Fujian Provincial Key Laboratory of Hematology, Fujian Medical University Union Hospital, Fuzhou 350001, Fujian Province, China. E-mail:
Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2013 Dec;21(6):1413-22. doi: 10.7534/j.issn.1009-2137.2013.06.010.
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 cells were also evaluated by MTT method. DNA ploidy 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 and 4.12 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 of MRP1, TOPOIIβ, 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.
本研究旨在探讨大黄素对耐药HL-60/ADR细胞多药耐药性（MDR）的逆转作用，并探究其潜在机制。采用MTT法评估HL-60/ADR细胞对大黄素及8种临床常用化疗药物的化疗耐药性。同时通过MTT法评估大黄素对HL-60/ADR细胞MDR的逆转作用。采用DNA倍体分析和DNA Ladder检测法，检测阿霉素（ADR）与大黄素联合作用对HL-60/ADR细胞的凋亡诱导作用。分别通过RT-PCR和Western Blot检测耐药相关基因和蛋白的表达变化。采用流式细胞术和共聚焦激光扫描显微镜检测ADR和柔红霉素（DNR）在细胞内的蓄积及亚细胞分布。结果显示，大黄素抑制HL-60/ADR细胞增殖，平均IC50值为24.09±1.72 μmol/L，与亲代HL-60细胞相似（平均IC50 = 23.18±0.87 μmol/L）。HL-60/ADR细胞对多种化疗药物耐药，如ADR、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）。低浓度大黄素与上述不同药物联合处理后，逆转倍数在1.58至4.12之间。ADR与大黄素联合显示出最佳逆转效果，联合处理后可检测到典型的亚二倍体峰（凋亡峰）和DNA Ladder。此外，大黄素下调了多药耐药相关蛋白1（MRP1）、拓扑异构酶IIβ（TOPOIIβ）、谷胱甘肽S转移酶π（GST π）和凋亡蛋白B细胞淋巴瘤-2（BCL-2）的mRNA和蛋白表达水平。此外，加入大黄素后，HL-60/ADR细胞内ADR和DNR的蓄积及亚细胞分布呈剂量依赖性增加。结论：大黄素对多药耐药的HL-60/ADR细胞具有逆转作用，可能是通过降低耐药相关基因的表达水平、增加化疗药物在细胞内的蓄积以及激活凋亡途径来实现的。
