姜黄素、红霉素逆转K562/A02细胞多药耐药机制的研究

目的研究姜黄素（curcumin，Cur）及红霉素（erythromycin，EM）对多药耐药（MDR）细胞株K562／A02的影响及作用机制。方法MTF法测定Cur、EM作用后K562／A02细胞对阿霉素（ADM）敏感性的变化。流式细胞仪测定细胞内柔红霉素的平均荧光强度（DNR MFI）。免疫组化法检测细胞膜上P—gP的表达。RT—PCR法检测细胞mdr1 mRNA水平：结果Cur、EM均可减低ADM对K562／A02细胞的IC50值，两药合用时逆转倍数可达11．3倍。K562／A02细胞内DNR MFI明显低于K562细胞（P〈0．01），Cur、EM均可明显增加K562／A02细胞内DNR MFI（P〈0．05），以两药合用时作用最为明显，Cur2．5μg／ml处理组细胞内DNR MFI略高于EM120μg／ml处理组，但差异无统计学意义（P〉0．05）。免疫组化检测结果显示K562／A02细胞P—gP表达明显高于K562细胞（P〈0．01），各组药物分别处理后，K562／A02细胞膜P—gP表达减低（P〈0．01），但仍高于K562细胞（P〈0．01）；各药物组处理5d细胞膜P—gP表达均低于3d组（P〈0．01），Cur与EM合用时细胞膜P—gP表达降低最为明湿，低于其它处理组（P〈0．01）。RT—PCR结果显示K562／A02细胞mdr1 mRNA水平明显高于K562细胞（P〈0．01），各组药物处理后，K562／A02细胞mdr1 mRNA水平均减低（P〈0．01），5d组低于3d组，但仍高于K562细胞（P〈0．01）；Cur与EM合用时K562／A02细胞mdr1 mRNA水平降低最为显著，Cur 2．5μg／ml处理5dK562／A02细胞mdr1 mRNA水平低于EM120μg／ml处理5d组（P〈0．01）。结论Cur、EM均可部分逆转K562／A02细胞的MDR，降低其P—gP的表达和功能，逆转作用有时间依赖性；两药联合应用时逆转作用明湿增强，Cur2．5μg／ml逆转作用略强于EM120μg／ml。

The study on reversing mechanism of multidrug resistance of K562/A02 cell line by curcumin and erythromycin

OBJECTIVE: To investigate the effects of curcumin (Cur) and erythromycin (EM) on multidrug resistance (MDR) reversal of K562/A02 cell line and their mechanism. METHODS: MTT assay was employed to determine the sensitivity of Cur, EM-treated K562/A02 cells to adriamycin (ADM). Flow cytometry was used to measure intracellular mean fluorescence intensity (MFI) of daunorubicin (DNR). P-gp expression was determined by immunohistochemistry. RT-PCR technique was used to examine the mdr1 mRNA level. RESULTS: IC(50) of ADM in K562/A02 cells was decreased when treated with Cur or EM, and the reversal times (RvT) was 4.9, 3.7 respectively. The RvT reached to 11.3 when treated with Cur (2.5 microg/ml) combined with EM (120 microg/ml). The DNR MFI in K562/A02 cells was significantly lower than that in K562 cells (P < 0.01), and was increased significantly when treated with Cur (2.5 microg/ml) or EM (120 microg/ml) (P < 0.05). There was no significant difference between DNR MFI of K562/A02 cells treated with Cur (2.5 microg/ml) or EM (120 microg/ml). Immunohistochemistry showed that P-gp expression was significantly higher in K562/A02 cells than in K562 cells (P < 0.01), and was reduced in K562/A02 cells treated with each (P < 0.01), though being still higher than that in K562 cells (P < 0.01). P-gp expression of K562/A02 cells treated with each drug for 5 days were lower than that for 3 days (P < 0.01), and lowered further when treated with Cur and EM together (P < 0.01). Mdr1 mRNA level in K562/A02 cells was higher than in K562 cells (P < 0.01), and was decreased when treated with each of the drugs (P < 0.01). The mdr1 mRNA level of K562/A02 cells treated with Cur (2.5 microg/ml) plus EM (120 microg/ml) was decreased most significantly than that treated with other group of drugs. After 5 day treatment the mdr1 mRNA level of K562/A02 cells with Cur (2.5 microg/ml) was lower than that with EM 120 microg/ml (P < 0.01). CONCLUSIONS: Either Cur or EM can partly reverse the multidrug resistance of K562/A02 cells and decrease the expression and function of P-gp in a time-dependent way. MDR reversing effect of Cur combined with EM is stronger than that of Cur or EM alone.