氯丙嗪对白血病细胞株K562/AO2多药耐药逆转作用的实验研究

Reversal of multidrug resistance in leukemic cell line K562/AO2 by chlordelazine in vitro

目的 研究氯丙嗪对阿霉素诱导的K5 6 2耐药细胞株 (K5 6 2 AO2 )多药耐药的逆转作用及机制 ,以便为临床应用提供实验依据。方法 (1)以四氮甲基唑蓝 (MTT)法检测氯丙嗪的细胞毒性及其对K5 6 2 AO2细胞的耐药逆转作用 ;(2 )以流式细胞术检测氯丙嗪处理前后K5 6 2 AO2细胞内柔红霉素 (DNR)的蓄积量 ;(3)以逆转录聚合酶链反应 (RT PCR)法检测氯丙嗪处理前后后K5 6 2 AO2细胞中mdr 1mRNA的表达水平。结果 (1)氯丙嗪在 3μg ml时对K5 6 2 AO2增殖的抑制作用小 ,抑制率<5 % ;(2 )氯丙嗪明显增加DNR对K5 6 2 AO2的毒性作用 (P <0 0 5 ) ,耐药逆转倍数为 1 90 1;(3)氯丙嗪可明显增强DNR在K5 6 2 AO2细胞内的蓄积 (P <0 0 5 ) ;(4)氯丙嗪对K5 6 2 AO2细胞mdr 1mRNA的表达水平无影响。结论 氯丙嗪通过增加K5 6 2 AO2细胞内DNR的蓄积显著提高K5 6 2 AO2细胞对DNR的敏感性 ,其逆转白血病细胞多药耐药的机制与mdr 1基因无关 。

Objective Some recent studies revealed that phenthiazine might be able to reverse tumor cell drug-resistance. Chlorderazin belongs to the phenthiazine compounds. The study aimed to investigate the reversing effect and mechanism of chlorderazin on multidrug resistance of leukemic cell line K562/AO2. Methods (1) The cytotoxicities of chlorderazin were assayed with the tetrazolium dye, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. (2) The reverse effect of chlorderazin on K562/AO2 cells was analyzed with MTT method. The multidrug resistance reversal index (RI)was equal to the ratio of control group IC 50/test group half inhibition concentration IC 50. (3) The intracellular daunorubicin (DNR) concentrations were measured by the flow cytometry. (4) Mdr1 mRNA expression was detected by reverse transcription-polymerase chain reaction (RT-PCR). The ratio of mdr-1/β-actin density was calculated. Results (1) Chlorderazin 3 μg/ml showed little toxicity to K562/AO2 cells and the suppression rate was less than 5%, so the concentration of 3 μg/ml chlorderazin was selected as the experiment concentration. (2)The cytotoxicities of DNR to K562/AO2 were enhanced by 3 μg/ml of chlorderazin (P<0.05) and RI was 1.901. (3) Chlorderazin of 3 μg/ml could increase the intracellular DNR accumulation significantly (P<0.05), and the fluorescence staining by the flow cytometry was higher (250.95±18.96)than the control group(112.75±15.78)and shift right in K562/AO2 cells treated with chlorderazin, and the difference was significant(P<0.05). (4) Chlorderazin has no significant influence to the expression level of mdr-1 mRNA. Both test group and control group showed a clear mdr-1 mRNA band located at the position of 157kb. The ratios of mdr-1/β-actin density were 0.414±0.012 in the test group and 0.447±0.027 in the control group, respectively, and the difference was not significant(P>0.05). Conclusion Chlorderazin could reverse the multidrug resistance by increasing the intracellular DNR accumulation in K562/AO2 cells. The effects had no correlation to the mdr-1 gene.Further study is needed.