Yin Li, Chen Bao-An, Cheng Jian, Ding Jia-Hua, Gao Chong, Sun Yun-Yu, Wang Jun, Zhao Gang, Gao Feng, Song Hui-Hui, Bao Wen, Wu Wei-Wei, Wang Fei, Liang Yi-Qiong, Xia Guo-Hua, Wang Xue-Mei
Department of Hematology, The Affiliated Zhongda Hospital, Southeast University Clinical Medical College, Nanjing 210009, Jiangsu Province, China.
Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2010 Feb;18(1):79-84.
This study was purposed to investigate the reversal effect of glucosylceramide synthase (GCS) inhibitor D, L-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) hydrochloride, on multidrug resistance in K562/A02 cells and its mechanism. The IC(50) (the half maximal inhibitory concentration) of PDMP was measured by MTT method. Cell apoptosis and intracellular daunorubicin (DNR) concentration were detected by flow cytometry. The expression of GCS and mdr1 genes were assayed by semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot. The results showed that the IC(50) of DNR in K562 and K562/A02 cells were 0.23 +/- 0.02 and 7.15 +/- 0.24 microg/ml respectively. When the concentration of PDMP was equal to or less than 20 micromol/L ( < / = 20 micromol/L), the obviously inhibitory effect on proliferation of K562 and K562/A02 cells was not observed, but both 20 micromol/L and 10 micromol/L PDMP could enhance the sensitivity of K562/A02 cells to DNR (p < 0.01) and the reversal multiples were 2.59 and 1.69 respectively. After treating with 20 micromol/L and 10 micromol/L PDMP for 48 hours, the concentration of DNR in K562/A02 cells increased (p < 0.05) and the apoptotic rate also was elevated (p < 0.01). The expressions of GCS and mdr1 genes were down-regulated at mRNA and protein levels after treating K562/A02 cells with 20 micromol/L PDMP for 48 hours. It is concluded that PDMP can enhance the sensitivity of K562/A02 cells to DNR by increasing cell apoptosis rate and accumulation concentration of DNR in cells, which may be related to down-regulated expressions of GCS and mdr1 genes.
本研究旨在探讨葡萄糖神经酰胺合酶（GCS）抑制剂盐酸D，L-苏式-1-苯基-2-癸酰胺基-3-吗啉代-1-丙醇（PDMP）对K562/A02细胞多药耐药性的逆转作用及其机制。采用MTT法测定PDMP的半数抑制浓度（IC50）。通过流式细胞术检测细胞凋亡和细胞内柔红霉素（DNR）浓度。采用半定量逆转录聚合酶链反应（RT-PCR）和蛋白质免疫印迹法检测GCS和mdr1基因的表达。结果显示，K562和K562/A02细胞中DNR的IC50分别为0.23±0.02和7.15±0.24μg/ml。当PDMP浓度≤20μmol/L时，未观察到对K562和K562/A02细胞增殖有明显抑制作用，但20μmol/L和10μmol/L的PDMP均可增强K562/A02细胞对DNR的敏感性（p<0.01），逆转倍数分别为- 2.59和1.69。用20μmol/L和10μmol/L的PDMP处理48小时后，K562/A02细胞内DNR浓度升高（p<0.05），凋亡率也升高（p<0.01）。用20μmol/L的PDMP处理K562/A02细胞48小时后，GCS和mdr1基因在mRNA和蛋白质水平的表达均下调。结论：PDMP可通过提高细胞凋亡率和细胞内DNR蓄积浓度增强K562/A02细胞对DNR的敏感性，其机制可能与GCS和mdr1基因表达下调有关。
