Xie Ping, Shen Yun-Fen, Shi Yuan-Ping, Ge Shu-Mei, Gu Zhong-Hua, Wang Jue, Mu Hui-Jun, Zhang Bin, Qiao Wei-Zhen, Xie Ke-Ming
Central Laboratory, No. 1 Hospital of Wuxi, Wuxi, Jiangsu 214002, China.
Leuk Res. 2008 Mar;32(3):475-80. doi: 10.1016/j.leukres.2007.07.006. Epub 2007 Aug 20.
Ceramide, as a second messenger, initiates one of the major signal transduction pathways in tumor apoptosis. Glucosylceramide synthase (GCS) catalyzes glycosylation of ceramide and produces glucosylceramide. Through GCS, ceramide glycosylation allows cellular escape from ceramide-induced programmed cell death. Here we investigated the expression of GCS in human leukemia cells and an association between GCS and multidrug resistance of leukemia cells. Using RT-PCR technique the level of GCS gene was detected in 65 clinical multidrug resistance/non-resistance cases with leukemia, and in K562 and K562/A02 cell lines. AlamarBlue Assay was applied to confirm the multidrug resistant of K562/A02 cells. PPMP, which is a chemical inhibitor for GCS, was used to determine the relationship between GCS and drug-resistance in K562/A02 cells. In addition, multidrug resistance gene (mdr1), Bcl-2 and Bax mRNA was also analyzed by RT-PCR. The expression of GCS and mdr1 mRNA in clinic multidrug resistance samples exhibited significantly increased compared with clinic drug sensitive group (P<0.05). There was the positive correlation both the expression of GCS and mdr1 genes in leukemia samples (P<0.01, gamma=0.7). AlamarBlue Assay showed that the K562/A02 cell line was 115-fold more resistant to adriamycin and 36-fold more resistant to vincristine compared with drug-sensitive K562 cell line. There also was significant expression difference of GCS and mdr1 genes between K562 and K562/A02 cells. Bcl-2 gene exhibited higher expressions whatever in clinic drug-resistance samples or K562/A02 cells, whereas the expressions of Bax gene were higher in drug-sensitive samples and K562 cells. PPMP increased sensitivity to adriamycin toxicity by inhibiting GCS in K562/A02 cells. Therefore, it is suggested that a high level of GCS in leukemia is possible contributed to multidrug resistance of leukemia cells. Abnormally expressions of the genes in associated with cell apoptosis might be one of the main molecular pathology mechanisms of multidrug resistance caused by GCS gene.
神经酰胺作为第二信使，启动肿瘤细胞凋亡的主要信号转导途径之一。葡萄糖神经酰胺合酶（GCS）催化神经酰胺的糖基化反应并生成葡萄糖神经酰胺。通过GCS，神经酰胺糖基化使细胞逃避神经酰胺诱导的程序性细胞死亡。在此，我们研究了GCS在人白血病细胞中的表达情况以及GCS与白血病细胞多药耐药性之间的关联。运用逆转录聚合酶链反应（RT-PCR）技术，检测了65例临床白血病多药耐药/非耐药病例以及K562和K562/A02细胞系中GCS基因的水平。采用alamarBlue检测法确认K562/A02细胞的多药耐药性。使用PPMP（一种GCS的化学抑制剂）来确定K562/A02细胞中GCS与耐药性之间的关系。此外，还通过RT-PCR分析了多药耐药基因（mdr1）、Bcl-2和Bax的mRNA。临床多药耐药样本中GCS和mdr1 mRNA的表达与临床药物敏感组相比显著增加（P<0.05）。白血病样本中GCS和mdr1基因的表达呈正相关（P<0.01，γ=0.7）。alamarBlue检测法表明，与药物敏感的K562细胞系相比，K562/A02细胞系对阿霉素的耐药性高115倍，对长春新碱的耐药性高36倍。K562和K562/A02细胞之间GCS和mdr1基因也存在显著的表达差异。无论在临床耐药样本还是K562/A02细胞中，Bcl-2基因均呈现较高表达，而Bax基因在药物敏感样本和K562细胞中的表达较高。PPMP通过抑制K562/A02细胞中的GCS增加了对阿霉素毒性的敏感性。因此，提示白血病中高水平的GCS可能导致白血病细胞的多药耐药性。与细胞凋亡相关基因的异常表达可能是GCS基因导致多药耐药的主要分子病理机制之一。
