ABSTRACT
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.
Subject(s)
Drug Resistance, Multiple , Glucosyltransferases/metabolism , Leukemia/enzymology , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Doxorubicin/pharmacology , Humans , bcl-2-Associated X Protein/metabolismABSTRACT
This study was purposed to explore the expression of glucosylceramide synthase (GCS) in human leukemia cells and its relationship with multidrug resistance. RT-PCR was used to analyze peripheral blood samples from 53 leukemia patients with multidrug resistance/non-resistance, and to detect the expression level of GCS gene in HL-60 cells and HL-60/ADR cells, the expression level was compared with the level of mdr-1. The expressions of GCS protein and P-gp protein in HL-60 cells and HL-60/ADR cells were assayed by Western blot analysis. The results showed that the relative optical density ratio of GCS gene amplified bands in samples of leukemia patients with drug-resistance was significantly higher than that in samples of leukemia patients with drug non-resistance group (P < 0.05), meanwhile the significant enhancement of optical density value of GCS gene amplified bands accompanied by high expression of mdr-1 gene. Their correlation showed positive (P < 0.01, r = 0.6). The GCS mRNA and protein were overexpressed in HL-60/ADR cells, and their expression levels were obviously higher than that in HL-60 cells, meanwhile the expression of mdr-1 mRNA and P-gp also significantly increased in HL-60/ADR cells. It is concluded that the high level of GCS in leukemia patients possibly is associated with multidrug resistance of leukemia cells.