ABSTRACT
<p><b>OBJECTIVE</b>To investigate the effect of glucosylceramide synthase (GCS) on P-glycoprotein (P-gp) expression via extracellular signal-regulated kinase (ERK) pathways in leukemia K562/A02 cell line.</p><p><b>METHODS</b>K562/A02 multidrug resistance cells were treated with GCS siRNA and U0126, respectively. Expression of multidrug resistance protein 1 (MDR1) mRNA was analyzed with qRT-PCR. Phosphorylated ERK1/2, total ERK1/2 protein and P-gp in different groups were measured with Western blotting.</p><p><b>RESULTS</b>After treated with U0126, P-ERK1/2 was decreased along with the increased U0126 concentration. P-ERK1/2 and P-gp were apparently down-regulated by U0126 at the concentrations of 20 μmol/L, 40 μmol/L and 60 μmol/L. After being transfected with GCS siRNA, GCS mRNA was inhibited by 70.50% (58.00%-76.00%) in K562/A02 cells. Compared with the negative control, both P-ERK1/2 and P-gp were inhibited significantly after RNAi for 72 hours (P<0.01 and P<0.05, respectively.</p><p><b>CONCLUSION</b>GCS may affect the expression of P-gp by ERK signal transduction pathway in leukemia cells.</p>
Subject(s)
Humans , ATP Binding Cassette Transporter, Subfamily B, Member 1 , Genetics , Metabolism , Cell Line, Tumor , Drug Resistance, Multiple , Drug Resistance, Neoplasm , Glucosyltransferases , Metabolism , K562 Cells , Leukemia , Genetics , Metabolism , MAP Kinase Signaling SystemABSTRACT
<p><b>OBJECTIVE</b>To investigate the correlation of glucosylceramide synthase (GCS) gene and multidrug resistance 1 (MDR1) gene in inducing multidrug resistance in human multidrug-resistant K562/A02 cell line, and search for a novel strategy for reversing multidrug resistance of leukemia cells.</p><p><b>METHODS</b>The expression levels of GCS and MDR1 mRNA in K562 and K562/A02 cells were assayed by RT-PCR. siRNAs targeting the GCS and MDR1 gene were transfected into K562/A02 cells with liposome, respectively. The differential expression of GCS and MDR1 mRNAs, as well as their correlation, were detected by RT-PCR and real time quantitative-PCR(QPCR).</p><p><b>RESULTS</b>The expression level of GCS and MDR1 mRNA was dramatically lower in drug-sensitive K562 cells compared with the K562/A02 cells. The GCS mRNA was inhibited by 73%(59%-82%) and MDR1 mRNA expression was down regulated by 67% (38%-82%) in K562/A02 cells after being transfected with GCS siRNA. The expression level of MDR1 mRNA was inhibited by 81%(63%-91%) and GCS mRNA expression had no apparent change in K562/A02 cells treated with MDR1 small interference RNA(siRNA).</p><p><b>CONCLUSION</b>Positive correlation was detected between the expression of GCS and MDR1 mRNA in K562/A02 cells and MDR1 mRNA expression was down regulated after silencing the GCS gene expression.</p>