ABSTRACT
AIM: To investigate whether glucosylceramide synthase (GCS) regulates apoptosis-related gene bcl-2 expression via MEK/ERK signaling pathway , thus enhancing drug resistance of K 562/A02 human leukemia multidrug resistant cell line.METHODS:siRNA targeting GCS was transfected into K562/A02 cells.Bcl-2, p-ERK and total ERK expression at mRNA and protein levels after GCS knockdown were detected by real-time PCR and Western blotting .After exposed to MEK-ERK pathway inhibitor U0126, the expression of Bcl-2 at mRNA and protein levels also was analyzed by real-time PCR and Western blotting , respectively.The viability of the cells was evaluated by CCK-8 assay.RESULTS:The expression of GCS and Bcl-2, as well as MEK/ERK signaling were significantly inhibited in K 562/A02 cells by GCS siRNA transfection compared with negative control group .Inactivation of MEK/ERK signaling due to U0126 treatment de-creased Bcl-2 mRNA and protein levels in a concentration-dependent manner , and sensitized K562/A02 cells to adriamy-cin.CONCLUSION:GCS may affect the expression of apoptosis-related gene bcl-2 by MEK/ERK signaling pathway , thus regulating multidrug resistance of human leukemia K 562/A02 cells.
ABSTRACT
<p><b>OBJECTIVE</b>To investigate whether transcription factor-kappaB (NF-κ B) is involved in the modulation of P-glycoprotein (P-gp) by glucosylceramide synthase (GCS) in a multidrug resistance leukemia cell line K562/A02 and to explore the relationship between NF-κ B and extracelluar signal-regulated kinase (ERK).</p><p><b>METHODS</b>K562/A02 cells were treated with GCSsiRNA, pyrrolidine dithiocarbamate (PDTC, a NF-κ B specific inhibitor) and U0126 (a MEK1/2 inhibitor), respectively. The expression of GCS and multidrug resistance protein 1 (MDR1) mRNA were analyzed with qRT-PCR. Various proteins of different groups were measured by Western blotting.</p><p><b>RESULTS</b>After transfected with GCSsiRNA for 48 h, GCS mRNA were reduced by 62% (51%-73%) and MDR1 mRNA was reduced by 52% (43%-61%) in the K562/A02 cells. Compared with the negative control, relative expression of NF-κ B p65 in nuclear and P-ERK1/2 were both down-regulated, and P-gp was also inhibited significantly at 72 h after transfected with GCSsiRNA (P< 0.05). In addition, the expression of P-gp was decreased at 24 h with 80 μ mol/L PDTC and 48 h with 20 μ mol/L PDTC. P-ERK1/2 was inhibited significantly when the cells were treated with 20 μ mol/L U0126 for 48 h. The expression of NF-κ B p65 in nuclear and P-gp were also down-regulated.</p><p><b>CONCLUSION</b>NF-κ B can modulate the effect of GCS on P-gp in K562/A02 cells. P-ERK1/2 can activate NF-κ B in above signal transduction pathway.</p>
Subject(s)
Humans , ATP Binding Cassette Transporter, Subfamily B, Member 1 , Genetics , Metabolism , Cell Line, Tumor , Drug Resistance, Multiple , Drug Resistance, Neoplasm , Glucosyltransferases , Genetics , Metabolism , K562 Cells , Leukemia , Genetics , NF-kappa B , Genetics , MetabolismABSTRACT
Purpose:To investigate the relation between the expression of cyclinB1 gene and drug resistance in patient with acute leukemia. Methods:Cyclin B1 gene mRNA expression in 13 cases of drug resistant and 14 drug non-resistant acute leukemia patients’ peripheral blood have been determinated by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR).The expression of mdr1 gene mRNA in these patients was measured with fluorescence -quantitative reverse transcription-polymerase chain reaction (RT-PCR). Results:The expression of cyclin B1 mRNA in the drug resistant group was significantly higher than the drug non-resistant group cyclin B1 mRNA (P