ABSTRACT
Ceramide metabolism is known to be an essential etiology for various diseases, such as atopic dermatitis and Gaucher disease. Glucosylceramide synthase (GCS) is a key enzyme for the synthesis of glucosylceramide (GlcCer), which is a main ceramide metabolism pathway in mammalian cells. In this article, we developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to determine GCS activity using synthetic non-natural sphingolipid C8-ceramide as a substrate. The reaction products, C8-GlcCer for GCS, could be separated on a C18 column by reverse-phase high-performance liquid chromatography (HPLC). Quantification was conducted using the multiple reaction monitoring (MRM) mode to monitor the precursor-to-product ion transitions of m/z 588.6 → 264.4 for C8-GlcCer at positive ionization mode. The calibration curve was established over the range of 0.625–160 ng/mL, and the correlation coefficient was larger than 0.999. This method was successfully applied to detect GCS in the human hepatocellular carcinoma cell line (HepG2 cells) and mouse peripheral blood mononuclear cells. We also evaluated the inhibition degree of a known GCS inhibitor 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) on GCS enzymatic activity and proved that this method could be successfully applied to GCS inhibitor screening of preventive and therapeutic drugs for ceramide metabolism diseases, such as atopic dermatitis and Gaucher disease.
Subject(s)
Animals , Humans , Mice , Calibration , Carcinoma, Hepatocellular , Cell Line , Chromatography, Liquid , Dermatitis, Atopic , Gaucher Disease , Mass Screening , Mass Spectrometry , Metabolism , MethodsABSTRACT
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
Objective To construct the plasmid containing short hairpin RNA (shRNA) of GCS in order to suppress the expression of GCS and to reverse the multidrug resistance in drug-resistant breast cancer cells. Methods Two reverse repeated motifs of sequence targeting GCS with 6 bp spacer were designed and synthesized in vitro,then they were inserted into the plasmid pSUPER. The recombinant plasmids were transfected into human breast cancer cells. GCS expression was detected by Western blot and immunocytochemistry. caspase-3 expression and its activity were assessed using Western blot and colorimetry,flow cytometry was performed to analyze the ratio of apoptosis. Results MTT method was used to evaluate the 50% inhibition concentration. Enzyme digestion analysis and DNA sequencing confirmed that the recombinant plasmids were successfully constructed. GCS protein content decreased 80%,82% respectively after transfection with recombinant plasmids. The positive rate of GCS expression reduced to 18.1%,16.7% respectively. caspase-3 expression and its activity were significantly higherand the apoptosis of cells increased dramatically. The drug resistance of breast cancer cells to antineoplastic drugs declined significantly. Conclusion shRNA can suppress GCS expression in human drug-resistant breast cancer cells effectively and restore the sensitivity to several antineoplastic drugs.