Chemotherapies And RNAi Inhibiting Expresseions Of Glucosylceramide Synthase Gene In Human K562/AO2 Cell Line And Their Role In Reversion Of Leukemia Multidrug Resistance

Objective To investigate the expressions of glucosylceramide synthase(GCS) mRNA which was inhibited by chemotherapies and RNAi in human leukaemia multidrug resistance cell line K562/AO2 and it's relation to multidrug resistanceMethods (1) The IC50 (the concentration causing 50â„…inhibition of cell growth) of ADR to K562 and K562/AO2 cells which had been treated by CsA and PPMP were assayed by Alamar BlueTM multifunction cell staining method. The expression level of GCS and mdr-1 message RNA was assayed by RT-PCR.(2)The realtime quantative PCR method were used to chose the just siRNA molecules targeting to glucosylceramide synthase mRNA and the inhibition rates of the expressions of GCS gene in K562/AO2 cells were measured by this method too;The expression of GCS protein were inspected by flow cytometry;RT- PCR method were used to evaluate the inspecial interference to the MDR1 mRNA ;and finally ,the cellulartoxicity trials were assayed by Alamar BlueTM multifunction cell staining method.Results (1)The combination of CsA and PPMP can obviously inhibit the expressions of GCS mRNA in K562/AO2 cells, and render cells sensitive to adriamycin. The low dosages combination of these two drugs not only has this effect but also has fewer cellulartoxicities. (2)The chosen siRNA molecules can successfully silence the expressions of glucosylceramide synthase and it's mRNA in K562/AO2 cells which targeting is very specialized and has little interference to MDR1 mRNA .The inhibited rate of GCS mRNA is 73.38% and the GCS protein is 18.41 folds. When the expressions of GCS gene is inhibited , K562/AO2 cells will restore it's sensitivity to adriamycin .the drug reversion multiple is 5.6 folds. Conclusion The combination between CsA and PPMP can reverse multidrug resistance of K562/AO2 cells by inhibiting the expression of GCS message RNA.When use siRNA targeting to GCS to'knock-down'GCS mRNA ,the K562/AO2 cells will restore it's sensitivity to adriamycin. These studies reveal that GCS gene has very intimate relations with the occurrences of MDR in leukaemia cells. GCS gene must act as a very important role in the occurrences of MDR in leukaemia. The high expressions of GCS gene is a novel mechanism of multidrug resistance and anti-GCS therapy as an innovative method can perhaps overcome multidrug resistance of leukaemia cells.