ABSTRACT
Aim To evaluate the effect of expression inhibition of spermine oxidase(SMO)on the actitumor activity of polyamine analogue CPENSpm (N~1-cyclopropylmethyl-N~(11)-ethylnorspermine).Methods siRNA technique was used to inhibit expression of SMO in human lung cancer line A549.QT-RT-PCR and enzyme activity assay was performed to determine the expression level of SMO.The cell proliferation was detected by MTT assay.The apoptosis of A549 cells were evaluated by DNA degradation and Sub-G_1/flow cytometry assay.Results The A549 cell line with silenced SMO expression was successfully obtained.Basic SMO mRNA and enzyme activity levels in the SMO-siRNA plasmid transfected cells were 0.53% and 14% lower than that in the control cells respectively. Treating A549 control cells by 10 μmol·L~(-1) CPENSpm for 24 hours resulted in a 10-folds up-regulation of SMO in mRNA level and 20-fold increase in enzyme activity,but this drug-induced SMO expression was obviously prevented in SMO-siRNA plasmid transfected cells.MTT assay demonstrated that SMO expression inhibition decreased the sensitivity of A549 cells to CPENSpm exposure(0～20 μmol·L~(-1)).DNA degradation and sub-G_1 assay proved a deceased ability of CPENSpm to induce apoptosis in SMO-siRNA plasmid transfected cells.Conclusion Up-regulation of SMO by CPENSpm is possibly one of the molecular basics for its antitumor activity.
ABSTRACT
Aim To investigate subcellular distribution and accumulation of ADR in the sensitive and multidrug-resistant HL-60 cells and its relation to multidrug resistance.Methods The subcellular distribution and accumulation of ADR were studied by confocal scanning laser microscope and flow cytometry.The effects of verapamil,BSO,brefeldin A and chloroquine on ADR distribution and accumulation in HL-60/ADR cells were also examined.Rhodamine123,NBD-ceramide and neutral red were used as fluorescent probes to stain the mitochondria,Golgi apparatus and lysosomes respectively were used to identify the subcellular compartments where ADR was sequestered.Results In drug-sensitive cell line HL-60,ADR fluorescence distributed evenly in the nucleus and cytoplasm,while in multidrug-resistant cell line HL-60/ADR,ADR fluorescence distributed in a punctated pattern in the cytoplasm and was reduced in the nucleus.The mode of ADR distribution in HL-60/ADR cells is highly similar to that of NBD-ceramide.BSO and brefeldin A,instead of verapamil and chloroquine could reverse the abnormal distribution and accumulation of ADR in HL-60/ADR cells.Conclusions The change of ADR distribution and reduction of ADR accumulation in multidrug-resistant cell line was involved in the mechanism of multidrug resistance.