| Objective : To investigate the effects of aclarubicin(ACR) on mitoxantrone(MIT) inducing Hela cell DNA stand breaks and apoptosis, as a further study on the mechanism of topoisomerase Ⅱ inhibitors. Method : Hela cells were maintained in RPMI-1640 medium supplemented with 10% newbrone calf serum(NCS), and 1% antibiotic-antimycotic solution (100units/ml penicillin, 100μg/ml streptomycin, 25μg/ml amphotericin B). They were grown at 37℃ in a humidified atmosphere of 5% CO2.Cells were pre-incubated for 20 min with 0.2μM ACR, then treated for one hour with the topo Ⅱ poisons MIT in different concentrations (1nM,5nM,10nM,50nM,100nM). The cells were harvested and used for the analysis of the following items: (1) Double fluorescence analysis Hela cells apoptosis ratio: Cultured cells were centrifuged and suspended in PBS, and then stained with a mixture of acridine orange/ethidium bromide. Fluorescent microscopy is used to identify non-viable cells whose nuclei were stained into bright orange, while viable cells were stained into bright green. Scoring the percentage of apoptotic cells, which theirnuclei became highly condensed or fragmented, and made quantitative analysis. (2) Single cell gel electrophoresis (SCGE): DNA strand breaks were evaluated using a SCGE (comet) assay. Cells (2-4×105) were suspended in 1ml phosphate buffered saline (PBS) following drugs treated, and 20μl of this cell suspension were added to 80μl of 1% low-melt agarose. The samples were then placed on a fully frosted slide precoated with 1% high-melt agarose. The slides were kept in the dark at 4°C for 5 min and then immersed in a lysis solution (100 mM Na2EDTA, 2.5 M NaCl, 10 mM Tris buffer, 1% Triton X 100) for 1 h. After treated by the electrophoresis buffer (300 mM NaOH, 1 mM Na2EDTA) for 20 min, electrophoresis was then performed in fresh solution for 30 min (25 V, 300mA). The samples were then immersed in a neutralizing solution (0.4 M Tris buffer, pH 7.5). following stained with ethidium bromide solution (1 μg/ml) for 20 min and destained with 10 mM MgSO4 for 20 min to decrease background staining, the slides were examined using a fluorescent microscope. (3) Immunohistochemical analysis: After 1 hour of incubation, cells were fixed with 4% paraformaldehyde for 60 minutes. The cells were then permeabilized with 1% Triton-X in PBS over night. After permeabilization, slides were incubated with a 1:100 dilution of topo Ⅱ polyclonal antibody for one hour (1% Triton-X in PBS). After several washes in PBS for 5 minutes, the sections were incubated with the secondary antibody andavidin-biotin-peroxidase complex. Through the slides were washed several times in PBS for 5 minutes, the reactions were visualized by using diaminobenzidine as chromogen. Negative controls were also included for ruling out nonspecific staining. All the items detected above were contrasted with the control group, which was not treated by any of the drugs.Results : The percentage of apoptotic cells detected by the acridine orange/ethidium bromide staining technique after treatment with drugs was evaluated, and the results demonstrated that apoptotic cells treated by ACR and MIT in the concentration of 10 nM,50 nM,100 nM were less than that of MIT used alone in the same concentration.DNA strand breakage was assessed after treatment with MIT for one hour. It was found that the length of comets tails were vary depending on the concentration of the drug. Treatment with 0.2 μmol/L ACR did not show a significant difference from control group. On the contrary, it was found that pre-incubation with ACR (0.2 μmol/L) could reduce the level of DNA breakage induced by MIT alone. Topo Ⅱ expression were observed at different groups. It demonstrated that there were higher expression of topoⅡin the experimental groups compared to the control group. Pre-incubation with ACR could reduced the topoⅡexpression resulted by MIT used alone. Conclusion : (1)ACR can antagonize the function ofMIT in reducing its cell apoptosi...
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