| Objectives:(1)To establish a simple,sensitive and clinical application method for quantitative detection of ADM;(2)To study the sequence of combination of TTD with ADM and the mechanismunderlying its reversal of MDR.Methods:(1)Concentration of ADM was carried out by fluorescence spectrophotometer;(2)Cell Counting kit-8(CCK-8)assay was used to detect the resistance index of K562/ADM,the cytotoxic effect of TTD and its reversal activity.The combination was divided into three groups according to the order of administration: the group which ADM and TTD were given at the same time,the group which TTD was added after ADM,and the group which ADM was added after TTD,and the cell action time of each group was48 h;(3)The established method by(1)was used to detect the ADM accumulation in K562 and K562/ADM cells;(4)Inorganic phosphorus release method was used to observe the effects of different ways of administration(TTD and ADM)on ATPase activity in K562/ADM cells;(5)Western Blot and q PCR were used to analyze the effects of different ways of administration(TTD and ADM)on the protein expression of P-gp and the transcription of MDR1 mRNA in K562/ADM cells,respectively.Results:(1)A method was established for the determination of ADM concentration in cells by fluorescence spectrophotometer.The concentration of ADM was in the range of0.05～10 μg/mL,in which there is a good linear relationship with a linear coefficient of0.9982 between the fluorescence intensity and the concentration.(2)Studies on the activity of MDR reversed by TTD: The IC50 values of K562 and K562/ADM cells to ADM were 1.06±0.10 μg/mL and 44.95±5.61 μg/mL,respectively,and the relative resistance coefficient was 42.41 folds.TTD at the concentration of 1.5μmol/L showed no significant cytotoxicity to K562 and K562/ADM cells and the inhibition rate was less than 10%.When 1.0 μmol /L TTD was combined with ADM,the IC50 value of K562/ADM cells was 12.06±1.27,and the relative resistance coefficient was 11.38,which was 2.73 times smaller than that of ADM alone.There was no significant difference between the IC50 value of K562 cells compared with ADM alone,and the 1.0 μmol/L TTD showed a good reversion activity;(3)Intracellular concentration of ADM was measured by fluorescence spectrophotometry: The combination of ADM with TTD at the same time has a more significant effect on K562/ADM cells with an increasing concentration of ADM,reaching59.84±0.06% and 74.41±0.03% of the average concentration of K562 cells,while there was no a significant increasing concentration of ADM in K562/ADM cells in the group which TTD was added for 48 h later than ADM;(4)The results of inorganic phosphorus release method showed that 1μmol/L TTD could significantly improve the ATPase activity in K562/ADM cells compared with ADM alone.In the combined group,administration of ADM with TTD simultaneously could significantly improve the ATPase activity in K562/ADM cells compared with single ADM group;(5)The results of q PCR showed that MDR1 mRNA in K562 cells was much lower than that in K562/ADM cells.ADM could increase the transcription level of MDR1 mRNA,and TTD could significantly reduce the transcription level of MDRl mRNA;(6)The results of Western Blot showed that there is a low expression of P-gp in K562 cells,while there is a strong expression of P-gp in K562/ADM cells.TTD could reduce expression of P-gp in K562/ADM cells.Conclusion:TTD can antagonize P-gp-mediated ADM resistance by down-regulating the expression of P-gp and competitively binding the P-gp site.Following the combination of ADM with TTD at the same time,the drug resistance of K562/ADM cells could be more effectively reversed. |
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