Effects Of Doxorubicin On The Level Of Reactive Oxygen Species And The Expression Of Carbonyl Reductase 1 In K562 Cells

ObjectiveThe level of Reactive oxygen species（ROS）and the expression of carbonyl reductase1（CRB1）mRNA in the K562 leukemia cell line under the action of doxorubicin（DOX）were studied,and some experimental bases were provided to study the relationship between CBRs and anthracyclines（ANTs）metabolism.Methods1.K562 leukemia cells were placed into 1640 medium containing 10%fetal bovine serum which was supplemented with 100 U/ml penicillin and 100mg/ml streptomycin,in a humidified atmosphere of 5%CO₂ at 37℃,and cell growth was observed;2.The logarithmic growth phase of K562 cells was randomly divided into 6 groups,and then,they were treated with 0,0.313,0.625,1.25,2.5,5.0μg/mlDOX for 12h、24h and48h,respectively.The proliferation of K562 cells was measured by CCK-8 test.3.The ROS levels in K562 cells were detected by flow cytometry（FCM）with the active oxygen detection kit after 48 hours in different concentrations of DOX.4.Annexin V-FITC/PI staining followed by flow cytometric analysis was used to detect apoptosis in exposure to different concentrations of DOX for 48 hours.5.RT-PCR was used to detect the relative expression level of CBR1 mRNA in K562cells after different concentrations of DOX for 48 hours.Results1.The survival rate of K562 cells decreased with the increase of DOX concentration and the prolongation of the time and showed concentration-dependent and time-dependent,the difference was statistically significance（p<0.05）.2.After treated with 0.313,0.625,1.25,2.5μg/ml of DOX for 48 hours,ROS levels were higher than that in the control group obviously.They were（119.73±9.45）%、（147.65±6.72）%、（167.78±3.30）%、（158.39±4.82）%.The level of ROS increased with the increase of the concentration,and the difference was statistically significance（p<0.05）.The ROS level in the 2.5μg/ml DOX was significantly higher than that in the control group,but with 1.25μg/ml DOX group had no significant difference（P=0.076）.3.After treated with 0,0.313,0.625,1.25μg/ml of DOX for 48 hours,cell apoptosis rates were（8.73±0.62）%、（26.01±1.71）%、（36.09±3.55）%、（47.89±3.94）%.The apoptosis rate increased with the increase of DOX concentration,and there was significant difference between groups（p<0.05）.4.After treated with 0,0.313,0.625,1.25,2.5μg/ml of DOX for 48 hours,RT-PCR results showed that the expression of CBR1 mRNA in K562 cell line was significantly higher than that in control group.They were（0.75±0.08）、（1.46±0.26）、（1.66±0.43）、（1.47±0.08）,respectively,and the difference was statistically significance（P<0.05）.But the difference of mRNA expression level in different concentration group has no statistically significance（P>0.05）.5.At 48 hours,the expression of CBR1 mRNA was not correlated with ROS level and apoptosis rate.Conclusion:1.DOX can inhibit the proliferation of K562 cells with concentration-dependent and time-dependent.However,drug stimulation with long time and high concentration did not improve the cytotoxicity of DOX,which suggesting that we should choose the appropriate drug concentration and time to minimize its side effects.2.The level of ROS in K562 cells increased with the concentration of DOX in a certain concentration and time range.In this process,the apoptosis rate was related to the level of ROS.3.DOX could upregulate the expression of CBR1 mRNA in K562 cells.