The Establishment Of Multidrug Resistant Cell Line Of Human Leukemic Via ROS Regulation And The Mechanism Research

Objective:to establish the K562 resistant model of human leukemia cells by incubation of H₂O₂ for a long time,and to investigate the mechanism of multidrug resistance induced by Reactive Oxygen Species?ROS?in tumor cells.Methods:The distribution of intracellular ROS in human leukemia cell K562 and drug resistant cell K562/A02 after DCFH-DA staining was observed by fluorescence inverted microscope.Flow cytometry was used to detect DCFH-DA-stained human leukemia cells.Different concentrations of H₂O₂ and incubation time were used to establish the model of resitant K562 cells.MTT assay was applied to determine the inhibition rate of Adriamycin?ADM?on these model cells.According to the screening results,the time and concentration of H₂O₂ were selected to establish the model.Finally,drug resitant cell model was named as K562/ROS.Morphological changes of K562/ROS cells were observed by inverted microscope.MTT assay was used to determine the inhibition rates of paclitaxel,cisplatin,cyclophosphamide,doxorubicin,and5-fluorouracil on K562/ROS cells.The IC₅₀ of each antitumor drug and the resistance index of model cells were calculated.K562,K562/ROS,and K562/A02 cells were stained with PI,and the cell cycle distributions of the three groups were detected by flow cytometry.K562,K562/ROS,and K562/A02 cells were stained with DCFH-DA.Flow cytometry was used to detect changes in ROS content in the three groups.The expression of P-glycoprotein?P-gp?in cells of three groups was detected by Western blot.The accumulation of Rh123 in K562,K562/ROS,K562/A02 cells were detected by flow cytometry to reflect the activity of P-gp.After immunostaining,the content and localization of P-gp and incorporated BrDU in K562,K562/ROS,and K562/A02 cells were observed using a laser confocal microscope.In addition,K562,K562/ROS and K562/A02 cells were incubated with PI3K and NF-?B inhibitors LY294002 and BAY117082.Then,MTT assay was used to detect the changes of ADM resistance in cells of three groups.The location of NF-?B in the K562,K562/ROS and K562/A02 cells were detected by laser confocal microscopy.Western blot was used to detect the expression of p-Akt,p-I?B,p-STAT3 and P-gp,thereby to analyze the mechanism of drug resistance in K562/ROS cells.Results:The fluorescence intensity of ROS in human leukemia drug-resistant cell K562/A02 was higher than that in sensitive cell K562,the fold of which was about 2.46±0.34 times.After the condition screen,0.1?mol/L H₂O₂ was chosen to incubate with K562 cells for 12 consecutive weeks to establish resistant K562/ROS cell line.The cell morphology of K562/ROS cells were changed significantly compared with their sensitive counterparts.The original round shape was changed to irregular shape with burr boundary.The adhesion of the cells to the bottom of the bottle increased,and the cells of suspension state decreased.The connection is blurred,the proliferation rate is accelerated,and the metabolic activity is increased.More importantly,K562/ROS cells showed multidrug resistant characteristics with the resitant indexs of 292.82,3.35,4.41,52.65,and 1.73,to paclitaxel,cisplatin,cyclophosphamide,doxorubicin,and 5-fluorouracil,respectively.Compared with K562,the ratio of G₂/M phase of drug-resistant K562/ROS cells increased significantly.The results of ROS assay showed that long-term incubation of 0.1?mol/L H₂O₂ increased the level of basal ROS in K562 cells.The results of Western blot and confocal laser scanning showed that long-term regulation of ROS could induce the expression of P-gp in K562 cells,and the increased P-gp mainly distributed in the cell membrane.The result of Rh123 Accumulation experiment suggested that expressed P-gp on K562/ROS cells with a strong efflux function.Mechanism study showed that PI3K and NF-?B inhibitors reversed the drug resistance of K562/ROS cells to different degrees,and the results were similar to those of K562/A02 cells.In addition,PI3K inhibitor LY294002 reduced the content of p-Akt,inhibited the nuclear translocation of NF-?B,and reduced the content of p-STAT3 and P-gp.While,NF-?B inhibitor BAY11-7082 inhibited the nuclear translocation of NF-?B,decreased the content of p-STAT3 and P-gp,but with no significant effect on p-Akt.Moreover,the combination of LY294002 and BAY11-7082could significantly down-regulate p-STAT3 and P-gp content in K562/ROS cells,which was similar to that of K562/A02 cells.Conclusion:The basal content of ROS in human leukemia-resistant strains was significantly higher than that of sensitive ones.The long-term incubation of ROS with certain concentration on K562 cells can promote multidrug resistance of the cells.The drug-resistant cells were named as K562/ROS.The main characteristics of K562/ROS cells were as follows:increased intracellular ROS content,changes in cell morphology,enhanced efflux protein function and expression,altered cell cycle distribution,and accelerated cell proliferation.The mechanism of drug resistance involved the over-activated PI3K pathway,which activated NF-?B and STAT3 pathway,leading to the upregulation of the functional P-gp expression on cell membrane.