| Objective:To investigate the effects of emodin combined with 3’-azido-3’-deoxythymidine(AZT)on proliferation,apoptosis and cycle of human chronic myeloid leukemia cell line K562 cells,and explore their molecular mechanisms in attempt to provide new insight for clinical combined targeted therapy for leukemiaMethod:The chronic myeloid leukemia K562 cell line was routinely cultured.The effects of different concentrations of emodin(8,16 and 32μM),AZT(80,160 and 320μM)and emodin combined with AZT were detected by MTT assay.Calcusyn 2.0 software was used to calculate the synergistic index(CI)of the two drugs.The apoptosis rate and cell cycle of K562 cells after treatment with emodin(32μM),AZT(320μM)and emodin combined with AZT were detected by flow cytometry.The expression of Egr-1 mRNA in K562 cells were detected by emodin(32μM),AZT(320μM)emodin combined with AZT through RT-PCRCells were transiently transfected by electroporation,and the cells were divided into blank control group,non-specific control group(transfection of non-specific sequence)and targeted transfected Egr-1 siRNA group.After 6 hours,transfected cells were observed by inverted fluorescence microscopy and cell transfection rate was detected by RT-PCR.The Cells were treated with emodin(32μM),AZT(320μM)and emodin combined with AZT after 72 hours,and the expression level of β-catenin protein in untransfected cells and Egr-1 siRNA cells was detected by Western blottingResult:Compared with the individual drug groups,emodin combined with AZT inhibited the proliferation of K562 cells 72 hours later,and had a significant synergistic effect(synergy index CI<1).Proliferation inhibition and synergy between the two drugs presented a concentration-dependent and time-dependent mannerThe apoptosis rate of K562 cells treated with emodin combined with AZT by flow cytometry was significantly higher than that of the control group(no drug intervention)and the single administration group(P<0.05).At the same time,after K562 cells were treated with emodin and AZT for 48 hours,Go/Gi phase cells increased and G2/M phase cells and S phase cells decreased(P<0.05).The cells were arrested at G0/G1 phase,and emodin combined with AZT significantly delayed cell cycle arrest of K562 compared with the single treatment group(P<0.05)RT-PCR results showed that the expression level of Egr-1 mRNA in K562 cells of emodin combined with AZT intervention group was up-regulated when compared with control group and AZT group(P<0.05).This indicates that the combination group has an enhanced effect on Egr-1 gene expressionTransfection of FAM fluorescent protein-labeled siRNA cells under a fluorescenced inverted microscope revealed that the transfected cells showed green fluorescence.The expression level of Egr-1 transfected by Egr-1 siRNA was detected by RT-PCR.Compared with the blank control and non-specific control(non-specific sequence),the expression level of Egr-1 transfected by the target siRNA was decreased(P<0.01)The expression of β-catenin protein in WNT/β-catenin signaling pathway was significantly decreased by immunoblotting in K562 cells treated with emodin(32μM),AZT(320μM)and emodin combined with AZT(32μM/320μM).The difference was statistically significant(P<0.05).Compared with untransfected K562 cells,the expression of β-catenin protein was significantly increased in the three groups of siRNA groups,and the difference was statistically significant(P<0.05).Compared with untransfected K562 cells,the expression levels of β-catenin protein in the three groups of non-specific transfected siRNA showed similar results,no significantly difference(P>0.05)Conclusion:Emodin combined with AZT could inhibit synergistically the proliferation of chronic myeloid leukemia K562 cells,promoted apoptosis and arrested cell cycle changes,which may be related to Egr-1 mediated WNT/β-catenin signaling pathway. |
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