Csn-B Combined With Imatinib Induced Apoptosis Of Chronic Myeloid Leukemia Cells Through Nur77/Pim-1/Drp1 Pathway

Objective:To investigate the anti-Cml activity of Nur77 specific agonist Csn-B combined with Imatinib by promoting Nur77 expression,and to explore the potential role of its Nur77/Pim-1/Drp1 signaling pathway.Methods:1.Through online tools GEO2R(https://www.ncbi.nlm.nih.gov/geo/geo2r/)for GSE43754 data set(sample,20 normal CML:patients=10:10)has carried on the variance analysis,to obtain the corresponding data.2.The cell activities of K562 cells were determined by CCK-8 assay after 24 h administration of Csn-B and Imatinib alone or in combination.3.Transwell method was used to detect the migration ability of K562 cells in each group after 24 h of single or combined administration of Csn-B and Imatinib.K562 cells attached to the bottom membrane of the chamber were observed under a microscope and photographed and counted.4.The apoptosis rate of K562 cells was detected by flow cytometry after 24 h treatment with Csn-B and Imatinib alone or in combination.5.Western blot assay was used to detect the expression levels of apoptosis-related proteins in K562 cells treated with Csn-B and Imatinib alone or in combination for 24 h.6.Reactive oxygen species(Ros)expression levels in K562 cells were detected after 24 h treatment with Csn-B and Imatinib alone or in combination.Results:1.By analyzing the GSE43754 data set,we found that Nur77 protein expression was significantly decreased in patients with chronic myeloid leukemia compared with normal people.2.CCK-8 results showed that the cell viability of K562 decreased with increasing concentrations of Csn-B and Imatinib at the same time,and the survival rate of K562 cells in the combination group was significantly lower than that in the monotherapy group(P<0.001).3.Transwell results showed that the migration ability of K562 cells was significantly decreased in the treatment group compared with the control group(P<0.001),and the migration inhibition effect was more obvious when the combined treatment of Csn-B and Imatinib was applied to K562 cells at the same time.4.Flow cytometry showed that the apoptosis rate of blank control group,Csn-B monotherapy group and Imatinib monotherapy group was 2.10%,9.45%and 9.94%,respectively,and that of combined treatment group was 17.54%,which was significantly higher than that of monotherapy groups(P<0.001).5.Western blot results showed that compared with the experimental control group,Csn-B and Imatinib alone or in combination could promote the protein expression levels of Nur77,P-drp1,S616 and Bax in K562 cells.It also inhibited the protein expression levels of Pim-1 and Bcl-2 in K562 cells,and the promotion and inhibition effects were more obvious after the combination of Csn-B and Imatinib.6.ROS results showed that compared with the experimental control group,the fluorescence intensity of Mitosox and Ros in K562 cells treated with Csn-B or Imatinib alone was increased,while the fluorescence intensity values of Mitosox and ROS in K562 cells treated with combined treatment were significantly increased(P<0.01,P<0.001).Conclusion:Csn-B combined with imatinib can enhance the expression of reactive oxygen species and induce apoptosis of K562 cells through Nur77/PAM-1/Drp1/pathway.