Effect Of Homoharringtonine On Proliferation And Apoptosis On Imatinib-resistant Chronic Myelogenous Leukemia Cells And The Relationship With Bcl-6/p53 Pathway

Objective To investigate the effect of homoharringtonine(HHT)on proliferation and apoptosis of imatinib-resistant chronic myelogenous leukemia(CML)cells and to detect the relationship with Bcl-6/p53 signaling pathway.Methods(1)The inhibitory effect of imatinib on K562 and K562/G01 cells were detected by Cell Counting Kit-8(CCK-8)in order to investigate the drug-resistant characteristic of K562/G01.(2)Using western blot and quantitative Real-time PCR(qPCR)detect the expression of the oncogene Bcl-6 protein and mRNA in the K562/G01 cells and primary bone marrow cells of CML blastic phase patients were detected.(3)The proliferative rate of K562/G01 cells treated with HHT was detected by CCK-8 and the cell apoptosis rate and cell cycle arrest rate were decteded by flow cytometry.(4)After being transfected with Bcl-6 siRNA using lipofectamine2000,the interference rate of transfected K562/G01 cells were detected with CCK-8 and western blot Cell apoptosis rate was determined by flow cytometry.The effect of Bcl-6 on proliferative and apoptosis of imatinib-resistant cells was observed.(5)qPCR was used to detect the expression of Bcl-6 and p53 mRNA in a variety of cell lines(K562 cells,K562/G01 cells,SUP-B15 cells,KU812 cells,293T cells)before and after treatment with HHT.(6)Western blot was used to detect the expression of Bcl-6 and p53 proteins in a.variety of cell lines(K562 cells,K562/G01 cells,SUP-B15 cells,KU812 cells,293T cells)before and after treatment with HHT.Results(1)The K562 cells were more sensitive to IM than the K562/G01 cells.The drug resistance intensity of K562/G01 cells was 19 times to the K562 cells.(2)The result of western blot indicated that the level of Bcl-6 protein was higher at K562/G01 cells than K562 cells.It also indicated that the level of Bcl-6 protein was higher at bone marrow mononuclear cells of CML blastic phase patients than cells of CML patients in chronic phase.(3)HHT could induce K562/G01 cells growth inhibition,promote apoptosis and produce cell cycle arrest.(4)Bcl-6-siRNA could down-regulated the expression of Bcl-6 mRNA and protein in K562/G01 cells.K562/G01 cells were sensitive to Bcl-6-induced growth inhibition and apoptosis.(5)After treatment with HHT,the expression of Bcl-6 protein in a variety of cell lines(K562 cells,K562/G01 cells,SUP-B15 cells,KU812 cells,293T cells)were down-regulated.At the same time,the expression of p53 protein were up-regulated.The expression of the proteins was dose-dependent.Conclusion The expression of Bcl-6 protein is higher in IM-resistant cell lines than IM non-resistant cell lines.HHT can suppress the growth and induce apoptosis of IM-resistant cells,the mechanism of which is associated with the down-regulation of Bcl-6 protein and up-regulation of p53 protein.