Mechanism Of Ouabain-induced Apoptosis In Jeko-1 Cells

Objective1.To study the effects of cardiac glycosides(digoxin,digitoxin.ouabain)on human hematological malignant cell lines proliferation and cell apoptosis,and select the sensitive cell line.2.To explore the antineoplastic mechanism of cardiac glycosides on sensitive cell line.Methods1.The human hematological malignant cell lines(CZ,Jeko-1,Mino,MM1-s,MM1-r cells)were cultured and then incubated for 24h,48h,72h in the presence or the absence(control)of the cardiac glycosides at different concentrations.Drug concentrations ranged as follows:digoxin(5ng/ml、10ng/ml、30ng/ml、50ng/ml),digitoxin(10ng/ml、20ng/ml、50ng/ml、100ng/ml),ouabain(1nM、20nM、50nM、100nM).After that,overall cell viability was assessed by means of the Cell Counting Kit-8(CCK-8)assay,and the IC50 doses were determined.2.The ability of cardiac glycosides to induce apoptosis was evaluated by Annexin-V and PI flow Cytometry Assay Kits,when exposed to a concentration of IC50 doses.3.The deletion of p53 gene and bcl-l/IgH rearrangement was detected by using fluorescence in situ hybridization(FISH).4.The expression of Na/K.ATPase α1,α2 subunit and c-myc protein in ouabain treated Jeko-1 cells were examined by the western blot,as well as the expression of α1 subunit protein in Jeko-1 cells and BM-MNCs.5.C-myc mRNA in ouabain treated Jeko-1 cells was examined by realtime PCR.Results1.Cardiac glycosides can inhibit cell proliferation and induce cell apoptosis in a time-and dose-dependent manner.Of all the cell types,human MCL cell line Jeko-1 was the most sensitive.The IC50 values of Jeko-1 for dogoxin,digitoxin and ouabain were 47.79±3.95ng/ml,80.89±1.84 ng/ml and 37.79±10.23 ng/ml,respectively.2.p53 gene deletion as well as Bcl-1/IgH rearrangement were demonstrated in Jeko-1 cell line.3.Compared with human bone marrow mononuclear cells,Jeko-1 cells overexpress Na/K-ATPase al subunit protein,and the protein level was increased by ouabain treatment in a time-and dose-dependent way.The expression of Na/K-ATPase a2 subunit in Jeko-1 cells was significantly reduced after ouabain treatment.C-myc wich was overexpressed in Jeko-1 cells was significantly reduced after ouabain treatment not only in protein level bot also in mRNA level.Conclusions1.Cardiac glycosides can inhibit human hematological malignant cell lines proliferation and induce apoptosis.Compared with other cell lines,Jeko-1 cell line showed hypersensitive to cardiac glycosides.P53 gene deletion as well as Bcl-1/IgH rearrangement were demonstrated in Jeko-1 cell line.2.Ouabain,as a specific ligand of Na/K-ATPase,might induce apoptosis in Jeko-1 cells by regulating Na/K-ATPase α1,α2 subunit and c-myc.