The Effects Of Ouabain On The Proliferation And Apoptosis Of Human Acute Lymphoblastic Leukemia Cell

Objective To investigate the effects of ouabain on the proliferation and apoptosis of human acute lymphoblastic leukemia cell line Jurkat and primary culture of acute lymphoblastic leukemia cell and explore its mechanism.Methods In vitro, exponential phase of human acute lymphoblastic leukemia cell line Jurkat was treated with different concentration of ouabain. MTT assay was used to to measure cell proliferation rate. The morphological changes in Jurkat cell line were observed after wright staining. Cell apoptosis was determined by flow cytometry after Annexin V/PI staining. Mitochondria membrane potential was measured by JC-1 assay. Cell membrane potential was labeled by DiBAC4(3) and measured by flow cytometry. Immunocytochemistry was used to examine the expression of NKA-α1 protein. The expression of Bcl-2 was detected by Western Blot. Primary culture of acute lymphoblastic leukemia cells were isolated by density gradients centrifugation and adhesive screening method. The vigor of cells was detected by MTT, and the apoptotic rate was measured by Annexin V/PI assay.Results MTT assay showed that ouabain could significantly inhibit the proliferation of Jurkat cell lines in a dose- and time-dependent manner (P<0.05). Wright's staining indicated dramatic proapoptotic morphological changes in Jurkat cell lines after ouabain treatment. Annexin V/PI assay revealed that cell apoptotic ratio of 10nmol/L,20nmol/L and 50nmol/L ouabain treatment was12.37±0.55% ,94.3±0.46%,98.6±0.79%. The difference was significant compared to the control group (P<0.05). JC-1 assay indicated that ouabain induced cell apoptosis accompanied with mitochondria membrane potential descent. Cell membrane potential detection showed that the treatment concentration of ouabain didn't induce the depolarization of jurkat cell membrane. Immuncytochemical method suggested ouabain down-regulated the expression of NKA-α1 protein in Jurkat cells. Western Blot showed the Bcl-2 expression decreaseed gradually.Low dose ouabain had the same effect on primary culture of acute lymphoblastic leukemia cell.Conclusion Ouabain can significantly inhibit the proliferation of human acute lymphoblastic leukemia cell line Jurkat in a dose- and time-dependent manner and induce its apoptosis in vitro. Via binding NKA-α1 protein, ouabain down-regulates its expression without inhibiting the function of ion transportation of Na,K-ATPase. It was connected with mitochondria pathway.