Effects Of Ouabain On Proliferation Regulations In Leukemia Cells And Related Molecular Mechanisms

PartⅠEffect of Ouabain at different concentrations on growth ofLeukemia Cells and expression of Na~+/K~+-ATPaseα1 subunitObjective Cardiotonic steroids (CTS) can bind to Na~+/K~+-ATPase to activatecomplex intracellular signaling cascades regulating the proliferation and apoptosis of cells.The aim of this study was to investigate the effects of ouabain at different concentrations ongrowth regulation in various kinds of leukemia cell lines.Through this investigation,weintend to explore the pathogenesis of leukemia,functions or effects of Na~+/K~+-ATPase as asignal transduction conductor and its effects on cell growth.Methods By using methyl thiazolyl tetrazolium (MTT) assay,the proliferation ratesof leukemia cell lines were observed after treating with ouabain atdifferent concentrations(1nmol/L,10nmol/L) after 6 hours,12 hours,24 hours incubation respectively.Theexpressions of Na~+/K~+-ATPaseα1 subunit of leukemia cells were evaluated byWestern-Blot.Results The MTT results showed that ouabain at 1nmol/L or 10nmol/L could notonly induce proliferation of lymphocytic leukemia B95 cell lines,but also megakaryocyticleukemia Meg01 cell lines.And these concentrations of ouabain could up-regulate theexpression of Na~+/K~+-ATPaseα1 subunit proved by Western-Blot.Compared with thecontrol groups,the differences of the proliferation and the expression of Na~+/K~+-ATPaseα1 subunit of the leukemia cell lines were significant among the groups of 1nmol/L or10nmol/L ouabain incubated for 24h (P＜0.05).The proliferation effect of leukemia celllines and the expression of Na~+/K~+-ATPaseα1 subunit were in dose dependent mannerwith ouabain concentration and incubation time.Conclusions Ouabain at 1nmol/L or 10nmol/L could promote proliferation of lymphocytic leukemia cell lines (B95) and megakaryocyfic leukemia cell lines (Meg01),the proliferation effect was in dose dependent manner with the concentration andincubation time of ouabain.It was concluded that Na~+/K~+-ATPaseα1 subunit might bindto ouabain to regulate the proliferation of leukemia cell lines of different origins. PartⅡSignal pathways in Ouabain-regulated proliferation ofLeukemia cellsObjective Cardiotonic steroids (CTSs) can bind to Na~+/K~+-ATPase and activateprotein kinase cascades,resulting in changes in cell proliferation,differentiation orapoptosis in a cell-specific manner.We explored the participation of ouabain-activatedsignaling pathways in growth regulation of leukemia cells.Methods Lymphocytic leukemia Jhhan cells and megakaryocytic leukemia M07ecells were incubated at different concentrations of ouabain (0,1 and 10 nmol/L) for 24hours.Cell proliferation was measured by methyl thiazolyl tetrazolium (MTT) assay.Toprobe the role of ouabain-induced signaling in control of cell growth,we employed c-Srckinase inhibitor PP2 and the MEK inhibitor PD98059,respectively.The expression ofNa~+/K~+-ATPaseα1 subunit of leukemia cells was evaluated by RT-PCR and westernblotting.Results 1 nmol/L and 10 nmol/L ouabain promoted proliferation of both Jhhan andM07e cells.Ouabain also could up-regulate the expression of Na~+/K~+-ATPaseα1 subunit.Addition of either PP2 or PD98059 blocked the effects of ouabain on cell proliferation andreduced the expression of Na~+/K~+-ATPaseα1 subunit of leukemia cells.Conclusion Ouabain activates Src and ERK1/2 pathways and regulates theproliferation of leukemia cells. PartⅢThe effects of Bcl-2 gene family in growth regulation ofleukemia cells by ouabainObjective The aim of this study was to investigate the effects of ouabain on growthregulation in B95 and Meg01 leukemia cell lines.Through this study,we plan to investigatethe effects of the Bcl-2 gene family participating in growth regulation induced by ouabain.Methods By using methyl thiazolyl tetrazolium (MTT) assay,the proiferation ratesof leukemia cell lines were observed after utilizing ouabain in various concentrations (1,10,100nmol/L).The Bcl-2,Bcl-XL,Bax mRNA expressions were evaluated by RT-PCRand the Bcl-2,Bcl-XL,Bax protein were evaluated through Western-Blot.Results The MTT results showed that low concentration of ouabain (1,10nmol/L)could promote the proliferation rates of lymphocytic leukemia B95 cell lines andmegakaryocytic leukemia Meg01 cell lines.And high concentration of ouabain (100nmol/L)could suppress the proliferation rates of B95 and Meg01 cell lines.The mRNA and proteinexpression of Bcl-XL,Bax were changed after ouabain treatment.Conclusion It is concluded that ouabain could regulate the growth of leukemia celllines.The growth regulation effects are mediated by Bcl-2 gene family signaling pathway.