The Mechanism Of Ouabain Induce The Apoptosis Of Raji Cells And Jurkat Cells

Background:Cardiotonic steroids(CTSs),specific ligand of the Na+/K+-ATPase(NKA)are concentration-dependent NKA inhibitors.More and more evidences showe that CTSs have an anti-tumor effect.Although combination chemotherapy and molecular targeted therapy improve the efficacy of hematological malignancies,there are still a lot of problems like the side effects and poor specificity.Exploring new anti-cancer drugs and discovering new targets for anti-tumor for hematological malignancies gain extensive attention in recent years.Researches indicate that CTSs also have significant anti-tumor effect on hematological malignancies,but the anti-tumor mechanism of CTSs is not quite clear yet.Aizman et.al show that Low doses of ouabain without inhibition the NKA could regulate cells proliferation and apoptosis through signaling pathways.Aperia et.al found that NKA and IP3R could form a signaling transduction domain by AnkyrinB,CTSs could adjust the signaling domains,regulate intracellular calcium oscillations,change the activity of nuclear transcription factor NF-κB,finally affect the growth of cells.That is to say CTSs may regulate the growth of cells through the NKA/IP3R-calcium oscillations-NF-κB signaling pathways.On the other hand,Xie showed that NKA could bind Src kinases directly to form a signaling transduction domain,which regulated the PI3K/AKT signaling pathway involved in cell growth.So CTSs may affect the cells proliferation and apoptosis through NKA/Src-PI3K/AKT-NF-κB signaling pathways.Xie further found that NKA/Src signaling pathway was related with the expression of IP3R,indicating that this two signaling pathway were interlinked.Our previous study showed that(Unpublished experimental data):CTSs induced apoptosis in various of hematological malignancies cell lines and primary culture of malignant cells.Raji cells and Jurkat cells were more sensitive to CTSs than other cells.Yet the anti-tumor mechanism of CTSs on hematological malignancies and why Raji cells and Jurkat cells are more sensitive remain unclear.c-myc,which is highly expressed in Raji cells,plays an important role in regulating the proliferation and apoptosis of Raji cells.The same is IL-2R to Jurkat cells.We wonder weather the mechanism of ouabain induce the apoptosis of Raji cells and Jurkat cells relevant with those two pathways?why Raji cells and Jurkat cells are sensitive to CTSs?Based on the previous discovery,we will further elaborate on the following issues:(1)to verify the effect of cardiac glycoside drug-ouabain on cell proliferation and apoptosis of Raji cells and Jurkat cells.(2)to explore weather ouabain induce the apoptosis of Raji cells and Jurkat cellsthe through the NKA/IP3R or NKA/Src signaling pathway;(3)to explore weather the sensitivities of Raji cells and Jurkat cells to ouabain is related with c-myc and IL-2R.Chapter 1 The mechanism of ouabain induce the apoptosis of Raji cells.Objective:Human Burkitt’s lymphoma is a highly malignant tumor of blood.Researches focus on finding efficient and specificity methods of treatments.Our previous study found that Burkitt’s lymphoma cell line(Raji cells)and primary culture of Burkitt’s lymphoma cells were sensitive to CTSs(Unpublished experimental data).The specificity characteristic of Raji cells is the genetic changes resulting the over-expression of oncogene c-myc.The c-myc is closely related to proliferation,differentiation and apoptosis in a variety of tumor cells.While the expression of c-myc is regulated by nuclear transcription factor(NF-κB).Recent studies showed that ouabain could regulate cell proliferation and apoptosis through NKA/IP3R-calcium oscillation-NF-κB pathway.Therefore,based on the previous studies we will explore the following tings:(1)to verify the effect of ouabain on cell proliferation and apoptosis of Raji cells;(2)to explore whether the mechanism of ouabain induce the apoptosis of Raji cells is connected with the NKA/IP3R-calcium oscillation-NF-κB signaling pathway.;(3)to study the effect of ouabain on c-myc expression in Raji cells.Methods:1.Raji cells were treated with different concentration of ouabain.2.The cell apoptosis was detected by Annexin-V and PI flow Cytometry Assay Kits3.The proliferation was detected by Cell Counting Kit-8(CCK-8)assay.4.The expression of related proteins(NKA-α1、IP3R、c-myc)was detected by Western blot and RT-PCR.5.The activity of NF-κB was tested with TransAM-p65 Kits.6.Using siRNA technology to reduced the expression of NKA in Raji cells.7.Raji cells with NKA-siRNA transfection were treated with different concentration of ouabain.Testing the apoptosis and proliferation of those cells.Detecting the expression of related proteins(IP3R、c-myc)of those cells.Results:1.Ouabain(25nM、50nM、100nM)induced apoptosis and proliferation inhibition of Raji cells in a concentration-dependent manner(p<0.05).2.The expression of NKA-al in Raji cells was increased after 48h ouabain treatment(p<0.05).On the contrary,IP3R and c-myc proteins along with the mRNA levels in Raji cells were significantly reduced after 48h ouabain treatment(p<0.05).3.The activity of NF-κB was also decreased by ouabian in a concentration-dependent manner in Raji cells(p<0.05).4.The expression of NKA-al was reduced after the siRNA which was transfected into Raji cells for 48h(p<0.05).This down-regulation of NKA-al decreased the ouabain effect on cell apoptosis and proliferation(p<0.05).5.The down-regulation of NKA-al in Raji cells also altered the effect of ouabain on IP3R and c-myc protein reductions(p<0.05).6.The effect of ouabain on NF-κB activity was decreased in down-regulation of NKA-al cells(p<0.05).Conclusion:1.Ouabain could inhibit cell proliferation and induce apoptosis of Raji cells,and the effects were concentration-dependent.2.ouabain inducing the apoptosis of Raji cells was associated with up-regulation of NKA-al subunit expression,down-regulation of IP3R,NF-kB activity weakened,When the expression of NKA-al subunit was weakened in Raji cells by NKA-al-siRNA,those effects of ouabain were weakened.These are indicated that the mechanism of ouabain anti-Raji cells may closely related to the NKA/IP3R-calcium oscillation-NF-κB signaling pathway.3.Ouabain could down-regulate the expression of c-myc in Raji cells.When the expression of NKA-al subunit was weakened,down-regulation of c-myc by ouabain was weakened too.That is to say c-myc may be one of the reasons that Raji cells are sensitive to ouabain.Chapter 2 The mechanism of ouabain induce apoptosis of Jurkat cells.Objective:T-cell acute lymphoblastic leukemia(T-ALL)is a kind of malignant disease with the abnormal proliferation of Precursor T lymphocytes.The efficacy of treatments is limited.T-ALL has a poor prognosis and the recurrence rate is high.Our previous study found that ouabain could induce apoptosis of Jurkat cells(acute T lymphoblastic leukemia cell line)(Unpublished experimental data).But the mechanism remains unclear.Jurkat cells are able to secrete specific T lymphocyte growth factor interleukin-2(IL-2),while IL-2 binding with the IL-2 receptor a subunit can affect the proliferation and differentiation of lymphocyte.Over-expression of IL-2R is occured in variety of tumor cells.Our previous studies showed that IL-2Rαin Jurkat cells were over-expressed(Unpublished experimental data).Gonzalez-Garcia et.al show that IL-2R involved in cell proliferation and apoptosis,and related with activation of multiple kinases,including Src kinases.Studis show that inositol 1,4,5-triphosphate receptor(IP3R)can affect intracellular calcium ion concentration,regulate the secretion of IL-2 within Jurkat cells involved in regulation of Jurkat cells proliferation and apoptosis.Xie et.al show that NKA and Src may form a Signal transduction domain.ouabain can regulate proliferation and apoptosis through NKA/Src-PI3K/AKT-nuclear transcription factor NF-κB signaling pathways.Aperia et.al show that NKA also can bind with IP3R as a signaling transduction domain,ouabain regulate the proliferation and apoptosis by NKA/IP3R-calcium oscillations-NF-κB signaling pathways.Xie et.al further show that the activation of NKA/Src signaling pathway by ouabain may enhance NKA/IP3R pathway,indicating that the two signaling pathways are interrelated.That is to say ouabain may regulate the proliferation and apoptosis of Jurkat cells through NKA/SRC/IP3R signaling pathway.There is no reports about the relationship between this pathway and Jurkat cells.As mentioned above,IL-2 secretion and the expression of IL-2R in Jurkat cells were associated with this signaling pathway,which we might speculate ouabain induces apoptosis of Jurkat is related with NKA/Src/IP3R-NF-κB signaling pathway.This study aimed to:(1)to verify the effect of ouabain on cell proliferation and apoptosis of Jurkat cells;(2)to explore whether the mechanism of ouabain induce the apoptosis of Jurkat cells is connected with the NKA/Src/IP3R-NF-κB signaling pathway.;(3)to study the effect of ouabain on IL-2R expression in Jurkat cells.Methods:1.Jurkat cells were treated with different concentration of ouabain.2.The cell apoptosis was detected by Annexin-V and PI flow Cytometry Assay Kits3.The proliferation was detected by Cell Counting Kit-8(CCK-8)assay.4.The expression of related proteins(IL-2R、Src、NKA-α1、IP3R)was detected by Western blot and RT-PCR.5.The activity of NF-κB was tested with TransAM-p65 Kits.6.Using siRNA technology to reduced the expression of NKA in Jurkat cells.7.Jurkat cells with NKA-siRNA transfection were treated with different concentration of ouabain.Testing the apoptosis and proliferation of those cells.Detecting the expression of related proteins(IP3R)of those cells.Results:1.Ouabain(25nM、50nM、100nM)induced the cell apoptosis and proliferation inhibition of Jurkat cells in a concentration-dependent manner(p<0.05).2.The expression of IL-2R,Src Y418,NKA-al and IP3R proteins in Jurkat cells were significantly reduced after 48h ouabain treatment(p<0.05).3.The activity of NF-κB was also decreased by ouabian in a concentration-dependent manner in Jurkat cells(p<0.05).4.The expression of NKA-al was reduced after the siRNA was transfected into Jurkat cells for 48h(p<0.05).This down-regulation of NKA-al decreased the ouabain effect on cell apoptosis and proliferation(p<0.05).5.The effect of ouabain on proteins reductions were altered in down-regulation of NKA-al cells(p<0.05).Conclusion:1.Ouabain could inhibit cell proliferation and induce apoptosis of Jurkat cells,the effects were concentration-dependent.2.ouabain inducing the apoptosis of Jurkat cells was associated with down-regulation of NKA-al subunit,Src(Y418),IP3R and NF-κB activity weakened,When the expression of NKA-al subunit was weakened in Jurkat cells by NKA-al-siRNA,those effects of ouabain were weakened.These are indicated that the mechanism of ouabain anti-Jurkat cells may closely related to the NKA/Src/IP3R-NF-κB signaling pathway.3.Ouabain could down-regulate the expression of IL-2R in Jurkat cells.IL-2R may be one of the reasons that Jurkat cells are sensitive to ouabain.