Inhibition Of Glycogen Synthase Kinase-3β-mediated Wnt And NF-ΚB Signaling Pathway Induces Apoptosis Of Acute Lymphoblastic Leukemia Cells

PART ONE THE LOCALIZATION AND EXPRESSIONOF GSK-3Β IN JURKAT CELL LINE AND BONEMARROW MONONUCLEAR CELLS OF CHILDHOODACUTE LYMPHOBLASTIC LEUKEMIAObjectives: To investigate the localization and expression of GSK-3βin Jurkat cells and bone marrow mononuclear cells (BMMNC) of childhoodacute lymphoblastic leukemia (ALL).Methods: The study was divided into the experimental group (Jurkatcell line and ALL cells) and the control group (ITP with normal bonemarrow). The mRNA and protein levels of GSK-3β were detected by RealTime PCR, immunofluorescence staining and Western blotting methods,respectively.Results:1. Real Time PCR analysis showed that both experimental andcontrol groups expressed GSK-3β mRNA. The expression levels of GSK-3βmRNA in Jurkat and ALL cells were8.53and5.19-fold compared to of the control group (P＜0.05).2. Immunofluorescence staining and Western blotting results showedthat GSK-3β was highly expressed in the cytoplasm and the nucleus of theexperimental group cells; in the control group, GSK-3β was only expressedin the cytoplasm but not expressed in the nucleus.Conclusions: GSK-3β was abnormally over-expressed in the nucleusof Jurkat cells and BMMNC of children with ALL. Nuclear accumulation ofGSK-3β seems to lay a theoretical foundation to regulate the intracellularsignaling molecules. PART TWO THE INHIBITION OF GSK-3ΒACTIVITY INDUCEED CELL APOPTOSIS OF ACUTELYMPHOBLASTIC LEUKEMIA CELL LINE JURKATEXPERIMENT A GSK-3Β INHIBITORS INDUCEDCELL APOPTOSIS IN JURKATObjectives: To explore the molecular mechanisms of apoptosisinduced by GSK-3β mediated Wnt/β-catenin and NF-κB signaling pathwayin Jurkat cells treated with GSK-3β inhibitors lithium chloride (LiCl) and SB216763.Methods: Jurkat cells were treated with LiCl and SB216763,respectively. Cell proliferation was detected by MTT assay to make the cellgrowth curve; Cell apoptosis was detected by Annexin V-PE/7-AAD doublestaining combined with flow cytometry; The localization and expression ofGSK-3β and its downstream signaling molecules β-catenin and NF-κB P65in Jurkat cells were detected by Western blotting; The changes ofdownstream target gene (survivin, c-myc and cyclinD1) expressions inWnt/β-catenin and NF-κB signaling pathways were detected by RT-PCR.Results:1.In the concentration of LiCl≥20mM, SB216763≥15μM, cellgrowth was obviously inhibited in a concentration-time-dependent manner.2. The mean number of Jurkat cells treated with LiCl at20,30,40mMconcentrations reached (10.89±1.32)%,(23.74±1.65)%,(38.26±4.37)%(NaCl as control, P＜0.05); The mean number of Jurkat cells treated withSB216763at different concentrations reached (14.71±2.53)%,(24.92±4.58)%,(43.25±4.72)%(DMSO as control, P＜0.05).3. Jurkat cells were treated with20mM LiCl, compared with the controlgroup, the expression of β-catenin protein in the cytoplasm and nucleusincreased by2.15and1.63-fold (P＜0.05); The expression of NF-κB P65protein in the cytoplasm and the nucleus had no obvious changes. WhileJurkat cells were treated with15μM SB216763, compared with the controlgroup, the expression of β-catenin protein in the cytoplasm and nucleus increased by3.27and2.84-fold (P＜0.05); The expression of NF-κB P65protein in the cytoplasm and nuclei had no significant changes.4. The expression of survivin mRNA was significantly reduced(P＜0.05), while the levels of c-myc and cyclin D1mRNA expression did notchange.Conclusions: GSK-3β inhibitors may slow down cell proliferation andpromote apoptosis of Jurkat cells. The mechanism may be GSK-3βinhibitors induced apoptosis in Jurkat cells by activated other signal systems,but not by β-catenin and NF-κB signaling pathways. EXPERIMENT B EXOGENOUS WNT-3A FOR CELLAPOPTOSIS IN JURKATObjectives: To explore the molecular mechanisms of apoptosisinduced by GSK-3β inhibition mediated Wnt/β-catenin and NF-κB signalingpathway in Jurkat cells through recombinant adenovirus vector-mediatedWnt-3a.Methods: Using recombinant DNA technology to build the Wnt-3arecombinant adenovirus expression vector Ad5-Wnt-3a. Jurkat cells weretransfected by the direct infection of recombinant adenovirus. Jurkat cellswere divided into three groups: the experimental group (Jurkat/Ad5-Wnt-3a group), the empty vector group (Jurkat/Ad5-GFP group) and the blankcontrol group (untransfected Jurkat group). The transfection or infectionefficiencies of cells in every group were detected by fluorescence and flowcytometry. The expression level of Wnt-3a mRNA in every group wasdetected by RT-PCR; The protein expression of β-catenin and GSK-3β wasdetected by Western blotting; Cell proliferation and cell cycle were detectedby MTT assay and by flow cytometry, respectively; Cell apoptosis wasmeasured by Annexin V-PE/7-AAD double staining combined with flowcytometry; The changes of downstream target gene (survivin, c-myc andcyclinD1) expression in Wnt/β-catenin signaling pathway were detected byRT-PCR.Results:1. Jurkat cells were transfected successfully by recombinedadenovirus Ad5-Wnt-3a, with MOI=60for the optimal transfection titer,and the transfection rate was (42.54±1.63)%.2. The experimental group expressed Wnt-3a gene efficiently aftertransfection of Ad5-Wnt-3a. The total protein expression of GSK-3βwassignificantly reduced; β-catenin protein expression was significantlyincreased2.38times compared with the control group; Actived β-catenincould significantly promote the proliferation of Jurkat cells (proliferationrate was about21.2%), and cause G0/G1phase cells down, S phase cellsincreased, but cell apoptosis was not obvious. There was no significant cellapoptosis. 3. RT-PCR results showed that the c-myc and cyclin D1mRNAexpression were (0.75±0.04) and (0.75±0.03) in the experimental group,significantly increased compared with empty vector group. The expressionof survivin mRNA did not change significantly.Conclusions: The exogenous Wnt-3a gene mediated by recombinantadenovirus vector could downregulate GSK-3β activity and activate thecanonical Wnt/β-catenin signaling pathway in Jurkat cell line. The activationof β-catenin promoted cell proliferation and cell cycle progression, but didnot affect cell apoptosis. Its mechanism may be β-catenin regulated theexpression s of downstream target genes c-myc and cyclin D1. PART THREE THE INHIBITION OF GSK-3ΒACTIVITY INDUCED CELLS APOPTOSIS IN CHILDRENWITH ACUTE LYMPHOBLASTIC LEUKEMIAObjectives: To explore the molecular mechanisms of apoptosisinduced by GSK-3β mediated Wnt/β-catenin and NF-κB signaling pathwayin ALL cells treated with GSK-3β inhibitors LiCl and SB216763.Methods: ALL cells were treated with LiCl and SB216763in vitroshort-term culture. The changes of GSK-3β and the localization andexpression level of the signaling molecules β-catenin and NF-κB P65were detected by Western blotting; Electrophoretic mobility shift assay (EMSA)was used to assess the binding of nuclear transcription factor NF-κB activityto the DNA; Annexin V-PE/7-AAD double staining combined with flowcytometry to detect cell apoptosis; Further, the changes of downstream targetgene (survivin, c-myc and cyclinD1) expression in Wnt/β-catenin andNF-κB signaling pathways was detected by RT-PCR.Results:1.ALL cells were treated with GSK-3β inhibitors LiCl andSB216763, the protein level of GSK-3β did not change in the cytoplasm, butits nuclear protein expression decreased significantly; The signaling criticaleffect molecular β-catenin protein expression in Wnt/β-catenin pathwayincreased significantly in the cytoplasm, while β-catenin expressiontranslocated into the nucleus increased slightly; The nuclear translocationprotein NF-κB P65in NF-κB signaling pathway was no significant changesin the cytoplasm/nucleus of cell, but the DNA binding activity of nuclearNF-κB P65was significantly reduced.2. SB216763concentration gradient increased (5,10,15μM) in theexperimental group, with an increasing apoptosis rate (36±5)%,(52±7)%and (70±4)%, respectively; LiCl in the cell sub-toxic concentrations with(5,10mM), induced apoptosis in ALL cells in a concentration dependentmanner.the control group were statistically significant.3. The apoptosis was not significantly affected and found to be15%to20%in the control cells with LiCl or SB216763treatment. However, Western blotting showed that expression of β-catenin protein translocatedinto the nucleus was significantly increased.4. RT-PCR showed that the downstream anti-apoptosis gene survivinexpression was reduced, but c-myc and cyclin D1mRNA expression levelsdid not change.Conclusions: The inhibition of GSK-3β activity in ALL primary cellsdid not change the cytoplasm/nucleus expression of the nuclear translocationprotein NF-κB P65, but reduced NF-κB P65transcriptional activity, whichpromoted apoptosis of ALL cells by decreasing the expression ofanti-apoptotic gene survivin. The level of β-catenin expression did notsignificantly affect cell apoptosis and proliferation, and the mechanismneeds further study. Apoptosis was not significantly affected in control cellstreated with GSK-3β inhibitors, suggesting that it may be associated withWnt/β-catenin signal activation.