| ObjectiveTo investigate the effect of PPARγ agonist pioglitazone on the growth, invasion andapoptosis of glioma cells, and try to disclose intrinsic molecular mechanism, to providetarget for explore new differentiation agents and experimental data for clinic utilization.Methods1. Detecting the effect of pioglitazone different concentrations and different actiontimes on the growth of glioma cells by CCK-8analysis. Detecting cell-migration bymonolayer wound healing assay after pioglitazone stimulation. Detecting the apoptosis ofcells under pioglitazone-treated by TUNEL method.2. The expressions of β-catenin and E-cadherin were analyzed by Western Blot, thesubcellular distribution of β-catenin was detected by immunofluorescent staining. DesignshRNA expression vector which can inhibit β-catenin gene expression specifically. Theeffects of β-catenin knockdown on the proliferation, migration and apoptosis of gliomacells were analyzed by CCK-8assay, wound healing assay and TUNEL method,respectively.3. To identify the role of PPARγ between antineoplastic of pioglitazone, CCK-8assayand Western Blot were applied to detected the proliferation of glioma cells and theexpression of PPARγ, β-catenin, cyclin D1, c-myc protein in the presence of PPARγantagonist GW9662.Results1. CCK-8assay showed pioglitazone significantly reduced the cellular viability ofglioma cells in a concentration-and time-dependent manner. Monolayer wound healingassay showed pioglitazone inhibited bordering glioma cells migration toward the center of the wound. MMP-2expression was significantly suppressed by pioglitazone treatment.TUNEL showed pioglitazone inducted glioma cells apoptotic cell death. Both proteinlevels of Bax and Bad were up-regulated in response to pioglitazone treatment.2. Western Blot showed pioglitazone induced β-catenin repression in does-dependentand time-dependent manner, but not change the total amount of E-cadherin.Iimmunofluorescence analysis indicated pioglitazone reduced the levels of β-cateninwithout altering the subcellular distribution of β-catenin. Knockdown of β-catenin resultingin decreased glioma cells proliferation, migration and promoted cell apoptosis. MMP-2was down-regulated, while Bad and Bax were up-regulated after β-catenin siRNAtransfection.3. Western Blot assay showed that pioglitazone increased PPARγ expression.Co-administration of GW9662reversed down-regulation of β-catenin, cyclin D1andc-myc which induced by pioglitazone.Conclusions1. PPARγ agonist pioglitazone can decrease glioma cells proliferation, migration andpromote cell apoptosis. These results indicated that pioglitazone displays antineoplasticeffects on glioma cells.2. Specifically knockdown of β-catenin resulted suppression of glioma cellsproliferation, migration and promotion of cell apoptosis. These results indicated thatβ-catenin plays a pivotal role in developing and progression of human glioma.3. Pioglitazone decreased the protein levels of β-catenin, cyclin D1and c-myc. Thesefindings indicated that pioglitazone displays antineoplastic effects in bothPPARγ-dependent and PPARγ-independent manner via regulates β-catenin expression andtranscriptional activity.
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