Expression Of PPARγ In Human Gliomas And Effect Of Its Agonist On The Growth And Apoptosis Of Glioma Cells

Object: Peroxisome Proliferator-Actived Receptors(PPAR),is a kind of nuclear hormone receptor,which plays important roles in regulating metabolisms.Three PPAR isforms are know to exist:PPARa,PPARβand PPARγ.After activation by ligand as is the case with nuclear receptors, PPARs binds to a specific element in the promoter region of target genes.Recently,PPARγwas found to express in tumor tissues and cells,which could be activated by its ligands and affect the differentiation,proliferation and apoptosis of tumor cells.The aim of this study is to investigate the significance of expression of PPARγin glioma tissuses and glioma cell line U-251,to determine the relationship between PPARγexpression and clinicopathological grade of glioma,and to discuss the inhibition effect of PPARγagonist Pioglitazone on the growth of glioma cells U-251 and its relation to apoptosis and apoptosis modulin Caspase-3,and the anti-glioma effect of PPARγagonist and the possible mechanism of it.Method: The expression of PPARγmRNA and PPARγprotein was detected in normal cerebral tissuses,human glioma Specimens and glioma cells line U-251 by using RT-PCR semi-quantity and immunohistochemistry methods.Detecting the effect of Pioglitazone different concentrations and different action times on the growth of glioma cells U-251 by MMT analysis.Detecting the morphological change of apoptosis by Cell culture and AO/EB double fluorescence stain staining ,detecting the rate of apoptosis in cells in different action times by flow cytometry and detecting the expression of cleaved Caspase-3 in cells in different concentrations by Western blot method.Results:1. The expression of PPARγmRNA and PPARγprotein have been detected in normal cerebral tissuses,human gliomas Specimens and glioma cells line U-251.The expression level of PPARγmRNA and PPARγprotein in human gliomas was considerably higher than those in normal cerebral tissuses(P<0.05);and the expression level of PPARγmRNA and PPARγprotein in gliomas of grade III～IV was considerably higher than those in gliomas of grade I～II.2. MTT analysiss:25,50,100,200μmol?L-1Pioglitazone all had the effect of PPARγ,which was time and dose-dependent(P<0.05).3. Morphological change of cells by cell culture and AO/EB staining: Cells had the typical manifestation of apoptosis,for example collapse,karyopycnosis and cytoplasm vacuolization.4. Flow cytometry quantitation:Pioglitazone could induce the apoptosis of glioma cells which was time-dependent.(P<0.05 ).5. Western blot:After Pioglitazone with different action times acted on glioma cells, cleaved Caspase-3 have not been detected in control group.The expression of cleaved Caspase-3 protein was detected in 24h group,the expression of cleaved Caspase-3 protein in 48h is the highest, and the expression in 72h was marked decline(P<0.05 ).Conclusion:1. The expression of PPARγhave been detected in human gliomas and normal cerebral tissuses,The expression of PPARγmay be correlation to the pathological grades of human gliomas PPARγmay be invoived in the tumorigenesis and progression of human gliomas.2. PPARγagonist Pioglitazone had the effect of inhibition on the growth of glioma cells U-251, which was time and dose-dependent in a range.3. PPARγagonist Pioglitazone could induce the apoptosis of glioma cells cells, which participated in its inhibition effect on the growth of glioma cells. 4. PPARγagonist Pioglitazone could up-regulate the expression of cleaved Caspase-3 protein, which showed that it induce the apoptosis of glioma cells through Caspase-dependent apoptosis pathway.The specific mechanism is not clear, which needs further investigation.