| Objective:To investigate the expression and significance of peroxisome proliferator-activated receptorγ(PPARγ) in human glioma tissues and cell lines SWO-38, U251 and SHG-44. To further investigate the effects of CDA-2 on expression of PPARγin SWO-38 cell and of CDA-2 on inducing SWO-38 cell differentiation in vitro. The mechanisms of inducing differentiation of PPARγwas also investigated to provide target for explore new differentiation agents and experimental data for clinic utilization.Methods:1. Immunohistochemical staining for PPARγwas performed using biopsy specimens of glioma with various histological types. Expression of PPARγand GFAP in glioma cell lines SWO-38, U251 and SHG-44 was also analyzed by using RT-PCR and western blot assay.2. Western blot assay was applied to detect the expression of PPARγin SWO-38 cell treated with rosiglitazone, ATRA. Expression of PPARγmRNA and protein also detected by RT-PCR and western blot treated by CDA-2 with various dose and time. The subcellular location of PPARγprotein was also detected by immunohistochemistry assay.3. To determine the effects of CDA-2 on cell viability and proliferation of human glioma cell, the exponentially proliferative SWO-38 cells were treated with 0-5 mg/ml CDA-2 for 24 hr, 48 hr and 72 hr, respectively, were determined by MTT and colony formation assay, as well as FCM assay. To elucidate the role of CDA-2 on inducing SWO-38 cell differentiation, cells on the sterile glass slide were stained with H&E and immunostained with GFAP. Moreover, we prepared the protein extract from the cells treated with 3 mg/ml CDA-2 for 72 hr to detect the expression of GFAP protein by western blot assay.4. To identify the role of PPARγon the differentiation of SWO-38 cells, MTT assay, colony formation assay and FCM assay, as well as western blot assay were applied to detected the changes of cell proliferation, cell cycle and expression of GFAP protein in the presence of PPARγantagonist GW9662. To further explore whether the mechanisms of CDA-2 on inducing SWO-38 cell differentiation was involved in PPARγ, we detected the expression of PTEN (p-PTEN) and COX -2 proteins with or without the presence of GW9662 by western blot assay.Results:1. Expression of PPARγin human glioma tissues and cell linesImmunohistochemical study showed that PPARγwas expressed in glioma tissues with positive rate of 37.5 % , whereas with positive expression rate of 76.9% in the tissues which is peripheral to tumors. Western blot and RT-PCR showed that PPARγwas expressed both in glioma cell lines SWO-38 and U251, while no expression was detected in SHG-44 cell. However, higher expression of GFAP was detected in SHG-44 cell.2. Expression of PPARγinduede by rosiglitazone , ATRA and CDA-2 on SWO-38 cellWestern blot showed that up-regulation of PPARγprotein after treated with rosiglitazone , ATRA. CDA-2 also induced both mRNA and protein expression of PPARγwith various dose from 0.5mg/ml to 3.0mg/ml. It also showed that strong staining of PPARγwas observed both in cytoplasm and nucleus of SWO-38 cells. When cells were exposed to CDA-2 for 6 hr, PPARγprotein was showed nuclear localization predominantly.3. Inducing differentiation of SWO-38 cell by CDA-2 was PPARγ-dependentThe results showed that CDA-2 caused an obvious morphological changes with multiple and elongated cytoplasmic processes and a significant increase in GFAP expression. Meanwhile, it also showed that CDA-2 significantly inhibited cell viability of SWO-38 in a dose- and time-dependent manner, cell cycle analysis also showed that SWO-38 cells treated with 3 mg/ml CDA-2 for 72 hr resulted in increasing the G0/G1 phase cells. In the presence of GW9662, The degression of cell viability and growth arrest with IC50 was 2.46±0.16 mg/ml and 1.79±0.18 mg/ml, respectively, and with increased S phase cells in contrast to control group. The level of GFAP was also partially reversed by GW9662 in contrast to control group.4. PPARγregulated the proteins expression during the SWO-38 cell differentiationThe results showed that CDA-2 caused an increasing expression of PTEN (p-PTEN) protein and a decreasing expression of COX-2 protein. Meanwhile, the expressions of PTEN (p-PTEN) and COX-2 proteins were partially reversed by GW9662 in contrast to control group. GW9662 has no effect on the expression of PPARγ.Conclusion:1. PPARγwas associated with carcinogens of glioma. Actived PPARγby agonist may be a novel approach to treatment of glioma. 2. CDA-2 demonstrates not only a significant inhibitory effect on proliferation of human glioma cell line in vitro and the inhibitory effect was in a time and dose-dependent manners, but also induces SWO-38 cell to differentiate and was a potential and natural anti-glioma drug.3. We demonstrated that not only high expression of PPARγin SWO-38 cell, but also PPARγprotein was translocation from plasm to nucleus predominantly, after exposure to CDA-2 for 6 h, which may imply the activation of PPARγ, suggesting the important role of PPARγin SWO-38 cell differentiation.4. The present study provided evidences that PPARγpathway was involved in human SWO-38 cell line glioma differentiation and PPARγwas able to functionally regulate downstream genes expression that affects SWO-38 cell differentiation, such as up-regulation of PTEN (p-PTEN), GFAP and down-regulation of COX-2.
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