The Growth Effects And Mechanisms Of Ligands For PPARγ On Pituitary Adenomas

Capter 1. Expression of peroxisome proliferator-activated receptor-r in human pituitary adenomas.Objective To evaluate the distribution and expression ofperoxisome proliferator-activated receptor r, (PPARr) in human pituitary adenomas, and investigate their interrelations between the expression levels of PPARr, and different adenoma groups.Methods Immunohistochemistry (IHC) was used to studied the expression of PPARr protein in 78 human pituitary adenomas, and reverse transcription polymerase chain reaction (RT-PCR) was used to confirm the expression levels of PPARr, mRNA in 33 human pituitary adenomas and 3 normal pituitary tissues.Results1. IHC showed that PPARr immunoreactivity was located mainly in cell cytoplasm and little in nucleus of pituitary adenoma cells.2. The positive percentage of PPARr, protein in invasive pituitary adenomas was 68.09% (32/47), it was significantly higher than that in non-invasive group (38.71%, 12/31).3. The expression level of PPARr mRNA in pituitary adenomas (2.988±0.183) was significantly higher than that in normal pituitary tissues (1.547±0.252). Compared with the latter, the expression level of PPARr, mRNA was significantly higher in multi-hormore secrecting adenoma, ACTH-, and FSH/LH-omas (P＜0.05). However, there was no significant difference between normal pituitary tissues and NF-, GH- or PRL-omas.4. The expression level of PPARr, mRNA in invasive pituitary adenomas was 3.947±0.431, it was significantly higher than that in non-invasive group (2.402±0.240), P＜0.05.5. In multi-hormore secrecting adenomas, FSH/LH-and NF-omas invasive adenomas, there was a higher level of PPARr mRNA than that in corresponding non-invasive adenomas respectively. However, there was no significant difference between the invasive and non-invasive group of PRL and GH-omas.Conclusion PPARr may play an important role in the growth and invasiveness of human pituitary adenomas and may be a novel therapeutic target for them. Capter 2. Inhibitory effects and the possible mechanism of ligands for peroxisome proliferator-activated receptor-r on the pituitary adenoma cells in vitroObjective To examine the effects of PPARr activation on the growth and GH secretion of pituitary adenoma cells in vitro, and to clarify the possible mechanism of PPARr activation on the therapy of pituitary adenomas.Methods1. GH3, a rat GH-secreting pituitary adenoma cell line, which expressed PPARr, was used in this study. GH3 cells were treated with Troglitazone and 15d-PGJ2, one of the synthetic and natural ligand of PPARr. The antiproliferative effects were evaluated by cell viability using MTT assay.2. The secretion of GH by GH3 cells, which were treated with Troglitazone and 15d-PGJ2 with various concentrations or different durations, was detected by semi-quantitative ELISA.3. The effects of PPARr activation on cell apoptosis and cell cycle were studied by electron-microscopy and flow cytometry respectively.4. After treated with different concentration of Troglitazone and 15d-PGJ2. the expression levels of apoptosis-associated genes, Bc1-2, Bax and caspase-3, were analyzed by Westem blot.Results1. MTT assay demonstrated that the proliferation of GH3 cells was significantly inhibited by Troglitazone and 15d-PGJ2 in dose-and time- dependent manner.2. Troglitazone and 15d-PGJ2 had potent inhibitory effect on the secretion of GH from GH3 cells with the manners of dose-and time- dependent also.3. Electron-microscopy showed that GH3 cells cultured with Troglitazone and 15d-PGJ2 caused nuclear and cytoplasmic shrinkage and condensation, and margination of chromatin against the nuclear membrane, and the formation of apoptotic bodies in GH3 cells.4. Flow cytometry demonstrated a significantly decreased fraction of G2 and S phase cells resulting from an increased accumulation of cells at G1 phase followed treatment with Troglitazone and 15d-PGJ2.5. The expression of caspase-3 and Bax, the pro-apoptotic members of Bc1-2 family were significantly up-regulated; and'Bc1-2, the anti- apoptotic gene, significantly decreased in a dose-dependent manner after treatment with Troglitazone and 15d-PGJ2.Conclusions1. Activation of PPARr exerts a negative regulatory effect on the growth of pituitary adenoma cells in vitro. 2. Activation of PPARr decreased the GH secretion from GH3 cells in dose-and time-dependent manner.3. Activation of PPARr induces apoptosis in GH3 cells.4. Activation of PPARr induces G1 phase cell cycle arrest and inhibits G2 and S phase development in GH3 cells.5. The inhibitory effect of PPARr activation on pituitary adenoma is probably associated with up-regulating the expression of caspase-3 and Bax, and down-regulating the expression of Bc1-2. These results suggest that PPARr might be a novel therapeutic target for the pituitary adenoma.