The Inhibitory Effects And Mechanism Of Fas Ligand/RGD-Fas Ligand On Pituitary Adenoma Cells

Objective To investigate the expressions of Fas, Fas ligand (FasL), and decoy receptor 3 (DcR3) in different endocrine types of human pituitary adenomas.Methods Immunohistochemistry (IHC) was applied to detect the protein expressions of Fas, FasL, and DcR3 in 98 human pituitary adenomas, and reverse transcription polymerase chain reaction (RT-PCR) was used to analyze the differences among mRNA expression levels of Fas, FasL, and DcR3 in 24 human pituitary adenomas and 2 normal pituitary tissues.Results1. The immunoreactivities of both Fas and DcR3 were located in cytoplasm and membrane of pituitary adenoma cells. The protein expression of FasL was not found in pituitary adenoma cells.The positive percentage of Fas protein expression in pituitary adenomas, either functioning or non-functioning, was significantly higher than that of DcR3 protein expression.The positive percentages of both Fas and DcR3 protein expressions in functioning adenomas were significantly higher than those in non-functioning group, respectively. The positive percentages of both Fas and DcR3 protein expressions in invasive adenomas were not significantly different from those in non-invasive group, respectively.2. In normal pituitary tissues, the mRNA expressions of Fas and FasL were detected while that of DcR3 was not found. In pituitary adenomas, however, the mRNA expressions of Fas and DcR3 were detected while that of FasL was not found.The expression levels of Fas mRNA in different endocrine types of adenomas were significantly lower than that in normal pituitary tissues, respectively. The expression levels of Fas mRNA in different endocrine types of adenomas were significantly higher than those of DcR3 mRNA in corresponding types of adenomas, respectively.There were no significantly different levels of Fas mRNA expression between the invasive adenomas (different endocrine types) and non-invasive group, respectively. Also, there were no significantly different levels of DcR3 mRNA between the invasive adenomas (different endocrine types) and non-invasive group, respectively.Conclusion The decreasing level of Fas expression, the loss of FasL expression, and the presence of DcR3 expression may play an important role in the genesis and growth of human pituitary adenomas. Fas may serve as a novel therapeutic target for exogenous FasL in the treatment of pituitary adenomas. Objective To study the effects of Fas ligand (FasL) and RGD-Fas ligand (RGD-FasL) on the growth of pituitary adenoma cells in vitro, and to clarify the possible mechanism.Methods1. The recombinant FasL gene and RGD-FasL gene were produced and the purified FasL protein and RGD-FasL protein were obtained.2. Rat pituitary adenoma cell lines GH3 and MMQ, and mouse pituitary adenoma cell line AtT20, were used in this study. The expressions of Fas and DcR3 mRNA in GH3/AtT20/MMQ cells were analyzed with RT-PCR and Flow cytometry.3. GH3/AtT20/MMQ cells were treated by drug (FasL or RGD-FasL) with various concentrations and different durations, respectively.(1) The antiproliferative effects were evaluated by cell viability using MTT assay.(2) The cell apoptosis was observed with optical and electron microscopies.(3) The DNA fragmentation pattern was visualized with agarose gel electrophoresis.(4) The effects of drugs on cell cycle were studied by Flow cytometry with PI staining.(5) The expression levels of apoptosis-associated genes, such as Caspase-8, Caspase-9, Caspase-3, RANKL, JNK2, and Bcl-2, were analyzed by Western blot.(6) Caspase inhibitory assay was used to confirm the mechanism for apoptosis.Results1. The purified recombinant FasL protein and RGD-FasL protein were obtained successfully.2. The expressions of Fas and DcR3 mRNA in GH3/AtT20/MMQ cells were confirmed with RT-PCR and Flow cytometry. The expression level of Fas mRNA in GH3 cells was lower than that in AtT20/MMQ cells, while the expression level of DcR3 mRNA in AtT20 cells was very low.3. MTT assay demonstrated that the proliferations of GH3/AtT20/MMQ cells were significantly inhibited by drug (FasL or RGD-FasL) in dose- and time- dependent manner. The inhibitory effect of RGD-FasL on cells was not significantly different from that of FasL. The positive correlation between drug concentration and antiproliferative rate was achieved with statistical analysis. The sensitivity of AtT20 cells to drugs was relatively higher, while that of GH3 cells was relatively lower.4. Optical and electron microscopies showed the typical apoptotic changes in GH3/AtT20/MMQ cells when treated by RGD-FasL. 5. A typical ladder pattern of internucleosomal DNA fragmentation was observed with agarose gel electrophoresis.6. Flow cytometry demonstrated an increased accumulation of cells at G1/G0 phase and a significantly decreased fraction of G2/S phase, as well as a significantly increased apoptosis index (AI).7. The expressions of Caspase-8, Caspase-9, Caspase-3, RANKL and JNK2 protein were significantly up-regulated; while that of Bcl-2 protein was significantly down-regulated.8. It was confirmed that the mechanism for GH3/AtT20/MMQ cells apoptosis was through Caspase pathway.Conclusions1. FasL and RGD-FasL induce cell apoptosis and cell cycle arrest through Caspase pathway, which inevitably leads to antiproliferative effects on pituitary adenoma cells.2. Regarding the inhibitory effect on pituitary adenoma cells, RGD-FasL has not significantly different from FasL, and may serve as a safer, tumor-targeted, clinically promising drug in treatment of pituitary adenomas because of tumoral pathological vessels targeted by RGD itself.