Study On The Mechanism Of Differential Sensitivities To Cabergoline And Bromocrip Tine In The Treatment Of Prolactinomas

Part I Differential sensitivities of pituitary adenoma cell lines to cabergoli:ne and bromocriptineObjective:The purposes of this study were to study the sensitivities of pituitary adenoma cell lines to cabergoline and bromocriptineMethods:Pituitary adenoma cells were treated with cabergoline(CAB)and bromocriptine(BRC)respectively,and cell viability was assessed using CCK8 assay,50%inhibitive concentration(IC50)of drug was examined.Results:After treatment with BRC(25,50 or 100?gM/l)for 48 h,the cell viability of GH3 and MMQ cells ranged from 82%to 20.2%(P<0.001)and 79.39%to 42.81%(P<0.001).After treatment with CAB(25,50 or 100?M/l)for 48 h,the cell viability of GH3 and MMQ cells ranged from 80.27%to 43.23%(P<0.001)and 56.86%to 11.17%(P<0.001).The IC50 value of BRC in the treatment of GH3 cells was lower than that of CAB and the IC50 value of CAB in the treatment of MMQ cells was lower than that of BRC.After treatment with the two drugs at the same concentration for 48 or 72 h,the cell viability of GH3 with BRC was lower than that of CAB,the cell viability of MMQ with CAB was lower than that of BRC.Conclusions:Sensitivity of GH3 cells to BRC is higher than CAB,sensitivity of MMQ cells to CAB is higher than BRC.Part ? Study on the mechanism of cell death of pituitary adenoma cell lines to cabergoline and bromocriptineObjective:The aim of this study is to investigate the mechanism of cell death of pituitary adenoma cell lines to CAB and BRCMethods:Transmission electron microscopy(TEM)was used to detect autophagosome.Flow cytometry was performed to evaluate apoptosis.Autophagy-and apoptosis-related protein expression level was examined by western blotting.The apoptosis and autophagy of pituitary adenoma cells were inhibited,and cell viability was assessed using CCK8 assay.Results:TEM revealed increased numbers of autolysosomes in the GH3 and MMQ cells treated with CAB and BRC compared with those in control cells respectively.The numbers of autolysosomes in the two cell types treated with CAB were higher than BRC.Western blotting showed an increase of the LC3-II/LC3-I ratio in CAB-and BRC-treated in GH3 and MMQ cells.The ratios of LC3-II/LC3-I in the two cell types treated with CAB were higher than BRC.3-MA reversed CAB-mediated cell death in GH3 and MMQ cells by inhibited the autophagy,but not rescue the cell viability of BRC induced.CAB and BRC both induced apoptosis,and the apoptosis rate of the two cell types treated with BRC were significantly higher than those of CAB.The cleaved-caspase-3 protein expression level was higher in BRC-treated GH3 and MMQ cells than those in CAB-treated.Z-VAD-FMK reversed CAB and BRC mediated cell death in GH3 and MMQ cells by inhibited the apoptosis.Conclusions:BRC-treated pituitary adenoma cells mainly underwent apoptosis.CAB-treated pituitary adenoma cells mainly underwent autophagic cell death.Part ? Study on the molecular mechanism of differential sensitivities to cabergoline and bromocriptine in the treatment of prolactinomasObjective:The aim of this study is to investigate the mechanism of cell death of pituitary adenoma cell lines to CAB and BRCMethods:Gene chip was used to screen the differential gene expression.The differentially regulated genes were validated by qRT-PCR.The expression of EGR1 and p-ERK1/2 were detected by Western blot.Knockdown of EGR1 was performed using lentivirus vectors containing siRNA transfection.The differentially regulated genes were validated by qRT-PCR.Knockdown of EGR1 was validated by RT-PCR.The expression of EGR1 were inhibited,and cell viability was assessed using CCK8 assay.The nude mouse model of prolactinoma transplanted with GH3 cell line was established to observe the effects of BRC on the transplanted tumors.The specimen of xenografts tumor was used to detected apoptosis and protein expression by TUNEL and immunohistochemical stainingResults:The results of microarray showed increased expression of EGR1 in GH3 cells treated with BRC.EGR1 mRNA expression level was upregulated by BRC-treated in GH3 and MMQ cells.BRC upregulated the expression of EGR1 and p-ERK1/2 in GH3 and MMQ cells.Knockdown of EGR1 could rescue the cell viability of pituitary adenoma cells treated with BRC.CAB downregulated the expression of p-AKT and p-mTOR in the two cell types.The transplantation tumor volume and mass of BRC-treated groups were significantly lower than the control group.The number of TUNEL positive cells was significantly increased in the BRC-treated group compared with the control group.The expression of EGR1 and ERK1/2 were also increased in BRC-treated group compared with untreated group by immunohistochemical staining.Conclusions:Our study provides evidence that BRC induces apoptosis of pituitary adenoma cell lines via ERK1/2-EGR1 pathway and CAB induce autophagy of pituitary adenoma cell lines via suppress AKT/mTOR pathway.