| Bromocriptine,the most commonly used dopamine agonists(DA)for prolactinoma,can effectively reduce tumor size of prolactinoma,but the mechanism was not fully understood.Apoptosis had been well-recognized to contribute to the tumor mass regression caused by bromocriptine.However,whether other types of non-apoptotic cell death involved in the bromocriptine-induced prolactinoma shrinkage had not been fully clarified.The newly discovered molecular mechanism of necroptosis provides the possibility to examine this programmed necrosis in the pharmacological function of bromocriptine.The aim of present study was to evaluate and investigate the underlying mechanism of necroptosis in involution of prolactinoma induced by bromocriptine.By immunohistochemistry,we found that the numbers of receptor-interacting serine-threonine kinase 3(RIP3)and phosphorylated mixed lineage kinase domain-like protein(p MLKL)-positive cells and their expression intensities were increased in patients with prolactinoma after bromocriptine therapy.For further exploring the mechanism of bromocriptine,prolactinoma cell line(MMQ cells)was adopted to study the mechanism of necroptosis in vitro.Cell viability and ATP level of MMQ cells were decreased,while reactive oxygen species(ROS)level was increased after bromocriptine treatment.The above effects could be partially reversed by Necrostatin-1,an inhibitor of necroptosis.Ultrastructural study further confirmed the necroptosis of MMQ cells,which was characterized by ruptured membrane,dissolved cytoplasm and especially the dramatically swollen mitochondria.Furthermore,we demonstrated that bromocriptine induced RIP3/MLKL-dependent necroptosis of prolactinoma cells and phosphoglycerate mutase family 5(PGAM5)/ Cyclophilin D(CypD)pathway was involved.The results suggested that necroptosis might be a promising target for clinical therapy for prolactinoma.This study is composed of the following four parts.Part 1: Expression of RIP3 and p MLKL could be induced by bromocriptine in human prolactinoma tissuesA total of 12 human prolcatinoma samples were randomly selected to incorporate into our study.Among these,6 samples were from patients treated with bromocriptine before surgery,and the other 6 samples were from patients who were not treated with bromocriptine.The data showed that the level of prolactin(PRL)was decreased and the tumor size was reduced after bromocriptine therapy,which is consistent with previous research.We then investigated whether the necroptosis was involved in this pharmacological function.Positive-staining signal of RIP3 can be found in both cytoplasm and cell membrane.Quantitative analysis indicated that the numbers of RIP3 positive cells and its immunofluorescence intensity(IFI)were significantly increased in patients with bromocriptine treatment,compared with patients without bromocriptine treatment.Meanwhile,p MLKL,another key mediator of necroptosis,was mainly located on cell membrane.The expression of p MLKL was significantly increased in prolactinoma tissues in patients treated with bromocriptine.These results demonstrated that bromocriptine induced the expression of RIP3 and MLKL(two key mediators of necroptosis)in prolactinoma,indicating that it might induce necroptosis.Part 2: Bromocriptine suppressed the cell viability of MMQ cells and induced the indicators of necroptosisCCK-8 assay was performed to detect the viability of MMQ cells.Firstly,the MMQ cells were treated with bromocriptine at different dosages from 0μM to 100 μM.We found that bromocriptine suppressed the viability of MMQ cells in a dose-dependent manner,and 100μM was the optimal concentration to have the maximum effect.We then tested that whether bromocriptine has a time-dependent effect on cell viability of MMQ cells.The data showed that there is no significant difference between cell viability after 24 h and 48h-treatment.Thus,100 μM of bromocriptine for 24 h-treatment was adopted for further experiments.Necrostatin-1 alone was also examined and no effects had been observed.Reduced cell viability can be caused by different forms of cell death.We then examined the cellular ROS and ATP level,two indicators of necroptosis,to test whether nercroptosis was involved in decreased viability of MMQ cells.The data showed that the ROS level of MMQ cells was increased 1.8-folds,while ATP level decreased 2.1-folds,and the effects can be significantly blocked by Necrostatin-1.Further,immunostaining data showed that the IFI of RIP3 and p MLKL in MMQ cells was significantly increased after bromocriptine treatment,and Necrostatin-1 significantly blocked the effect.The results suggested that bromocriptine could induce RIP3/MLKL-dependent necroptosis of MMQ cells.To further confirm the bromocriptine’s function is dependent on RIP3/MLKL signal,three pairs of si RNA against RIP3 and MLKL were first tested for their silencing efficiency respectively,and RIP3-3 and MLKL-1 was found to have the maximum silencing efficiency.Further data showed that knocking down RIP3 and MLKL could significantly reverse the changes of cellular ROS and ATP,two indicators of necroptosis,of MMQ cells induced by bromocriptine.These data indicated that bromocriptine-induced necroptosis of MMQ cells was at least partially dependent on RIP3/MLKL signal.Part 3: Ultrastructural study confirmed the necroptosis of MMQ cells induced by bromocriptineTo further confirm the necroptosis of MMQ cells after bromocriptine treatment,electron microscope was used to study the ultrastructural changes of MMQ cells.The data showed that the numbers of necroptotic cells was dramatically increased in cells treated with bromocriptine.The necroptotic cells can be distinguished with significant features,such as intracellular edema,lysed cytoplasmic material and ruptured cell membrane.Besides these,a remarkably increased numbers of swollen mitochondria was found in MMQ cells after bromocriptine treatment,compared with the control.These effects can be inhibited by addition of Necrostatin-1.The above results verified the pro-necroptotic effects of bromocriptine on prolactinoma cells.Part 4: PGAM5-CypD pathway involved in bromocriptine induced-necroptosis of prolactinoma cellsIn our electron microscopic study,the morphological features of necroptotic MMQ cells was characterized not only by the ruptured membrane and lysed cytoplasm,but also the significantly injured,massively swollen mitochondria,which indicated that mitochondrial dysfunction was involved in bromocriptine-induced necroptosis of MMQ cells.Considering the PGAM5 and CypD,two downstream signal molecules of RIP3,mediate the swelling and rupture of the mitochondria in pathological conditions,we then tested whether the PGAM5/CypD pathway was involved in bromocriptine-induced necroptosis of MMQ cells.The result of western blot showed that Necrostatin-1 significantly decreased the expression of PGAM5 and p-CypD of MMQ cells induced by bromocriptine,which was in accordance with the alteration of RIP3 and p MLKL.In addition,there was no effect of Bromocriptine on total MLKL and CypD expression levels.To further investigate the role of PGAM5/CypD pathway in bromocriptine-induced RIP3/MLKL-dependent necroptosis,the protein-protein interaction of p-CypD and RIP3 was then verified in bromocriptine-treated MMQ cells.Further,si RNA against CypD and PGAM5 was transfected into MMQ cells-treated by bromocriptine.Three pairs of si RNA against CypD and PGAM5 were first tested for their silencing efficiency,and CypD-3 and PGAM5-2 was found to have the maximum silencing efficiency.Further data showed that knocking down CypD and PGAM5 could significantly inhibit the necroptotic promotion of MMQ cells by bromocriptine,shown by the indicators of ROS and ATP.These data indicated that PGAM5/CypD signaling was involved in bromocriptine-induced necroptosis of MMQ cells. |
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