Study On The Effect And Mechanism Of Regulating Androgen Receptor Through Keap1/Nrf2 Pathway Combined With Bromocriptine In The Treatment Of Pituitary Prolactin-secreting Adenomas

Objective: To discuss the expression of Androgen Receptor(AR)in drug-resistant pituitary prolactin-secreting adenoma and its correlation with Bromocriptine(Brc)treatment sensitivity.To explore the effect and mechanism of regulating AR and coordinating Brc in the treatment of pituitary prolactin-secreting adenoma through in vivo and in vitro experiments.To explore the therapeutic effect of AR antagonist combined with Brc on pituitary prolactin-secreting adenoma.Methods: The clinical data of 52 patients with drug-resistant pituitary prolactin-secreting adenoma were retrospectively analyzed.The correlation between the expression level and the sensitivity to Brc treatment was compared among groups after AR staining.Lentivirus transfection was used to knock down the AR in pituitary adenoma cells.m RNA expression levels were detected by RT-q PCR.The expression levels of related proteins were detected by western blotting assay.PRL secretion levels in cell medium supernatant and blood of nude mice were detected by ELISA.Cell proliferative activity and drug toxicity were determined by CCK-8 method.Plate cloning was used to evaluate cell proliferation.The cell cycle and apoptosis were detected by flow cytometry.Dual luciferase and Ch IP experiments verified the regulation of Nrf2 transcription by AR.Subcutaneous graft and intraperitoneal injection were used to evaluate tumor growth rate and drug efficacy in nude mice.CDK4 immunofluorescence staining was performed after tumor sections to evaluate tumor proliferation.Results: Compared with the weakly positive and moderately positive AR expression groups,the proportion of tumor shrinkage after Brc treatment in the strongly positive group was smaller [(29.5±9.2)% vs.(25.9±9.8)% vs.(17.3±8.2)%,respectively].The decrease rate of serum PRL was lower [(50.3±17.3)% vs.(42.5±14.2)% vs.(30.4±15.0)%,respectively].All the differences were statistically significant(all P <0.01).In vivo and in vitro experiments,it was found that AR knockdown inhibited the proliferation of GH3 cells and MMQ cells in pituitary adenoma,promoted cell apoptosis,and reduced the secretion level of PRL.These effects were more significant after the effect of Brc.Knockdown of AR promotes endogenous apoptosis and iron death of pituitary adenoma cells by amplifying intracellular ROS signaling.It was subsequently confirmed that AR could bind to Nrf2 promoter in nucleus and promote Nrf2 transcription,thus up-regulating Nrf2 expression level,and knocking down AR could inhibit Keap1/Nrf2 pathway.Finally,in vitro and in vivo experiments showed that Enzalutamide(Enza)combined with Brc could significantly inhibit the proliferation of pituitary adenoma cells,promote cell apoptosis and reduce the level of PRL secretion.Conclusion: The expression level of AR in drug-resistant pituitary prolactin-secreting adenoma was negatively correlated with its sensitivity to Brc treatment.Inhibition of AR can inhibit Keap1/Nrf2 pathway by regulating Nrf2 transcription,thus amplifying ROS signal and coordinating with Brc in the treatment of pituitary prolactin-secreting adenoma.AR antagonist Enza combined with Brc can significantly inhibit the proliferation and promote the apoptosis of pituitary adenoma cells.Part Ⅰ:Correlation research between androgen receptor expression and sensitivity to bromocriptine therapy in pituitary prolactin-secreting adenomasObjective: Pituitary prolactin-secreting adenomas are the most common functional pituitary adenomas,and dopamine agonists are the first choice for its treatment.However,about 20~30% of patients develop drug resistance during clinical treatment with Brc.At present,the mechanism of resistance of patients with prolactin-secreting adenomas to bromocriptine treatment is unclear.In this study,the correlation between AR expression level in drug-resistant pituitary prolactin-secreting adenoma and Brc treatment sensitivity was analyzed and explored by comparing the differences in the sensitivity of patients with pituitary prolactin-secreting adenomas to bromocriptine treatment.Methods: The clinical data of 52 patients with drug-resistant pituitary prolactin-secreting adenomas who underwent transsphenoidal microresection at Department of Neurosurgery,Tongji Hospital,Tongji Medical College,Huazhong University of Science and Technology from January 2017 to June 2022 were retrospectively analyzed.All patients were regularly treated with Brc(mean dose was 11.5mg/d,mean time was 8.7 months)before surgery,and finally underwent transsphenoidal microsurgery to remove the adenomas.The adenoma samples were embedded,sectioned and stained by immunohistochemistry.According to the proportion and intensity of AR cell expression,the adenoma samples were divided into weakly positive group,moderate positive group and strongly positive group(12 cases,13 cases and 27 cases,respectively).By comparing the clinical characteristics,efficacy and prognosis of the three groups of patients,the correlation between the expression level of AR in pituitary prolactin-secreting adenomas and the sensitivity of Brc treatment was analyzed and discussed.Results: There were no significant differences in gender,age,adenoma size before and after treatment and serum PRL level among the three groups(all P > 0.05),and the baseline was basically the same and comparable.Compared with the other two groups,the degree of adenoma shrinkage in patients with strongly AR expression was smaller after Brc treatment [(0.6±0.2)cm vs.(0.4±0.1)cm vs.(0.3±0.1)cm,respectively].The proportion of reduction was lower [(29.5±9.2)% vs.(25.9±9.8)% vs.(17.3±8.2)%,respectively].The serum PRL level decreased even less [(836.4±631.7)ng/m L vs.(527.4±685.3)ng/m L vs.(166.2±352.3)ng/m L,respectively].The proportion of decrease was smaller [(50.3±17.3)% vs.(42.5±14.2)% vs.(30.4±15.0)%,respectively].The differences were statistically significant(all P < 0.01).In addition,the Knosp grade of adenomas in the strongly positive AR expression group was significantly higher than that in the other two groups,and the difference was statistically significant(P < 0.001).In terms of short-term immediate remission rate and long-term follow-up remission rate,the AR expression strongly positive group was lower than the other two groups,but the difference was not statistically significant(all P > 0.05).For patients with no remission after surgery,the effective rate of Brc therapy was lower in the group with strongly AR expression,but the difference was not statistically significant(P > 0.05).Conclusion: The expression level of AR in pituitary prolactin-secreting adenomas is correlated with its sensitivity to Brc treatment.The higher the expression level of AR,the more resistance to Brc treatment,and the lower the sensitivity.For patients with drug-resistant pituitary prolactin-secreting adenomas,AR may become a potential therapeutic target,but its related mechanism needs to be further studied.Part Ⅱ:Correlation between androgen receptor knockdown and sensitivity of pituitary adenomas cells to bromocriptine therapyObjective: Based on the content of the first part of the study,it was found that the sensitivity of patients with drug-resistant pituitary prolactin-secreting adenomas to Brc was correlated with the differential expression of AR.Subsequently,the correlation between AR in GH3 cells and MMQ cells of rat pituitary gland knockdown and its sensitivity to Brc treatment was further explored.Methods: AR gene expression was inhibited in GH3 cells and MMQ cells by lentivirus sh RNA transfection.RT-q PCR was used to detect the expression level of AR m RNA and verify the knock-down effect.Western blotting was used to detect the protein expression level of AR and verify the knock-down effect.The secretion level of PRL in cells culture medium supernatant and blood of nude mice were detected by ELISA.Cells proliferation and activity were determined by CCK-8 method.The cells proliferation ability were evaluated by plate clone formation assay.Flow cytometry was used to detect cells cycle and apoptosis.Tumor growth rate and drug efficacy were evaluated by subcutaneous tumor grafting and intraperitoneal injection in nude mice.CDK4 immunofluorescence staining was performed to evaluate tumor proliferation.Results: Firstly,stable transfection cell lines with AR knockdown were successfully constructed in GH3 cells and MMQ cells of rat pituitary adenomas,and the effect of AR knockdown was verified by RT-q PCR and Western blot.Subsequently,the proliferation and apoptosis of rat pituitary adenomas GH3 cells and MMQ cells after AR knockdown were inhibited,the proportion of S-phase cells in the cells cycle was decreased,the level of PRL secretion was decreased,and the proportion of apoptosis(including early apoptosis and late apoptosis)was increased.These effects were more significant after Brc treatment,and the sensitivity of cells to Brc increased significantly.In vivo tumor-forming experiments in nude mice showed that the tumor volume,tumor weight,CDK4 expression level and blood PRL level in the AR knockdown group were significantly lower than those in the control group,and the differences were more significant after treatment with Brc.Conclusion: The decreased expression of AR in rat pituitary adenomas GH3 cells and MMQ cells can inhibit cell proliferation,reduce PRL secretion level and promote cell apoptosis in vitro and in vivo.These effects are more significant in the treatment of Brc,and the cells are more sensitive to Brc treatment.Part Ⅲ:Mechanism of inhibiting androgen receptor in pituitary adenoma cells can enhance the sensitivity of bromocryptine therapyObjective: In the second part of the study,we found that reducing the expression level of AR in rat pituitary adenoma cell lines significantly enhanced the effects of Brc on the inhibition of proliferation,promotion of apoptosis and reduction of PRL secretion level of rat pituitary adenoma cells,but the specific mechanism was unclear.This study aims to explore the mechanism by which inhibiting AR in pituitary adenomas can enhance the sensitivity of Brc therapy.Methods: Western blotting was used to detect the protein expression levels of caspase-3,Bcl-2,Bax,GPX4,SLC7A11,Keap1 and Nrf2.The orange probe for oxidative stress detection(live cell)was used to detect intracellular reactive oxygen species(ROS)level.N-Acetyl-L-cysteine(NAC)was used as ROS inhibitor and dimethyl fumarate as Nrf2 agonist.Cell proliferation and activity were determined by CCK-8 method.The cell proliferation ability was evaluated by plate clone formation assay.Cell cycle and apoptosis were detected by flow cytometry.The level of PRL secretion in cell culture medium supernatant was detected by ELISA.The m RNA expression levels of Keap1 and Nrf2 were detected by RT-q PCR.Dual luciferase assay and Ch IP assay were used to verify the regulation of Nrf2 transcription by AR.Results: Knockdown of AR in GH3 and MMQ cells of rat pituitary adenomas promotes endogenous apoptosis and iron death,and one effect is mediated by amplification of intracellular ROS signaling.After the introduction of ROS inhibitor NAC,it could be found that the effects of Brc on inhibiting cell proliferation,promoting cell apoptosis and reducing PRL secretion level were rescued,which were more significant in the AR knockdown group.Subsequently,we found that knockdown of AR resulted in increased expression level of Keap1 and decreased expression level of Nrf2.However,dimethyl fumarate can significantly inhibit ROS signal amplification caused by AR knockdown.We further found that AR can bind to specific base sequences of Nrf2 through database query.Double luciferase experiment and Ch IP experiment proved that AR,as a nuclear transcription factor,can bind to the specific region of Nrf2 promoter in the nucleus and promote the transcription of Nrf2,so as to up-regulate the expression level of Nrf2.Conclusion: Inhibiting AR in GH3 cells and MMQ cells of rat pituitary adenoma can enhance the sensitivity of Brc therapy.This effect is mainly achieved by regulating Nrf2 transcription and inhibiting Keap1/Nrf2 pathway,thus amplifying ROS signaling mediated cell endogenous apoptosis and iron death.Part Ⅳ:Effect of androgen receptor antagonist combined with bromocriptine on proliferation and apoptosis of pituitary adenoma cellsObjective: To investigate the effect of AR antagonist combined with Brc on proliferation,apoptosis and PRL secretion of rat pituitary adenomas GH3 and MMQ cells in vitro and in vivo.Methods: Cell proliferation,activity and Enza toxicity were determined by CCK-8 assay.The effects of drugs on cell proliferation were evaluated by plate cloning assay.The levels of PRL secretion in cell culture medium supernatant and blood of nude mice were detected by ELISA.Flow cytometry was used to detect the cell cycle and apoptosis after drug treatment.To detect intracellular ROS levels after drug treatment,an orange probe was used to detect oxidative stress(living cells).Western blotting was used to detect the expression of related proteins.The tumor growth rate and drug efficacy were evaluated by subcutaneous tumor grafting and intraperitoneal injection of Enza and Brc in nude mice.CDK4 immunofluorescence staining was performed after tumor sections were taken to evaluate the tumor proliferation level.Results: The optimum concentration of Enza was determined as 5μM by toxicity test.The results of cell proliferation,apoptosis and PRL secretion experiments showed that low concentration of Enza combined with Brc could significantly inhibit the proliferation of pituitary tumor cells,promote cell apoptosis and reduce the level of PRL secretion.Through the orange probe of oxidative stress detection,it was found that the ROS signal in pituitary tumor cells was significantly amplified by combination therapy,which was higher than that in the single therapy group.At the same time,Western blot experiments were performed to verify that the combined drug could significantly increase the endogenous apoptosis and ferric death levels of pituitary tumor cells,and significantly inhibit Keap1/Nrf2 pathway.The results of in vivo experiment showed that the tumor volume and tumor weight of the combined drug group was significantly lower than that of the control group.After immunofluorescence staining,the expression level of CDK4 in the combined drug group was significantly lower than that in the control group.Conclusion: Compared with Brc alone,low concentration AR antagonist Enza combined with Brc could significantly inhibit the proliferation of pituitary adenoma cells,promote apoptosis and reduce the secretion of PRL in vitro and in vivo.