Bromocriptine Induces Autophagy-dependent Cell Death In Pituitary Adenomas

Objective:Prolactinoma is a type of high incidence carcinoma that accounts for approximately 40%of all pituitary adenomas.Pituitary adenoma treatment strategies include surgery,radiotherapy and drug treatment,which mainly involves dopamine agonists(DA).Preoperative DA therapy reduces the likelihood of successful adenoma removal due to fibrosis.However,DAs are effective for prolactinoma treatment;these drugs mainly include bromocriptine(BRC),cabergoline(CAB)and pergolide.BRC effectively reduces prolactin levels,decreases tumor size and restores gonadal function in patients with prolactinoma.BRC is also effective for other tumors,such as nonfunctional pituitary adenoma,ACTH-secreting pituitary adenomas and acromegaly.CAB is used for long-term medication given its long half-life and low risk.However,CAB costs at least twice as much as BRC and did not demonstrate superior performance in other outcomes.Consequently,BRC treatment is widely applied in China and other developing countries.BRC induces apoptosis in pituitary adenoma cells;other studies have demonstrated that CAB activates ACD pathways and suppresses tumor growth.However,modern studies cannot elucidate the cytotoxic mechanism of DA for pituitary adenoma.This study provides a novel research direction for the drug treatment of pituitary adenomas.Methods:The slices for immunohistochemical pathology were randomly selected from 37 paraffin-embedded prolactinoma pituitary adenoma tissue sections(15 males and 22 females)in Binzhou Medical University Hospital.In total,17 patients were exclusively treated with BRC.The other 20 patients were not treated with BRC.We examined the expression of LC3 and Bcl-2 by immunohistochemical staining in the human pituitary adenomas.GH3 and MMQ rat pituitary adenomas cell lines were purchased from the ATCC.GH3 and MMQ cells were treated by BRC and/or rapamycin(RAPA).Cell viability were measured by CCK-8,the cell cycle were measured flow cytometry,ELISA measured prolactin secretion,the conversion ratio of LC3-I to LC3-II and the ratio of Bcl-2/Bax were detected by Western blot.Immunofluorescence measured LC3 expression after drug treatment.Results:LC3 was significantly expressed in prolactin(PRL)-secreting pituitary adenomas in which the patients were exclusively treated with BRC.LC3 expression was negative in prolactin(PRL)-secreting pituitary adenomas in which patients were not treated with BRC.The positive expression rate of normal brain tissue was 0%.BRC suppressed the viability of BRC-treated MMQ and GH3 cells in a time-and dose-dependent manner.Treatment with 60 μmol/L BRC for 24 h induced cell death in MMQ cells up to 50%.However,110 μmol/L BRC was required to produce a similar effect in GH3 cells.Cell viability was similar in MMQ cells treated with RAPA and low concentrations of BRC and those treated with a high concentration of BRC.Evidence showed the induction of cell-cycle arrest at the Go-Gi phase(MMQ:control G0-G1,73.66%± 3.50%vs.BRC-treated G0-G1,93.47%± 3.02%;GH3:control G0-G1,73.93%± 0.50%vs.BRC-treated G0-G1,92.23%± 0.50%).RAPA alone cannot effectively arrest the cells at the G0-G1 phase(MMQ:control G0-G1,73.66%± 3.50%vs.RAPA-treated G0-G1,79.92%± 5.28%;GH3:control G0-G1,73.93%± 0.50%vs.RAPA-treated G0-G1,79.83%± 6.22%).BRC treatment also decreased the number of cells in S phase(MMQ:control,17.68%±1.23%vs.BRC-treated,2.44%± 0.16%;GH3:control,20.58%± 1.33%vs.BRC-treated,5.39%±0.50%).RAPA alone cannot effectively decrease the number of cells in S phase(MMQ:control,17.68%± 1.23%vs.RAPA-treated,17.28%±2.16%;GH3:control,20.58%±1.33%vs.RAPA-treated,16.19%±2.50%).The cells were arrested in the G0-G1 phase(MMQ:control G0-G1,73.66%± 3.50%vs.BRC and RAPA-treated G0-G1,93.67%± 2.89%;GH3:control G0-G1,73.93%± 0.50%vs.BRC and RAPA-treated G0-G1,92.91%± 0.64%).In addition,the number of cells in S phase decreased(MMQ:control,17.68%± 1.23%vs.BRC and RAPA-treated,2.6%± 0.23%;GH3:control,20.58%± 1.33%vs.BRC and RAPA-treated,3.85%± 0.37%).PRL concentrations were 1123±26 ng/L,1092±23 ng/L,299±15 ng/L,and 285± 12 ng/L in the supernatant of MMQ cells and 786±25 ng/L,775±28,202±13 ng/L,and 189±14 ng/L in the supernatant of GH3 cells treated with control,RAPA alone,BRC alone and BRC&RAPA,respectively.MMQ and GH3 cells were treated with different concentrations of BRC for 24-h induction.As the concentration increased,the conversion ratio of LC3-I to LC3-II also increased and the Bcl-2/BAX ratio did not significantly change in dose-and time-dependent manners.By contrast,the conversion ratio of LC3-I to LC3-II increased with time when a 60 μmol/L concentration was used.Immunofluorescence assays revealed low LC3 expression in normal MMQ and GH3 cells.LC3 expression was significantly enhanced after 24 h of induction with BRC.BRC and RAPA are more likely to induce cell autophagy.The conversion ratio of LC3-I to LC3-II was significantly increased in MMQ cells treated with a combination of BRC(40 μmol/L)and RAPA(100 nmol/L)compared with BRC treatment alone(60 μmol/L)or RAPA treatment alone(100 nmol/L).Conclusion:BRC-treated pituitary adenoma cells mainly underwent autophagic cell death(ACD)rather than apoptosis.