Bromocriptine and cabergoline induce cell death in prolactinoma cells via the ERK/EGR1 and AKT/mTOR pathway respectively

Abstract

The treatment of hyperprolactinemia is based on the use of dopamine agonists, mainly bromocriptine (BRC) and cabergoline (CAB). They reduce tumour size effectively and restore gonadal function. However, there is a difference in drug sensitivity between CAB and BRC in patients with prolactinoma, although the underlying mechanisms are still unknown. Thus, we investigated whether there are differences in tumour sensitivity to CAB and BRC and their possible differential mechanisms in two prolactinoma cell lines. In our study, we found that GH3 cells are more sensitive to BRC and that MMQ cells are more sensitive to CAB. Moreover, BRC and CAB elicited cell death via different pathways; BRC induced prolactinoma cell death mainly through the apoptosis pathway, and CAB induced pituitary prolactinoma cell death mainly via the autophagic cell death pathway. Using gene microarray analysis, we found that BRC induces the apoptosis of prolactinoma cells through the ERK/EGR1 signalling pathway, whereas CAB induces autophagic death by inhibiting the AKT/mTOR signalling pathway. Our study showed the difference in tumour sensitivity and differential mechanisms in BRC- and CAB-treated prolactinoma cells, which provides a theoretical basis for the accurate treatment of prolactinoma.

Fig. 1. CAB and BRA decreased the viability of GH3 and MMQ cells.

a–d Cell survival was determined by the CCK8 assay. MMQ and GH3 cells were treated with CAB and BRC at concentrations of 0, 25, 50, and 100 μM, respectively, for 24, 48, and 72 h. e GH3 and MMQ cells were treated with CAB or BRC at a concentration of 50 μM for 48 h, and cell viability was tested with the CCK8 kit

Fig. 2. Comparison of CAB- and BRC-induced autophagy in prolactinoma cells.

a, b Electron microscopy images with enlargements. Arrows show autophagic vacuoles in GH3 and MMQ cells treated with BRC (50 μM) or CAB (50 μM) for 48 h. c, d Western blot analysis of LC3-I and LC3-II in GH3 and MMQ cells with or without BRC (50 μM) or CAB (50 μM) treatment at different concentrations

Fig. 3. CAB promoted autophagy-induced cell death in prolactinoma cells.

a Electron micrographs of GH3 and MMQ cells treated with BRC or CAB for 48 h, with or without 3-MA (5 mM). Histogram shows the autolysosome structures from multiple experiments in a total of 50 cells (mean ± SD). b, c Immunoblot analysis of LC3-I and LC3-II in GH3 and MMQ cells with or without BRC (50 μM) or CAB (50 μM) treatment and with or without 3-MA (5 mM). d GH3 and MMQ cells were treated with CAB and BRC at a concentration of 50 μM for 48 h, with or without 3-MA (5 mM), and cell viability was tested with the CCK8 kit

Fig. 4. The comparison of CAB- and BRC-induced apoptosis in prolactinoma cells.

a, b MMQ and GH3 cells were treated with CAB (50 μM) or BRC (50 μM) as indicated for the apoptosis assay by Annexin V-FTIC and PI double staining. c MMQ and GH3 cells were treated with CAB (50 μM) or BRC (50 μM), and the total proteins were analysed by Western blot using antibodies against cleaved caspase-3 and β-actin

Fig. 5. BRC promoted apoptosis-induced cell death in prolactinoma cells.

a MMQ and GH3 cells were treated with CAB (50 μM) or BRC (50 μM) with or without Z-VAD-FMK (50 μM), as indicated for the apoptosis assay by Annexin V-FTIC and PI double staining. b, c MMQ and GH3 cells were treated with CAB (50 μM) or BRC (50 μM) with or without Z-VAD-FMK (50 μM), and the total proteins were analysed by Western blot using antibodies against cleaved caspase-3 and β-actin. d MMQ and GH3 cells were treated with CAB (50 μM) in the presence or absence of Z-VAD-FMK (50 μM) at 50 μM for 48 h, and cell viability was determined by CCK8

Fig. 6. Differential mRNA levels and pathways participating in BRC- and CAB-induced cell death.

a Differential mRNA expression in GH3 cells treated with or without BRC (50 μM). Differential mRNA expression in MMQ cells treated with or without CAB (50 μM). b Pathway enrichment analysis of BRC-treated GH3 cells. c Pathway enrichment analysis of CAB-treated MMQ cells

Fig. 7. BRC activates ERK1/2 and upregulates the expression of EGR1 in prolactinoma cells.

a Immunoblot analysis of EGR1 and p-ERK1/2 in GH3 and MMQ cells treated with DMSO or BRC (50 μM) for 48 h. b RT-qPCR analysis of the mRNA expression of EGR1 in GH3 and MMQ cells treated with BRC (50 μM) for 48 h. c, d Immunoblot analysis and RT-PCR of EGR1 expression after GH3 and MMQ cells were transfected with the control (Ctrl) or the siRNA against EGR1 for three days. e Cell viability of MMQ and GH3 cells treated with the control (Ctrl) or with the siRNA against EGR1. f, g GH3 and MMQ cells were treated with CAB at concentrations of 25, 50, and 100 μM, followed by immunoblot analysis for p-Akt, p-mTOR and β-actin

Fig. 8. Subcutaneous xenograft model and drug therapy in nude mice.

a Schematic representation of the experimental procedure employed to study tumours in nude mice. Tumour formation was observed, and tumourigenic nude mice were randomly divided into a control group and a BRC treatment group (n = 5). b Representative images of xenograft tumours in nude mice. c The tumour volume on the nude mouse. d Representative images of the tumour samples from each group. e The tumour weight of each group. f The TUNEL staining and immunohistochemical analyses of EGR1 and p-ERK in tumour samples of each group. g The proposed mechanism of BRC- and CAB-induced cell death in prolactinoma cells