Prolactinoma Cells Research Articles

Network pharmacology analysis and molecular mechanism of paeoniflorin and its metabolite in prolactinoma cells.

Prolactinoma was the most common functionalpituitary neuroendocrine tumor tissue type, which was caused by excessive proliferation of pituitary prolactin (PRL) cells. Drug therapy of dopamine receptoragonists was generally considered as the prior treatment for prolactinoma patients. However, there were still prolactinoma patients who were resistant to dopamine agonists. Studies have been reported that paeoniflorin can inhibit the secretion of PRL in prolactinoma cells lacking dopamine D2 receptor (D2R) expression, and paeoniflorin can be metabolized into albiflorin by intestinal flora in rats. The effect of albiflorin on prolactinoma has not been reported yet. In this study, network pharmacology was used to analyze the mechanism of paeoniflorin and its metabolite albiflorin as multi-target therapy for prolactinoma, and the experimental verification was carried out. In order to clarify the complex relationship among paeoniflorin, albiflorin and prolactinoma, we constructed a component-target-disease network, and further constructed interaction network, MMP9, EGFR, FGF2, FGFR1 and LGALS3 were screened as the core targets. Kyoto encyclopedia of genes and genomes (KEGG) analysis showed that paeoniflorin and albiflorin may be involved in various pathways in the treatment of prolactinoma, included relaxin signaling pathway and PI3K-Akt signaling pathway. Molecular docking analysis showed that paeoniflorin and albiflorin had good binding activity with MMP9. Western blotting results showed that paeoniflorin and albiflorin could significantly reduce the expression of MMP9, and ELISA results showed that paeoniflorin and albiflorin could significantly reduce the concentration of PRL in GH3 cells, and the reduce degree of albiflorin was stronger than paeoniflorin at 50μM, which indicated that albiflorin might be a potential drug to treat prolactinoma, which can regulate prolactinoma through MMP9 and reduce the concentration of PRL. Our study provided a new therapeutic strategy for prolactinoma.

Bromocriptine sensitivity in bromocriptine-induced drug-resistant prolactinomas is restored by inhibiting FGF19/FGFR4/PRL.

At present, various treatment strategies are available for pituitary adenomas, including medications, surgery and radiation. The guidelines indicate that pharmacological treatments, such as bromocriptine (BRC) and cabergoline (CAB), are important treatments for prolactinomas, but drug resistance is an urgent problem that needs to be addressed. Therefore, exploring the mechanism of drug resistance in prolactinomas is beneficial for clinical treatment. In our research, BRC-induced drug-resistant cells were established. Previous RNA sequencing data and an online database were used for preliminary screening of resistance-related genes. Cell survival was determined by Cell Counting Kit-8 (CCK-8) assay, colony formation assays and flow cytometry. Quantitative real-time polymerase chain reaction (qRT‒PCR), western blotting, immunohistochemistry, immunofluorescence and Co-immunoprecipitation (Co-IP) were used to assess the molecular changes and regulation. The therapeutic efficacy of BRC and FGFR4 inhibitor fisogatinib (FISO) combination was evaluated in drug-resistant cells and xenograft tumors in nude mice. Consistent with the preliminary results of RNA sequencing and database screening, fibroblast growth factor 19 (FGF19) expression was elevated in drug-resistant cells and tumor samples. With FGF19 silencing, drug-resistant cells exhibited increased sensitivity to BRC and decreased intracellular phosphorylated fibroblast growth factor receptor 4 (FGFR4) levels. After confirming that FGF19 binds to FGFR4 in prolactinoma cells, we found that FGF19/FGFR4 regulated prolactin (PRL) synthesis through the ERK1/2 and JNK signaling pathways. Regarding the effect of targeting FGF19/FGFR4 on BRC efficacy, FISO and BRC synergistically inhibited the growth of tumor cells, promoted apoptosis and reduced PRL levels. Overall, our study revealed FGF19/FGFR4 as a new mechanism involved in the drug resistance of prolactinomas, and combination therapy targeting the pathway could be helpful for the treatment of BRC-induced drug-resistant prolactinomas.

THU553 Anti-tumor Effect Of Rosiglitazone Via Upregulating 15-PDGH In Prolactin Secreting Adenoma

Abstract Disclosure: C. Kang: None. J. Oh: None. E. Wang: None. S. Lee: None. J. Nam: None. J. Hong: None. E. Lee: None. C. Ku: None. Background: Rosiglitazone, a synthetic peroxisome proliferator-activated receptor γ (PPAR γ) ligand, are used to treat type II diabetes. Over the last few years, PPAR γ has received much attention for its ability to exert variable tumors. Objective: The aim of this study is to investigate the anti-tumor effect of rosiglitazone on prolactin secreting pituitary adenomas and its mechanism. Materials & Methods: Lactotroph cell line (GH4, MMQ) were treated with Phosphate-buffered saline (PBS) or rosiglitazone. In vitro analysis such as qPCR, immunoblot, prolactin (PRL) ELISA, Cell proliferation assay, and Fluorescence-activating cell sorting (FACS) analysis were performed to examine the effect of rosiglitazone on Lactotroph cell lines. Result: To investigate the direct effect of rosiglitazone on PRL secretion of the pituitary adenomas, GH4 and MMQ cells were treated with rosiglitazone 1, 5, 10, 50 μM for 72 hr. Both PRL mRNA and secretion levels were significantly decreased by rosiglitazone in a dose-dependent manner. In previous studies, 15-PDGH has been implicated as a tumor suppressor gene with the property that inhibits the tumor growth. Interestingly, 5 μM rosiglitazone upregulated the mRNA and protein levels of 15-PDGH in both GH4 (4.73-fold) and MMQ (4.04-fold) cells. Next, we investigated whether rosiglitazone had any effects on the proliferation of prolactinoma cells. Proliferation of the GH4 and MMQ cells were significantly decreased by rosiglitazone in a dose-dependent manner after treatment. Rosiglitazone potently induced cell cycle arrest in sub-G1inGH4 and MMQ cells. Furthermore, rosiglitazone treatment significantly increased both early (2.31-fold) and late apoptosis (1.9-fold) of GH4 and MMQ cells. Conclusion: These results collectively position 15-PDGH as a potential new therapeutic target for prolactinomas and implicate rosiglitazone as a possible alternative pharmacological approach for prolactinomas. Presentation: Thursday, June 15, 2023

Letrozole Decreased Testosterone-Induced Cell Proliferation and Prolactin Secretion also Increased Apoptosis in MMQ and GH3 Rat Prolactinoma Cell Lines.

Aromatase enzyme plays an essential role in estrogen-induced tumorigenesis. It is expressed in the normal pituitary and more significantly in prolactinoma tissues. The aim of this study was to investigate the effects of an aromatase inhibitor, letrozole, on MMQ and GH3 rat prolactinoma cell lines and evaluate the possible mechanism of action. MMQ and GH3 cells were characterized with demonstrating aromatase enzyme and estrogen receptor alpha expression by PCR and immunofluorescence staining. After dose optimization for testosterone (T) and letrozole (L), four groups were established: only the testosteron-treated group (T) to detect cell proliferation; only letrozole-treated group (L) to investigate apoptotic effects; testosterone and letrozole concomitant-treated group to demonstrate inhibition of testosterone induced cell proliferation with letrozole treatment s(T + L) and control group (C) with no treatment. The proliferation rate of cells was determined by WST-1. For the detection of apoptotic and necrotic cells, Annexin V and caspase-3 labeling was used. Prolactin and estrogen levels were measured with ELISA, and the mRNA expression of aromatase and Esr1 was also determined. Testosterone induced the proliferation of MMQ and GH3 cells and further increased prolactin and estradiol levels. Adding letrozole to testosterone resulted in decreased cellular proliferation and even induced apoptosis. Also, letrozole administration significantly decreased prolactin and estradiol levels. However, letrozole alone had no effects on proliferation and apoptosis. Gene expression of aromatase and Esr1 was also significantly decreased by letrozole treatment. This in vitro study demonstrated that treatment of testosterone proliferating cells with letrozole resulted in decreased prolactin levels and cell proliferation and induced apoptosis, and further loss of aromatase and Esr1 mRNA expression were observed. Although this is an in vivo study, the results showed unique and novel findings which may easily be adapted to clinical use for further verification.

The effects of hyponatremia on prolactinoma cell populations

The effects of hyponatremia on prolactinoma cell populations

β-Catenin is reduced in membranes of human prolactinoma cells and it is inhibited by temozolomide in prolactin secreting tumor models.

Prolactinomas are the most frequent pituitary tumor subtype. Despite most of them respond to medical treatment, a proportion are resistant and become a challenge in clinical management. Wnt/β-Catenin pathway has been implicated in several cancers including pituitary tumors and other sellar region malignancies. Interestingly, Wnt/β-Catenin inhibition augments the cytotoxicity of the chemotherapeutic agent Temozolomide (TMZ) in different cancers. TMZ is now being implemented as rescue therapy for aggressive pituitary adenoma treatment. However, the molecular mechanisms associated with TMZ action in pituitary tumors remain unclear. Our aims in the present study were to evaluate differential β-Catenin expression in human resistant prolactinomas and Wnt/β-Catenin signaling activation and involvement in Prolactin (PRL) secreting experimental models treated with TMZ. We first evaluated by immunohistochemistry β-Catenin localization in human resistant prolactinomas in which we demonstrated reduced membrane β-Catenin in prolactinoma cells compared to normal pituitaries, independently of the Ki-67 proliferation indexes. In turn, in vivo 15 mg/kg of orally administered TMZ markedly reduced PRL production and increased prolactinoma cell apoptosis in mice bearing xenografted prolactinomas. Intratumoral β-Catenin strongly correlated with Prl and Cyclin D1, and importantly, TMZ downregulated both β-Catenin and Cyclin D1, supporting their significance in prolactinoma growth and as candidates of therapeutic targets. When tested in vitro, TMZ directly reduced MMQ cell viability, increased apoptosis and produced G2/M cell cycle arrest. Remarkably, β-Catenin activation and VEGF secretion were inhibited by TMZ in vitro. We concluded that dopamine resistant prolactinomas undergo a β-Catenin relocalization in relation to normal pituitaries and that TMZ restrains experimental prolactinoma tumorigenicity by reducing PRL production and β-Catenin activation. Together, our findings contribute to the understanding of Wnt/β-Catenin implication in prolactinoma maintenance and TMZ therapy, opening the opportunity of new treatment strategies for aggressive and resistant pituitary tumors.

Forskolin Stimulates Estrogen Receptor (ER) α Transcriptional Activity and Protects ER from Degradation by Distinct Mechanisms.

Estradiol action is mediated by estrogen receptors (ERs), a and ß. Estradiol binding initiates ER-mediated transcription and ER degradation, the latter of which occurs via the ubiquitin-proteasome pathway. Inhibition of proteasome activity prevents estradiol-induced ERα degradation and transactivation. In ER-positive GH3 cells (a rat pituitary prolactinoma cell line), forskolin, acting via protein kinase A (PKA), stimulates ERα transcriptional activity without causing degradation, and proteasome inhibition does not block forskolin-stimulated transcription. Forskolin also protects liganded ERα from degradation. In the current study, we first examined ERα and ERβ transcriptional activity in ER-negative HT22 cells and found that forskolin stimulated ERα-, but not ERβ-dependent transcription, through the ligand-binding domain (LBD). We also identified four mutations (L396R, D431Y, Y542F, and K534E/M548V) on the ERα LBD that selectively obliterated the response to forskolin. In GH3 cells, transfected ERα mutants and ERβ were protected from degradation by forskolin. Ubiquitination of ERα and ERβ was increased by forskolin or estradiol. ERα ubiquitination was diminished by a mutated ubiquitin (K48R) that prevents elongation of polyubiquitin chains for targeting the proteasome. Increased ERα ubiquitination was not affected by the deletion of the A/B domain but significantly diminished in the F domain deletion mutant. Our results indicate distinct and novel mechanisms for forskolin stimulation of ERα transcriptional activity and protection from ligand-induced degradation. It also suggests a unique mechanism by which forskolin increases unliganded and liganded ERα and ERβ ubiquitination but uncouples them from proteasome-mediated degradation regardless of their transcriptional responses to forskolin.

ACT001 reverses resistance of prolactinomas via AMPK-mediated EGR1 and mTOR pathways.

Dopamine agonist (DA) is the first choice for the treatment of prolactinomas, and drug resistance is unavoidable during treatment due to the heterogeneity of tumors. The two prolactinoma cell lines (GH3 cells and MMQ cells) were found to have different sensitivity and responding modes to the cabergoline (CAB) and bromocriptine (BRC). In this research, we disclosed the capability of ACT001, a derivative of parthenolide analogs, to activate AMPK by increasing the intracellular reactive oxygen species (ROS) level and AMP/ATP ratio to reverse DA resistance through dual pathways in prolactinoma cells. The results indicated that ACT001 could reverse the CAB resistance in GH3 cells by inhibiting the mTOR signaling pathway, inducing cell death through autophagy, and reverse the BRC resistance in MMQ cells by activating the EGR1 signaling pathway, inducing cell death through apoptosis. Our results suggested that ACT001 is a promising therapeutic compound for treating DA-resistant prolactinomas.

Over-expression of miR-124 impairs proliferation, invasion and epithelial-mesenchymal transition in pituitary prolactinoma by targeting PHD finger protein 19


 
 
 
 Purpose: To determine the effect of miR-124 in pituitary prolactinoma.
 Methods: The viability and proliferation of prolactinoma cells were investigated using Cell Counting Kit-8 and 5-ethynyl-2′-deoxyuridine staining assays. Cell migration and invasion were investigated using the transwell assay. The epithelial-mesenchymal transition was investigated using western blotting. The target gene of miR-124 was verified by the luciferase activity assay.
 Results: The viability and proliferation of prolactinoma cells were repressed by miR-124 over-expression (p < 0.01). Forced miR-124 expression suppressed prolactinoma cell migration and invasion (p < 0.01). E-cadherin expression was enhanced, while N-cadherin and vimentin were reduced, by miR- 124 over-expression (p < 0.01). PHF19 (plant homeodomain-like finger protein 19) contains an miR-124 binding site, and PHF19 over-expression enhanced cell proliferation, promoted cell migration and invasion, reduced E-cadherin expression and enhanced N-cadherin and vimentin expression in prolactinoma cells. Additionally, miR-124 mimic-induced suppression of prolactinoma cell growth and metastasis was attenuated by forced PHF19 expression.
 Conclusion: MiR-124 retards prolactinoma cell growth and metastasis by reducing PHF19, providing a promising therapeutic target for prolactinoma.
 
 
 

Curcumin Sensitizes Prolactinoma Cells to Bromocriptine by Activating the ERK/EGR1 and Inhibiting the AKT/GSK-3β Signaling Pathway In Vitro and In Vivo.

Although bromocriptine (BRC) as first-line drug is recommended for treating patients with prolactinoma, a minority of patients with prolactinoma are resistance to BRC. Moreover, our previous study showed the difference in drug sensitivity in BRC-treated rat prolactinoma cells, MMQ cells are more resistant to BRC, and GH3 cells are more sensitive to BRC. Curcumin (Cur) has been shown to inhibit proliferation of prolactinoma cell lines. The aim of this study is to further investigate whether Cur could enhance the growth-inhibitory effect of BRC resistance on prolactinoma cell lines and its possible mechanism. CCK-8 kit was used to test cell growth. Cell cycle analysis and apoptosis were performed by flow cytometry. Electron microscopy was used to test autophagosome. The mRNA expression profiles were analyzed using the Affymetrix Gene-Chip array. Western blot was used to test protein expression. Our data showed that Cur enhanced the growth-inhibitory effect of BRC on GH3 and MMQ cell proliferation. BRC and Cur both induced cell apoptosis, and Cur could significantly increase the apoptosis of BRC on pituitary adenoma cells through the ERK/EGR1 signaling pathway. Moreover, Cur could enhance the autophagic cell death (ACD) of BRC on tumor cells by inhibiting the AKT/GSK-3β signaling pathway. The same results were confirmed invivo study. Taken together, Cur sensitizes rat prolactinoma cells to BRC by activating the ERK/EGR1 and inhibiting the AKT/GSK-3β signaling pathway.

MicroRNA‑29‑3p regulates the β‑catenin pathway by targeting IGF1 to inhibit the proliferation of prolactinoma cells.

The present study aimed to analyze the effects and underlying mechanisms of microRNA (miR)-29-3p on the proliferation and secretory abilities of prolactinoma cells by targeting insulin-like growth factor (IGF)-1/β-catenin. The relationship between miR-29a-3p and the survival of prolactinoma cells was analyzed with the Kaplan-Meier method in reference to The Cancer Genome Atlas. The expression levels of miR-29a-3p and IGF-1 in MMQ and GH3 cells were detected. A dual-luciferase reporter gene assay was performed to verify the combination of miR-29a-3p and IGF-1. Cells were transfected with a miR-29a-3p mimic and/or IGF-1 pcDNA3.1 to analyze the effects on the proliferation, apoptosis and secretion of prolactin (PRL) and growth hormone (GH) of prolactinoma cells. The effects on β-catenin in the cytoplasm and nucleus were investigated by western blot analysis. The results showed that miR-29a-3p expression was low in MMQ and GH3 cells. Overexpression miR-29a-3p inhibited IGF-1 mRNA and protein expression. miR-29a-3p inhibited cell proliferation and PRL and GH expression, and promoted apoptosis by inhibiting IGF-1. Increasing the expression of miR-29a-3p increased β-catenin levels in the cytoplasm, whereas IGF-1 promoted β-catenin activation and entry into the nucleus, and reversed the inhibitory effects of miR-29a-3p on β-catenin. To conclude, miR-29a-3p inhibited the proliferation and secretory abilities of prolactinoma cells by inhibiting nuclear translocation of β-catenin via a molecular mechanism that is inseparable from IGF-1.

MicroRNA-7a2 Regulates Prolactin in Developing Lactotrophs and Prolactinoma Cells.

Prolactin production is controlled by a complex and temporally dynamic network of factors. Despite this tightly coordinated system, pathological hyperprolactinemia is a common endocrine disorder that is often not understood, thereby highlighting the need to expand our molecular understanding of lactotroph cell regulation. MicroRNA-7 (miR-7) is the most highly expressed miRNA family in the pituitary gland and the loss of the miR-7 family member, miR-7a2, is sufficient to reduce prolactin gene expression in mice. Here, we used conditional loss-of-function and gain-of-function mouse models to characterize the function of miR-7a2 in lactotroph cells. We found that pituitary miR-7a2 expression undergoes developmental and sex hormone-dependent regulation. Unexpectedly, the loss of mir-7a2 induces a premature increase in prolactin expression and lactotroph abundance during embryonic development, followed by a gradual loss of prolactin into adulthood. On the other hand, lactotroph development is delayed in mice overexpressing miR-7a2. This regulation of lactotroph function by miR-7a2 involves complementary mechanisms in multiple cell populations. In mouse pituitary and rat prolactinoma cells, miR-7a2 represses its target Raf1, which promotes prolactin gene expression. These findings shed light on the complex regulation of prolactin production and may have implications for the physiological and pathological mechanisms underlying hyperprolactinemia.

Pimozide augments bromocriptine lethality in prolactinoma cells and in a xenograft model via theSTAT5/cyclinD1 and STAT5/Bcl‑xL signaling pathways.

As hyperprolactinemia is observed in patients with bromocriptine-resistant prolactinoma, prolactin (PRL) has been implicated in the development of bromocriptine resistance. Since PRL primarily mediates cell survival and drug resistance via the Janus kinase-2 (JAK2)/signal transducer and activator of transcription 5A (STAT5) signaling pathway, the STAT5 inhibitor, pimozide, may inhibit cell proliferation and reverse bromocriptine resistance in prolactinoma cells. In the present study, compared with bromocriptine or pimozide alone, the combination of pimozide and bromocriptine exerted enhanced reduction in cell growth and proliferation, and increased apoptosis and cell cycle arrest in bromocriptine-resistant prolactinoma cells. A reduction in phospho-STAT5, cyclin D1 and B-cell lymphoma extra-large (Bcl-xL) expression levels were observed in cells treated with the combination of drugs. In addition, pimozide suppressed spheroid formation of human pituitary adenoma stem-like cells, and reduced the protein expression of the cancer stem cell markers, CD133 and nestin. Pimozide did not exert any additional antitumor activity in STAT5-knockdown primary culture cells of human bromocriptine-resistant prolactinomas. Furthermore, Pimozide combined with bromocriptine treatment significantly reduced human prolactinoma xenograft growth. Western blot and immunohistochemical analyses also demonstrated significant inhibition of cell proliferation and stem cell marker proteins in vivo. Collectively, these data indicated that pimozide treatment reduced prolactinoma growth by targeting both proliferating cells and stem cells, at least in part, by inhibiting the STAT5/Bcl-xL and STAT5/cyclin D1 signaling pathways.

Paeoniflorin ameliorates antipsychotic-induced hyperprolactinemia in rats by attenuating impairment of the dopamine D2 receptor and TGF-β1 signaling pathways in the hypothalamus and pituitary

Ethnopharmacological relevancePaeoniflorin, a prominent component in some Chinese formulas for hyperprolactinemia-associated disorders, has been found to inhibit prolactin secretion in prolactinoma cells. AimTo examine the efficacy of paeoniflorin on hyperprolactinemia and the underlying mechanisms of action. Materials and methodsHyperprolactinemia in female rats was generated by administration of olanzapine (5 mg/kg, by a gavage method, once daily, × 13 weeks). The rats were co-treated with paeoniflorin (10 and 50 mg/kg). Prolactin and TGF-β1 concentrations were detected by ELISA. Protein expression was determined by Western blot. The effect in MMQ cells was also examined. ResultsPaeoniflorin inhibited olanzapine-induced increases in plasma prolactin concentration and prolactin protein overexpression in the pituitary and hypothalamus of rats. Further, paeoniflorin restored olanzapine-induced downregulation of pituitary and hypothalamic dopamine D2 receptor (D2R) protein expression. More importantly, paeoniflorin attenuated olanzapine-suppressed protein expression of transforming growth factor (TGF)-β1 and its downstream genes, type II TGF-β receptor, type I TGF-β receptor and phosphorylated SMAD3 in the tissues. However, paeoniflorin did not affect plasma TGF-β1 concentration and hepatic TGF-β1 protein expression. In accord, olanzapine-induced increase in prolactin concentration, upregulation of prolactin protein expression, and downregulation of protein expression of the D2R and TGF-β1 signals in MMQ cells were attenuated. ConclusionsThis study demonstrates that paeoniflorin ameliorates olanzapine-induced hyperprolactinemia in rats by attenuating impairment of the D2R and TGF-β1 signaling pathways in the hypothalamus and pituitary. Our findings may provide evidence to support the use of paeoniflorin-contained Chinese herbs and formulas for hyperprolactinemia and its associated disorders.

Estrogen receptor α/prolactin receptor bilateral crosstalk promotes bromocriptine resistance in prolactinomas.

Prolactinomas are the most common type of functional pituitary adenoma. Although bromocriptine is the preferred first line treatment for prolactinoma, resistance frequently occurs, posing a prominent clinical challenge. Both the prolactin receptor (PRLR) and estrogen receptor α (ERα) serve critical roles in the development and progression of prolactinomas, and whether this interaction between PRLR and ERα contributes to bromocriptine resistance remains to be clarified. In the present study, increased levels of ERα and PRLR protein expression were detected in bromocriptine-resistant prolactinomas and MMQ cells. Prolactin (PRL) and estradiol (E2) were found to exert synergistic effects on prolactinoma cell proliferation. Furthermore, PRL induced the phosphorylation of ERα via the JAK2-PI3K/Akt-MEK/ERK pathway, while estrogen promoted PRLR upregulation via pERα. ERα inhibition abolished E2-induced PRLR upregulation and PRL-induced ERα phosphorylation, and fulvestrant, an ERα inhibitor, restored pituitary adenoma cell sensitivity to bromocriptine by activating JNK-MEK/ERK-p38 MAPK signaling and cyclin D1 downregulation. Collectively, these data suggest that the interaction between the estrogen/ERα and PRL/PRLR pathways may contribute to bromocriptine resistance, and therefore, that combination treatment with fulvestrant and bromocriptine (as opposed to either drug alone) may exert potent antitumor effects on bromocriptine-resistant prolactinomas.

Identification of driver genes and key pathways of prolactinoma predicts the therapeutic effect of genipin.

The purpose of the present study was to identify the potential targets and markers for diagnosis, therapy and prognosis in patients with prolactinoma at the molecular level and to determine the therapeutic effects of genipin in prolactinoma. The gene expression profiles of GSE2175, GSE26966 and GSE36314 were obtained from the Gene Expression Omnibus database. The differentially expressed genes (DEGs) were identified after comparing between gene expression profiles of the prolactinoma tissues and normal tissues. Then, Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis and protein-protein interaction (PPI) network analysis were conducted. In addition, in vitro, scratch assay, colony-forming assay, Cell Counting Kit 8 (CCK8) assay and flow cytometry were performed to verify the functional effects of genipin. An aggregate of 12,695, 3,847 and 5,310 DEGs were identified from GSE2175, GSE26966 and GSE36314, respectively. The results of GO and KEGG analysis showed that the DEGs significant and important for prolactinoma were mostly involved with ‘spindle pole’ and ‘oocyte meiosis’. A total of 20 genes were selected as hub genes with high degrees after PPI network analysis, including mitogen-activated protein kinase 1 (MAPK1), MYC, early growth response 1 (EGR1), Bcl2 and calmodulin 1 (CALM1). CCK8 assay, colony-forming assay and scratch assay were performed to verify the anti-prolactinoma effect of genipin. The results of flow cytometry showed that apoptosis was increased by genipin. MAPK1, MYC, EGR1, Bcl2 and CALM1 were screened as main hub genes. Genipin upregulated the expression level of EGR1 and p21 (downstream mediator of EGR1) and EGR1, inhibited the proliferation and migration of prolactinoma cells. Genipin is a promising drug for treatment of patients with prolactinoma.

FOXP1-induced lncRNA CLRN1-AS1 acts as a tumor suppressor in pituitary prolactinoma by repressing the autophagy via inactivating Wnt/\u03b2-catenin signaling pathway

As the commonest type of functional pituitary tumor, prolactinoma takes up around 40–60% of functional pituitary tumors. Despite dedications attributed to the treatment of prolactinoma, complete cure remains difficult. Hence, it is of significance to bring to light the underlying mechanism of prolactinoma. Long noncoding RNAs (lncRNAs) are a group of transcripts which can regulate various biological processes. In the present study, we explored an lncRNA that was differentially downregulated in prolactinoma samples. LncRNA clarin 1 antisense RNA 1 (CLRN1-AS1) was downregulated in 42 patient samples and inactivated the Wnt/β-catenin signaling pathway. Functionally, CLRN1-AS1 suppressed cell proliferation, promoted apoptosis, and inhibited autophagy. Subcellular fractionation assay revealed that CLRN1-AS1 was located in the cytoplasm of prolactinoma cells. Based on bioinformatics analysis and mechanism experiments, we determined that CLRN1-AS1 acted as a competing endogenous RNA (ceRNA) by sponging miR-217 to upregulate the dickkopf WNT signaling pathway inhibitor 1 (DKK1). Furthermore, Forkhead box P1 (FOXP1) was verified to be a transcription suppressor of CLRN1-AS1. In summary, this study revealed that FOXP1-induced CLRN1-AS1 regulated cellular functions in pituitary prolactinoma by sponging miR-217 to release the DKK1/Wnt/β-catenin signaling pathway.

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

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.

MiR-93-5p targets Smad7 to regulate the transforming growth factor-β1/Smad3 pathway and mediate fibrosis in drug-resistant prolactinoma

Prolactinoma is a common subtype of pituitary tumors. Dopamine receptor agonists are the preferred treatment for prolactinoma; however, with this therapy, drug resistance often occurs. In our previous work, we found that partial resistant prolactinomas showed increased fibrosis and that the transforming growth factor (TGF)-β1/Smad3 signaling pathway mediated fibrosis and was involved in drug resistance. Additionally, the success of surgery is known to be heavily influenced by the consistency of the pituitary adenoma. Therefore, in this study, we aimed to clarify the mechanisms of fibrosis in prolactinoma. Using high-throughput sequencing for analysis of microRNAs, we found that miR-93-5p was significantly upregulated in prolactinoma samples with a high degree of fibrosis compared with that in samples without fibrosis. Furthermore, we found that miR-93-5p was negatively correlated with the relative expression of Smad7 and positively correlated with the relative expression of TGF-β1 in clinical prolactinoma samples. In addition, luciferase reporter assays showed that miR-93-5p could downregulate the Smad7 gene, an important inhibitor of the TGF-β1/Smad3 signaling pathway, and activate TGF-β1/Smad3 signaling-mediated fibrosis in a feed-forward loop. Moreover, miR-93-5p could enhance the drug resistance of prolactinoma cells by regulation of TGF-β1/Smad3-dependent fibrosis. Taken together, our findings demonstrated that miR-93-5p may be a potential therapeutic target for inhibiting fibrosis and reducing drug resistance in prolactinoma cells.

PGAM5-CypD pathway is involved in bromocriptine-induced RIP3/MLKL-dependent necroptosis of prolactinoma cells

Bromocriptine, the most commonly used dopamine (DA) receptor agonists 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 (pMLKL)-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.