Pituitary Adenoma Cells Research Articles

SCP2-mediated cholesterol membrane trafficking promotes the growth of pituitary adenomas via Hedgehog signaling activation

BackgroundMetabolic reprogramming is an important characteristic of tumors. In the progression of pituitary adenomas (PA), abnormal glucose metabolism has been confirmed by us before. However, whether cholesterol metabolism is involved in the process of PA remains unclear. This study aimed to investigate whether abnormal cholesterol metabolism could affect the progression of PA.MethodsWe analyzed the expression of sterol carrier protein 2 (SCP2) in 40 surgical PA samples. In vitro experiments and xenograft models were used to assess the effects of SCP2 and cholesterol on proliferation of PA. The incidence of hypercholesterolemia between 140 PA patients and 100 heathy controls were compared.ResultsWe found an upregulation of SCP2 in PA samples, especially in tumors with high proliferation index. Forced expression of SCP2 promoted PA cell lines proliferation in vitro. Furthermore, SCP2 regulated cholesterol trafficking from cytoplasm to membrane in GH3 cells, and extracellularly treating GH3 cells and primary PA cells with methyl-β-cyclodextrin/cholesterol complex to mimic membrane cholesterol concentration enhanced cell proliferation, which suggested a proliferative effect of cholesterol. Mechanistically, cholesterol induced activation of PKA/SUFU/GLI1 signaling via smoothened receptor, which was well-known as Hedgehog signaling, resulting in inhibiting apoptosis and promoting cell cycle. Accordingly, activation of Hedgehog signaling was also confirmed in primary PA cells and surgical PA samples. In vivo, SCP2 overexpression and high cholesterol diet could promote tumor growth. Intriguingly, the incidence of hypercholesterolemia was significantly higher in PA patients than healthy controls.ConclusionsOur data indicated that dysregulated cholesterol metabolism could promote PA growth by activating Hedgehog signaling, supporting a potential tumorigenic role of cholesterol metabolism in PA progression.

SRC fine-tunes ADAM10 shedding activity to promote pituitary adenoma cell progression.

A disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) is a metalloproteinase known to modulate the progression of several types of tumor. However, the role played by ADAM10 in pituitary adenomas is currently unknown, and what factors orchestrate the activation of ADAM10 in this kind of tumor is also unclear. Here, we found that SRC kinase is an ADAM10-interacting partner and that SRC kinase activity is required for this interaction. As a new positive regulator promoting the shedding activity of ADAM10, SRC could compete with calmodulin 1 (CALM1) for ADAM10 binding in a mutually exclusive manner. Strikingly, the interaction between ADAM10 and CALM1 is regulated by SRC activity. Furthermore, we proved that the cytoplasmic region of ADAM10 is required for the shedding activity of ADAM10 upon SRC activation. As a proof-of-concept, we discovered that the combination of ADAM10 and SRC inhibitors can inhibit cell proliferation and migration to a great extent. Thus, our findings shed light on a novel therapeutic strategy to block the tumorigenesis and migration of pituitary adenoma.

AFAP1-AS1 Promotes Proliferation of Pituitary Adenoma Cells through miR-103a-3p to Activate PI3K/AKT Signaling Pathway

We previously found that AFAP1-AS1 regulates the cell growth of pituitary tumor cells; however, the mechanism still remains unclear. Here, we investigated whether AFAP1-AS1 acts as a competing endogenous RNA of miR-103a-3p to regulate pituitary adenoma growth via the PI3K/AKT pathway. The bind between AFAP1-AS1 and rno-miR-103a-3p was measured by luciferase reporter assay, and rno-miR-103a-3p expression was measured by quantitative reverse transcription polymerase chain reaction. Proliferation, cell cycle, and apoptosis were measured by cell counting kit 8 and flow cytometry. Rat growth hormone (GH) and prolactin (PRL) levels in culture supernatant of GH3 and MMQ cells were measured by enzyme-linked immunosorbent assay. AFAP1-AS1 binds to rno-miR-103a-3p in rat pituitary adenoma cells. Additionally, rno-miR-103a-3p overexpression suppressed rat pituitary adenoma cell proliferation, induced cell apoptosis, arrested cell cycle in the G/S phase, reduced GH and PLR secretion, and inhibited the PI3K/AKT signaling pathway. Activated PI3K/AKT signaling pathway revised the effect of rno-miR-103a-3p overexpression on proliferation and GH and PLR secretion. Coexpression of both si-AFAP1-AS1 and rno-miR-103a-3p inhibitor promoted cell proliferation and cell cycle progression, reduced cell apoptosis, enhanced GH and PLR secretion, and activated thePI3K/AKT signaling pathway in rat pituitary adenoma cells. We found that AFAP1-AS1 and miR-103a-3p could be a potential therapeutic target for pituitary adenoma.

Atiprimod induce apoptosis in pituitary adenoma: Endoplasmic reticulum stress and autophagy pathways.

Pituitary adenoma is the most common tumor with a high recurrence rate due to a hormone-dependent JAK/signal transducer and activator of transcriptions (STAT) signaling. Atiprimod, a novel compound belonging to the azaspirane class of cationic amphiphilic drugs, has antiproliferative, anticarcinogenic effects in multiple myeloma, breast, and hepatocellular carcinoma by blocking STAT3 activation. Therapeutic agents' efficiency depends on endoplasmic reticulum (ER) stress-autophagy regulation during drug-mediated apoptotic cell death decision. However, the molecular machinery of dose-dependent atiprimod treatment regarding ER stress-autophagy has not been investigated yet. Thus, our aim is to investigate the ER stress-autophagy axis in atiprimod-mediated apoptotic cell death in GH-secreting rat cell line (GH3) pituitary adenoma cells. Dose-dependent atiprimod treatment decreased GH3 cell viability, inhibited cell growth, and colony formation. Upregulation of Atg5, Atg12, Beclin-1 expressions, cleavage of LC-3II and formation of autophagy vacuoles were determined only after 1 µM atiprimod exposure.In addition, atiprimod-triggered ER stress was evaluated by BiP, C/EBP-homologous protein (CHOP), p-PERK upregulation, and Ca+2 release after 1 µM atiprimod exposure. Concomitantly, increasing concentration of atiprimod induced caspase-dependent apoptotic cell death via modulating Bcl2 family members. Moreover, by N-acetyl cycteinc pretreatment, atiprimod triggered reactive oxygen species generation and prevented apoptotic induction. Concomitantly, dose-dependent atiprimod treatment decreased both GH and STAT3 expression in GH3 cells. In addition, overexpression of STAT3 increased atiprimod-mediated cell viability loss and apoptotic cell death through suppressing autophagy and ER stress key molecules expression profile. In conclusion, a low dose of atiprimod exposure triggers autophagy and mild-ER stress as a survival mechanism, but increased atiprimod dose induced caspase-dependent apoptotic cell death by targeting STAT3 in GH3 pituitary adenoma cells.

Canine Ependymoma: Diagnostic Criteria and Common Pitfalls.

Reports of canine ependymoma are generally restricted to single case reports with tumor incidence estimated at 2% to 3% of primary central nervous system (CNS) tumors. While most commonly reported in the lateral ventricle, tumors can occur anywhere in the ventricular system and in extraventricular locations. Rosettes and pseudorosettes are a common histologic feature; however, these features can be mimicked by other CNS neoplasms. Thirty-seven potential ependymoma cases were identified in a retrospective database search of 8 institutions, and a histologic review of all cases was conducted. Of 37 cases, 22 candidate cases were further subjected to a consensus histologic and immunohistochemical review, and only 5 of 37 (13.5%) were conclusively identified as ependymoma. The neuroanatomic locations were the lateral ventricle (3/5), third ventricle (1/5), and mesencephalic aqueduct (1/5). Subtypes were papillary (4/5) and tanycytic (1/5). Histologic features included rosettes (5/5), pseudorosettes (5/5), ependymal canals (2/5), tanycytic differentiation (1/5), blepharoplasts (1/5), ciliated cells (1/5), and high nuclear to cytoplasmic ratio (5/5). Immunolabeling for GFAP (4/4) and CKAE1/3 (3/4) was found in pseudorosettes, rosettes, and scattered individual neoplastic cells. Diffuse but variably intense cytoplasmic S100 immunolabeling was detected in 3 of 4 cases. Olig2 intranuclear immunolabeling was observed in less than 1% of the neoplastic cells (3/3). Tumors that had pseudorosettes and mimicked ependymoma included oligodendroglioma, choroid plexus tumor, pituitary corticotroph adenoma, papillary meningioma, and suprasellar germ cell tumor. These findings indicate that canine ependymoma is an extremely rare neoplasm with histomorphologic features that overlap with other primary CNS neoplasms.

DEPTOR inhibits cell proliferation and confers sensitivity to dopamine agonist in pituitary adenoma.

DEP domain-containing mechanistic target of rapamycin (mTOR)-interacting protein (DEPTOR) is an important modulator of mTOR, a highly conserved kinase whose hyperactivation is critically involved in a variety of human tumors. The role of DEPTOR playing in pituitary adenoma (PA) is largely unknown. Here, we reported that DEPTOR was downregulated in PA tissues, especially dopamine-resistant prolactinomas. Consistently, overexpression of DEPTOR inhibited pituitary tumor GH3 and MMQ cells proliferation in vitro and in vivo, and sensitized GH3 and MMQ cells to cabergoline (CAB), a dopamine agonist (DA). Conversely, knockdown of DEPTOR promoted GH3 and MMQ cells proliferation, and conferred cells resistance to CAB. Mechanistically, DEPTOR inhibited both mTOR Complex 1 (mTORC1) and 2 (mTORC2) activities in PA cells. In addition, DEPTOR expression level was increased to suppress mTOR kinase activity via decreasing E3 ubiquitin ligase, βTrCP1, in response to CAB. Furthermore, DEPTOR enhanced autophagy-dependent cell death to confer cells sensitivity to CAB. Taken together, our results suggest that DEPTOR may be a potential target for the treatment of PAs.

SOX2 mediates crosstalk between Sonic Hedgehog and the Wnt/β-catenin signaling pathway to promote proliferation of pituitary adenoma cells.

The Sonic hedgehog (Shh) and Wnt/β-catenin signaling pathways are important regulators of early tissue patterning and stem cell propagation, and aberrant regulation of these two signaling pathways has been associated with a number of types of cancer. The identification of adult stem cells in the pituitary raised the question of whether these two signaling pathways are involved in pituitary adenoma formation. In the present study, it was identified that treating a human pituitary adenoma cell line with Shh was not able to promote the proliferation of cancer cells, but Shh was able to upregulate the expression of sex-determining region Y box 2 (SOX2) which is characteristic of stem cells. The addition of Shh into β-catenin-expressing cells also promoted cell proliferation. On the other hand, addition of Wnt3a into or overexpression of β-catenin in SOX2-expressing cancer cells was able to promote cell proliferation, Further investigation revealed that SOX2 is required to mediate crosstalk between the Shh and Wnt/β-catenin signaling pathways to promote the proliferation of pituitary adenoma cells.

Antitumor effects of growth hormone-releasing hormone (GHRH) antagonists in ACTH and GH-secreting pituitary adenoma cell lines

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Osilodrostat Is a Potential Novel Steroidogenesis Inhibitor for the Treatment of Cushing Syndrome: An In Vitro Study.

Metyrapone and ketoconazole, frequently used steroidogenesis inhibitors for treatment of Cushing syndrome, can be associated with side effects and limited efficacy. Osilodrostat is a CYP11B1 and CYP11B2 inhibitor, with unknown effects on other steroidogenic enzymes. To compare the effects of osilodrostat, metyrapone, and ketoconazole on adrenal steroidogenesis, and pituitary adenoma cells in vitro. HAC15 cells, 17 primary human adrenocortical cell cultures, and pituitary adenoma cells were incubated with osilodrostat, metyrapone, or ketoconazole (0.01 to 10 µM). Cortisol and ACTH were measured using chemiluminescence immunoassays, and steroid profiles by liquid chromatography-mass spectrometry. In HAC15 cells, osilodrostat inhibited cortisol production more potently (IC50: 0.035 µM) than metyrapone (0.068 µM; P < 0.0001), and ketoconazole (0.621 µM; P < 0.0001). IC50 values of osilodrostat and metyrapone for basal cortisol production varied with a 25- and 18-fold difference, respectively, with comparable potency. Aldosterone production was inhibited more potently by osilodrostat vs metyrapone and ketoconazole. Osilodrostat and metyrapone treatment resulted in strong inhibition of corticosterone and cortisol, 11-deoxycortisol accumulation, and modest effects on adrenal androgens. No pituitary-directed effects of osilodrostat were observed. Under our study conditions, osilodrostat is a potent cortisol production inhibitor in human adrenocortical cells, comparable with metyrapone. All steroidogenesis inhibitors showed large variability in sensitivity between primary adrenocortical cultures. Osilodrostat might inhibit CYP11B1 and CYP11B2, in some conditions to a lesser extent CYP17A1 activity, and a proximal step in the steroidogenesis. Osilodrostat is a promising treatment option for Cushing syndrome, and in vivo differences with metyrapone are potentially driven by pharmacokinetic differences.

TSP-1 is downregulated and inversely correlates with miR-449c expression in Cushing's disease.

The pathogenesis of Cushing's disease, which is caused by pituitary corticotroph adenoma, remains to be studied. Secreted angioinhibitory factor thrombospondin‐1 (TSP‐1) is an adhesive glycoprotein that mediates cell‐to‐cell and cell‐to‐matrix interactions and is associated with platelet aggregation, angiogenesis and tumorigenesis. We have found that the expression of TSP‐1 is significantly lower in human pituitary corticotroph tumours compared with normal adenohypophysis. This study aims to elucidate the role of TSP‐1 in regulating the tumour function of pituitary adenomas. Forced overexpression of TSP‐1 in a murine AtT20 pituitary corticotroph tumour cell line decreased corticotroph precursor hormone proopiomelanocortin (POMC) transcription and adrenocorticotropic hormone (ACTH) secretion. Functional studies showed that TSP‐1 overexpression in pituitary adenoma cells suppressed proliferation, migration and invasion. We have demonstrated that TSP‐1 is a direct target of miR‐449c. Further study showed that miR‐449c activity enhanced tumorigenesis by directly inhibiting TSP‐1 expression. Low expression of lncTHBS1, along with low expression of TSP‐1, was associated with the high expression of miR‐449c in Cushing's disease patients. Furthermore, RNA‐immunoprecipitation associates miR‐449c with lncTHBS1 suggesting that lncTHBS1 might be a negative regulator of miR‐449c. Taken together, this study has demonstrated that lncTHBS1 might function as competing endogenous RNA for miR‐449c, which could suppress the development of Cushing's disease.

Benzene affects the response to octreotide treatment of growth hormone secreting pituitary adenoma cells

Growth hormone (GH) secreting pituitary adenomas are the main cause of acromegaly. Somatostatin analogs are the gold standard of medical therapy; however, resistance represents a big drawback in acromegaly management. We recently demonstrated that benzene (BZ) modifies the aggressiveness of GH-secreting rat pituitary adenoma cells (GH3), increasing GH secretion and altering the synthesis of molecules involved in the somatostatin signaling pathway. Based on these pieces of evidence, this study aimed to evaluate the effects of BZ on octreotide (OCT) efficacy in GH-secreting adenoma cells. In GH3 cells, BZ counteracted the anti-proliferative action of OCT. GH gene expression, unmodified by OCT, remained high in BZ-treated cells as well as after treatment with the association of both. GH secretion, reduced by OCT, was increased after treatment with BZ alone or when the pollutant was used with OCT. The combination of BZ and OCT greatly reduced the gene expression of ZAC1 and SSTR2; and this reduction was also present at a protein level. BZ caused an increase in the protein level of the transcription factor STAT3 and in its phosphorylated form. In the presence of BZ, OCT lost the ability to reduce the phosphorylated protein levels. Finally, in primary cultures of human pituitary adenoma cells, BZ caused an increase in GH secretion. OCT decreased GH secretion, but the addition of BZ reversed the OCT effect. In conclusion, our results suggest that BZ may have an important role in the resistance of pituitary adenomas to the pharmacological treatment with somatostatin analogs.

Role and mechanisms of a three-dimensional bioprinted microtissue model in promoting proliferation and invasion of growth-hormone-secreting pituitary adenoma cells

Growth-hormone-secreting pituitary adenoma (GHSPA) is a benign tumour with a high incidence and large economic burden, which greatly affects quality of life. The aetiological factors are yet to be clarified for GHSPA. Conventional two-dimensional (2D) monolayer culture of tumour cells cannot ideally reflect the growth status of tumours in the physiological environment, and insufficiencies of in vitro models have severely restricted the progress of cancer research. Three-dimensional (3D) bioprinting technology is being increasingly used in various fields of biology and medicine, which allows recapitulation of the in vivo growth environment of tumour cells. In this study, a GHSPA microtissue model was established using 3D bioprinting. Tumour cells in the 3D environment exhibited more active cell cycle progression, secretion, proliferation, invasion, and tumourigenesis compared with those in the 2D environment. Furthermore, the molecular mechanisms of the 3D-printed microtissue model were explored. We demonstrated that the 3D-printed microtissue provides an excellent in vitro model at the tissue level for oncological research and may facilitate in-depth studies on the aetiology, treatment, drug resistance, and long-term prognosis of GHSPA .

Lapatinib decreases the ACTH production and proliferation of corticotroph tumor cells.

Cushing's disease is almost always caused by hypersecretion of adrenocorticotropic hormone (ACTH) from a pituitary adenoma. A mutation in the deubiquitinase gene USP8 has been found in human ACTH-producing pituitary adenoma cells. This mutational hotspot hyperactivates USP8, rescuing epidermal growth factor receptor (EGFR) from lysosomal degradation and ensuring its sustained signaling in Cushing's disease. An EGFR inhibitor would be an effective anti-tumor agent in EGFR-related tumors. We investigated the effect of a potent dual tyrosine kinase inhibitor, lapatinib, on ACTH production and cell proliferation in AtT-20 mouse corticotroph tumor cells. Lapatinib decreased proopiomelanocortin (Pomc) mRNA levels and ACTH levels in AtT-20 cells and also inhibited cell proliferation, induced apoptosis, and decreased pituitary tumor-transforming gene 1 (Pttg1), a hallmark of pituitary tumors, mRNA levels. KSN/Slc nude mice were subcutaneously inoculated with AtT-20 cells. After 1 week, the mice were randomized either to control or lapatinib groups. The inhibitor decreased the tumor weight of AtT-20 allografts in vivo versus control mice. Lapatinib also significantly decreased Pomc and Pttg1 mRNA levels in the tumor and plasma ACTH and corticosterone levels in vivo. Thus, lapatinib decreases the ACTH production and proliferation of corticotroph tumor cells. An EGFR-targeting therapy could be an important treatment for Cushing's disease.

MiR-16 regulates proliferation and apoptosis of pituitary adenoma cells by inhibiting HMGA2.

Previous studies have revealed that elevated expression of high mobility group A2 (HMGA2) is closely associated with the occurrence of pituitary adenomas (PAs). The expression of microRNA (miR)-16 is deregulated in PA tissues. Bioinformatics analysis has demonstrated that there is a complementary region between seed region of miR-16 and 3'-untranslated region (3'-UTR) of HMGA2 gene. In the present study, it was investigated whether miR-16 may regulate the expression of HMGA2 and whether it is involved in the pathogenesis of PAs. A total of 52 patients with PAs were recruited. Normal brain tissues obtained from 12 patients with traumatic brain injury were used as controls. The association between miR-16 and HMGA2 was validated using dual-luciferase reporter gene assay. HP75 cells were cultured in vitro and divided into the following groups: miR control, miR-16 mimic, small interfering RNA (si)-negative control, si-HMGA2 and miR-16 mimic+si-HMGA2 groups. The expression of miR-16 and HMGA2 in HP75 cells was determined using qRT-PCR and western blot. Cell proliferation was detected using the Cell Counting Kit-8 assay and apoptosis was detected using the TdT-UTP nick end labeling assay. Compared with normal pituitary tissues, the expression of miR-16 in PA tissues was significantly decreased, while the mRNA and protein levels of HMGA2 were significantly increased. miR-16 targeted the 3'-UTR of HMGA2 gene and regulated the expression of HMGA2. Transfection with siRNAs targeting HMGA2 and/or miR-16 mimics inhibited the expression of HMGA2 and the proliferative ability of HP75 cells, whereas it increased apoptosis of HP75 cells. The downregulation of miR-16 and upregulation of HMGA2 were involved in the pathogenesis of PAs. Thus, it is hypothesized that miR-16 inhibited the proliferation and promoted apoptosis of HP75 cells by inhibiting HMGA2 expression.

Effects of anti-estrogens on cell invasion and survival in pituitary adenoma cells: A systematic study

Although the molecular mechanisms underlying the formation of pituitary adenomas are largely unknown, it is clear that estrogen plays a key role in the pathogenesis of pituitary adenomas. Though this is exemplified by an investigation of fulvestrant in the pituitary adenoma cell line GH3, no systematic studies on the effects of selective estrogen receptor modulators (SERMs) on functional properties of pituitary adenoma cell lines to modulate cell migration, cell invasion, and cell survival are available. Here we analyzed the effects of fulvestrant and three SERMs, bazedoxifene, clomifene, and raloxifene, on pituitary adenomas cell lines AtT20, TtT/GF, and GH3. In cell survival assays, clomifene was shown to be the most potent compound in all three cell lines with IC50 values ranging between 2, 6, and 10 μM, respectively, depending on the cell type. Raloxifene and bazedoxifene were also effective but to a lower extent. Also, all SERMs affected migratory and invasive behavior of pituitary adenoma cells. Mechanistically, treatment of cells with SERMs caused cell apoptosis, as demonstrated by Caspase 3/7 activity and western blot assays. In addition, western blots demonstrate activation of p53 in TtT/GF cells and loss of ERK1/2 activation in AtT20 cells. In contrast, fulvestrant was only effective in GH3 cells. Thus, the general applicability of SERMs for pituitary adenoma cells might be promising in clinical applications for the treatment of pituitary adenomas.

Magmas inhibition as a potential treatment strategy in malignant glioma.

Magmas (mitochondria-associated protein involved in granulocyte-macrophage colony-stimulating factor signal transduction) is a nuclear gene that encodes the mitochondrial import inner membrane translocase subunit Tim16. Magmas is highly conserved, ubiquitously expressed in mammalian cells, and is essential for cell viability. Magmas expression levels are increased in prostate cancers and pituitary adenomas. Moreover, silencing Magmas by RNAi sensitizes pituitary adenoma cells to pro-apoptotic stimuli and induces a G0/G1 accumulation. The aim of this study was to examine whether inhibition of Magmas by small molecule inhibitors could be beneficial for the treatment of malignant gliomas. We evaluated the expression of Magmas in patient-derived glioblastoma tissue samples and xenograft models. We studied the feasibility of a small molecule Magmas inhibitor (BT#9) as a therapeutic agent in stable human glioma cell lines and high-grade patient derived glioma stem-like cells. Magmas was overexpressed in tissue sections from glioma patients and xenografts. In vivo studies revealed that BT#9 could cross the blood-brain barrier in the animal model. Magmas inhibition by BT#9 in glioma cell lines significantly decreased cell proliferation, induced apoptosis along with vacuole formation, and blocked migration and invasion. In addition, BT#9 treatment decreased the respiratory function of glioma cells, supporting the role that Magmas serves as a reactive oxygen species regulator. This is the first study on the role of Magmas in glioma. Our findings suggest that Magmas plays a key role in glioma cell survival and targeting Magmas by small molecule inhibitors may be a therapeutic strategy in gliomas.

Overexpression of microRNA-34a Attenuates Proliferation and Induces Apoptosis in Pituitary Adenoma Cells via SOX7

Pituitary adenomas constitute one of the most common intracranial tumors and are typically benign. However, the role of the tumor suppressor microRNA-34a (miR-34a), which is implicated in other cancers, in pituitary adenoma pathogenesis remains largely unknown. miR-34a expression was compared between GH4C1 cancer cells and normal cells derived from the pituitary gland of Rattus norvegicus, and the effects of miR-34a on GH4C1 cell proliferation and apoptosis were examined. miR-34a target genes were identified and analyzed computationally. The mRNA levels of the miR-34a target genes were measured using qRT-PCR, and the protein levels of the differentially expressed targets were assessed by western blotting. miR-34a expression was significantly lower in GH4C1 cells, whereas miR-34a overexpression significantly inhibited GH4C1 cell proliferation and promoted cell apoptosis though SRY-box 7 (SOX7). Our data facilitate the development of a better understanding of the pathogenesis and treatment of pituitary adenomas by elucidating the crucial role of miR-34a in the development of pituitary adenomas.

Survivin as a potential therapeutic target of acetylsalicylic acid in pituitary adenomas.

Acetylsalicylic acid (ASA) is known as a cancer preventing agent, but there is no data available regarding the effect of ASA on pituitary cells.We investigated 66 nonfunctioning (NFPA) and growth hormone (GH)-producing adenomas and 15 normal pituitary samples. Functional assays (cell viability, proliferation, flow cytometry cell cycle analysis, caspase-3 activation and DNA degradation) were applied to explore the effect of ASA, YM155 (survivin inhibitor), survivin-targeting siRNA and TNF-related apoptosis-inducing ligand (TRAIL) in RC-4B/C and GH3 cells. Pituitary adenoma xenografts were generated in immunocompromised mice.We found that survivin was overexpressed and TRAIL was downregulated in NFPAs compared to normal pituitary tissue. ASA decreased proliferation but did not induce apoptosis in pituitary cells. Additionally, ASA treatment decreased cells in S phase and increased cells in G2/M phase of the cell cycle. Inhibition of survivin using an inhibitor or siRNA-mediated silencing reversed the ASA-induced growth inhibition partially. In addition, we also found survivin-independent effects of ASA on the cell cycle that were mediated through inhibition of cyclin A, cyclin dependent kinase 2 (CDK2) and phospho-CDK2. We also aimed to test the effect of acetylsalicylic acid in an animal model using RC-4 B/C cells, but in contrast to GH3 cells, RC-4 B/C cells failed to adhere and grow a xenograft.We concluded that ASA inhibited the growth of pituitary adenoma cells. Survivin inhibition is a key mechanism explaining its antineoplastic effects. Our results suggest that inhibition of survivin with small molecules or ASA could serve as potential therapeutic agents in NFPA.

Combined effects of resveratrol and radiation in GH3 and TtT/GF pituitary adenoma cells.

Resveratrol and radiation decrease viability in various tumor cells. This study aims to investigate combined effects of resveratrol and radiation on viability, induction of apoptosis and necrosis, and expression of apoptosis modulators in rodent GH3 and TtT/GF pituitary adenoma cells in vitro. Cells were incubated with 10-100µM resveratrol. Medium and medium with ethanol served as controls. After 2h, cells were irradiated with 0-5 Gray (Gy) and further incubated for 48-72h. Cell viability was quantified using a hemocytometer. Cell death was assessed with an enzyme-linked immunosorbent assay (ELISA) that detects free nucleosomes in cell lysates and free nucleosomes released to the culture medium. Expression of B-cell lymphoma-2 protein (BCL-2) and BCL-2 associated Xprotein (BAX) was measured using quantitative real time-polymerase chain reaction (qRT-PCR) to analyze changes in BAX/BCL-2 ratio. Resveratrol and irradiation with 4 Gy alone and in combination significantly decreased cell viability (p = 0.017 and less). In the ELISA, 10 μM resveratrol significantly induced apoptosis in TtT/GF cells at 0 Gy (p < 0.001), but not at 3 or 5 Gy. In the ELISA, 10 μM resveratrol significantly induced necrosis in GH3 cells at 0, 3 and 5 Gy (p < 0.001). While qRT-PCR did not demonstrate a significant effect of 10 µM resveratrol or radiation on expression of BAX or BCL-2, a significant increase in the BAX/BCL-2 ratio was found after irradiation with 5 Gy in GH3 cells (p = 0.0027). While moderate irradiation solely led to inhibited proliferation, resveratrol induced cell death in rodent pituitary adenoma cells.

Grifolic acid induces GH3 adenoma cell death by inhibiting ATP production through a GPR120-independent mechanism

BackgroundGrifolic acid is a derivative of grifolin, an antitumor natural compound, and it was reported as an agonist of free fatty acid receptor GPR120. Little is known about its antitumor effects and the involvement of GPR120.MethodsGH3 cells, the rat anterior pituitary adenoma cells, were cultured and the cell death was measured by MTT assay and Annexin V/PI staining. The mitochondrial membrane potential (MMP) of GH3 cells was measured by JC-1 staining. Cellular ATP levels and the intracellular NAD/NADH ratio were measured. GPR120 expression in GH3 cells was observed by RT-PCR and Western Blot, and siRNA was used to inhibit GPR120 expression in GH3 cells.ResultsGrifolic acid dose- and time-dependently induced the necrosis of GH3 cells. Grifolic acid significantly reduced the mitochondrial membrane potential (MMP) and decreased cellular ATP levels in GH3 cells. In contrast, the MMP of isolated mitochondria was not decreased by grifolic acid. The intracellular NAD/NADH ratio was significantly increased by grifolic acid. GPR120 is expressed in GH3 cells, but GPR120 agonists such as EPA, GW9508 and TUG891 did not affect the viability of GH3 cells. Moreover, GPR120 siRNA knockdown showed no significant influence on grifolic acid-induced GH3 cell death.ConclusionGrifolic acid induces GH3 cell death by decreasing MMP and inhibiting ATP production, which may be due to the inhibition of NADH production through a GPR120-independent mechanism.