Pituitary Adenoma Cell Cultures Research Articles

TB-02 Comprehensive analysis of expandable benign pituitary adenomas without genetic manipulations

Background: Since most pituitary adenomas are benign, there is no model of pituitary adenomas that can be continuously cultured without genetic manipulation. In this study, we analyzed the genetic characteristics of the established cell lines by continuous culture of benign pituitary adenomas without genetic manipulation. Method: The pituitary adenoma was continuously cultured by modifying the conditional reprogramming (CR) method, and the morphology was observed and fluorescent immunostaining (IF) was performed. In addition, genetic characteristics were comprehensively analyzed, differences from the surgical samples were analyzed by enrichment analysis using GSEA, and network analysis was performed using cytoscape based on the protein-protein interaction database. (Results) 1) Of the 14 cases of pituitary adenoma that had undergone primary culture (median MIB-1 labeling index 1.5%), 12 cases were capable of continuous culture for more than 2 months. (6 months or more: 5 cases) 2) After 2 weeks of culturing, the cell morphology changed dramatically. These cultured cells were positive for follicular cell marker s100, ectodermal nervous system markers Nestin, Notch1, and mesodermal marker Vimentin. In these cells, synaptophysin, which was strongly positive in the surgical specimen collected at the same time as the culture, disappeared. 3) Enrichment analysis revealed that gene expression such as epithelial mesenchymal transition(EMT), angiogenesis, IL6 signaling via NFkB, extracellular degradation, integrin cell surface interaction increased in cultured cells, and gene expression such as neurotransmitter including synaptophysin decreased significantly.(Conclusion) 1) The modified CR method allowed continuous culture of pituitary adenoma cells. 2) The cultured cells showed characteristic change of EMT, which was seen in invasive pituitary adenoma.

Pituispheres Contain Genetic Variants Characteristic to Pituitary Adenoma Tumor Tissue.

The most common type of pituitary neoplasms is benign pituitary adenoma (PA). Clinically significant PAs affect around 0.1% of the population. Currently, there is no established human PA cell culture available and when PA tumor cells are cultured they form two distinct types depending on culturing conditions either free-floating aggregates also known as pituispheres or cells adhering to the surface of cell plates and displaying mesenchymal stem-like properties. The aim of this study was to trace the origin of sphere-forming and adherent pituitary cell cultures and characterize the potential use of these surgery derived cell lines as PA model. We carried out a paired-end exome sequencing of patients' tumor and germline DNA using Illumina NextSeq followed by characterization of corresponding PA cell cultures. Variation analysis revealed a low amount of somatic mutations (mean = 5.2, range 3–7) in exomes of PAs. Somatic mutations of the primary surgery material can be detected in the exomes of respective pituispheres, but not in exomes of respective mesenchymal stem-like cells. For the first time, we show that the genome of pituispheres represents genome of PA while mesenchymal stem cells derived from the PA tissue do not contain mutations characteristic to PA in their genome, therefore, most likely representing normal cells of pituitary or surrounding tissues. This finding indicates that pituispheres can be used as a human model of PA cells, but combination of cell culturing techniques and NGS needs to be employed to adjust for disability to propagate spheres in culturing conditions.

Common tools for pituitary adenomas research: cell lines and primary cells

Pituitary tumor is the common primary brain tumor in humans. For further studying the pathogenesis and new therapeutic targets of pituitary adenoma, cell lines and primary cells are necessary tools. Different from primary cells that have short survival time and hormone secretion maintenance time, cell lines would be endowed with immortal characteristics under the help of gene modification. This review is to explore whether these cell lines still have similar pathophysiological changes in pituitary adenoma cells and methods to prolong the lifespan of pituitary adenoma primary cells. In the cell lines summarized in the review, HP75, PDFS, HPA and GX were derived from human pituitary adenomas. It was found that the cell lines commonly used in articles published between January 2014 and July 2019 were GH3, AtT20, MMQ, GH4C1, HP75 and TtT/GF. Besides, it was glad that many methods had been used to prolong the lifespan and maintain characteristics of pituitary adenoma primary cells. The paper reviews most of pituitary adenoma cell lines that have been successfully established since 1968 and the relevant situation of primary culture of pituitary adenoma cells. Obviously, it requires us to make more efforts to obtain human pituitary adenoma cell lines and prolong the lifespan of pituitary adenoma primary cells with maintaining their morphology and ability to secret hormones.

Effects of metformin on cell growth and AMPK activity in pituitary adenoma cell cultures, focusing on the interaction with adenylyl cyclase activating signals.

For a few years we have been investigating AMP-activated protein kinase (AMPK) as a target for drug therapy of GH-secreting pituitary adenomas. Aim of this study was to investigate the direct effects of metformin, which causes AMPK activation in different cell types, on rat pituitary adenoma cell growth and on related cell signalling pathways. Our results suggest that metformin can exert a growth-inhibitory activity in rat pituitary tumor cells mediated by AMPK activation, although multiple mechanisms are most likely involved. Membrane proteins, including growth factor receptors, are valuable targets of AMPK. The inhibition of the mTOR-p70S6 kinase signalling pathway plays a role in the suppressive effect of metformin on pituitary tumor cell growth. Metformin did not affect the MTT reduction activity in energetic stress conditions. Finally, metformin was still able to induce AMPK activation and to inhibit cell growth in cells treated with forskolin and in transfected cells overexpressing GHRH-receptor and treated with GHRH. Hence, adenylyl cyclase over-activation does not account for the lack of response of some human pituitary tumors to AMPK-activating compounds invitro.

Lactate dehydrogenase A promotes the invasion and proliferation of pituitary adenoma

Lactate dehydrogenase A (LDHA) has been reported to be involved in the initiation and progression of tumors. However, the potential role of LDHA in pituitary adenoma (PA) remains unknown. In this study, we showed that the expression levels of LDHA mRNA and protein were significantly elevated in invasive PA samples, and positively correlated with higher Ki-67 index. Overexpression of LDHA in a PA cell line (GH3) promoted glucose uptake through the upregulation of glucose transporter-1 (Glut1), lactate secretion and induced cellular invasion by upregulation of matrix metalloproteinase2 (MMP2). LDHA also promoted GH3 cell proliferation through induction of cell cycle progression via activation of the Akt-GSK-3β-cyclinD1 pathway. Accordingly, oxamate-induced inhibition of LDHA suppressed glucose uptake, lactate secretion, invasion and proliferation in GH3 cells via down regulation of Glut1 and MMP2 expression and inhibition of the Akt-GSK-3β-cyclinD1 pathway. Moreover, oxamate induced GH3 cell apoptosis by increasing mitochondrial reactive oxygen species (ROS) generation. In vivo, LDHA overexpression promoted tumor growth, and oxamate delayed tumor growth. In primary PA cell cultures, oxamate also effectively suppressed invasion and proliferation. Our data indicate that LDHA is involved in promoting the progression of PA, and oxamate might be a promising therapeutic agent for the treatment of PA.

Efficacy of 5-aminolevulinic acid based photodynamic therapy in pituitary adenomas—experimental study on rat and human cell cultures

BackgroundIncomplete resection of pituitary adenomas may result in recurrence. As adjuvant irradiation is not riskless, alternative treatment options should be investigated. 5-aminolevulinic acid based photodynamic therapy (5-ALA based PDT) showed promising results for malignant gliomas. The present study examined the efficacy of 5-ALA PDT in vitro on benign pituitary adenoma cell cultures. MethodsIn group I experiments were performed on immortalized rat pituitary adenoma cells (GH3). The cultured cells were treated with different 5-ALA concentrations ranging from 7.5–16.5μg/ml. In Group II human pituitary adenoma cell cultures were obtained from surgically resected adenoma tissue (n=15). These were incubated with 5-ALA concentrations from 12.5–100μg/ml. The concentration ranges had been determined in preliminary dose-finding tests. For both groups incubation time was four hours and PDT was performed by exposition to laser light (635nm, 625s, 18.75J/cm2). Cell viability was examined by WST-1 assay. ResultsIn both groups PDT showed a 5-ALA concentration-dependent effect on cell death. In group I lower 5-ALA concentrations were necessary to destroy all cells as compared to group II. Moreover, in group II, the different subtypes of human adenomas showed different sensitivities to 5-ALA-based PDT (secreting vs. non-secreting). Especially corticotroph adenomas were highly sensitive to 5-ALA PDT. ConclusionsThe GH3 cell line was an useful in vitro model to optimize different PDT parameters. Human pituitary adenoma cells could also be killed by 5-ALA PDT, however this required higher 5-ALA concentrations. Furthermore, the results suggested different 5-ALA sensitivities between different human adenoma cell types. More experiments are necessary to confirm these preliminary results.

Inflammatory triggers of angiogenic factor production in pituitary tumor cells

In pituitary adenomas, which are known to display decreased vascular density in comparison to normal pituitary tissue, the exact role of angiogenesis in tumor transformation and progression remains unclear. As our previous studies performed on pituitary adenoma cell cultures have shown an increased basal production of the pro-angiogenic and pro-inflammatory cytokine IL-8 compared to normal pituitaries, we have chosen to take a closer look at the possible role of inflammation-modulated angiogenesis in pituitary adenomas.

RSUME is involved in the regulation of angiogenic factor production and growth of human neuroendocrine tumors

RSUME is a small RWD-containing sumoylation enhancing protein that shows maximum expression in pituitary and pancreas. RSUME is up regulated under hypoxic conditions and is critically involved in the production of angiogenic factors in pituitary adenoma cell cultures. We have therefore tested whether also in a neuroendocrine pancreatic cell line (Bon-1 cells) RSUME is involved in tumorigenic processes. RSUME was found to be expressed in Bon-1 cells under basal cell culture conditions and both RSUME mRNA synthesis and protein expression was stimulated under hypoxia-mimicking conditions (CoCl2 treatment). Subsequently HIF-1a expression as well as the release of VEGF-A into the cell culture supernatant was strongly enhanced. Knock-down of RSUME by siRNA strongly impaired CoCl2-induced HIF-1a production and VEGF-A secretion indicating a critical role of RSUME in angiogenic factor production by Bon-1 cells. Moreover, RSUME seems to play a role in Bon-1 cell proliferation as moderate but significant growth suppression was observed in Bon-1 cells in which RSUME was knocked down. These results suggest a role of RSUME in the progression of human neuroendocrine pancreatic tumors, which has to be studied in tissue and primary cell cultures of these tumors.

Inhibitory effect of adenovirus-mediated D2S gene plus bromocriptine therapy on primary cultures of human non-functioning pituitary adenoma cells

To explore the inhibitory effect of non-functioning pituitary adenoma (NFPA) cells after a combined treatment of adenovirus mediated D2S gene and bromocriptine in vitro. Adenovirus containing dopamine 2 receptor short isoform (D2S) gene was used to infect NFPA cells. The transfection of D2S gene into NFPA cells was confirmed by immunofluorescence. And cell apoptosis of infected cells treated by bromocriptine was evaluated with CCK-8 assay in vitro. When D2S gene transfection and bromocriptine was used in combination, the survival rate of NFPA cells significantly decreased (40 ± 5)% versus the control group (97 ± 5)% and the pAd-EGFP transfection combined bromocriptine treatment group (90 ± 9)% (P < 0.05). The combined treatment of adenovirus-mediated D2S gene and bromocriptine can effectively induce the apoptosis of NFPA cells on primary culture and increase the sensitivity of NFPA to dopamine agonist.

Curcumin suppresses HIF1A synthesis and VEGFA release in pituitary adenomas

Curcumin (diferuloylmethane), a polyphenolic compound derived from the spice plant Curcuma longa, displays multiple actions on solid tumours including anti-angiogenic effects. Here we have studied in rodent and human pituitary tumour cells the influence of curcumin on the production of hypoxia inducible factor 1α (HIF1A) and vascular endothelial growth factor A (VEGFA), two key components involved in tumour neovascularisation through angiogenesis. Curcumin dose-dependently inhibited basal VEGFA secretion in corticotroph AtT20 mouse and lactosomatotroph GH3 rat pituitary tumour cells as well as in all human pituitary adenoma cell cultures (n=32) studied. Under hypoxia-mimicking conditions (CoCl(2) treatment) in AtT20 and GH3 cells as well as in all human pituitary adenoma cell cultures (n=8) studied, curcumin strongly suppressed the induction of mRNA synthesis and protein production of HIF1A, the regulated subunit of the hypoxia-induced transcription factor HIF1. Curcumin also blocked hypoxia-induced mRNA synthesis and secretion of VEGFA in GH3 cells and in all human pituitary adenoma cell cultures investigated (n=18). Thus, curcumin may inhibit pituitary adenoma progression not only through previously demonstrated anti-proliferative and pro-apoptotic actions but also by its suppressive effects on pituitary tumour neovascularisation.

RSUME is implicated in HIF-1-induced VEGF-A production in pituitary tumour cells

The recently cloned small RWD-domain containing protein RSUME was shown to increase protein levels of hypoxia-inducible factor-1α (HIF-1α). The latter is the oxygen-regulated subunit of HIF-1, the most important transcription factor of the cellular adaptive processes to hypoxic conditions. It is also a major regulator of vascular endothelial growth factor-A (VEGF-A), which is critically involved in the complex process of tumour neovascularisation. In this study, the expression and role of RSUME in pituitary tumours was studied. We found that RSUME mRNA was up-regulated in pituitary adenomas and significantly correlated with HIF-1α mRNA levels. Hypoxia (1% O(2)) or treatment with hypoxia-mimicking CoCl(2) enhanced RSUME and HIF-1α expression, induced translocation of HIF-1α to the nuclei and stimulated VEGF-A production both in pituitary tumour cell lines and primary human pituitary adenoma cell cultures. When RSUME expression was specifically down-regulated by siRNA, the CoCl(2)-induced increase VEGF-A secretion was strongly reduced which was shown to be a consequence of the RSUME knockdown-associated reduction of HIF-1α synthesis. Thus, RSUME plays an important role in initiating pituitary tumour neovascularisation through regulating HIF-1α levels and subsequent VEGF-A production and may therefore be critically involved in pituitary adenoma progression.

Curcumin acts as anti-tumorigenic and hormone-suppressive agent in murine and human pituitary tumour cells in vitro and in vivo

Curcumin (diferuloylmethane) is the active ingredient of the spice plant Curcuma longa and has been shown to act anti-tumorigenic in different types of tumours. Therefore, we have studied its effect in pituitary tumour cell lines and adenomas. Proliferation of lactosomatotroph GH3 and somatotroph MtT/S rat pituitary cells as well as of corticotroph AtT20 mouse pituitary cells was inhibited by curcumin in monolayer cell culture and in colony formation assay in soft agar. Fluorescence-activated cell sorting (FACS) analysis demonstrated curcumin-induced cell cycle arrest at G2/M. Analysis of cell cycle proteins by immunoblotting showed reduction in cyclin D(1), cyclin-dependent kinase 4 and no change in p27(kip). FACS analysis with Annexin V-FITC/7-aminoactinomycin D staining demonstrated curcumin-induced early apoptosis after 3, 6, 12 and 24 h treatment and nearly no necrosis. Induction of DNA fragmentation, reduction of Bcl-2 and enhancement of cleaved caspase-3 further confirmed induction of apoptosis by curcumin. Growth of GH3 tumours in athymic nude mice was suppressed by curcumin in vivo. In endocrine pituitary tumour cell lines, GH, ACTH and prolactin production were inhibited by curcumin. Studies in 25 human pituitary adenoma cell cultures have confirmed the anti-tumorigenic and hormone-suppressive effects of curcumin. Altogether, the results described in this report suggest this natural compound as a good candidate for therapeutic use on pituitary tumours.

Overexpression of Stromal Cell–Derived Factor 1 and Its Receptor CXCR4 Induces Autocrine/Paracrine Cell Proliferation in Human Pituitary Adenomas

Hypothalamic or locally produced growth factors and cytokines control pituitary development, functioning, and cell division. We evaluated the expression of the chemokine stromal cell-derived factor 1 (SDF1) and its receptor CXCR4 in human pituitary adenomas and normal pituitary tissues and their role in cell proliferation. The expression of SDF1 and CXCR4 in 65 human pituitary adenomas and 4 human normal pituitaries was determined by reverse transcription-PCR, immunohistochemistry, and confocal immunofluorescence. The proliferative effect of SDF1 was evaluated in eight fibroblast-free human pituitary adenoma cell cultures. CXCR4 mRNA was expressed in 92% of growth hormone (GH)-secreting pituitary adenomas (GHoma) and 81% of nonfunctioning pituitary adenomas (NFPA), whereas SDF1 was identified in 63% and 78% of GHomas and NFPAs, respectively. Immunostaining for CXCR4 and SDF1 showed a strong homogenous labeling in all tumoral cells in both GHomas and NFPAs. In normal tissues, CXCR4 and SDF1 were expressed only in a subset of anterior pituitary cells, with a lower expression of SDF1 compared with its cognate receptor. CXCR4 and SDF1 were not confined to a specific cell population in the anterior pituitary but colocalized with discrete subpopulations of GH-, prolactin-, and adrenocorticorticotropic hormone-secreting cells. Conversely, most of the SDF1-containing cells expressed CXCR4. In six of eight pituitary adenoma primary cultures, SDF1 induced a statistically significant increase in DNA synthesis that was prevented by the treatment with the CXCR4 antagonist AMD3100 or somatostatin. CXCR4 and SDF1 are overexpressed in human pituitary adenomas and CXCR4 activation may contribute to pituitary cell proliferation and, possibly, to adenoma development in humans.

Estradiol stimulates vascular endothelial growth factor and interleukin-6 in human lactotroph and lactosomatotroph pituitary adenomas.

Estrogens are considered to be critically involved in lactotroph and lactosomatotroph pituitary tumor development. In addition to direct effects, estradiol-induced tumor formation may involve alterations in growth factor and cytokine production. We have studied whether estradiol stimulates the production of the angiogenic vascular endothelial growth factor and the potential tumor progression factor interleukin-6 in 5 lactotroph (LA) and 5 lactosomatotroph (LSA) human pituitary adenoma cell cultures. All tumors secreted heterogenous basal amounts of VEGF (18.0 +/- 1.4 to 425 +/- 26 pg/ml per 24 h) and IL-6 (18.1 +/- 1.5 to 604 +/- 17 pg/ml per 24 h). Estradiol (100 nM) significantly enhanced VEGF release in all LA and LSA cell cultures (47 to 168 % above basal). IL-6 secretion was stimulated in 3 out of 5 LA and in all LSA cell cultures (31 to 287 % above basal). In cell cultures obtained from tumors from which sufficient cells could be isolated, a dose-dependent effect of estradiol (1 to 100 nM) on VEGF and IL-6 production was observed. Stimulation of IL-6 and/or VEGF secretion by estradiol in the majority of human lactotroph and lactosomatotroph adenoma cell cultures studied, suggests that estrogens may contribute to adenoma expansion through the stimulation of these auto-/paracrine-acting adenoma progression factors.

Is there a functional role of bacterial endotoxin receptor (Tlr4) in pituitary tumor progression?

Members of the Toll receptor (Tlr) family are critically involved in the innate immune response after bacterial infections. Effects of bacterial endotoxins (LPS) of gram-negative bacteria are mediated predominantly by Tlr 4. We have shown that LPS stimulated IL-6 secretion in pituitary folliculostellate (FS) cells via Tlr4 receptors and the p38α MAP kinase/ nuclear factor-κB pathway. However, we detected Tlr4 expression also in corticotroph (AtT20) and gonadotroph (αT3–1) cell lines suggesting that Tlr4 is also expressed in endocrine epithelial pituitary tumour cells. Therefore we looked for Tlr4 expression in 3 normal human pituitaries and in 67 pituitary adenomas. By in situ hybridisation and immunohistochemistry, sporadic Tlr4 mRNA and protein expression was detected in all endocrine cell types of aIl 3 normal pituitaries. A highly variable expression of Tlr4 mRNA and protein was observed in epithelial cells in 30% of hormone-inactive adenomas, in 47% of somatotroph tumors, in 33% of lactotroph neoplasias and in 50% of corticotroph and thyreotroph adenomas. Since most pituitary adenomas produce IL-6, the effect of LPS on intratumoral IL-6 secretion was investigated in primary cell cultures of pituitary adenomas. In 9 IL-6-producing adenomas expressing Tlr4, the IL-6 release was enhanced dramatically by LPS in a dose- and -time-dependent manner. The specific p38α MAP kinase inhibitor SB203580 dose-dependently suppressed the LPS-induced IL-6 secretion indicating that the signal transduction pathway might be similar to FS cells. Moreover, dexamethasone dose-dependently inhibited LPS-induced IL-6 production in all pituitary adenoma cell cultures. Since IL-6 is supposed to represent a growth factor of pituitary adenoma cells, we speculate that bacterial endotoxins could support pituitary tumour progression in a subset of Tlr4-expressing adenomas during infectious or inflammatory processes and that cortisol may act protective under these conditions.

In vitro corticotropin-releasing hormone (CRH) stimulation of adrenocorticotropin release from corticotroph adenoma cells: effect of prolonged exposure to CRH and its interaction with cortisol.

To examine if down-regulation of CRH-induced ACTH release occurs in corticotroph adenoma cells as well as CRH-glucocorticoid interactions in these cells, we established primary cultures of pituitary adenoma cells obtained by transphenoidal surgery from five patients with Cushing's disease. To prevent binding of glucocorticoids by serum proteins, we used a serum-free medium containing insulin, transferrin, selenium, and epidermal growth factor. The latter was found to be essential for both basal and CRH-stimulated ACTH secretion. CRH acutely stimulated, in a dose-dependent manner, ACTH release by all adenomas studied, with an IC50 of 0.5 X 10(-9) mol/L. Prolonged exposure (10 days) to a half-maximal stimulatory concentration of CRH led to continuous stimulation of ACTH secretion. A 4-day incubation with cortisol induced a dose-dependent decrease in both basal and long term CRH-stimulated ACTH release, with no difference in the IC50 (1 X 10(-8) mol/L). These data suggest that long term exposure to CRH does not desensitize corticotroph adenoma cells. Thus, it is unlikely that long-acting analogs of CRH will be useful in the treatment of Cushing's disease. ACTH secretion from corticotroph adenomas is restrained by glucocorticoids; the sensitivity of these cells to the negative effect of glucocorticoids is not modified by long term stimulation with CRH.

Effect of dexamethasone on growth hormone (GH) response to growth hormone releasing hormone in acromegaly.

The effect of dexamethasone on the GH response to GH-releasing hormone (GHRH) was studied in vivo in six patients with acromegaly as well as in vitro in monolayer cultures of GH-secreting pituitary tumor cells obtained from three of these patients. Oral administration of 9 mg/day dexamethasone for 2 days decreased plasma GH responses to iv injection of 100 micrograms GHRH-(1-44 amide) in all six patients. Blood glucose levels were significantly increased, while plasma somatomedin-C levels were significantly decreased by this regimen of dexamethasone treatment. On the other hand, 2-day pretreatment with 50 nM dexamethasone of monolayer cultures of pituitary adenoma cells potentiated GH release basally and/or in response to 100 pM to 1 nM GHRH in vitro. These results indicate that the potentiating action of 2-day treatment of dexamethasone in vitro is overwhelmed in vivo by some extra-pituitary action, probably on the central nervous system, of glucocorticoids in patients with acromegaly.