GH3 Cells Research Articles

17β-estradiol binding to ERα promotes the progression of prolactinoma through estrogen-response element-induced CaBP-9k up-regulation.

17β-estradiol (E2) is considered to be an important instigator of prolactinoma, and can positively regulate the expression of calbindin-D9k (CaBP-9k) which contains an estrogen responsive element (ERE) via estrogen receptors (ERs). However, the detailed mechanism of E2 in promoting CaBP-9k expression and their roles in prolactinoma progression remain unclear. Here, we aimed to characterize it. The luciferase gene reporter assay with luc-ERE transfection showed that E2 treatment significantly enhanced the transcriptional level of CaBP-9k, whereas CaBP-9k activity was reduced when GH3 and MMQ cells were treated with AZD9496, an antagonist of ERα. E2 treatment increased the protein expressions of CaBP-9k and ERα but not ERβ, whereas this effect was also abolished when cells were treated with AZD9496. Besides, immunoprecipitation (IP) and immunofluorescence assays demonstrated that CaBP-9k could directly interact with ERα not ERβ, and Chromatin IP (ChIP) assay showed that ERα could bind to ERE of the CaBP-9k promoter. Moreover, cell counting kit-8 (CCK-8) and flow cytometry assays showed that E2 treatment significantly enhanced cell viability and inhibited cell apoptosis, but these effects were all abolished when ERα was down-regulated by short hairpin RNA (shRNA) or inhibited by AZD9496, as well as CaBP-9K suppression in both GH3 and MMQ cell lines. Taken together, these findings indicated that E2 stimulation promoted prolactin cell proliferation and inhibited cell apoptosis through ERα-induced CaBP-9k up-regulation, which then accelerated the advanced progression of prolactinoma.

Characterization in Dual Activation by Oxaliplatin, a Platinum-Based Chemotherapeutic Agent of Hyperpolarization-Activated Cation and Electroporation-Induced Currents.

Oxaliplatin (OXAL) is regarded as a platinum-based anti-neoplastic agent. However, its perturbations on membrane ionic currents in neurons and neuroendocrine or endocrine cells are largely unclear, though peripheral neuropathy has been noted during its long-term administration. In this study, we investigated how the presence of OXAL and other related compounds can interact with two types of inward currents; namely, hyperpolarization-activated cation current (Ih) and membrane electroporation-induced current (IMEP). OXAL increased the amplitude or activation rate constant of Ih in a concentration-dependent manner with effective EC50 or KD values of 3.2 or 6.4 μM, respectively, in pituitary GH3 cells. The stimulation by this agent of Ih could be attenuated by subsequent addition of ivabradine, protopine, or dexmedetomidine. Cell exposure to OXAL (3 μM) resulted in an approximately 11 mV rightward shift in Ih activation along the voltage axis with minimal changes in the gating charge of the curve. The exposure to OXAL also effected an elevation in area of the voltage-dependent hysteresis elicited by long-lasting triangular ramp. Additionally, its application resulted in an increase in the amplitude of IMEP elicited by large hyperpolarization in GH3 cells with an EC50 value of 1.3 μM. However, in the continued presence of OXAL, further addition of ivabradine, protopine, or dexmedetomidine always resulted in failure to attenuate the OXAL-induced increase of IMEP amplitude effectively. Averaged current-voltage relation of membrane electroporation-induced current (IMEP) was altered in the presence of OXAL. In pituitary R1220 cells, OXAL-stimulated Ih remained effective. In Rolf B1.T olfactory sensory neurons, this agent was also observed to increase IMEP in a concentration-dependent manner. In light of the findings from this study, OXAL-mediated increases of Ih and IMEP may coincide and then synergistically act to increase the amplitude of inward currents, raising the membrane excitability of electrically excitable cells, if similar in vivo findings occur.

Characterization of Effectiveness in Concerted Ih Inhibition and IK(Ca) Stimulation by Pterostilbene (Trans-3,5-dimethoxy-4'-hydroxystilbene), a Stilbenoid.

Pterostilbene (PTER), a natural dimethylated analog of resveratrol, has been demonstrated to produce anti-neoplastic or neuroprotective actions. However, how and whether this compound can entail any perturbations on ionic currents in electrically excitable cells remains unknown. In whole-cell current recordings, addition of PTER decreased the amplitude of macroscopic Ih during long-lasting hyperpolarization in GH3 cells in a concentration-dependent manner, with an effective IC50 value of 0.84 μM. Its presence also shifted the activation curve of Ih along the voltage axis to a more hyperpolarized potential, by 11 mV. PTER at a concentration greater than 10 μM could also suppress l-type Ca2+ and transient outward K+ currents in GH3 cells. With the addition of PTER, IK(Ca) amplitude was increased, with an EC50 value of 2.23 μM. This increase in IK(Ca) amplitude was attenuated by further addition of verruculogen, but not by tolbutamide or TRAM-39. Neither atropine nor nicotine, in the continued presence of PTER, modified the PTER-stimulated IK(Ca). PTER (10 μM) slightly suppressed the amplitude of l-type Ca2+ current and transient outward K+ current. The presence of PTER (3 μM) was also effective at increasing the open-state probability of large-conductance Ca2+-activated K+ (BKCa) channels identified in hippocampal mHippoE-14 neurons; however, its inability to alter single-channel conductance was detected. Our study highlights evidence to show that PTER has the propensity to perturb ionic currents (e.g., Ih and IK(Ca)), thereby influencing the functional activities of neurons, and neuroendocrine or endocrine cells.

低表达miR-210-3p调控大鼠垂体瘤GH3与MMQ细胞株增殖与迁移

低表达miR-210-3p调控大鼠垂体瘤GH3与MMQ细胞株增殖与迁移

Therapeutic Effect of a Novel Chimeric Molecule Targeting Both Somatostatin and Dopamine Receptors on Growth Hormone-Secreting Pituitary Adenomas.

BackgroundAcromegaly is a rare disease primarily caused by growth hormone (GH)-secreting pituitary adenomas, and its treatment is costly. Moreover, some patients are unresponsive to treatment. Hence, there are increasing efforts to develop new drugs with improved effectiveness for this disease. BIM23B065 is a novel chimeric molecule that acts on both somatostatin and dopamine receptors. This study aimed to investigate the effects of BIM23B065 compared with those of a somatostatin receptor analog and a dopamine agonist.MethodsThe effects of BIM23B065 on the proliferation, GH and insulin-like growth factor-1 (IGF-1) levels, and extracellular signal-regulated kinase (ERK) 1/2 and cyclic AMP response element binding (CREB) phosphorylation of GH3 cells were investigated with MTS assay, enzyme-linked immunosorbent assay, and Western blotting, respectively. The dosage and treatment duration of BIM23B065 were tested in animal models of GH-secreting pituitary adenoma. The effect of BIM23B065 (3 mg/kg/day) on changes in IGF-1 levels before and after treatment was further investigated.ResultsIn vitro, BIM23B065 treatment decreased GH release in the culture media and downregulated ERK 1/2 and CREB phosphorylation to 22% and 26%, respectively. In vivo, IGF-1 expression decreased to 50 % after 4 weeks of treatment with BIM23B065 using an osmotic pump implant. Moreover, magnetic resonance imaging results showed that the tumor size decreased significantly following treatment with BIM23B065 for 4 weeks.ConclusionThe novel chimeric molecule was effective in decreasing IGF-1 and GH levels and may serve as an effective therapeutic agent for acromegaly.

Characterization of the Inhibitory Effect of Gastrodigenin and Gastrodin on M-type K+ Currents in Pituitary Cells and Hippocampal Neurons.

Gastrodigenin (HBA) and gastrodin (GAS) are phenolic ingredients found in Gastrodia elata Blume (GEB), a traditional Chinese herbal medicine. These compounds have been previously used to treat cognitive dysfunction, convulsion, and dizziness. However, at present, there is no available information regarding their potential ionic effects in electrically excitable cells. In the current study, the possible effects of HBA and GAS on different ionic currents in pituitary GH3 cells and hippocampal mHippoE-14 neurons were investigated using the patch-clamp technique. The addition of HBA or GAS resulted in the differential inhibition of the M-type K+ current (IK(M)) density in a concentration-dependent manner in GH3 cells. HBA resulted in a slowing of the activation time course of IK(M), while GAS elevated it. HBA also mildly suppressed the density of erg-mediated or the delayed-rectifier K+ current in GH3 cells. Neither GAS nor HBA (10 µM) modified the voltage-gated Na+ current density, although they suppressed the L-type Ca2+ current density at the same concentration. In hippocampal mHippoE-14 neurons, HBA was effective at inhibiting IK(M) density as well as slowing the activation time course. Taken together, the present study provided the first evidence that HBA or GAS could act on cellular mechanisms, and could therefore potentially have a functional influence in various neurologic disorders.

Pituitary tumour fibroblast-derived cytokines influence tumour aggressiveness.

Tumour-associated fibroblasts (TAFs) are key elements of the tumour microenvironment, but their role in pituitary neuroendocrine tumours (PitNETs) has been little explored. We hypothesised that TAF-derived cytokines may play a role in tumour aggressiveness and that their release can be inhibited by somatostatin analogues. TAFs were isolated and cultured from 16 PitNETs (11 clinically non-functioning tumours and 5 somatotropinomas). The fibroblast secretome was assessed with a 42-plex cytokine array before and after multiligand somatostatin receptor agonist pasireotide treatment. Angiogenesis and epithelial-to-mesenchymal transition pathway assessment included CD31, E-cadherin and ZEB1 expression. GH3 cells treated with TAF- or skin fibroblast-conditioned medium were assessed for migration, invasion and cell morphology changes. PitNET TAFs secreted significant amounts of cytokines including CCL2, CCL11, VEGF-A, CCL22, IL-6, FGF-2 and IL-8. TAFs from PitNETs with cavernous sinus invasion secreted higher IL-6 levels compared to fibroblasts from non-invasive tumours (P = 0.027). Higher CCL2 release from TAFs correlated with more capillaries (r = 0.672, P = 0.004), and TAFs from PitNETs with a higher Ki-67 tended to secrete more CCL2 (P = 0.058). SST1 is the predominant somatostatin receptor in TAFs, and pasireotide decreased TAF-derived IL-6 by 80% (P < 0.001) and CCL2 by 35% (P = 0.038). GH3 cells treated with TAF-conditioned medium showed increased migration and invasion compared to cells treated with skin fibroblast-conditioned medium, with morphological and E-cadherin and ZEB1 expression changes suggesting epithelial-to-mesenchymal transition. TAF-derived cytokines may increase PitNET aggressiveness, alter angiogenesis and induce epithelial-to-mesenchymal transition changes. Pasireotide's inhibitory effect on TAF-derived cytokines suggest that this effect may play a role in its anti-tumour effects.

Evidence for Effective Inhibitory Actions on Hyperpolarization-Activated Cation Current Caused by Ganoderma Triterpenoids, the Main Active Constitutents of Ganoderma Spores.

The triterpenoid fraction of Ganoderma (Ganoderma triterpenoids, GTs) has been increasingly demonstrated to provide effective antioxidant, neuroprotective or cardioprotective activities. However, whether GTs is capable of perturbing the transmembrane ionic currents existing in electrically excitable cells is not thoroughly investigated. In this study, an attempt was made to study whether GTs could modify hyperpolarization-activated cation currents (Ih) in pituitary tumor (GH3) cells and in HL-1 atrial cardiomyocytes. In whole-cell current recordings, the addition of GTs produced a dose-dependent reduction in the amplitude of Ih in GH3 cells with an IC50 value of 11.7 µg/mL, in combination with a lengthening in activation time constant of the current. GTs (10 µg/mL) also caused a conceivable shift in the steady-state activation curve of Ih along the voltage axis to a more negative potential by approximately 11 mV. Subsequent addition of neither 8-cyclopentyl-1,3-dipropylxanthine nor 8-(p-sulfophenyl)theophylline, still in the presence of GTs, could attenuate GTs-mediated inhibition of Ih. In current-clamp voltage recordings, GTs diminished the firing frequency of spontaneous action potentials in GH3 cells, and it also decreased the amplitude of sag potential in response to hyperpolarizing current stimuli. In murine HL-1 cardiomyocytes, the GTs addition also suppressed the amplitude of Ih effectively. In DPCPX (1 µM)-treated HL-1 cells, the inhibitory effect of GTs on Ih remained efficacious. Collectively, the inhibition of Ih caused by GTs is independent of its possible binding to adenosine receptors and it might have profound influence in electrical behaviors of different types of electrically excitable cells (e.g., pituitary and heart cells) if similar in vitro or in vivo findings occur.

Soluble Guanylyl Cyclase Alpha1 Subunit: A New Marker for Estrogenicity of Endocrine Disruptor Compounds.

Endocrine-disrupting chemicals (EDCs) include widespread naturally occurring and synthetic substances in the environment that adversely affect humans and wildlife. Because of the increasing numbers of EDCs, screening methods and ideal biomarkers to determine EDC potencies at relevant environmental concentrations need to be drastically improved. Soluble guanylyl cyclase α1 subunit (sGCα1) is an abundant cytosolic protein ubiquitously expressed in most tissues. We previously showed that sGCα1 is specifically and highly up-regulated by estrogen (E2) in vivo and in vitro, even though it lacks estrogen-responsive elements. The aim of the present study was to evaluate sGCα1 protein expression as a potential marker for xenoestrogenic EDC exposure in the E2-responsive lactosomatotroph-derived pituitary cell line GH3. Cells were incubated with a wide variety of EDCs such as heavy metals and a metalloid, synthetic E2 derivatives, plastic byproducts, and pesticides at a range of doses including those with proven xenoestrogenic activity. We demonstrated that E2 increased sGCα1 expression in GH3 cells as well as in other E2-responsive tumor cell lines. Moreover, this effect was fully dependent on estrogen receptor (ER) activation. Importantly, sGCα1 protein levels were strongly up-regulated by all the EDCs tested, even by those exhibiting low or null ER binding capacity. We provide evidence that the in vitro sGCα1 protein assay may be a very sensitive and powerful tool to identify compounds with estrogenic activity, which could improve current mammalian-based screening methods. Environ Toxicol Chem 2019;38:2719-2728. © 2019 SETAC.

Chemokines modulate the tumour microenvironment in pituitary neuroendocrine tumours

Non-tumoural cells within the tumour microenvironment (TME) influence tumour proliferation, invasiveness and angiogenesis. Little is known about TME in pituitary neuroendocrine tumours (PitNETs). We aimed to characterise the role of TME in the aggressive behaviour of PitNETs, focusing on immune cells and cytokines. The cytokine secretome of 16 clinically non-functioning PitNETs (NF-PitNETs) and 8 somatotropinomas was assessed in primary culture using an immunoassay panel with 42 cytokines. This was correlated with macrophage (CD68, HLA-DR, CD163), T-lymphocyte (CD8, CD4, FOXP3), B-lymphocyte (CD20), neutrophil (neutrophil elastase) and endothelial cells (CD31) content, compared to normal pituitaries (NPs, n = 5). In vitro tumour–macrophage interactions were assessed by conditioned medium (CM) of GH3 (pituitary tumour) and RAW264.7 (macrophage) cell lines on morphology, migration/invasion, epithelial-to-mesenchymal transition and cytokine secretion. IL-8, CCL2, CCL3, CCL4, CXCL10, CCL22 and CXCL1 are the main PitNET-derived cytokines. PitNETs with increased macrophage and neutrophil content had higher IL-8, CCL2, CCL3, CCL4 and CXCL1 levels. CD8+ T-lymphocytes were associated to higher CCL2, CCL4 and VEGF-A levels. PitNETs had more macrophages than NPs (p < 0.001), with a 3-fold increased CD163:HLA-DR macrophage ratio. PitNETs contained more CD4+ T-lymphocytes (p = 0.005), but fewer neutrophils (p = 0.047) with a 2-fold decreased CD8:CD4 ratio. NF-PitNETs secreted more cytokines and had 9 times more neutrophils than somatotropinomas (p = 0.002). PitNETs with higher Ki-67 had more FOXP3+ T cells, as well as lower CD68:FOXP3, CD8:CD4 and CD8:FOXP3 ratios. PitNETs with “deleterious immune phenotype” (CD68hiCD4hiFOXP3hiCD20hi) had a Ki-67 ≥ 3%. CD163:HLA-DR macrophage ratio was positively correlated with microvessel density (p = 0.015) and area (p < 0.001). GH3 cell-CM increased macrophage chemotaxis, while macrophage-CM changed morphology, invasion, epithelial-to-mesenchymal transition and secreted cytokines of GH3 cells. PitNETs are characterised by increased CD163:HLA-DR macrophage and reduced CD8:CD4 and CD8:FOXP3 T cell ratios. PitNET-derived chemokines facilitate macrophage, neutrophil and T cell recruitment into the tumours which can determine aggressive behaviour.

Hormonal Regulation of Glucocorticoid Inactivation and Reactivation in αT3-1 and LβT2 Gonadotroph Cells.

The regulation of reproductive function by glucocorticoids occurs at all levels of the hypothalamo-pituitary-gonadal axis. Within the pituitary, glucocorticoids have been shown to directly alter gene expression in gonadotrophs, indicating that these cell types are sensitive to regulation by the glucocorticoid receptor. Whilst the major glucocorticoid metabolising enzymes, 11β-hydroxysteroid dehydrogenase (11βHSD; HSD11B1 and HSD11B2), have been described in human pituitary adenomas, the activity of these enzymes within different pituitary cell types has not been reported. Radiometric conversion assays were performed in αT3-1, LβT2 (gonadotrophs), AtT-20 (corticotrophs) and GH3 (somatolactotrophs) anterior pituitary cell lines, using tritiated cortisol, corticosterone, cortisone or 11-dehydrocorticosterone as substrates. The net oxidation of cortisol/corticosterone and net reduction of cortisone/11-dehydrocorticosterone were significantly higher in the two gonadotroph cells lines compared with the AtT-20 and GH3 cells after 4 h. Whilst these enzyme activities remained the same in αT3-1 and LβT2 cells over a 24 h period, there was a significant increase in glucocorticoid metabolism in both AtT-20 and GH3 cells over this same period, suggesting cell-type specific activity of the 11βHSD enzyme(s). Stimulation of both gonadotroph cell lines with either 100 nM GnRH or PACAP (known physiological regulators of gonadotrophs) resulted in significantly increased 11β-dehydrogenase (11βDH) and 11-ketosteroid reductase (11KSR) activities, over both 4 and 24 h. These data reveal that gonadotroph 11βHSD enzyme activity can act to regulate local glucocorticoid availability to mediate the influence of the HPA axis on gonadotroph function.

Reduction of miR-212 contributes to pituitary adenoma cell invasion via targeting c-Met.

The current study aimed to evaluate the expression and role of miR-212 in the progression of pituitary adenoma (PA), thereby providing a theoretical basis and potential therapy methods for PA patients. Our data showed that miR-212 levels were significantly reduced in PA tissues than normal pituitary tissues. However, no significant difference was identified in the serum of PA patients and healthy control. In addition, the expression of miR-212 in invasive PA was significantly lower than that in noninvasive and normal pituitary tissues. Moreover, the level of miR-212 was decreased with the increase of tumor invasion. Meanwhile, the expression of miR-212 in giant adenomas was significantly lower than that in macroadenomas and microadenomas. Furthermore, inhibition of miR-212 significantly enhanced the proliferation and invasive capacity of GH3 cells. Dual luciferase reporter assay and western blot analysis confirmed that c-Met was a target gene of miR-212. More importantly, upregulation of c-Met significantly prompted PA cell proliferation mainly as a result of the enhanced level of phosphorylation of AKT. This effect could be abolished when c-Met was silenced in GH3 cells. In summary, reduced miR-212 expression in PA contributed to abnormal cancer cell proliferation and invasion mainly by targeting c-Met.

High Effectiveness in Actions of Carfilzomib on Delayed-Rectifier K+ Current and on Spontaneous Action Potentials.

Carfilzomib (CFZ, Kyprolis®) is widely recognized as an irreversible inhibitor of proteasome activity; however, its actions on ion currents in electrically excitable cells are largely unresolved. The possible actions of CFZ on ionic currents and membrane potential in pituitary GH3, A7r5 vascular smooth muscle, and heart-derived H9c2 cells were extensively investigated in this study. The presence of CFZ suppressed the amplitude of delayed-rectifier K+ current (I K(DR)) in a time-, state-, and concentration-dependent manner in pituitary GH3 cells. Based on minimal reaction scheme, the value of dissociation constant for CFZ-induced open-channel block of I K(DR) in these cells was 0.33 µM, which is similar to the IC50 value (0.32 µM) used for its efficacy on inhibition of I K(DR) amplitude. Recovery from I K(DR) block by CFZ (0.3 µM and 1 µM) could be well fitted by single exponential with 447 and 645 ms, respectively. The M-type K+ current, another type of K+ current elicited by low-threshold potential, was slightly suppressed by CFZ (1 µM). Under current-clamp condition, addition of CFZ depolarized GH3 cells, broadened the duration of action potentials as well as raised the firing frequency. In A7r5 vascular smooth muscle cells or H9c2 cardiac cells, the CFZ-induced inhibition of I K(DR) remained efficacious. Therefore, our study led us to reflect that CFZ or other structurally similar compounds should somehow act on the activity of membrane KV channels through which they influence the functional activities in different types of electrically excitable cells such as endocrine, neuroendocrine cells, smooth muscle cells, or heart cells, if similar in vivo findings occur.

CCNB1 affects cavernous sinus invasion in pituitary adenomas through the epithelial\u2013mesenchymal transition

BackgroundTo investigate the relationship between cyclin B1 (CCNB1) gene expression and cavernous sinus invasion in pituitary adenomas.MethodsTwenty-four pituitary adenoma tissue samples were examined by RT-qPCR and Western blot to assess the mRNA expression levels and protein levels of CCNB1, E-cadherin and N-cadherin. Correlation analyses between the expression levels of E-cadherin, N-cadherin and CCNB1 were performed. After lentivirus-mediated knockdown of CCNB1 in rat pituitary adenoma cell lines (GH3 and GT1-1), cell function changes were studied. The relationship between CCNB1 and epithelial-mesenchymal transition (EMT) was further verified by animal experiments.ResultsCCNB1 and N-cadherin gene expression were significantly higher in the invasive pituitary adenomas than in the non-invasive pituitary adenomas. Conversely, E-cadherin expression in the invasive pituitary adenomas was significantly lower. CCNB1 gene expression was downregulated in the GH3 and GT1-1 pituitary adenoma cell lines; N-cadherin expression was also decreased, but E-cadherin expression was increased. These results were confirmed in vivo. After downregulation of CCNB1, cell invasion and migration was significantly reduced in Transwell experiments.ConclusionHigh CCNB1 expression in pituitary adenoma affects cavernous sinus invasion through EMT.

Cytoskeleton Protein Filamin A Is Required for Efficient Somatostatin Receptor Type 2 Internalization and Recycling through Rab5 and Rab4 Sorting Endosomes in Tumor Somatotroph Cells

The high expression of somatostatin receptor 2 (SST₂) in growth hormone (GH)-secreting tumors represents the rationale for the clinical use of somatostatin analogs (SSAs) in acromegaly. Recently, the cytoskeletal protein Filamin A (FLNA) has emerged as key modulator of the responsiveness of GH-secreting pituitary tumors to SSAs by regulating SST₂ signaling and expression. The aim of this study was to explore FLNA involvement in SST₂ intracellular trafficking in tumor somatotroph cells. By biotinylation assay, we found that FLNA silencing abolished octreotide-mediated SST₂ internalization in rat GH3 cell line (28.0 ± 2.7 vs. 4 ± 4.3% SST₂ internalization, control versus FLNA small interfering RNAs (siRNA) cells, respectively, p < 0.001) and human GH-secreting primary cultured cells (70.3 ± 21.1 vs. 24 ± 19.2% SST₂ internalization, control versus FLNA siRNA cells, respectively, p < 0.05). In addition, confocal imaging revealed impaired SST₂ recycling to the plasma membrane in FLNA silenced GH3 cells. Coimmunoprecipitation and immunofluorescence experiments showed that FLNA, as well as β-arrestin2, is timely dependent recruited to octreotide-stimulated SST₂ receptors both in rat and human tumor somatotroph cells. Although FLNA expression knock down did not prevent the formation of β-arrestin2-SST₂ complex in GH3 cells, it significantly impaired efficient SST₂ loading into cytosolic vesicles positive for the early endocytic and recycling markers Rab5 and 4, respectively (33.7 ± 8.9% down to 25.9 ± 6.9%, p < 0.05, and 28.4 ± 7.4% down to 17.6 ± 5.7%, p < 0.01, for SST₂-Rab5 and SST₂-Rab4 colocalization, respectively, in control versus FLNA siRNA cells). Altogether these data support an important role for FLNA in the mediation of octreotide-induced SST₂ trafficking in GH-secreting pituitary tumor cells through Rab5 and 4 sorting endosomes.

Integrated thyroid endocrine disrupting effect on zebrafish (Danio rario) larvae via simultaneously repressing type II iodothyronine deiodinase and activating thyroid receptor-mediated signaling following waterborne exposure to trace azocyclotin

As a widely used organotin acaricide nowadays, azocyclotin (ACT) could induce thyroidal endocrine disruption in fishes and amphibians, but its dominant disrupting mode remains unknown. In this study, zebrafish were firstly exposed to ACT (0.18–0.36 ng/mL) from 2 hpf (hours post fertilization) to 30 dpf (days post fertilization), and a series of developmental toxicological endpoints and thyroid hormones were measured. Result showed that no developmental toxicity to zebrafish was found in 0.18 and 0.24 ng/mL groups except decreased body weight (30 dpf, 0.24 ng/mL). However, exposed to 0.36 ng/mL ACT led to reductions in heartbeat (48 hpf), hatching rate (72 hpf) and bodyweight (30 dpf). General tendencies of decreases in free T3 but increases in free T4 and reductions in ratio of free T3/T4 were also found, inferring that type II deiodinase (Dio2) was repressed. This inference was confirmed by Western analysis that Dio2 expression reduced by 42.7% after 0.36 ng/mL ACT treatment. Moreover, RNA-Seq analysis implied that exposed to 0.36 ng/mL ACT altered the genome-wide gene expression profiles of zebrafish. Totally 5660 genes (involving 3154 down-regulated and 2596 up-regulated genes) were differentially expressed, and 13 deferentially expressed genes including down-regulated dio2 were significantly enriched in thyroid hormone signaling pathway. Subsequently, an in vitro thyroid receptor-reporter gene assay using GH3 cells was performed to further explore the potential disrupting mechanism. Result showed that luciferase activity slightly increased after exposure to ACT alone or ACT combined with low level T3, but was suppressed when combined with high level T3. It indicted there probably existed a competitive relationship in some extent between ACT and T3 in vivo. Overall, the present study provided preliminary evidences that long-term exposure to trace ACT repressed Dio2 expression, declined T3 and then activated thyroid receptor-mediated signaling, thereby leading to integrated thyroid endocrine disruption in zebrafish larvae.

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.

Roles of differential expression of miR-543-5p in GH regulation in rat anterior pituitary cells and GH3 cells.

Growth hormone (GH) is an important hormone released by the pituitary gland that plays a key role in the growth and development of organisms. In our study, TargetScan analysis and the dual luciferase reporter assays were used to predict and screen for miRNAs that might act on the rat Gh1 gene, and we identified miR-543-5p. Then, the GH3 cell line and the primary rat pituitary cells were transfected with miRNA mimic, inhibitor, and siRNA. We detected the Gh1 gene expression and the GH secretion by real-time PCR and ELISAs, respectively, to verify the regulatory effect of miR-543-5p on GH secretion. The results showed that miR-543-5p can inhibit Gh1 mRNA expression and reduce GH secretion. MiR-543-5p inhibitor upregulated Gh1 mRNA expression and increased GH secretion compared with the negative control. In summary, miR-543-5p downregulates Gh1 expression, resulting in a decrease in GH synthesis and secretion, which demonstrates the important role of miRNAs in regulating GH and animal growth and development.

Regulating the CCNB1 gene can affect cell proliferation and apoptosis in pituitary adenomas and activate epithelial-to-mesenchymal transition.

The aim of the present study was to investigate the role and potential regulatory mechanisms of cyclin B1 (CCNB1) in the proliferation, apoptosis and epithelial-to-mesenchymal transition (EMT) in pituitary adenomas. A total of 24 specimens were included in the present study. The expression levels of CCNB1 protein in two normal pituitary and 22 pituitary adenoma tissues were determined by western blotting. CCNB1 was knocked-down by lentiviral-mediated infection of short hairpin RNA (shRNA) in GH3 and MMQ cell lines. The proliferation, cell cycle and apoptosis of GH3 and MMQ cell lines were detected using a Cell Counting Kit-8 and flow cytometer. Reverse transcription-quantitative PCR was utilized to detect the expression level of CCNB1 gene and EMT markers. In the present study, resveratrol (RES) was used as an inhibitor of CCNB1. The protein expression level of CCNB1 in pituitary adenomas was higher than that in normal pituitary tissue, as assessed by western blot analysis. In addition, the expression level of CCNB1 in invasive pituitary adenomas was higher when comparing invasive pituitary adenomas and non-invasive pituitary adenomas. Knockdown of CCNB1 resulted in significant decreases in cell viability and proliferation, arrested cell cycle at the G2/M phase and increased apoptosis. In addition, knockdown of CCNB1 significantly decreased the expression levels of the mesothelial cell marker N-cadherin (P<0.001), but significantly increased the expression levels of the epithelial cell markers E-cadherin (P<0.01) and p120-catenin (P<0.001). Further analyses identified that RES inhibited the expression level of CCNB1, and RES treatment exhibited a similar effect as CCNB1 shRNA infection. The present study suggested that suppressing the expression level of CCNB1 could regulate the proliferation and apoptosis of pituitary tumor cells and alter the expression level of various EMT markers. In addition, RES treatment could be used as an inhibitor of CCNB1. The present study also identified the molecular mechanisms underlying CCNB1 role in EMT.

Ubenimex induces apoptotic and autophagic cell death in rat GH3 and MMQ cells through the ROS/ERK pathway.

PurposeUbenimex, an aminopeptidase N (APN) inhibitor, is widely known for its use as an adjunct therapy for cancer therapy. However, in recent studies, it has also conferred antitumour effects in many cancers, but its anticancer mechanism is largely unknown. This study aims to investigate the specific anticancer activities and mechanisms of ubenimex in GH3 and MMQ cells.Materials and methodsIn this study, we investigated the anticancer effects of ubenimex in GH3 and MMQ cells. Cell viability and cell death were assessed by the Cell Counting Kit-8 kit (CCK-8) and a LIVE/DEAD cell imaging kit. Apoptosis and intracellular reactive oxygen species (ROS) generation were assessed by flow cytometry and fluorescence microscopy. Autophagosome formation was detected by transmission electron microscopy, and autophagic flux was measured with mRFP-GFP-LC3 adenoviral transfection. The protein expression level was detected by Western blotting.ResultsThe results revealed that treatment with ubenimex induced apoptotic and autophagic cell death in GH3 and MMQ cells, which resulted in decreased viability, an increased proportion of apoptotic cells, and autophagosome formation. Further experiments showed that ubenimex induced ROS generation and activated the ROS/ERK pathway. The ROS scavenger NAC could attenuate ubenimex-induced apoptosis and autophagy.ConclusionOur studies revealed that ubenimex exerted anticancer effects by inducing apoptotic and autophagic cell death in GH3 and MMQ cells, rendering it a possible effective adjunctive therapy for pituitary treatment.