Celastrol Induces Apoptosis and Autophagy via the AKT/mTOR Signaling Pathway in the Pituitary ACTH-secreting Adenoma Cells.

OBJECTIVE: Pituitary adrenocorticotropic hormone (ACTH)-secreting adenoma is a relatively intractable endocrine adenoma that can cause a range of severe metabolic disorders and pathological changes involving multiple systems. Previous studies have shown that celastrol has antitumor effects on a variety of tumor cells via the AKT/mTOR signaling. However, whether celastrol has pronounced antitumor effects on pituitary ACTH-secreting adenoma is unclear. This study aimed to identify a new effective therapeutic drug for pituitary ACTH-secreting adenoma. METHODS: Mouse pituitary ACTH-secreting adenoma cells (AtT20 cells) were used as an experimental model in vitro and to establish a xenograft tumor model in mice. Cells and animals were administered doses of celastrol at various levels. The effects of celastrol on cell viability, migration, apoptosis and autophagy were then examined. Finally, the potential involvement of AKT/mTOR signaling in celastrol's mechanism was assessed. RESULTS: Celastrol inhibited the proliferation and migration of pituitary adenoma cells in a time- and concentration-dependent manner. It blocked AtT20 cells in the G0/G1 phase, and induced apoptosis and autophagy by downregulating the AKT/mTOR signaling pathway. Similar results were obtained in mice. CONCLUSION: Celastrol exerts potent antitumor effects on ACTH-secreting adenoma by downregulating the AKT/mTOR signaling in vitro and in vivo.

Clinicopathological Characteristics of Plurihormonal Pituitary Adenoma.

Background: As the pathogenesis of plurihormonal pituitary adenoma (PPA) is unclear and the diagnostic criteria are inconsistent, clinicians still find it challenging to diagnose. To analyze the relationship between clinical and pathological characteristics in PPA. Methods: The clinical data of patients with 70 PPAs admitted during 2008-2010 and 2019-2020 were collected and analyzed. In particular, hormone examination using cell culture supernatant was performed to confirm PPA cases from 2019 to 2020. Results: PPA accounted for 13% of all pituitary cases recorded in the same period. There were 30 men and 40 women. Fifty-three percent of patients had one endocrine manifestation, and 1% presented with two endocrine symptoms. However, none of the patients had three endocrine manifestations. The level of one and two types of hormones was elevated in 52 (74.3%) and 5 (7.1%) patients, respectively and that of three types of hormones was increased only in one patient. Immunohistochemical staining for PRL + TSH or FSH/LH was most commonly performed (n = 17), followed by that for PRL + GH + ACTH and PRL + GH + TSH or FSH/LH (n = 14) and PRL + ACTH (n = 10). The primary culture results in vitro were consistent with the pathological findings in five (41.7%) patients. Moreover, 4 of 12 patients diagnosed with PPA during 2019-2020 tested positive for SOX2. Conclusion: The pathogenesis of PPA remains elusive due to the lack of specific clinical symptoms and endocrine changes. Examination of hormones on tumor culture supernatant is helpful for its diagnosis.

MicroRNAs in Dopamine Agonist-Resistant Prolactinoma.

Dopamine agonists (DAs) are preferred for the treatment of prolactinomas and are usually very effective. Nonetheless, 20-30% of bromocriptine- and approximately 10% of cabergoline-treated individuals exhibit resistance to DAs. In addition, the mechanism underlying this phenomenon remains elusive. In this study, we summarize the major findings regarding the role of microRNAs (miRNAs) in the pathogenesis of DA-resistant prolactinoma (DARP). Currently available evidence suggests that miRNAs are usually dysregulated in DARP and that, although controversial, the dysregulated miRNAs target the transforming growth factor (TGF)-ß, dopamine 2 receptor (D2R), or estradiol (E2)/estrogen receptor (ER) signaling pathways to mediate the therapeutic effect of DAs. These findings provide new incentives for research on innovative strategies for predicting patients' responsiveness to dopamine therapies and for developing treatment approaches. Unfortunately, recent studies tended to focus exclusively on the differential miRNA expression profiles between DARP and dopamine-sensitive prolactinoma, and no definitive consensus has been reached regarding the role of these miRNAs in the modulation mechanism. Therefore, current and future efforts should be directed toward the exploration of the mechanism underlying the dysregulation of miRNAs as well as of the target proteins that are affected by the dysregulated miRNAs. Furthermore, the modulation of the expression of dysregulated miRNAs, which target the D2R, TGF-ß, or E2/ER signaling pathways, might be a promising alternative to treat patients with DARP and improve their prognosis.

Exploiting D2 receptor ß-arrestin2-biased signalling to suppress tumour growth of pituitary adenomas.

BACKGROUND AND PURPOSE: Dopamine agonists targeting D2 receptor have been used for decades in treating pituitary adenomas. There has been little clear evidence implicating the canonical G protein signalling as the mechanism by which D2 receptor suppresses the growth of pituitary tumours. We hypothesize that ß-arrestin2-dependent signalling is the molecular mechanism dictating D2 receptor inhibitory effects on pituitary tumour growth. EXPERIMENTAL APPROACH: The involvement of G protein and ß-arrestin2 in bromocriptine-mediated growth suppression in rat MMQ and GH3 tumour cells was assessed. The anti-growth effect of a ß-arrestin2-biased agonist, UNC9994, was tested in cultured cells, tumour-bearing nude mice and primary cultured human pituitary adenomas. The effect of G protein signalling on tumour growth was also analysed by using a G protein-biased agonist, MLS1547, and a Gßγ inhibitor, gallein, in vitro. KEY RESULTS: ß-arrestin2 signalling but not G protein pathways mediated the suppressive effect of bromocriptine on pituitary tumour growth. UNC9994 inhibited pituitary tumour cell growth in vitro and in vivo. The suppressive function of UNC9994 was obtained by inducing intracellular reactive oxygen species generation through downregulating mitochondrial complex I subunit NDUFA1. The effects of Gαi/o signalling and Gßγ signalling via D2 receptor on pituitary tumour growth were cell-type-dependent. CONCLUSION AND IMPLICATIONS: Given the very low expression of Gαi/o proteins in pituitary tumours and the complexity of the responses of pituitary tumours to G protein signalling pathways, our study reveals D2 receptor ß-arrestin2-biased ligand may be a more promising choice to treat pituitary tumours with improved therapeutic selectivity.

Down-regulation of Aquaporin-1 mediates a microglial phenotype switch affecting glioma growth.

Aquaporin 1 (AQP1), a transmembrane protein that forms water channels, has previously been shown to facilitate growth and progression of many types of tumors by modulating tumor cell migration, proliferation and angiogenesis. Here, we determined the impact of AQP1 expression in the tumor environment on the progression of brain tumors. Primary microglia from wild type(WT) and AQP1 knockout(KO) mice were used to test AQP1 effect on microglia function by using Western blot, quantative PCR, in an experimental in vivo mouse glioma model and organotypic brain slice culture. Deletion of AQP1 in the host tissue significantly reduced the survival of the mice implanted with GL261 glioma cells. The density of glioma-associated microglia/macrophages was almost doubled in AQP1KO mice. We found that factors secreted from GL261 cells decrease microglial AQP1 expression via the MEK/ERK pathway, and that inhibition of this pathway with Trametinib reduced tumor growth and prolonged the survival of tumor bearing mice, an effect which required the presence of microglia. Deletion of AQP1 in cultured microglia resulted in an increase in migratory activity and a decrease in TLR4-dependent innate immune responses. Our study demonstrates a functional relevance of AQP1 expression in microglia and hints to AQP1 as a potential novel target for glioma therapy.

Identification and characterization of microglia/macrophages in the granuloma microenvironment of encephalic schistosomiasis japonicum.

BACKGROUND: Egg-induced immune response and granuloma formation are thought to be the basis of central nervous system (CNS)-related clinical symptoms of Schistosoma japonicum. Microglia/macrophages are the major immune cells involved in detection and subsequent elimination of pathogens and injured tissue in the brain. However, little is known about their role in the pathogenesis of neuroschistosomiasis. The main purpose of the study is to clarify the pathological involvement of microglia/macrophages in the pathogenesis of neuroschistosomiasis (NS). METHODS: Staining techniques were applied to the granuloma tissues excised from 4 patients, as well as mice model which was established by microinjecting viable S. japonicum eggs into the brain. Clinical features of the patients and neurological symptoms in mice were also collected and analyzed in terms of their correlation with microglia/macrophages. RESULTS: Microglia/macrophages constituted the major portions of the granulomas surrounding the eggs in both all human cases and S. japonicum egg-injected mice. Granuloma persisted in all patients accompanied by unremitted neurological symptoms, while in mice granuloma formation initiated on day 3, peaked on day 7 and subsided on day 30 post injection with S. japonicum eggs. No neurological abnormalities were observed in egg-injected mice except for significant weight decrease on day 3 compared with saline-injected control. M1 polarization of microglia/macrophages was confirmed in egg-injected mice 3 days post injection and in all human cases. M2 polarization was absent in human patients despite the duration of complaints but dominated in the whole progression of egg-induced pathology in mice until the elimination of eggs and subsidence of neuroinflammation on day 30 post injection. CONCLUSIONS: Microglia/macrophages participated actively in the granuloma microenvironment of encephalic schistosomiasis japonicum in both human and mice. The polarization pattern of microglia/macrophages coincided with the symptomatic features in human cases and S. japonicum egg-injected mice, indicating M2 instead of M1 activation as a probably more important mediator in the battle against egg-induced pathology and concomitant manifestations. These new findings will shed light on the pathogenesis of NS from a brand-new perspective, and may contribute to the immunotherapy development for such disease, favoring perhaps M2 polarization of microglia/macrophages as a feasible strategy.

Docosahexaenoic acid induces glial cell-line derived neurotrophic factor release in C6 glioma cells: Implications of antidepressant effects for docosahexaenoic acid.

Dietary deficiency of n-3 polyunsaturated fatty acids (PUFAs) is involved in the pathophysiology and etiology of major depressive disorder. Supplementation with docosahexaenoic acid (DHA) exerts antidepressant-like effect; however, the molecular mechanism of DHA action remains unclear. Here we examined the effects of DHA on the modulation of glial cell line-derived neurotrophic factor (GDNF), which is essential for neural development, plasticity, neurogenesis, and survival. We demonstrated that DHA treatment significantly increased GDNF release in a concentration dependent manner in rat C6 glioma cells (C6 cells) and primary cultured rat astrocytes, which is also associated with increased expression of GDNF mRNA. Furthermore, the DHA-induced GDNF production was inhibited by mitogen activated protein kinase (MEK) inhibitor and protein kinase C (PKC) inhibitor, but not protein kinase A (PKA) inhibitor and p38 mitogen-activated protein kinase (MAPK) inhibitor. DHA-induced extracellular signal-regulated kinase (ERK) activation is dependent on the PKC, as demonstrated by its reversibility after pretreatment with PKC inhibitor. Moreover, fibroblast growth factor receptor (FGFR inhibitor) but not epidermal growth factor receptor (EGFR) inhibitor blocked the activation of ERK induced by DHA treatment. DHA-induced GDNF production was also blocked by FGFR inhibitor, suggesting that FGFR is also involved in ERK activation-mediated GDNF production induced by DHA. Our study demonstrates that DHA-induced release of GDNF, mediated by PKC and FGFR-dependent on ERK activation, may contribute to the antidepressant-like effect of DHA.

Docosahexaenoic acid alters Gsα localization in lipid raft and potentiates adenylate cyclase.

OBJECTIVE: Supplementation with docosahexaenoic acid (DHA), an ω-3 polyunsaturated fatty acid (PUFA), recently has become popular for the amelioration of depression; however the molecular mechanism of DHA action remains unclear. The aim of this study was to investigate the mechanism underlying the antidepressant effect of DHA by evaluating Gsα localization in lipid raft and the activity of adenylate cyclase in an in vitro glioma cell model. METHODS: Lipid raft fractions from C6 glioma cells treated chronically with DHA were isolated by sucrose gradient ultracentrifugation. The content of Gsα in lipid raft was analyzed by immunoblotting and colocalization of Gsα with lipid raft was subjected to confocal microscopic analysis. The intracellular cyclic adenosine monophosphate (cAMP) level was determined by cAMP immunoassay kit. RESULTS: DHA decreased the amount of Gsα in lipid raft, whereas whole cell lysate Gsα was not changed. Confocal microscopic analysis demonstrated that colocalization of Gsα with lipid raft was decreased, whereas DHA increased intracellular cAMP accumulation in a dose-dependent manner. Interestingly, we found that DHA increased the lipid raft level, instead of disrupting it. CONCLUSIONS: The results of this study suggest that DHA may exert its antidepressant effect by translocating Gsα from lipid raft and potentiating the activity of adenylate cyclase. Importantly, the reduced Gsα in lipid raft by DHA is independent of disruption of lipid raft. Overall, the study provides partial preclinical evidence supporting a safe and effective therapy using DHA for depression.