Ubiquitination of p21 by E3 ligase RNF135 promotes the proliferation of human glioblastoma cells.

Glioblastoma multiforme (GBM) is a highly heterogeneous tumor of the central nervous system with a high mortality rate. The upregulation of RING finger protein 135 (RNF135), an E3 ligase, has been observed in GBM, but the associated mechanisms have not been fully elucidated. The aim of the present study was to identify the substrate of RNF135 and study its functions in GBM. Bioinformatics analyses were performed. In addition, RNF135 was overexpressed or knocked down in human U87 and U251 GBM cells, and the effect on cell proliferation was analyzed using Cell Counting Kit-8 and colony formation assays. Furthermore, the interaction of RNF135 with its potential substrate was analyzed using glutathione S-transferase, yeast two-hybrid, immunoprecipitation (IP), co-IP and immunoblotting assays. Bioinformatics analysis indicated that RNF135 serves as a marker of poor prognosis in GBM. The overexpression of RNF135 was demonstrated to promote the proliferation of GBM cells, while the knockdown of RNF135 inhibited GBM cell growth. In addition, the results of the interaction experiments indicate that RNF135 interacts with p21 and mediates the degradation of p21 by ubiquitination. The major site of RNF135-mediated p21 ubiquitination was identified as K163. Further experiments demonstrated that RNF135 promotes the proliferation of GBM cells mainly via p21. In summary, these findings suggest that RNF135 has potential as a therapeutic target for the treatment of GBM.

[Clinical observation of microsurgical removal of the hemilateral tuberculum sellae meningiomas through contralateral eyebrow arch approach].

The current study aimed to investigate the clinical feasibility of microscopic resection of hemilateral tuberculum sellae meningiomas (TSM) via the contralateral eye brow arch approach. The clinical data of 34 patients with TSM who underwent microsurgery from January 2016 to June 2021 in the Neurosurgery Department of Ruijin Hospital Affiliated to Shanghai Jiaotong University School of Medicine and the First Affiliated Hospital of Henan University were collected and reviewed. The postoperative visual acuity improvement rate was 88.5% (23/26), and the total tumor resection rate was 88.2% (30/34); the postoperative visual acuity improvement in patients with total tumor resection was better than that of patients with partial resection [90.9% (20/22) vs 3/4]. Meanwhile, the postoperative visual acuity improvement in patients with the superior optic nerve and laterl-superior optic nerve was better than that of patients with the lateral optic nerve type (12/14, 8/8 vs 3/4). Supraorbital skin numbness occurred in 3 cases after operation, and the symptoms disappeared during follow-up; 2 cases had mild disturbance of hormone level, and urine output of 2 cases increased after operation, which returned to normal level after symptomatic treatment; 1 case had subcutaneous effusion which was absorbed after treatment. There were no complications such as olfactory disturbance and intracranial infection. During follow-up for 3-60 (33±6) months, recurrence occurred in 2 cases and reoperation was performed. For the hemilateral TSM, according to the preoperative evaluation of the origin of the TSM and the side with visual impairment, the contralateral eyebrow approach is selected to fully expose the tumor base below the optic nerve. It is beneficial to fully resect the tumor under direct vision, and the symptoms of postoperative visual impairment are significantly improved, indicating that the current surgical method can be used in the clinical setting.

ACT001 inhibits pituitary tumor growth by inducing autophagic cell death via MEK4/MAPK pathway.

ACT001, derived from traditional herbal medicine, is a novel compound with effective anticancer activity in clinical trials. However, little is known regarding its role in pituitary adenomas. Here, we demonstrated that ACT001 suppressed cell proliferation and induced cell death of pituitary tumor cells in vitro and in vivo. ACT001 was also effective in suppressing the growth of different subtypes of human pituitary adenomas. The cytotoxic mechanism ACT001 employed was mainly related to autophagic cell death (ACD), indicated by autophagosome formation and LC3-II accumulation. In addition, ACT001-mediated inhibitory effect decreased when either ATG7 was downregulated or cells were cotreated with autophagy inhibitor 3-methyladenine (3-MA). RNA-seq analysis showed that mitogen-activated protein kinase (MAPK) pathway was a putative target of ACT001. Specifically, ACT001 treatment promoted the phosphorylation of JNK and P38 by binding to mitogen-activated protein kinase kinase 4 (MEK4). Our study indicated that ACT001-induced ACD of pituitary tumor cells via activating JNK and P38 phosphorylation by binding with MEK4, and it might be a novel and effective anticancer drug for pituitary adenomas.

Proteomic Analysis of Perihematoma Tissue from Patients with Intracerebral Hemorrhage Using iTRAQ-Based Quantitative Proteomics.

Intracerebral hemorrhage is a complicated disorder with limited proven prognostic and therapeutic targets and elusive mechanisms. With proteomic methods, we aimed to explore the global protein expression profile of perihematomal tissue from ICH patients and identify potential pathophysiological pathways and protein markers. Using iTRAQ-labeling quantitative proteomics technology, four ICH brain sample and four non-ICH brain samples were analyzed. Among the 3740 quantifiable proteins, 884 were dysregulated in the patients compared to those in the controls (p < 0.05). After bioinformatics analysis, the differentially expressed proteins were found to be mostly involved in hemostatic processes, nutrient metabolism signaling pathways, and antioxidation pathways. Moreover, fibronectin 1 was revealed to be at the center of the protein-protein interaction networks. In summary, the potential pathways and brain protein markers that could potentially be used to predict the prognosis of ICH were obtained from the altered proteomic profile of perihematomal tissue. Thus, these data may yield novel insights into the mechanisms of ICH-induced secondary brain injury.

Autophagy and Pituitary Adenoma.

Pituitary adenomas (PAs) are common, benign intracranial tumors that are usually effectively controlled with surgery, pharmacotherapy or radiotherapy. Some PAs against which conventional treatment is ineffective are great clinical challenges at present. Autophagy is a widespread physiological process in cells. Through autophagy, cells can degrade damaged or redundant proteins and organelles and achieve the recycling of intracellular substances to maintain the homeostasis of the intracellular environment. An increasing number of studies have demonstrated the importance of autophagy in tumor therapy. Both radiotherapy and chemotherapy can induce autophagy, which plays different roles in the course of therapy. In recent years, there has been growing interest in the role of autophagy during the treatment of PAs. This chapter reviews the recent progress of research on autophagy in PA and the autophagic mechanisms in the treatment of PA.

Prolactin-Secreting Lung Adenocarcinoma Metastatic to the Pituitary Mimicking a Prolactinoma: A Case Report.

BACKGROUND AND IMPORTANCE: Metastasis to the pituitary gland is uncommon in patients with systemic disseminated cancer. Individual articles have reported cases of pituitary metastasis mimicking a prolactinoma, but no case of a prolactin-secreting tumor metastasizing to the pituitary mimicking a prolactinoma has been reported so far. CLINICAL PRESENTATION: This article reports a 67-yr-old man with a recent onset of headaches, ophthalmoplegia, hypopituitarism, and hyperprolactinemia who was initially diagnosed with prolactinoma and given bromocriptine in the local hospital. Because of vomiting after taking drugs, he was transferred to our hospital for further diagnosis and treatment. Serum prolactin was elevated up to 1022 ng/mL, and pituitary magnetic resonance imaging revealed a 2.9 × 2.8 × 2.3 cm sellar mass with pituitary apoplexy, for which endoscopic transsphenoidal surgery was performed. Postoperative pathology and western blotting disclosed a prolactin-positive metastatic lung adenocarcinoma. Whole exome sequencing revealed a number of gene mutations including KRAS, PIK3CA, ALK, and CTNNB1. The patient died of deterioration of the lung disease 3 mo after the initial diagnosis. CONCLUSION: To the best of our knowledge, this is the first report of a prolactin-secreting tumor metastasizing to the pituitary mimicking a prolactinoma as confirmed by both immunohistochemistry and western blot. Prolactin secretion is rare and elusive, and may associate with specified gene mutations.

MicroRNA-93 mediates cabergoline-resistance by targeting ATG7 in prolactinoma.

To date, the management of dopamine agonist (DA)-resistant prolactinomas remains a major clinical problem. Previously, we determined that miRNA-93 expression increases in DA-resistant prolactinomas; however, the role of miRNA-93 in the DA resistance remains largely unexplored. Hence, this study aimed to investigate the susceptibility of tumor cells to cabergoline (CAB) and the autophagy changes in MMQ and GH3 cells after miRNA-93 overexpression or inhibition. We used bioinformatics to identify the potential target of miRNA-93. Subsequently, we analyzed the correlation between miRNA-93 and autophagy-related 7 (ATG7) using protein expression analysis and luciferase assays. Furthermore, the change in the effect of miRNA-93 was measured after ATG7 overexpression. miRNA-93 expression was elevated in DA-resistant prolactinomas, whereas the expression of its identified target, ATG7, was downregulated. miRNA-93 overexpression suppressed the cytotoxic effect of CAB in MMQ and GH3 cells. In contrast, miRNA-93 downregulation enhanced CAB efficiency and promoted cell autophagy, eventually resulting in apoptosis. These results were further confirmed in vivo xenograft models in nude mice. ATG7 overexpression could reverse the inhibitory effect of miRNA-93 on CAB treatment. Taken together, our results suggest that miRNA-93 mediates CAB resistance via autophagy downregulation by targeting ATG7 and serves as a promising therapeutic target for prolactinoma.

Intractable Hiccups Associated with Chiari Type I Malformation: Case Report and Literature Review.

BACKGROUND: The authors report the case of a 34-year-old man who presented with intractable hiccups. The imaging examination showed that the patient was suffering from syringomyelia associated with Chiari type I malformation. CASE DESCRIPTIONS: The patient underwent posterior fossa decompression combined with bilateral tonsillectomy and duroplasty. The intractable hiccups completely resolved 1 week after operation and had not recurred at 2 months after surgery. Postoperative magnetic resonance imaging showed the atrophy of the tonsils of the cerebellum and disappearance of the cavities of the spinal cord. CONCLUSIONS: Intractable hiccups as the main symptoms of Chiari type I malformation are extremely rare in the clinic. Decompression surgery should be an appropriate method to relieve the symptoms.

A Novel Invasive-Related Biomarker in Three Subtypes of Nonfunctioning Pituitary Adenomas.

OBJECTIVE: To identify biomarkers key to invasiveness of the 3 subtypes of nonfunctioning pituitary adenomas (NFPAs) and provide a guidance for therapeutic decision making and identification of potential adjuvant drugs. METHODS: Fifty NFPA tumor tissues obtained from transsphenoidal surgery were used in the study. Three invasive NFPAs and 4 noninvasive NFPAs were used for gene expression microarray analyses. In addition, there are 5 invasive NFPAs and 4 noninvasive NFPAs used for proteomic analyses. Invasive-related biomarkers were identified by bioinformatics analysis by integrating the transcriptomics and proteomics data sets. All 3 subtypes of NFPAs (null cell adenomas, oncocytomas, and gonadotroph adenomas) were used to validate differentially expressed candidate biomarkers by means of quantitative real-time reverse transcription polymerase chain reaction and Western blot. The level of EZR was downregulated in pituitary adenoma cell line GH3 to investigate the invasive effect of EZR on GH3 cells by using the RNA interference technique. RESULTS: Eight genes involved in the invasion function were found by bioinformatics analysis, and the EZR gene was identified as a novel invasive-related biomarker in the 3 subtypes of NFPAs. The expression level of EZR was found higher in terms of invasiveness than the noninvasive ones of the 3 subtypes of NFPAs. Moreover, the knockdown of EZR inhibited the invasion of GH3 cells in vitro. CONCLUSIONS: EZR is a novel biomarker in terms of invasion among the 3 subtypes of NFPAs, and it is a promising guide for therapeutic decision making as well.

Treatment of Middle Cranial Fossa Arachnoid Cysts: A Systematic Review and Meta-Analysis.

OBJECTIVE: To review the literature and analyze the efficacy and safety of 3 surgical methods (neuroendoscopic fenestration, microsurgical fenestration, and cystoperitoneal shunting) for middle cranial fossa arachnoid cysts (MCFACs). METHODS: We searched MEDLINE, PubMed, and Cochrane Central electronic databases and collected studies of patients with MCFACs treated with 1 of 3 surgical methods. Eligible studies reported the rate of clinical symptoms improvement (RCSI), rate of cyst reduction (RCR), rate of total complications (RTC), rate of short-term complications (RSTC), rate of long-term complications (RLTC), and other parameters. RESULTS: Eighteen studies met the criteria. MCFACs were divided into 3 groups on the basis of surgical method: RCSI in group I (237 patients, neuroendoscopic fenestration) was 90% (95% confidence interval [CI]: 83%-95%); RCR: 76% (95% CI: 67%-84%); RTC: 28% (95% CI: 22%-34%); RSTC: 23% (95% CI: 17%-30%); and RLTC: 6% (95% CI: 3%-11%). RCSI in group II (144 patients, microsurgical fenestration) was 87% (95% CI: 75%-96%); RCR: 87% (95% CI: 70%-97%); RTC: 49% (95% CI: 30%-68%); RSTC: 44% (95% CI: 21%-68%); RLTC: 3% (95% CI: 0%-12%). RCSI in group III (93 patients, cystoperitoneal shunting) was 93% (95% CI: 66%-99%); RCR: 93% (95% CI: 66%-99%); RTC: 20% (95% CI: 5%-42%); RSTC: 10% (95% CI: 0%-31%); RLTC: 15% (95% CI: 9%-23%). RLTC differed significantly between the 3 groups (P = 0.005); RTC and RSTC between group I and group II (P = 0.002). CONCLUSIONS: All 3 surgical methods are effective for MCFACs, but considering safety, neuroendoscopic fenestration may be the best initial procedure.

[Clinical misdiagnosis analysis of valproate encephalopathy].

OBJECTIVE: To analyze the clinical and laboratory characteristics of postoperative patients of valproate encephalopathy (VHE), summarize the diagnostic and treatment experiences, discuss the reason of misdiagnosis and improve the level of early diagnosis. METHODS: A total of 12 VHE patients diagnosed after an application of valproate were recruited from January 2010 to April 2013. The characteristics of clinical manifestations and laboratory examinations were retrospectively analyzed. RESULTS: Among them, the misdiagnoses included intracranial hemorrhage (n = 1), secondary brain edema (n = 1), postoperative cerebral infarction (n = 1), postoperative epileptic deterioration (n = 4), electrolyte disorder (n = 2), intracranial infection (n = 1), vasospasm (n = 1) and non-specific (n = 1). All patients had disturbance of consciousness associated with elevated blood ammonia. The symptoms of VHE were not correlated with the dosage and concentration of valproate. VHE was more likely to occur in patients treated with valproic acid sodium injection or other antiepileptic drugs. The symptoms dramatically improved after a withdrawal of valproate. CONCLUSION: VHE should be considered in postoperative neurosurgical valproate-treated patients with unknown disturbance of consciousness. Timely diagnosis is needed and valproate should be withdrawn to avoid serious consequences.

Evidence of brain tumor stem progenitor-like cells with low proliferative capacity in human benign pituitary adenoma.

Tumor stem cells have been implicated as cancer-initiating cells in malignant brain tumors. However, whether benign brain tumors also contain tumor stem cells are largely unexplored. Here, we investigated whether tumor stem-like cells were present in pituitary adenoma similar to malignant brain tumors. By immunocytochemistry, we found that pituitary adenoma tissues expressed neural stem cell marker. These cells could form neurospheres in vitro, expressed neural stem/progenitor cell markers and generated daughter cells with the capacity to differentiate into three neural lineages. Importantly, compared with non-invasive pituitary adenomas, we found that CD133 expression was significantly increased in invasive pituitary adenomas, suggesting that the proliferative capacity was correlated with the malignance of pituitary adenomas. Finally, invasive pituitary adenomas cells displayed lower proliferative ability than glioblastoma. Our data indicate that a subpopulation of stem/progenitor-like cells are present in pituitary adenomas, and these cells may be responsible for benign tumor initiation and maintenance.

Mouse glioma immunotherapy mediated by A2B5+ GL261 cell lysate-pulsed dendritic cells.

Immunotherapy strategies targeting glioma stem-like cells (GSCs) hold promise for improving outcomes in glioblastoma patients. We used the A2B5 monoclonal antibody to classify GSCs derived from the mouse GL261 glioma cell line, and A2B5+ GL261 cell lysate-pulsed dendritic cells (DCs) were used to treat mouse glioma. We found that such DCs elicited a stronger specific cytotoxic T lymphocyte response against A2B5+ GL261 cells than A2B5- GL261 cell lysate-pulsed DCs. The effect of A2B5+ GL261 cell lysate-pulsed DCs in vivo depended on when the vaccination was started. In the tumor cell adaptation phase, C57BL/6 mice had an immune response against GL261, and vaccination enhanced the immune response and prevented glioma formation in 37.5% of mice. In contrast, after glioma formation, the immune response against GL261 was decreased, and vaccination had no therapeutic effect. Our findings suggest that vaccination with A2B5+ GL261 cell lysate-pulsed DCs only has some glioma preventive effect.

Dopamine receptor D2S gene transfer improves the sensitivity of GH3 rat pituitary adenoma cells to bromocriptine.

Bromocriptine, a dopamine agonist (DA), has been used in the treatment of prolactinomas. Recent studies have indicated that dopamine 2 receptor short isoform (D2S) may play an important role in suppressing PRL synthesis and prolactinoma cell growth under DA treatment. In the current study, we investigated the role of D2S in the therapeutic action of bromocriptine in GH3 using both in vitro and in vivo approaches. Infection of adenovirus-D2S increased D2S expression in GH3 cells (P<0.05). D2S expression significantly decreased the GH3 cell viability subjected to bromocriptine treatment in vitro (P<0.05). In nude mice, adenovirus-D2S transfection sensitized GH3 xenograft to bromocriptine treatment evidenced by the significant inhibition of D2S expressed tumor growth as compared with vector control. Furthermore, decrease of Bcl-2 expression, increase of Bax, and active Caspase-3 were found in D2S expressed GH3 xenograft subjected to bromocriptine treatment. In summary, our study indicates that D2S expression plays a critical role in the therapeutic action of bromocriptine in pituitary adenomas and that adenovirus-mediated D2S gene transfer combined with bromocriptine may provide a novel treatment for DA-resistant prolactinomas.

Notch4 is activated in endothelial and smooth muscle cells in human brain arteriovenous malformations.

Up-regulation of Notch4 was observed in the endothelial cells in the arteriovenous malformations (AVMs) in mice. However, whether Notch4 is also involved in brain AVMs in humans remains unclear. Here, we performed immunohistochemistry on normal brain vascular tissue and surgically resected brain AVMs and found that Notch4 was up-regulated in the subset of abnormal vessels of the brain AVM nidus, compared with control brain vascular tissue. Two-photon confocal images show that Notch4 was expressed not only in the endothelial but also in the smooth muscle cells of the vascular wall in brain AVMs. Western blotting shows that Notch4 was activated in brain AVMs, but not in middle cerebral artery of normal human brain, which was confirmed by immunostaining. Our findings suggest a possible contribution of Notch4 signalling to the development of brain AVMs in human.

Differential effects of nerve growth factor on expression of dopamine 2 receptor subtypes in GH3 rat pituitary tumor cells.

Nerve growth factor (NGF) can increase expression of dopamine 2 receptors (D2R) in GH3 rat pituitary tumor cells (GH3 cells). As D2R exists in long (D2L) and short (D2S) isoforms, effects of NGF on D2R subtypes have not been accurately evaluated. In this study, we compared mRNA levels of D2R subtypes in GH3 cells treated with or without NGF with real-time RT-PCR. In addition, we also evaluated the relationship between GH3 cell growth after bromocriptine treatment and mRNA levels of D2R subtypes. We found that D2R total, D2L, and D2S mRNA in GH3 cells were significantly increased after NGF treatment, compared with the vehicle group. Moreover, NGF increased the ratio of D2S to D2L. GH3 cell survival rate after bromocriptine treatment was negatively correlated with D2R total mRNA, and D2S may be more potent than D2L in inhibiting cell growth. Cell apoptosis rate was highly elevated in GH3 cells treated with NGF and bromocriptine, compared with the control group or the group treated with NGF or bromocriptine alone. Our data provide preliminary evidence that the effect of NGF was more prominent on expression of D2S than D2L, in addition, D2S might have a greater impact suppressing GH3 cells growth than D2L.

[Differential expression analysis of prolactinoma-related microRNAs].

OBJECTIVE: To explore the relationship between the prolactinoma-related microRNAs (miRNA) and the development, growth and hormone secretion of prolactinoma. METHODS: The technique of Solexa sequencing was employed to analyze the differential expressions of prolactinoma and normal anterior pituitary gland samples. And the stem-loop real-time polymerase chain reaction (PCR) was utilized for confirmation. RESULTS: According to the differentially expressed profiles of miRNAs, 4 miRNAs were down-regulated (miR-130a, miR-199b-3p, miR-200b, miR-125b, P < 0.05) and 6 miRNAs up-regulated (miR-342-3p, miR-432, miR-23b, miR-493, miR-493(*), miR-664(*), P < 0.05). The expression levels of miR-493(*) and miR-432 had a significant positive correlation with the serum level of prolactin (r = 0.47, P < 0.05; r = 0.528, P < 0.01) while miR-342-3p a significantly positive correlation with the invasiveness (r = 0.402, P < 0.05). CONCLUSION: miRNAs are differentially expressed between normal anterior pituitary gland and prolactinomas, between invasive and localized prolactinomas and among different hormone secretion levels. It suggests that miRNAs may be involved in the physiological process of development, growth and hormone secretion of prolactinoma.

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

OBJECTIVE: 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. METHODS: 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. RESULTS: 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). CONCLUSION: 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.