Endosome associated trafficking regulator 1 promotes tumor growth and invasion of glioblastoma multiforme via inhibiting TNF signaling pathway.

PURPOSE: Endosome associated trafficking regulator 1 (ENTR1) is a novel endosomal protein, which can affect multiple cellular biological behavior by remodeling plasma membrane structures. However, little is known regarding its function and underlying mechanisms in glioblastoma multiforme. METHODS: Expression profile and clinical signature were obtained from The Public Database of human tumor. Immunohistochemical staining and western blotting assays were used to measure ENTR1 expression level. Human primary GBM tumor cells and human GBM cell lines A172, U87 and U251 were used to clarify the precise role of ENTR1. CCK-8 assays, wound healing and transwell invasion assays were designed to investigate cell viability, invasion and migration of GBM cells, respectively. Underlying molecular mechanisms of ENTR1 were determined via RNA-seq analysis. Tumor formation assay was used to validate the influence of ENTR1 in vivo. RESULTS: Compared with normal brain tissues, ENTR1 was highly expressed in gliomas and correlated with malignant grades of gliomas and poor overall survival time. The proliferation and invasion of GBM cells could be weaken and the sensitivity to temozolomide (TMZ) chemotherapy increased after knocking down ENTR1. Overexpression of ENTR1 could reverse this effect. RNA-seq analysis showed that tumor necrosis factor (TNF) signaling pathway might be a putative regulatory target of ENTR1. Tumor formation assay validated that ENTR1 was a significant factor in tumor growth. CONCLUSION: Our results indicated that ENTR1 played an important role in cell proliferation, invasion and chemotherapeutic sensitivity of GBM, suggesting that ENTR1 might be a novel prognostic marker and significant therapeutic target for GBM.

A pyroptosis gene-based prognostic model for predicting survival in low-grade glioma.

Background: Pyroptosis, a lytic form of programmed cell death initiated by inflammasomes, has been reported to be closely associated with tumor proliferation, invasion and metastasis. However, the roles of pyroptosis genes (PGs) in low-grade glioma (LGG) remain unclear. Methods: We obtained information for 1,681 samples, including the mRNA expression profiles of LGGs and normal brain tissues and the relevant corresponding clinical information from two public datasets, TCGA and GTEx, and identified 45 differentially expressed pyroptosis genes (DEPGs). Among these DEPGs, nine hub pyroptosis genes (HPGs) were identified and used to construct a genetic risk scoring model. A total of 476 patients, selected as the training group, were divided into low-risk and high-risk groups according to the risk score. The area under the curve (AUC) values of the receiver operating characteristic (ROC) curves verified the accuracy of the model, and a nomogram combining the risk score and clinicopathological characteristics was used to predict the overall survival (OS) of LGG patients. In addition, a cohort from the Gene Expression Omnibus (GEO) database was selected as a validation group to verify the stability of the model. qRT-PCR was used to analyze the gene expression levels of nine HPGs in paracancerous and tumor tissues from 10 LGG patients. Results: Survival analysis showed that, compared with patients in the low-risk group, patients in the high-risk group had a poorer prognosis. A risk score model combining PG expression levels with clinical features was considered an independent risk factor. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses indicated that immune-related genes were enriched among the DEPGs and that immune activity was increased in the high-risk group. Conclusion: In summary, we successfully constructed a model to predict the prognosis of LGG patients, which will help to promote individualized treatment and provide potential new targets for immunotherapy.

Brusatol Inhibits Proliferation and Invasion of Glioblastoma by Down-Regulating the Expression of ECM1.

Brusatol (Bru), a Chinese herbal extract, has a variety of anti-tumor effects. However, little is known regarding its role and underlying mechanism in glioblastoma cells. Here, we found that Bru could inhibit the proliferation of glioblastoma cells in vivo and in vitro. Besides, it also had an inhibitory effect on human primary glioblastoma cells. RNA-seq analysis indicated that Bru possibly achieved these effects through inhibiting the expression of extracellular matrix protein 1 (ECM1). Down-regulating the expression of ECM1 via transfecting siRNA could weaken the proliferation and invasion of glioblastoma cells and promote the inhibitory effect of Bru treatment. Lentivirus-mediated overexpression of ECM1 could effectively reverse this weakening effect. Our findings indicated that Bru could inhibit the proliferation and invasion of glioblastoma cells by suppressing the expression of ECM1, and Bru might be a novel effective anticancer drug for glioblastoma cells.

Radiomic features as a risk factor for early postoperative seizure in patients with meningioma.

PURPOSE: This study aim to identify the clinical risk factors of and to develop a radiomics-based predictive model for early postoperative seizure. METHODS: We retrospectively assessed 322 operative patients with meningioma who met the inclusion criteria from January 2014 to December 2016 at The First Affiliated Hospital of Wenzhou Medical University. Univariate and multivariate analyses were performed to determine the predictive value of clinical variables. Magnetic resonance imaging (MRI) was performed to obtain the radiomic score (Rscore) for early postoperative seizure. Radiological features were evaluated using the AK software. The minimal redundancy (mRMR) and least absolute shrinkage and selection operator (LASSO) methods were used to assess for radiomic features, and the Rscore was obtained based on radiomic characteristics using a specific formula. RESULTS: In total, 260 patients who met the inclusion criteria were finally enrolled in this study. Among them, 20 experienced early postoperative seizure. Logistic regression analysis showed that Rscore was associated with a significantly high risk of seizure (p<0.000). Receiver operating characteristic (ROC) curve analysis revealed that the area under the ROC curve of the Rscore was 0.92 (95% confidence interval: 0.853-0.987). The model had a high accuracy for predicting early postoperative seizure. CONCLUSIONS: The Rscore was found to be associated with a high risk of early postoperative seizures. Thus, a higher Rscore (>-1.644) can identify high-risk patients requiring intensive care.

Case Report: Next-Generation Sequencing Reveals Tumor Origin in a Female Patient With Brain Metastases.

BACKGROUND: Brain metastasis mainly originates from lung cancer. Napsin A and TTF-1 factors have frequently been detected in lung adenocarcinoma cases. Brain metastasis tumors with napsin A and TTF-1 positive are easily classified as lung adenocarcinoma origin. However, some thyroid cancers also exhibit these clinical features. Besides, lung is the most common metastasis of undifferential thyroid cancer. Therefore, it requires development of novel diagnostic tools to aid in distinguishing between pulmonary and thyroid origin. PATIENT FINDINGS: We reported a case that was initially diagnosed as brain metastatic lung cancer based on immunohistochemistry results. Analysis of next-generation sequencing (NGS) data from the brain lesion revealed that the cancer may have originated from the thyroid. We detected combo mutations in TERT promoter mutation, RET fusion and TP53, which are common in undifferential thyroid cancer (UTC), but rare for lung cancer. These results, coupled with identification of PAX8, indicated that this patient had UTC. Additionally, her three sons, despite being asymptomatic, were all diagnosed with papillary thyroid carcinoma. SUMMARY: The patient received anlotinib treatment and showed good clinical outcomes. One month after anlotinib treatment, the pulmonary nodules were found to be controlled, and the thyroid tumor drastically reduced, and tracheal compression relieved. She continued anlotinib treatment for the following two months, but died one month later because the treatment stopped owing to financial reasons. All her sons underwent total thyroidectomy with lymph node dissection. CONCLUSIONS: Although NGS has been reported to assist in diagnosis of the origin of some tumors, this is the first evidence of NGS for the determination of the origin of thyroid tumors. To our knowledge, this is the first time that a combination of multiple mutations has been used to help determine the origin of a tumor, compared with the previous single mutant gene. Moreover, this is the first evidence on the use of anlotinib for treatment of UTC with distant metastasis. Besides, all three sons of the patient had thyroid carcinoma in subsequent examinations, indicating high-risk for familial non-medullary thyroid cancer in UTC patients and necessity for performing thyroid ultrasound testing in other family members.

Calpain suppresses cell growth and invasion of glioblastoma multiforme by producing the cleavage of filamin A.

BACKGROUND: Filamin A is the most widely expressed isoform of filamin in mammalian tissues. It can be hydrolyzed by Calpain, producing a 90-kDa carboxyl-terminal fragment (ABP90). Calpeptin is a chemical inhibitor of Calpain, which can inhibit this effect. It has been shown that ABP90 acts as a transcription factor which is involved in mediating cell signaling. However, the significance of ABP90 and its clinical signature with underlying mechanisms have not been well studied in glioblastoma multiforme (GBM). METHODS: ABP90 protein was measured in 36 glioma patients by Western blot. Human GBM cell lines U87 and A172 were used to clarify the precise role of ABP90. CCK-8 assay was used to analyze the cell viability. Transwell invasion assay and wound healing assay were used to analyze the migration and invasion. Expression of matrix metalloproteinase 2/tissue inhibitors of metalloproteinase 2 (MMP2/TIMP2) protein was analyzed by Western blot. RESULTS: ABP90 protein expression was lower in GBM tissues. The patients with low ABP90 protein expression had a shorter OS time (p = 0.046). After being treated with Calpain, the expression of ABP90 was upregulated, which led to a decline of cell viability, enhanced the efficacy of temozolomide and restrained the cell invasion. Calpeptin could inhibit the effect. The mechanism might be involved in the balance of MMP2/TIMP2. CONCLUSIONS: Our present data suggest that ABP90 expression is a significant prognostic factor and may play an important role in cell viability, chemotherapeutic sensitivity and invasion of GBM.

Temozolomide promotes immune escape of GBM cells via upregulating PD-L1.

Glioblastoma multiforme (GBM) is the most common malignant primary brain tumor with poor prognosis, and currently effective therapeutic strategies are still limited. Although temozolomide (TMZ) is commonly used for GBM therapy and its mechanism was well characterized, while its side effects were required comprehensive investigation. In the present study, we revealed that TMZ-challenged GBM cells strongly suppressed pro-inflammatory cytokines expression in activated periphery blood mononuclear cells (PBMC), which depended on enhanced transcription of CD274 (encoding PD-L1), but not other immune checkpoints, such as CD276, HVEM and galectin-9. Moreover, abundance of membranous PD-L1 was also increased in TMZ-treated GBM cells. When PD-L1 expression was knocked down by short hairpin RNA (shRNA), inhibitory effect of TMZ-treated GBM cells on PBMC became weakened, suggesting that PD-L1 was crucial for immune inhibition capacity of TMZ-treated GBM cells. Additionally, actinomycin D reduced PD-L1 expression in GBM cells after TMZ challenge, indicating that PD-L1 induction occurred at transcriptional level. The immunoblotting results demonstrated that STAT3 signaling was involved in TMZ-mediated PD-L1 induction, and attenuated expression of PD-L1 was observed using STAT3 inhibitor VI or STAT3 shRNA. Finally, the animal study showed that combination of TMZ and PD-1 antibody therapy strongly inhibited tumor growth and achieved the improved survival rate of GBM mice. Accordingly, this study revealed the classical chemotherapy drug TMZ promoted GBM cells immune escape, even TMZ combine with PD-1 antibody treatment not further improve survival ratio of recurrent GBM patients compared with traditional therapy methods, while our animal study provided evidence that combination of TMZ and PD-1 antibody was a promising way to treat GBM, these contradictory results indicate improving the PD-1 antibody delivery efficiency can exert strong combinational therapy outcomes.

miR­34a derived from mesenchymal stem cells stimulates senescence in glioma cells by inducing DNA damage.

Insights into the roles of microRNAs (miRNAs/miRs) in development and disease, particularly in cancer, have made miRNAs attractive tools and targets for novel therapeutic approaches in the treatment of glioma. miR­34a, as a well­known tumor suppressor miRNA, is closely related with cellular senescence. Mesenchymal stem cells (MSCs) are a major component of the tumor microenvironment and possess the ability to deliver exogenous miRs to glioma cells to exert anti­tumor effects. The present study investigated whether modified MSCs with miR­34a possess an anti­tumor function in glioma cells. A Transwell system was used to co­culture U87 glioma cells and MSCs overexpressing miR­34a, and cell proliferation and senescence assessed. The expression of senescence­related genes p53, Cdkn1a, and Cdkn2c were tested using reverse transcription­quantitative polymerase chain reaction and protein expression levels of sirtuin 1 (SIRT1) and γ­H2A histone family, member X were detected by western blotting. Telomerase activity of U87 cells was examined using the Telo TAGGG Telomerase PCR ELISA PLUS kit. The results demonstrated that the delivered exogenous miR­34a from MSCs significantly decreased expression of the target gene SIRT1. In addition, the delivered miR­34a decreased the proliferation of glioma cells and provoked the expression of senescence­related genes p53, Cdkn1a, and Cdkn2c. In addition, upregulation of miR­34a induced DNA damage, shortened telomere length and impaired telomerase activity. However, these pro­senescent effects were reversed by forced SIRT1 upregulation. In conclusion, the results demonstrated a novel role for miR­34a, inducing glioma cell senescence, whereas miR­34a modulation of SIRT1, inducing DNA damage, is crucial for miRNA replacement therapy in glioma treatment.

Subdural Hemorrhage from Cerebral Amyloid Angiopathy-Related Intracerebral Hemorrhage: A Risk Factor for Postoperative Hemorrhage.

OBJECTIVE: Surgical treatment for cerebral amyloid angiopathy (CAA)-related intracerebral hemorrhage (ICH) is controversial. A subset of CAA-related ICH with associated subdural hemorrhage (SDH) has been reported. This study aimed to evaluate clinical results and surgical outcomes of this type of ICH with associated SDH. METHODS: Study participants included 98 patients with CAA-related ICH who met Boston criteria. Patients were divided into an SDH group and a control (no SDH) group. Clinical and neuroimaging features and surgical outcomes of the 2 groups were compared. RESULTS: Lobular shape of hematoma was found significantly more often in the SDH group (65.7% [23/35]) compared with the control group (25.4% [16/63]; P < 0.001). Subarachnoid hemorrhage was found significantly more often in the SDH group (34.3% [12/35]) compared with the control group (7.9% [5/63]; P = 0.001). The rate of postoperative hemorrhage was significantly higher in the SDH group (61.5% [8/13]) than in the control group (16.2% [6/37]; P = 0.006). The frequency of occurrence of postoperative hemorrhage was significantly higher in the SDH group (13/13) than in the control group (6/37; P = 0.017). A good surgical outcome occurred in none (0/12) of the patients in the SDH group, whereas a good surgical outcome occurred in 51.9% (14/27) of patients in the control group (P = 0.006). CONCLUSIONS: Patients with CAA-related ICH with associated SDH more frequently have postoperative hemorrhage and have a worse surgical outcome. These findings are useful in choosing therapeutic methods and preoperative planning of surgical strategy.

HSP47 is associated with the prognosis of laryngeal squamous cell carcinoma by inhibiting cell viability and invasion and promoting apoptosis.

Heat shock protein 47 (HSP47) is a 47 kDa collagen binding protein that has a close relationship with the development and progression of tumours. However, little is known concerning the expression profile of HSP47 in laryngeal squamous cell carcinoma (LSCC) patients and there is still insufficient data concerning the underlying mechanisms. The aim of the present study was to explore the expression of HSP47 in LSCC and provide an overview of its association with tumourigenicity and clinical prognosis. The expression of HSP47 in LSCC and adjacent non-cancerous laryngeal tissues was assessed via western blotting and immunohistochemical studies. The prognostic significance of HSP47 expression was analysed using a Kaplan-Meier survival curve. To investigate the influence of HSP47 on the viability, invasion and apoptosis of a LSCC cell line, we performed an in vitro analysis with plasmid vectors and small interfering RNA (siRNA). Our results showed that HSP47 protein expression in the LSCC tissues was markedly decreased compared to that noted in the adjacent non-cancerous tissues, and low expression of HSP47 was correlated with poor prognosis in LSCC patients. Upregulation of HSP47 via plasmid vectors inhibited the proliferation, reduced the invasive ability, increased the sensitivity to cisplatin chemotherapy, promoted apoptosis, and induced the G1 phase arrest of LSCC cells in vitro. The expression of apoptosis-regulating proteins was also altered when HSP47 was upregulated, involving increased expression of cleaved caspase-7/-8/-9, PARP, and Bax and decreased expression of Bcl-2. Our present data suggest that HSP47 is an important prognostic factor and an attractive therapeutic target in LSCC due to its influence on the biological behaviour of LSCC cells.

MicroRNA-342 inhibits the progression of glioma by directly targeting PAK4.

Glioma is an extremely aggressive and lethal type of brain tumour that originates from glial cells. MicroRNA (miRNA) dysregulation has been implicated in the occurrence and progression of many human cancers, including glioma. Thus, some specific miRNAs are potential therapeutic targets for glioma diagnosis, therapy and prognosis. MicroRNA-342 (miR­342) has been reported to be abnormally expressed in various types of cancer. However, the precise roles of miR­342 in glioma remain unknown. The present study showed that miR­342 is relatively downregulated in glioma tissues and cell lines compared with that in adjacent normal tissues and normal human astrocytes. We observed that low miR­342 expression levels are correlated with advanced WHO grades and low KPS scores of glioma patients. In addition, the results of the functional assays demonstrated that miR­342 overexpression inhibits the proliferation and invasion of glioma cells and induces apoptosis. Further investigation revealed that P21 activated kinases 4 (PAK4) is a direct target of miR­342 in glioma. PAK4 was significantly upregulated in glioma tissues and inversely correlated with miR­342 expression. Moreover, PAK4 knockdown can mimic the effects of miR­342 on glioma cell proliferation, invasion and apoptosis. Notably, restoration of expression of PAK4 reversed the suppressive effects induced by the miR­342 in the glioma cells. The upregulation of miR­342 inactivated the AKT and ERK pathways in glioma. These findings may contribute to the understanding of the molecular mechanism underlying the carcinogenesis and progression of glioma, and to provide novel therapeutic target for the treatment of glioma patients.

Antiproliferative and apoptosis-inducing activity of schisandrin B against human glioma cells.

BACKGROUND: Malignant glioma is the most devastating and aggressive tumour in the brain and is characterised by high morbidity, high mortality and extremely poor prognosis. The main purpose of the present study was to investigate the effects of schisandrin B (Sch B) on glioma cells both in vitro and in vivo and to explore the possible anticancer mechanism underlying Sch B-induced apoptosis and cell cycle arrest. METHODS: The anti-proliferative ability of Sch B on glioma cells were assessed by MTT and clony formation assays. Flow cytometric analysis was used to detect cell cycle changes. Apoptosis was determined by Hoechst 33342 staining and annexin V/PI double-staining assays. The mitochondrial membrane potential was detected by Rhodamine 123 staining. The in vivo efficacy of Sch B was measured using a U87 xenograft model in nude mice. The expressions of the apoptosis-related and cell cycle-related proteins were analysed by western blot. Student's t-test was used to compare differences between treated groups and their controls. RESULTS: We found that Sch B inhibited growth in a dose- and time-dependent manner as assessed by MTT assay. In U87 and U251 cells, the number of clones was strongly suppressed by Sch B. Flow cytometric analysis revealed that Sch B induced cell cycle arrest in glioma cells at the G0/G1 phase. In addition, Sch B induced glioma cell apoptosis and reduced mitochondrial membrane potential (ΔΨm) in a dose-dependent manner. Mechanically, western blot analysis indicated that Sch B induced apoptosis by caspase-3, caspase-9, PARP, and Bcl-2 activation. Moreover, Sch B significantly inhibited tumour growth in vivo following the subcutaneous inoculation of U87 cells in athymic nude mice. COCLUSIONS: In summary, Sch B can reduce cell proliferation and induce apoptosis in glioma cells and has potential as a novel anti-tumour therapy to treat gliomas.

Endogenous neurotrophin-3 promotes neuronal sprouting from dorsal root ganglia.

In the present study, we investigated the role of endogenous neurotrophin-3 in nerve terminal sprouting 2 months after spinal cord dorsal root rhizotomy. The left L1-5 and L7-S2 dorsal root ganglia in adult cats were exposed and removed, preserving the L6 dorsal root ganglia. Neurotrophin-3 was mainly expressed in large neurons in the dorsal root ganglia and in some neurons in spinal lamina II. Two months after rhizotomy, the number of neurotrophin-3-positive neurons in the spared dorsal root ganglia and the density of neurite sprouts emerging from these ganglia were increased. Intraperitoneal injection of an antibody against neurotrophin-3 decreased the density of neurite sprouts. These findings suggest that endogenous neurotrophin-3 is involved in spinal cord plasticity and regeneration, and that it promotes axonal sprouting from the dorsal root ganglia after spinal cord dorsal root rhizotomy.

Killing effect of interleukin-13 receptor alpha 2 (IL-13Ralpha2) sensitized DC-CTL cells on human glioblastoma U251 cells.

Recent studies show that IL-13Ralpha2, a brain tumor-associated antigen for IL-13, may play a role in immunotherapy for glioblastoma. Thus, we stimulated the lymphocyte by monocyte-derived dendritic cells (DCs). The DCs were pulsed with IL-13Ralpha2 in vitro and then co-cultured with lymphocytes. After inducing cytotoxic T cells (CTLs) and co-culturing with U251 cells for 24 h in 96 wells, Cell Count Kit-8 (CCK-8) was added to every well equally. The optical density (OD) value was detected and recorded after 2 h. The DCs efficiently presented the antigen to the CTLs, resulting in CTLs activation and proliferation. The induced CTLs showed specific cytotoxic against U251 cells (P<0.01). The results demonstrated that IL-13Ralpha2 induced CTLs could kill glioma U251 in vitro, which suggests that IL-13 Ralpha2 might have such an impact in vivo and thus recombinant IL-13Ra2 protein might be used as an anti-tumor vaccine, providing a promising new strategy for the treatment of brain malignant gliomas.