Novel cancer-fighting role of ticagrelor inhibits GTSE1-induced EMT by regulating PI3K/Akt/NF-κB signaling pathway in malignant glioma.

Background: Glioma is the most common malignant brain tumor of the central nervous system. Despite of the improvement of therapeutic strategy, the prognosis of malignant glioma patients underwent by STUPP strategy is still unexpected. Previous studies have suggested that ticagrelor exerted chemotherapeutic effects by inhibition of epithelial-mesenchymal transition (EMT) in various diseases including tumors. However, whether ticagrelor can exhibit the antitumor efficiency in glioma by affecting the EMT process is still unclear. In this study, we investigated the cancer-fighting role of ticagrelor and demonstrated its chemotherapeutic mechanism in glioma. Materials and methods: The MTT assay was performed to detect the cytotoxicity of ticagrelor in glioma cells. We evaluated the expression of Ki67 in glioma cells by immunofluorescence assay after ticagrelor treatment. We conducted wound healing assay and transwell assay to determine the effects of ticagrelor on the migration and invasion of glioma cells. RNA-seq analysis was conducted to examine potential target genes and alternative signaling pathways for ticagrelor treatment. The expression levels of key EMT -related proteins were examined by Western blot experiment. Results: Ticagrelor inhibited the proliferation, migration and invasion of glioma cells with a favorable toxicity profile in vitro. Ticagrelor downregulated the expression of GTSE1 in glioma cells. RNA-seq analysis explored that GTSE1 acted as the potential target gene for ticagrelor treatment. Upregulation of GTSE1 antagonized the inhibitory effect of ticagrelor on the invasion of glioma and EMT progression by regulation of PI3K/Akt/NF-κB signaling pathway. And ticagrelor also exhibited the similar chemotherapeutic effect of glioma in vivo. Conclusions: Ticagrelor as a potential chemotherapeutic option induced the inhibition of the GTSE1-induced EMT progression by regulation of PI3K/AKT/NF-κB signaling pathway.

MiRNAs as potential therapeutic targets and biomarkers for non-traumatic intracerebral hemorrhage.

Non-traumatic intracerebral hemorrhage (ICH) is the most common type of hemorrhagic stroke, most often occurring between the ages of 45 and 60. Hypertension is most often the cause of ICH. Less often, atherosclerosis, blood diseases, inflammatory changes in cerebral vessels, intoxication, vitamin deficiencies, and other reasons cause hemorrhages. Cerebral hemorrhage can occur by diapedesis or as a result of a ruptured vessel. This very dangerous disease is difficult to treat, requires surgery and can lead to disability or death. MicroRNAs (miRNAs) are a class of non-coding RNAs (about 18-22 nucleotides) that are involved in a variety of biological processes including cell differentiation, proliferation, apoptosis, etc., through gene repression. A growing number of studies have demonstrated miRNAs deregulation in various cardiovascular diseases, including ICH. In addition, given that computed tomography (CT) and/or magnetic resonance imaging (MRI) are either not available or do not show clear signs of possible vessel rupture, accurate and reliable analysis of circulating miRNAs in biological fluids can help in early diagnosis for prevention of ICH and prognosis patient outcome after hemorrhage. In this review, we highlight the up-to-date findings on the deregulated miRNAs in ICH, and the potential use of miRNAs in clinical settings, such as therapeutic targets and non-invasive diagnostic/prognostic biomarker tools.

Irregular shape as an independent predictor of prognosis in patients with primary intracerebral hemorrhage.

The utility of noncontrast computed tomography markers in the prognosis of spontaneous intracerebral hemorrhage has been studied. This study aimed to investigate the predictive value of the computed tomography (CT) irregularity shape for poor functional outcomes in patients with spontaneous intracerebral hemorrhage. We retrospectively reviewed all 782 patients with intracranial hemorrhage in our stroke emergency center from January 2018 to September 2019. Laboratory examination and CT examination were performed within 24 h of admission. After three months, the patient's functional outcome was assessed using the modified Rankin Scale. Multinomial logistic regression analyses were applied to identify independent predictors of functional outcome in patients with intracerebral hemorrhage. Out of the 627 patients included in this study, those with irregular shapes on CT imaging had a higher proportion of poor outcomes and mortality 90 days after discharge (P < 0.001). Irregular shapes were found to be significant independent predictors of poor outcome and mortality on multiple logistic regression analysis. In addition, the increase in plasma D-dimer was associated with the occurrence of irregular shapes (P = 0.0387). Patients with irregular shapes showed worse functional outcomes after intracerebral hemorrhage. The elevated expression level of plasma D-dimers may be directly related to the formation of irregular shapes.

Safety Analysis of Simultaneous Cranioplasty and Ventriculoperitoneal Shunt Placement.

AIM: To investigate the safety of combined cranioplasty (CP) and ventriculoperitoneal shunt (VPS) placement. Furthermore, we investigated whether the sequence of these procedures affects the postoperative complication rates associated with staged CP and VPS placement. MATERIAL AND METHODS: We retrospectively investigated patients who developed communicating hydrocephalus after decompressive craniectomy and subsequently underwent VPS placement and CP at the hospital at which this study was performed between January 2009 and December 2019. Patients were categorized into group 1 (simultaneous CP and VPS placement) and group 2 (CP and VPS placement performed separately). Group 2 was subcategorized into subgroup 2a (CP performed before VPS placement) and subgroup 2b (VPS placement performed before CP). The Student?s t and Chi square tests were used to analyze intergroup differences. RESULTS: This study included 86 patients; 22 in group 1 and 64 in group 2 (24 patients in subgroup 2a and 40 patients in subgroup 2b). No statistically significant difference was observed in the overall complication rates between groups 1 and 2 (36.4% vs. 28.1%, P=0.591). However, the incidence of infections was significantly higher in group 1 than in group 2 (22.7% vs. 4.7%, P=0.024). Subgroup analysis showed that the overall complication rate was signi?cantly lower in subgroup 2a than in subgroup 2b (12.5% vs. 37.5%, P=0.031). CONCLUSION: Simultaneous CP and VPS placement is associated with a high incidence of infections. Moreover, compared with initial CP, initial VPS placement is associated with a significantly higher risk of overall complications in patients who undergo a staged procedure.

Exosomal miR-2276-5p in Plasma Is a Potential Diagnostic and Prognostic Biomarker in Glioma.

INTRODUCTION: Exosomal microRNAs (miRNAs) play an essential role in near and distant intercellular communication and are potential diagnostic and prognostic biomarkers for various cancers. This study focused on evaluation of exosomal miR-2276-5p in plasma as a diagnostic and prognostic biomarker for glioma. METHODS: Plasma exosomes from 124 patients with glioma and 36 non-tumor controls were collected and subjected to quantitative real-time polymerase chain reaction (qRT-PCR) analysis for the exosomal miR-2276-5p expression. Bioinformatic analyses were performed to identify a gene target, and CGGA and TCGA databases were checked for evaluation of prognostic relevance. RESULTS: The exosomal miR-2276-5p in glioma patients had a significantly decreased expression, compared with non-glioma patients (p < 0.01). Receiver operating characteristics (ROC) curve analyses were observed to regulate the diagnostic sensitivity and specificity of miR-2276-5p in glioma; the area under the curve (AUC) for miR-2276-5p was 0.8107. The lower expression of exosomal miR-2276-5p in patients with glioma correlated with poorer survival rates. RAB13 was identified as the target of miR-2276-5p which was high in glioma patients, especially those with higher tumor grades and correlated with poor survival. CONCLUSION: The circulating exosomal miR-2276-5p is significantly reduced in the plasma of glioma patients, and thus, it could be a potential biomarker for patients with glioma for diagnostic and/or prognostic purposes.

Diagnostic and prognostic potential of circulating miRNAs for intracranial aneurysms.

Intracranial aneurysm (IA) is an abnormal focal dilation of an artery in the brain that results from a weakening of the inner muscular layer of a blood vessel wall. IAs represent the most common etiology of nontraumatic subarachnoid hemorrhage (SAH). Despite technological advances in the treatment and use of new diagnostic methods for IAs, they continue to pose a significant risk of mortality and disability. Thus, early recognition of IA with a high risk of rupture is crucial for the stratification of patients with such a formidable disease. MicroRNAs (miRNA) are endogenous noncoding RNAs of 18-22 nucleotides that regulate gene expression at the post-transcriptional level through interaction with 3'-untranslated regions (3'UTRs) of the target mRNAs. MiRNAs are involved in the pathogenesis of IAs, including in the mechanisms of formation, growth, and rupture. It is known that in many biological fluids of the human body, such as blood or cerebrospinal fluid (CSF), numerous miRNAs, called circulating miRNAs, have been detected. The expression profile of circulating miRNAs represents a certain part of the cells in which they are modified and secreted in accordance with the physiological or pathological conditions of these cells. Circulating miRNAs can be secreted from cells into human biological fluids in extracellular vesicles or can be bound to Ago2 protein, which makes them resistant to the effects of RNAse. Therefore, circulating miRNAs are considered as new potential biomarkers of interest in many diseases, including IA.

Circulating MicroRNAs as Potential Noninvasive Biomarkers of Spontaneous Intracerebral Hemorrhage.

BACKGROUND: Spontaneous intracerebral hemorrhage (ICH) is a common and severe neurological disorder that has been associated with high rates of mortality and morbidity. It is urgent to find new biomarkers for the early diagnosis and prevention of ICH. In recent years, micro-RNAs (miRNAs) have been proved to play an important role in vascular damage and inflammation in cerebrovascular diseases, including ICH. In the peripheral blood, circulating miRNAs will be present at a remarkably steady level. In the present study, we explored the circulating plasma microRNA (miR)-181b, miR-223, miR-155, and miR-145 as new potential biomarkers for the diagnosis of ICH. METHODS: The plasma samples from 106 patients with ICH and 50 patients without ICH (control group) were collected and subjected to quantitative real-time polymerase chain reaction analyses for the expression levels of circulating miR-181b, miR-223, miR-155, and miR-145. RESULTS: The expression levels of plasma circulating miR-145 (P < 0.001), miR-223, and miR-155 were increased in patients with ICH compared with those in the control group (P < 0.05). However, the expression of plasma circulating miR-181b was decreased in patients with ICH compared with that in the control group (P < 0.001). Receiver operating characteristic curve analyses were performed to determine the diagnostic sensitivity and specificity of miR-145 and miR-181b to detect ICH. The area under the curve for miR-145 was 0.766 (95% confidence interval, 0.689-0.838) and for miR-181b was 0.78 (95% confidence interval, 0.70-0.86), suggesting that circulating miR-145 and miR-181b can be used to differentiate patients with ICH from those without ICH. CONCLUSION: Our results have shown that measurement of circulating miR-181b, miR-223, miR-155, and miR-145 in plasma samples could serve as a potential noninvasive tool for ICH detection.

Oncogenic Ras is downregulated by ARHI and induces autophagy by Ras/AKT/mTOR pathway in glioblastoma.

BACKGROUND: Glioblastoma is a disease with high heterogeneity that has long been difficult for doctors to identify and treat. ARHI is a remarkable tumor suppressor gene in human ovarian cancer and many other cancers. We found over-expression of ARHI can also inhibit cancer cell proliferation, decrease tumorigenicity, and induce autophagic cell death in human glioma and inhibition of the late stage of autophagy can further enhance the antitumor effect of ARHI through inducing apoptosis in vitro or vivo. METHODS: Using MTT assay to detect cell viability. The colony formation assay was used to measure single cell clonogenicity. Autophagy associated morphological changes were tested by transmission electron microscopy. Flow cytometry and TUNEL staining were used to measure the apoptosis rate. Autophagy inhibitor chloroquine (CQ) was used to study the effects of inhibition at late stage of autophagy on ARHI-induced autophagy and apoptosis. Protein expression were detected by Western blot, immunofluorescence and immunohistochemical analyses. LN229-derived xenografts were established to observe the effect of ARHI in vivo. RESULTS: ARHI induced autophagic death in glioma cells, and blocking late-stage autophagy markedly enhanced the antiproliferative activites of ARHI. In our research, we observed the inhibition of RAS-AKT-mTOR signaling in ARHI-glioma cells and blockade of autophagy flux at late stage by CQ enhanced the cytotoxicity of ARHI, caused accumulation of autophagic vacuoles and robust apoptosis. As a result, the inhibition of RAS augmented autophagy of glioma cells. CONCLUSION: ARHI may also be a functional tumor suppressor in glioma. And chloroquine (CQ) used as an auxiliary medicine in glioma chemotherapy can enhance the antitumor effect of ARHI, and this study provides a novel mechanistic basis and strategy for glioma therapy.

Peiminine Inhibits Glioblastoma in Vitro and in Vivo Through Cell Cycle Arrest and Autophagic Flux Blocking.

BACKGROUND/AIMS: Glioblastoma multiforme (GBM) is the most devastating and widespread primary central nervous system tumour in adults, with poor survival rate and high mortality rates. Existing treatments do not provide substantial benefits to patients; therefore, novel treatment strategies are required. Peiminine, a natural bioactive compound extracted from the traditional Chinese medicine Fritillaria thunbergii, has many pharmacological effects, especially anticancer activities. However, its anticancer effects on GBM and the underlying mechanism have not been demonstrated. This study was conducted to investigate the potential antitumour effects of peiminine in human GBM cells and to explore the related molecular signalling mechanisms in vitro and in vivo Methods: Cell viability and proliferation were detected with MTT and colony formation assays. Morphological changes associated with autophagy were assessed by transmission electron microscopy (TEM). The cell cycle rate was measured by flow cytometry. To detect changes in related genes and signalling pathways in vitro and in vivo, RNA-seq, Western blotting and immunohistochemical analyses were employed. RESULTS: Peiminine significantly inhibited the proliferation and colony formation of GBM cells and resulted in changes in many tumour-related genes and transcriptional products. The potential anti-GBM role of peiminine might involve cell cycle arrest and autophagic flux blocking via changes in expression of the cyclin D1/CDK network, p62 and LC3. Changes in Changes in flow cytometry results and TEM findings were also observed. Molecular alterations included downregulation of the expression of not only phospho-Akt and phospho-GSK3ß but also phospho-AMPK and phospho-ULK1. Furthermore, overexpression of AKT and inhibition of AKT reversed and augmented peiminine-induced cell cycle arrest in GBM cells, respectively. The cellular activation of AMPK reversed the changes in the levels of protein markers of autophagic flux. These results demonstrated that peiminine mediates cell cycle arrest by suppressing AktGSk3ß signalling and blocks autophagic flux by depressing AMPK-ULK1 signalling in GBM cells. Finally, peiminine inhibited the growth of U251 gliomas in vivo. CONCLUSION: Peiminine inhibits glioblastoma in vitro and in vivo via arresting the cell cycle and blocking autophagic flux, suggesting new avenues for GBM therapy.

Nitazoxanide, an antiprotozoal drug, inhibits late-stage autophagy and promotes ING1-induced cell cycle arrest in glioblastoma.

Glioblastoma is the most common and aggressive primary brain tumor in adults. New drug design and development is still a major challenge for glioma treatment. Increasing evidence has shown that nitazoxanide, an antiprotozoal drug, has a novel antitumor role in various tumors and exhibits multiple molecular functions, especially autophagic regulation. However, whether nitazoxanide-associated autophagy has an antineoplastic effect in glioma remains unclear. Here, we aimed to explore the underlying molecular mechanism of nitazoxanide in glioblastoma. Our results showed that nitazoxanide suppressed cell growth and induced cell cycle arrest in glioblastoma by upregulating ING1 expression with a favorable toxicity profile. Nitazoxanide inhibited autophagy through blockage of late-stage lysosome acidification, resulting in decreased cleavage of ING1. A combination with chloroquine or Torin1 enhanced or impaired the chemotherapeutic effect of nitazoxanide in glioblastoma cells. Taken together, these findings indicate that nitazoxanide as an autophagy inhibitor induces cell cycle arrest in glioblastoma via upregulated ING1 due to increased transcription and decreased post-translational degradation by late-stage autophagic inhibition.

Oxymatrine induces cell cycle arrest and apoptosis and suppresses the invasion of human glioblastoma cells through the EGFR/PI3K/Akt/mTOR signaling pathway and STAT3.

Oxymatrine (OM), a natural quinolizidine alkaloid extracted from the traditional Chinese herb Sophora flavescens, has been revealed to produce antitumor activities in various cancer cell lines, including glioblastoma lines, in vitro. However, the mechanisms by which OM exerts its antitumor effect against glioma are poorly understood. The aim of this study was to investigate the role of OM in the proliferation, apoptosis and invasion of glioma cells and to reveal the underlying mechanisms. The effects of OM on U251MG cells in vitro were determined using a Cell Counting Kit­8 (CCK­8) assay, flow cytometric analysis, Annexin V­FITC/PI staining, DAPI staining, a terminal deoxynucleotidyl transferase­mediated dUTP nick end­labeling (TUNEL) assay, a Transwell assay and western blotting. Our data indicated that OM inhibited proliferation, arrested the cell cycle at the G0/G1 phase, decreased the expression levels of G1 cell cycle regulatory proteins (cyclin D1, CDK4 and CDK6), inhibited invasion and induced apoptosis in glioma cells. Additional investigations revealed that the expression levels of p­STAT3 and key proteins in the EGFR/PI3K/Akt/mTOR signaling pathway, such as p­EGFR, p­Akt and p­mTOR, were markedly decreased after OM treatment, while the total STAT3, EGFR, Akt and mTOR levels were not affected. These findings indicated that the EGFR/PI3K/Akt/mTOR signaling pathway and STAT3 suppression may be a potential mechanism of the OM­mediated antitumor effect in glioblastoma cells and that EGFR may be a target of OM. Hence, OM may be a promising drug and may offer a novel therapeutic strategy for malignant gliomas in the future.

Isogambogenic Acid Inhibits the Growth of Glioma Through Activation of the AMPK-mTOR Pathway.

BACKGROUND/AIMS: Glioma is the most devastating cancer in the brain and has a poor prognosis in adults. Therefore, there is a critical need for novel therapeutic strategies for the management of glioma patients. Isogambogenic acid, an active compound extracted from the Chinese herb Garcinia hanburyi, induces autophagic cell death. METHODS: Cell viability was detected with MTT assays. Cell proliferation was assessed using the colony formation assay. Morphological changes associated with autophagy and apoptosis were tested by TEM and Hoechst staining, respectively. The apoptosis rate was measured by flow cytometry. Western blot, immunofluorescence and immunohistochemical analyses were used to detect protein expression. U87-derived xenografts were established for the examination of the effect of isogambogenic acid on glioma growth in vivo. RESULTS: Isogambogenic acid induced autophagic death in U87 and U251 cells, and blocking late-stage autophagy markedly enhanced the antiproliferative activities of isogambogenic acid. Moreover, we observed the activation of AMPK-mTOR signalling in isogambogenic acid-treated glioma cells. Furthermore, the activation of AMPK or the inhibition of mTOR augmented isogambogenic acid-induced autophagy. Inhibition of autophagy attenuated apoptosis in isogambogenic acid-treated glioma cells. Finally, isogambogenic acid inhibited the growth of U87 glioma in vivo. CONCLUSION: Isogambogenic acid inhibits the growth of glioma via activation of the AMPK-mTOR signalling pathway, which may provide evidence for future clinical applications in glioma therapy.

Inhibition of autophagy induced by quercetin at a late stage enhances cytotoxic effects on glioma cells.

Glioma is the most common primary brain tumor in the central nervous system (CNS) with high morbidity and mortality in adults. Although standardized comprehensive therapy has been adapted, the prognosis of glioma patients is still frustrating and thus novel therapeutic strategies are urgently in need. Quercetin (Quer), an important flavonoid compound found in many herbs, is shown to be effective in some tumor models including glioma. Recently, it is reported that adequate regulation of autophagy can strengthen cytotoxic effect of anticancer drugs. However, it is not yet fully clear how we should modulate autophagy to achieve a satisfactory therapeutic effect. 3-Methyladenine (3-MA) and Beclin1 short hairpin RNA (shRNA) were used to inhibit the early stage of autophage while chloroquine (CQ) to inhibit the late stage. MTT assay was implemented to determine cell viability. Transmission electron microscopy, western blot, and immunohistochemistry were adopted to evaluate autophagy. Western blot, flow cytometry, and immunohistochemistry were used to detect apoptosis. C6 glioma xenograft models were established to assess the therapeutic effect (the body weight change, the median survival time, and tumor volume) in vivo. Quercetin can inhibit cell viability and induce autophagy of U87 and U251 glioma cells in a dose-dependent manner. Inhibition of early-stage autophagy by 3-MA or shRNA against Beclin1 attenuated the quercetin-induced cytotoxicity. In contrast, suppression of autophagy at a late stage by CQ enhanced the anti-glioma efficiency of quercetin. Therapeutic effect of quercetin for malignant glioma can be strengthened by inhibition of autophagy at a late stage, not initial stage, which may provide a novel opportunity for glioma therapy.

Methionine and cystine double deprivation stress suppresses glioma proliferation via inducing ROS/autophagy.

Cancer cells are highly dependent on methionine and cystine (Met-Cys) for survival and proliferation. However, the molecular mechanism is not fully clear. The present study is to investigate the effects of Met-Cys deprivation on glioma cells proliferation. The results showed that Met-Cys double deprivation had synergistic action on elevating ROS level, decreased GSH level and inhibition of glioma cell proliferation. Moreover, both of them deprivation triggered autophagy of glioma cells both in vitro and in vivo. Importantly, Met-Cys double restriction diet inhibited growth of glioma. These results provided a new regulation mechanism of Met-Cys metabolism on affecting glioma cell proliferation, suggesting that targeting Met-Cys metabolism may be a potential strategy for glioma therapy.

Calcitriol enhances 5-aminolevulinic acid-induced fluorescence and the effect of photodynamic therapy in human glioma.

BACKGROUND: Glioma recurrence frequently occurs close to the marginal area of the surgical cavity as a result of residual infiltrating glioma cells. Fluorescence-guided surgery with 5-aminolevulinic acid (ALA) for resection of gliomas has been used as an effective therapeutic approach to discriminate malignant tissue from brain tissue and to facilitate patient prognosis. ALA-based photodynamic therapy is an effective adjuvant treatment modality for gliomas. However, insufficient protoporphyrin IX (PpIX) accumulation may limit the applicability of fluorescence-guided resection and photodynamic therapy in the marginal areas of gliomas. METHODS: To be able to understand how to overcome these issues, human glioma cells and normal astrocytes were used as the model system. Glioma cells and astrocytes were preconditioned with calcitriol for 48 hours and then incubated with ALA. Changes in ALA-induced PpIX fluorescence and cell survival after light exposure were assessed. Furthermore, expression of porphyrin synthetic enzymes in pretreated glioma cells was analyzed. RESULTS: Calcitriol can be administered prior to ALA as a non-toxic preconditioning regimen to significantly enhance ALA-induced PpIX levels and fluorescence. This increase in PpIX level was detected preferentially in glioma versus normal cells. Also, calcitriol pretreated glioma cells exhibited increased cell death following ALA-based photodynamic therapy. Furthermore, mechanistic studies documented that expression of the porphyrin synthesis enzymes coproporphyrinogen oxidase was increased by calcitriol at the mRNA level. CONCLUSION: We demonstrated for the first time a simple, non-toxic and highly effective preconditioning regimen to selectively enhance PpIX fluorescence and the response of ALA-PDT in glioma cells. This finding suggests that the combined treatment of glioma cells with calcitriol plus ALA may provide an effective and selective therapeutic modality to enhance ALA-induced PpIX fluorescent quality for improving discrimination of tumor tissue and PDT efficacy.