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Cancer and its associated conditions have significant impacts on public health at many levels worldwide, and cancer is the leading cause of death among adults. Peroxisome proliferator-activated receptor α (PPARα)-specific agonists, fibrates, have been approved by the Food and Drug Administration for managing hyperlipidemia. PPARα-specific agonists exert anti-cancer effects in many human cancer types, including glioblastoma (GBM). Recently, we have reported that the hypoxic state in GBM stabilizes hypoxia-inducible factor-1 alpha (HIF-1α), thus contributing to tumor escape from immune surveillance by activating the expression of the pH-regulating protein carbonic anhydrase IX (CA9). In this study, we aimed to study the regulatory effects of the PPARα agonist fibrate on the regulation of HIF-1α expression and its downstream target protein in GBM. Our findings showed that fenofibrate is the high potency compound among the various fibrates that inhibit hypoxia-induced HIF-1α and CA9 expression in GBM. Moreover, fenofibrate-inhibited HIF-1α expression is mediated by HO-1 activation in GBM cells through the AMP-activated protein kinase (AMPK) pathway. In addition, fenofibrate-enhanced HO-1 upregulation activates SIRT1 and leads to subsequent accumulation of SIRT1 in the nucleus, which further promotes HIF-1α deacetylation and inhibits CA9 expression. Using a protein synthesis inhibitor, cycloheximide, we also observed that fenofibrate inhibited HIF-1α protein synthesis. In addition, the administration of the proteasome inhibitor MG132 showed that fenofibrate promoted HIF-1α protein degradation in GBM. Hence, our results indicate that fenofibrate is a useful anti-GBM agent that modulates hypoxia-induced HIF-1α expression through multiple cellular pathways.

Simple Summary The present study showed that a prescribed psychotropic medicine paliperidone inhibits GBM growth and prolongs survival in mouse brain tumor model and decreased the programmed death ligand 1 expression. Using the 3D co-culture also found that dopamine receptor D2 regulates the interaction of GBM-macrophage-induced PD-L1 expression in GBMs. In addition, the expression of DRD2 and PD-L1 in GBM modulates tumor-associated macrophage polarization. Our results also indicated that there is a contact-independent mechanism of PD-L1 induction in GBM upon interaction between GBM and monocytes. The present study also found that the interaction of GBM-macrophage-enhanced PD-L1 expression in GBM occurred by modulating the ERK and STAT3 signaling pathways. In addition, the inhibition of DRD2 reduces the upregulation of PD-1 expression, and it is regulating signaling in GBM. Abstract A previous study from our group reported that monocyte adhesion to glioblastoma (GBM) promoted tumor growth and invasion activity and increased tumor-associated macrophages (TAMs) proliferation and inflammatory mediator secretion as well. The present study showed that prescribed psychotropic medicine paliperidone reduced GBM growth and immune checkpoint protein programmed death ligand (PD-L)1 expression and increased survival in an intracranial xenograft mouse model. An analysis of the database of patients with glioma showed that the levels of PD-L1 and dopamine receptor D (DRD)2 were higher in the GBM group than in the low grade astrocytoma and non-tumor groups. In addition, GFP expressing GBM (GBM-GFP) cells co-cultured with monocytes-differentiated macrophage enhanced PD-L1 expression in GBM cells. The enhancement of PD-L1 in GBM was antagonized by paliperidone and risperidone as well as DRD2 selective inhibitor L741426. The expression of CD206 (M2 phenotype marker) was observed to be markedly increased in bone marrow-derived macrophages (BMDMs) co-cultured with GBM. Importantly, treatment with paliperidone effectively decreased CD206 and also dramatically increased CD80 (M1 phenotype marker) in BMDMs. We have previously established a PD-L1 GBM-GFP cell line that stably expresses PD-L1. Experiments showed that the expressions of CD206 was increased and CD80 was mildly decreased in the BMDMs co-cultured with PD-L1 GBM-GFP cells. On the other hands, knockdown of DRD2 expression in GBM cells dramatically decreased the expression of CD206 but markedly increased CD80 expressions in BMDMs. The present study suggests that DRD2 may be involved in regulating the PD-L1 expression in GBM and the microenvironment of GBM. Our results provide a valuable therapeutic strategy and indicate that treatments combining DRD2 antagonist paliperidone with standard immunotherapy may be beneficial for GBM treatment.

Glioblastoma (GBM), the most aggressive brain tumor, has a poor prognosis due to the ease of migration to surrounding healthy brain tissue. Recent studies have shown that bradykinin receptors are involved in the progression of various cancers. However, the molecular mechanism and pathological role of bradykinin receptors remains unclear. We observed the expressions of two major bradykinin receptors, B1R and B2R, in two different human GBM cell lines, U87 and GBM8901. Cytokine array analysis showed that bradykinin increases the production of interleukin (IL)-8 in GBM via B1R. Higher B1R levels correlate with IL-8 expression in U87 and GBM8901. We observed increased levels of phosphorylated STAT3 and SP-1 in the nucleus as well. Using chromatin immunoprecipitation assay, we found that STAT3 and SP-1 mediate IL-8 expression, which gets abrogated by the inhibition of FAK and STAT3. We further demonstrated that IL-8 expression and cell migration are also regulated by the SP-1. In addition, expression levels of STAT3 and SP-1 positively correlate with clinicopathological grades of gliomas. Interestingly, our results found that inhibition of HDAC increases IL-8 expression. Moreover, stimulation with bradykinin caused increases in acetylated SP-1 and p300 complex formation, which are abrogated by inhibition of FAK and STAT3. Meanwhile, knockdown of SP-1 and p300 decreased the augmentation of bradykinin-induced IL-8 expression. These results indicate that bradykinin-induced IL-8 expression is dependent on B1R which causes phosphorylated STAT3 and acetylated SP-1 to translocate to the nucleus, hence resulting in GBM migration.

Glioblastoma (GBM) is the most common and lethal brain tumor with high inflammation. GBM cells infiltrate microglia and macrophages and are surrounded by pro-inflammatory cytokines. Interleukin (IL)-1β, which is abundantly expressed in the tumor microenvironment, is involved in tumor progression. Intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 mediate cell-cell interactions, and these cell adhesion molecules (CAMs) can be regulated by cytokines in immune cells or cancer cells in the inflammatory tumor microenvironment. In this study, we found that ICAM-1 and VCAM-1 expression was induced when GBM cells were treated with IL-1β, and that adhesive interaction between monocytes and GBM cells increased accordingly. The levels of soluble CAMs (sICAM-1 and sVCAM-1) were also increased in the supernatants induced by IL-1β. Furthermore, the conditioned media contained sICAM-1 and sVCAM-1, which further promoted IL-6 and CCL2 expression in differentiated macrophages. IL-1β downregulated Src homology 1 domain-containing protein tyrosine phosphatase (SHP-1) in GBM. The expression of ICAM-1 and VCAM-1 was regulated by p38, AKT, and NF-κB signaling pathways, which were modulated by SHP-1 signaling. The present study suggests that IL-1β-induced protein expression of ICAM-1 and VCAM-1 in GBM may modulate the adhesive interaction between GBM and monocytes. In addition, IL-1β also induced the soluble form of ICAM-1 and VCAM-1 in GBM, which plays a key role in the regulation of tumor-associated monocyte/macrophage polarization.

Carbonic anhydrases (CAs) are acid&ndash, base regulatory proteins that modulate a variety of physiological functions. Recent findings have shown that CAIX is particularly upregulated in glioblastoma multiforme (GBM) and is associated with a poor patient outcome and survival rate. An analysis of the GSE4290 dataset of patients with gliomas showed that CAIX was highly expressed in GBM and was negatively associated with prognosis. The expression of CAIX under hypoxic conditions in GBM significantly increased in protein, mRNA, and transcriptional activity. Importantly, CAIX upregulation also regulated GBM motility, monocyte adhesion to GBM, and the polarization of tumor-associated monocytes/macrophages (TAM). Furthermore, the overexpression of CAIX was observed in intracranial GBM cells. Additionally, epidermal growth factor receptor/signal transducer and activator of transcription 3 regulated CAIX expression under hypoxic conditions by affecting the stability of hypoxia-inducible factor 1&alpha, In contrast, the knockdown of CAIX dramatically abrogated the change in GBM motility and monocyte adhesion to GBM under hypoxic conditions. Our results provide a comprehensive understanding of the mechanisms of CAIX in the GBM microenvironment. Hence, novel therapeutic targets of GBM progression are possibly developed.

Sirtuin 1 (SIRT1) has been reported to be involved in the mechanisms underlying longevity and has also been indicated as a valuable regulator of age-related neurological disorders. Some natural products increase SIRT1 activity and stimulate deacetylation of various proteins. In the present study, SIRT1 overexpression by genetic modification or treatment with SIRT1 activators significantly inhibited the secretion of nitric oxide and expression of inducible nitric oxide synthase, cyclooxygenase 2, and proinflammatory mediator-interleukin 1β-in microglia. SIRT1 activation also decreased the levels of K379 acetyl-p53 and the protein inhibitor of activated Stat 1 expression in microglial cells. In addition, it dramatically promoted M2 polarization of microglia, which enhanced cell motility and altered phagocytic ability. We also used minocycline, a well-known inhibitor of microglial activation, to study the mechanism of SIRT1 signaling. Minocycline treatment decreased neuroinflammatory responses and promoted M2 polarization of microglia. It also reduced the acetyl-p53 level in the brain tissues in an inflammatory mouse model. Our findings demonstrated that SIRT1 participates in the maintenance of microglial polarization homeostasis and that minocycline exerts regulatory effects on SIRT1 activation. Therefore, our results indicate that SIRT1 activation may be a useful therapeutic target for the treatment of neuroinflammation-associated disorders.

Glioblastoma (GBM) is characterized by severe hypoxic and acidic stress in an abnormal microenvironment. Monocarboxylate transporter (MCT)4, a pH-regulating protein, plays an important role in pH homeostasis of the glycolytic metabolic pathways in cancer cells. The present study showed that GBM exposure to hypoxic conditions increased MCT4 expression. We further analyzed the glioma patient database and found that MCT4 was significantly overexpressed in patients with GBM, and the MCT4 levels positively correlated with the clinico-pathological grades of gliomas. We further found that MCT4 knockdown abolished the hypoxia-enhanced of GBM cell motility and monocyte adhesion. However, the overexpression of MCT4 promoted GBM cell migration and monocyte adhesion activity. Our results also revealed that MCT4-regulated GBM cell motility and monocyte adhesion are mediated by activation of the serine/threonine-specific protein kinase (AKT), focal adhesion kinase (FAK), and epidermal growth factor receptor (EGFR) signaling pathways. Moreover, hypoxia mediated the acetylated signal transducer and activator of transcription (STAT)3 expression and regulated the transcriptional activity of hypoxia inducible factor (HIF)-1&alpha, in GBM cell lines. In a GBM mouse model, MCT4 was significantly increased in the tumor necrotic tissues. These findings raise the possibility for the development of novel therapeutic strategies targeting MCT4.

Tumor-associated macrophages (TAMs) originate as circulating monocytes, and are recruited to gliomas, where they facilitate tumor growth and migration. Understanding the interaction between TAM and cancer cells may identify therapeutic targets for glioblastoma multiforme (GBM). Vascular cell adhesion molecule-1 (VCAM-1) is a cytokine-induced adhesion molecule expressed on the surface of cancer cells, which is involved in interactions with immune cells. Analysis of the glioma patient database and tissue immunohistochemistry showed that VCAM-1 expression correlated with the clinico-pathological grade of gliomas. Here, we found that VCAM-1 expression correlated positively with monocyte adhesion to GBM, and knockdown of VCAM-1 abolished the enhancement of monocyte adhesion. Importantly, upregulation of VCAM-1 is dependent on epidermal-growth-factor-receptor (EGFR) expression, and inhibition of EGFR effectively reduced VCAM-1 expression and monocyte adhesion activity. Moreover, GBM possessing higher EGFR levels (U251 cells) had higher VCAM-1 levels compared to GBMs with lower levels of EGFR (GL261 cells). Using two- and three-dimensional cultures, we found that monocyte adhesion to GBM occurs via integrin α4β1, which promotes tumor growth and invasion activity. Increased proliferation and tumor necrosis factor-α and IFN-γ levels were also observed in the adherent monocytes. Using a genetic modification approach, we demonstrated that VCAM-1 expression and monocyte adhesion were regulated by the miR-181 family, and lower levels of miR-181b correlated with high-grade glioma patients. Our results also demonstrated that miR-181b/protein phosphatase 2A-modulated SP-1 de-phosphorylation, which mediated the EGFR-dependent VCAM-1 expression and monocyte adhesion to GBM. We also found that the EGFR-dependent VCAM-1 expression is mediated by the p38/STAT3 signaling pathway. Our study suggested that VCAM-1 is a critical modulator of EGFR-dependent interaction of monocytes with GBM, which raises the possibility of developing effective and improved therapies for GBM.

Natural products have historically been regarded as an important resource of therapeutic agents. Resveratrol and melatonin have been shown to increase SIRT1 activity and stimulate deacetylation. Glioblastoma multiforme (GBM) is the deadliest of malignant types of tumor in the central nervous system (CNS) and their biological features make treatment difficult. In the glioma microenvironment, infiltrating immune cells has been shown to possess beneficial effects for tumor progression. We analyzed SIRT1, CCL2, VCAM-1 and ICAM-1 in human glioma cell lines by immunoblotting. The correlation between those markers and clinico-pathological grade of glioma patients were assessed by the Gene Expression Omnibus (GEO) datasets analysis. We also used monocyte-binding assay to study the effects of melatonin on monocyte adhesion to GBM. Importantly, overexpression of SIRT1 by genetic modification or treatment of melatonin significantly downregulated the adhesion molecular VCAM-1 and ICAM-1 expression in GBM. CCL2-mediated monocyte adhesion and expression of VCAM-1 and ICAM-1 were regulated through SIRT1 signaling. SIRT1 is an important modulator of monocytes interaction with GBM that gives the possibility of improved therapies for GBM. Hence, this study provides a novel treatment strategy for the understanding of microenvironment changes in tumor progression.

Glioblastoma (GBM) is the most commonly occurring tumor in the cerebral hemispheres. Currently, temozolomide (TMZ), an alkylating agent that induces DNA strand breaks, is considered the frontline chemotherapeutic agent for GBM. Despite its frontline status, GBM patients commonly exhibit resistance to TMZ treatment. We have recently established and characterized TMZ-resistant human glioma cells. The aim of this study is to investigate whether curcumin modulates cell apoptosis through the alternation of the connexin 43 (Cx43) protein level in TMZ-resistant GBM. Overexpression of Cx43, but not ATP-binding cassette transporters (ABC transporters), was observed (approximately 2.2-fold) in TMZ-resistant GBM cells compared to the Cx43 levels in parental GBM cells. Furthermore, at a concentration of 10[Formula: see text][Formula: see text]M, curcumin significantly reduced Cx43 protein expression by about 40%. In addition, curcumin did not affect the expression of other connexins like Cx26 or epithelial-to-mesenchymal transition (EMT) proteins such as [Formula: see text]-catenin or [Formula: see text]E-catenin. Curcumin treatment led to an increase in TMZ-induced cell apoptosis from 4% to 8%. Importantly, it did not affect the mRNA expression level of Cx43. Concomitant treatment with the translation inhibitor cycloheximide (CHX) exerted additional effects on Cx43 degradation. Treatment with the autophagy inhibitor 3-MA (methyladenine) did not affect the curcumin-induced Cx43 degradation. Interestingly, treatment with the proteasome inhibitor MG132 (carbobenzoxy-Leu-Leu-leucinal) significantly negated the curcumin-induced Cx43 degradation, which suggests that curcumin-induced Cx43 degradation occurs through the ubiquitin-proteasome pathway.

Prior studies suggest that individual differences in stress responses contribute to the pathogenesis of neuropsychiatric disorders. In the present study, we investigated the role of small ubiquitin-like modifier (SUMO) E3 ligase protein inhibitor of activated STAT1 (PIAS1) in mediating stress responses to chronic social defeat stress (CSDS). We found that mRNA and protein levels of PIAS 1 were decreased in the hippocampus of high-susceptibility (HS) mice but not in low-susceptibility (LS) mice after CSDS. Local overexpression of PIAS1 in the hippocampus followed by CSDS exposure promoted stress resilience by attenuating social avoidance and improving anxiety-like behaviors. Viral-mediated gene transfer to generate a conditional knockdown of PIAS1 in the hippocampus promoted social avoidance and stress vulnerability after subthreshold microdefeat. HS mice displayed decreased levels of glucocorticoid receptor (GR) expression, and GR SUMOylation in the hippocampus was associated with stress vulnerability. Furthermore, cytokine/chemokine levels were changed predominantly in the hippocampus of HS mice. These results suggest that hippocampal PIAS1 plays a role in the regulation of stress susceptibility by post-translational modification of GRs.

Inhibition of microglial over-activation is an important strategy to counter balance neurodegenerative progression. We previously demonstrated that the adenosine monophosphate-activated protein kinase (AMPK) may be a therapeutic target in mediating anti-neuroinflammatory responses in microglia. Brain-derived neurotrophic factor (BDNF) is one of the major neurotrophic factors produced by astrocytes to maintain the development and survival of neurons in the brain, and have recently been shown to modulate homeostasis of neuroinflammation. Therefore, the present study focused on BDNF-mediated neuroinflammatory responses and may provide an endogenous regulation of neuroinflammation. Among the tested neuroinflammation, epigallocatechin gallate (EGCG) and minocycline exerted BDNF upregulation to inhibit COX-2 and proinflammatory mediator expressions. Furthermore, both EGCG and minocycline upregulated BDNF expression in microglia through AMPK signaling. In addition, minocycline and EGCG also increased expressions of erythropoietin (EPO) and sonic hedgehog (Shh). In the endogenous modulation of neuroinflammation, astrocyte-conditioned medium (AgCM) also decreased the expression of COX-2 and upregulated BDNF expression in microglia. The anti-inflammatory effects of BDNF were mediated through EPO/Shh in microglia. Our results indicated that the BDNF-EPO-Shh novel-signaling pathway underlies the regulation of inflammatory responses and may be regarded as a potential therapeutic target in neurodegenerative diseases. This study also reveals a better understanding of an endogenous crosstalk between astrocytes and microglia to regulate anti-inflammatory actions, which could provide a novel strategy for the treatment of neuroinflammation and neurodegenerative diseases.

Glioblastoma multiforme (GBM) is the most common type of primary and malignant tumor occurring in the adult central nervous system. Temozolomide (TMZ) has been considered to be one of the most effective chemotherapeutic agents to prolong the survival of patients with glioblastoma. Many glioma cells develop drug-resistance against TMZ that is mediated by increasing O-6-methylguanine-DNA methyltransferase (MGMT) levels. The expression of connexin 43 was increased in the resistant U251 subline compared with the parental U251 cells. The expression of epithelial–mesenchymal transition (EMT)-associated regulators, including vimentin, N-cadherin, and β-catenin, was reduced in the resistant U251 subline. In addition, the resistant U251 subline exhibited decreased cell migratory activity and monocyte adhesion ability compared to the parental U251 cells. Furthermore, the resistant U251 subline also expressed lower levels of vascular cell adhesion molecule (VCAM)-1 after treatment with recombinant tumor necrosis factor (TNF)-α. These findings suggest differential characteristics in the drug-resistant GBM from the parental glioma cells.

Traditional assessment tools, such as ''Learning and Study Strategy Scale Inventory (LASSI)'', are typically pen-and-paper tests that require responses to a multitude of questions. This may easily lead to student's resistance, fatigue and unwillingness to complete the assessment. To improve the situation, a hybrid data mining technique was applied to analyze the LASSI surveys of freshmen students at Tamkang University. The most significant contribution of this research is in dynamically reducing the number of questions while the LASSI assessment is proceeding. To verify the appliance of the proposed method, a web-based LASSI self-assessment system (Web-LSA) was developed. This system can be used as a guide to determine study disturbances for high-risk groups, and can provide counselors with fundamental information on which to base follow-up counseling services to its users.

The expression of matrix metalloproteinase-13 (MMP-13) has been shown to be elevated in some pathophysiological conditions and is involved in the degradation of extracellular matrix in astrocytes. In current study, the function of MMP-13 was further investigated. The conditioned medium (CM) collected from activated microglia increased interleukin (IL)-18 production and enhanced MMP-13 expression in astrocytes. Furthermore, treatment with recombinant IL-18 increased MMP-13 protein and mRNA levels in astrocytes. Recombinant IL-18 stimulation also increased the enzymatic activity of MMP-13 and the migratory activity of astrocytes, while administration of MMP-13 or pan-MMP inhibitors antagonized IL-18-induced migratory activity of astrocytes. In addition, administration of recombinant IL-18 to astrocytes led to the phosphorylation of JNK, Akt, or PKCδ, and treatment of astrocytes with JNK, PI3 kinase/Akt, or PKCδ inhibitors significantly decreased the IL-18-induced migratory activity. Taken together, the results suggest that IL-18-induced MMP-13 expression in astrocytes is regulated by JNK, PI3 kinase/Akt, and PKCδ signaling pathways. These findings also indicate that IL-18 is an important regulator leading to MMP-13 expression and cell migration in astrocytes.

Microglial activation has been widely demonstrated to mediate inflammatory processes that are crucial in several neurodegenerative disorders. Pharmaceuticals that can deliver direct inhibitory effects on microglia are therefore considered as a potential strategy to counter balance neurodegenerative progression. Caffeic acid phenethyl ester (CAPE), a natural phenol in honeybee propolis, is known to possess antioxidant, anti-inflammatory and anti-microbial properties. Accordingly, the current study intended to probe the effects of CAPE on microglia activation by using in vitro and in vivo models. Western blot and Griess reaction assay revealed CAPE significantly inhibited the expressions of inducible nitric oxide synthase (NOS), cyclooxygenase (COX)-2 and the production of nitric oxide (NO). Administration of CAPE resulted in increased expressions of hemeoxygenase (HO)-1and erythropoietin (EPO) in microglia. The phosphorylated adenosine monophosphate-activated protein kinase (AMPK)-α was further found to regulate the anti-inflammatory effects of caffeic acid. In vivo results from immunohistochemistry along with rotarod test also revealed the anti-neuroinflammatory effects of CAPE in microglia activation. The current study has evidenced several possible molecular determinants, AMPKα, EPO, and HO-1, in mediating anti-neuroinflammatory responses in microglial cells.

Complete-exchange multicasting is quite useful for numerous applications. We explicate a load-balanced complete-exchange multicast protocol (LCM), based on passive clustering, for wireless ad-hoc networks. From a logical viewpoint, LCM constructs multiple cluster structures to balance the traffic loads among all group members. It further avoids overloading of nodes or traffic congestion on the route. In LCM, an innovative scheme is devised to deal with the scenario in which a node plays different roles for different cluster structures. Simulation results show that LCM not only preserves all the advantages of passive clustering, but also performs very efficiently in complete-exchange multicasting. Consequently, LCM is really a feasible solution for load-balanced complete-exchange multicasting, especially for dense networks.