Rat C6 Glioma Cells Research Articles

VEGF affects mitochondrial ROS generation in glioma cells and acts as a radioresistance factor.

Vascular endothelial growth factor (VEGF) is closely related to angiogenesis. Anticancer therapy by inhibiting VEGF signaling is well established. However, the role of VEGF in cell-cell communication during the response to ionizing radiation is not well understood. Here, we examined the role of VEGF on radiosensitivity of cells. The addition of recombinant VEGF (rVEGF) on cultured rat C6 glioma cells showed a radioprotective effects on X-ray irradiation and reduced oxidative stress. These effects were also observed by endogenous VEGF in supernatant of C6 glioma cells. Reduction of oxidative stress by VEGF is suggested to underlie the radioprotective effects. The mechanism of VEGF-induced reduction of oxidative stress was indicated by a decreased oxygen consumption rate (OCR) in mitochondria. However, the number of DNA double-strand breaks (DSB) immediately after irradiation was not reduced by the treatment with VEGF. These results suggest that VEGF plays a role in cell survival after irradiation by controlling the oxidative condition through mitochondrial function that is independent of the efficiency of DSB induction.

AMPK activation attenuates cancer-induced bone pain by reducing mitochondrial dysfunction-mediated neuroinflammation.

Bone metastasis of cancer cells leads to severe pain by disrupting bone structure and inducing central sensitization. Neuroinflammation in the spinal cord plays a decisive role in the maintenance and development of pain. In the current study, male Sprague-Dawley (SD) rats are used to establish a cancer-induced bone pain (CIBP) model by intratibial injection of MRMT-1 rat breast carcinoma cells. Morphological and behavioral analyses verify the establishment of the CIBP model, which represents bone destruction, spontaneous pain and mechanical hyperalgesia in CIBP rats. Activation of astrocytes marked by upregulated glial fibrillary acidic protein (GFAP) and enhanced production of the proinflammatory cytokine interleukin-1β (IL-1β) are accompanied by increased inflammatory infiltration in the spinal cord of CIBP rats. Furthermore, activation of the NOD-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasome is consistent with increased neuroinflammation. Adenosine monophosphate-activated protein kinase (AMPK) activation is involved in attenuating inflammatory pain and neuropathic pain. Intrathecal injection of the AMPK activator AICAR in the lumbar spinal cord reduces dynamin-related protein 1 (Drp1) GTPase activity and suppresses NLRP3 inflammasome activation. This effect consequently alleviates pain behaviors in CIBP rats. Cell research on C6 rat glioma cells indicates that AICAR treatment restores IL-1β-induced impairment of mitochondrial membrane potential and elevation of mitochondrial reactive oxygen species (ROS). In summary, our findings indicate that AMPK activation attenuates cancer-induced bone pain by reducing mitochondrial dysfunction-mediated neuroinflammation in the spinal cord.

Cytotoxicity and Antitumor Activity of Arglabin and its Derivatives.

At present, more than 8000 sesquiterpene lactones have been isolated and described from natural sources, a significant part of which has cytotoxicity and antitumor activity. One of the practically available sesquiterpene lactones is arglabin, which, as a renewable material, is used for the synthesis of new compounds. The article presents data on the study of cytotoxicity and antitumor activity of the arglabin and its derivatives using molecular modeling methods and, in the experiment in vitro and in vivo. The aim of this work is to study the cytotoxicity and antitumor activity of new compounds based on the sesquiterpene lactone arglabin using molecular modeling and experimental pharmacology. ChemDraw programs and a set of AutoDock programs were used for computer simulation. Molecular docking was carried out using the Maestro graphical interface of the Schrödinger Suite software package (Schrödinger, LLC, New York, NY, 2017). Docking modes standard precision and XP (extra precision) were used. In in vitro experiments, the antitumor activity of compound samples was studied in models of 60 human tumor cell lines, and clonogenic C6 rat glioma cells. The antitumor activity of the samples was studied in experiments in vivo on white outbred rats with transplanted tumors and was evaluated by the inhibition of tumor growth and the magnitude of the increase in average life expectancy. When studying the antitumor activity on 60 cell lines of tumor cells (NCI60), clonogenic cells of C6 rat glioma, a high antitumor activity of some arglabin derivatives was established. The connection between the structure of arglabin derivatives and their inhibitory effect on farnesyl protein transferase, topoisomerases -I and -II was studied.

Commentary on 'Identification and characterization of select oxysterols as ligands for GPR17'.

Commentary on 'Identification and characterization of select oxysterols as ligands for GPR17'.

Exogenous Ceramide Treatment Induce Death and Cytotoxicity in Glioma Cells.

To evaluate the cytotoxic and proapoptotic effects of C6 ceramide on the C6 rat glioma cell line. The C6 rat glioma cell line was evaluated. Using a confocal microscope and the appropriate software, the cytotoxic effects of C6 ceramide were identified using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H tetrazolium bromide (MTT) colorimetric experiments. Transmission electron microscopy (TEM) was utilized to examine the ultrastructural changes following treatment with IC50 concentrations of C6 ceramide. Condensation and fragmentation of nuclei and DNA laddering was observed, indicating apoptotic cell death. C6 ceramide induced apoptosis and effectively caused cytotoxicity in the C6 glioblastoma cells. MTT assay demonstrated > 90% cell death after short-term application of C6 ceramide, confirming its apoptosis-triggering effect. Apoptosis was also confirmed via confocal microscopy and TEM. Glioblastoma cells undergo apoptosis when exposed to C6 ceramide, which makes it a potential chemotherapeutic agent for the treatment of this aggressive brain cancer.

HIF-1α Causes LCMT1/PP2A Deficiency and Mediates Tau Hyperphosphorylation and Cognitive Dysfunction during Chronic Hypoxia.

Chronic hypoxia is a risk factor for Alzheimer's disease (AD), and the neurofibrillary tangle (NFT) formed by hyperphosphorylated tau is one of the two major pathological changes in AD. However, the effect of chronic hypoxia on tau phosphorylation and its mechanism remains unclear. In this study, we investigated the role of HIF-1α (the functional subunit of hypoxia-inducible factor 1) in tau pathology. It was found that in Sprague-Dawley (SD) rats, global hypoxia (10% O2, 6 h per day) for one month induced cognitive impairments. Meanwhile it induced HIF-1α increase, tau hyperphosphorylation, and protein phosphatase 2A (PP2A) deficiency with leucine carboxyl methyltransferase 1(LCMT1, increasing PP2A activity) decrease in the rats' hippocampus. The results were replicated by hypoxic treatment in primary hippocampal neurons and C6/tau cells (rat C6 glioma cells stably expressing human full-length tau441). Conversely, HIF-1α silencing impeded the changes induced by hypoxia, both in primary neurons and SD rats. The result of dual luciferase assay proved that HIF-1α acted as a transcription factor of LCMT1. Unexpectedly, HIF-1α decreased the protein level of LCMT1. Further study uncovered that both overexpression of HIF-1α and hypoxia treatment resulted in a sizable degradation of LCMT1 via the autophagy--lysosomal pathway. Together, our data strongly indicated that chronic hypoxia upregulates HIF-1α, which obviously accelerated LCMT1 degradation, thus counteracting its transcriptional expression. The increase in HIF-1α decreases PP2A activity, finally resulting in tau hyperphosphorylation and cognitive dysfunction. Lowering HIF-1α in chronic hypoxia conditions may be useful in AD prevention.

In Vitro Cytotoxic Effects and Mechanisms of Action of Eleutherine Isolated from Eleutherine plicata Bulb in Rat Glioma C6 Cells.

Gliomas are the most common primary malignant brain tumors in adults, and have a poor prognosis, despite the different types of treatment available. There is growing demand for new therapies to treat this life-threatening tumor. Quinone derivatives from plants have received increased interest as potential anti-glioma drugs, due to their diverse pharmacologic activities, such as inhibiting cell growth, inflammation, tumor invasion, and promoting tumor regression. Previous studies have demonstrated the anti-glioma activity of Eleutherine plicata, which is related to three main naphthoquinone compounds-eleutherine, isoeleutherine, and eleutherol-but their mechanism of action remains elusive. Thus, the aim of this study was to investigate the mechanism of action of eleutherine on rat C6 glioma. In vitro cytotoxicity was evaluated by MTT assay; morphological changes were evaluated by phase-contrast microscopy. Apoptosis was determined by annexin V-FITC-propidium iodide staining, and antiproliferative effects were assessed by wound migration and colony formation assays. Protein kinase B (AKT/pAKT) expression was measured by western blot, and telomerase reverse transcriptase mRNA was measured by quantitative real-time polymerase chain reaction (qRT-PCR). Eleutherine reduced C6 cell proliferation in a dose-dependent manner, suppressed migration and invasion, induced apoptosis, and reduced AKT phosphorylation and telomerase expression. In summary, our results suggest that eleutherine has potential clinical use in treating glioma.

New class of fused [3,2-b][1,2,4]triazolothiazoles for targeting glioma in vitro

Glioma is aggressive malignant tumor with limited therapeutic interventions. Herein we report the synthesis of fused bicyclic 1,2,4-triazolothiazoles by a one-pot multi-component approach and their activity against C6 rat and LN18 human glioma cell lines. The target compounds 2-(6-phenylthiazolo[3,2–b][1,2,4]triazol-2-yl) isoindoline-1,3-diones and (E)-1-phenyl-N-(6-phenylthiazolo[3,2–b][1,2,4]triazol-2-yl) methanimines were obtained by the reaction of 5-amino-4H-1,2,4-triazole-3-thiol with substituted phenacyl bromide, phthalic anhydride, and different aromatic aldehydes in EtOH/HCl under reflux conditions. In C6 rat glioma cell lines, compounds 4g and 6i showed good cytotoxic activity with IC50 values of 8.09 and 8.74 μM, respectively, resulting in G1 and G2-M phase arrest of the cell cycle and activation of apoptosis by modulating phosphorylation of ERK and AKT pathway.

Impact of PEGylated Liposomal Doxorubicin and Carboplatin Combination on Glioblastoma.

Glioblastoma is an incurable cancer with a 5-year survival chance of less than 5%. Chemotherapy is a therapeutic approach to treating the disease; however, due to the presence of the blood–brain barrier (BBB), the probability of success is low. To overcome this issue, nanoparticles are promising carriers for crossing the BBB and delivering drugs to the tumor. In this study, the anticancer efficacy of doxorubicin (DOX) and carboplatin (CB) loaded into polyethylene glycol (PEG)ylated liposome nanoparticles (PEG-Lip) and in treating brain cancer was evaluated in vitro and in vivo. The results demonstrated that PEG-Lip-DOX/CB with a size of 212 ± 10 nm was synthesized that could release the loaded drugs in a controlled manner, from which 56.3% of the loaded drugs were released after 52 h. In addition, PEG-Lip-DOX/CB could significantly increase the cytotoxicity effects of the drugs against rat glioma C6 cells (IC50: 8.7 and 12.9 µM for the drugs-loaded nanoparticles and DOX + CB, respectively). The in vivo results also demonstrated that PEGylated liposomes, compared to non-PEGylated liposomes (Lip) and DOX + CB, were more efficient in increasing the therapeutic effects and decreasing the side effects of the drugs, in which the survival times of the glioblastoma-bearing rats were 39, 35, and 30 days in the PEG-Lip-DOX/CB, Lip-DOX/CB, and DOX + CB receiver groups, respectively. In addition, the weight loss was found to be 8.7, 10.5, and 13%, respectively, in the groups. The results of the toxicity evaluation were also confirmed by histopathological studies. Overall, the results of this study demonstrated that the encapsulation of DOX and CB into PEG-Lip is a promising approach to improving the properties of DOX and CB in terms of their therapeutic effects and drug side effects for the treatment of glioblastoma.

Exposure of aluminium to C6 glioma cells modulates molecular and functional neurotoxic markers.

The risk of aluminium exposure to humans is very high as it may get into the human body through excessive dietary contaminants, inhalation of fine particulate matter, or through parenteral routes as a vaccine adjuvant and so forth. The increased level of aluminium in brain tissue has been shown to be associated with several neurodegenerative and neurotoxic adverse effects, including AD. However, the exact mechanism of aluminium-induced neurotoxicity is still unclear. Therefore, our study aimed to investigate the mechanism of neurotoxic and neurodegenerative effects through in vitro exposure of aluminium in rat glioma C6 cell line. The findings of our study have indicated that aluminium chloride exposure may lead to alteration in acetylcholine levels, increased oxidative imbalanceand induction of molecular structural and functional markers of neuronal inflammation. This study also demonstrated that aluminium exposure may lead to the induction of caspase-3 along with apoptotic cell death and a significant increase in amyloid-beta and hyperphosphorylated tau levels in C6 cells. Thus, this study may provide a mechanistic understanding of the regulation of neuroinflammatory and neurodegenerative biomarkers due to aluminium exposure.

The Effects of Methanol Extracts of Hyssopus officinalis on Model of Induced Glioblastoma Multiforme (GBM) in Rats.

Given the complexity of pathophysiological processes of brain tumors, ineffective therapies, and high mortality rate, new therapeutic options with less toxicity are necessary. Hyssopus officinalis (hyssop) is an aromatic plant with important biological activities. The aim of this study is to assess the anti-cancer effect of hyssop extract on damages of glioblastoma multiforme. In this study, total flavonoids, phenolic content, and quantification of phenolic compound of hyssop extracts were analyzed. In vitro antioxidant properties of hyssop extract were also examined. In addition, cell viability, apoptosis, and cell cycle were evaluated in C6 glioma cell culture. In vivo, the rats were divided randomly into four main groups: intact, control, vehicle, and treatment groups. 1 × 106 C6 rat glioma cells were implanted into the right caudate nucleus of the rat's brain. The treatment group received the methanol extract of hyssop (100mg/kg) for 7days. Evolution of locomotor activity, tumor volume, survival rate, activities of antioxidant enzymes (superoxide dismutase (SOD) and catalase (CAT)), vascular endothelial growth factor (VEGF) expression, TUNEL-positive cells, p53 and p21 mRNA expression, and histological alterations were performed. The results showed that the methanol extract of hyssop increased the apoptosis and reduced the cell division of C6 glioma cells in cell culture. Moreover, methanol extract decreased the tumor volume and prolonged survival. Also, the activity of SOD and CAT enzymes was reduced in tumor tissue and enhanced in surrounding tissue. TUNEL-positive cells were increased in methanol extract of hyssop group. The expression of p53 and p21 mRNA was upregulated in the treatment group. Moreover, the histological analysis indicated a considerable decrease in invasion of tumor cells and inflammation in the hyssop-treated rats. According to the achieved results, it can be stated that hyssop has sufficient potential to inhibit damage of brain tumors, at least in part, by affecting the oxidative stress and cell proliferation pathways.

Influence of the properties of different graphene-based nanomaterials dispersed in polycaprolactone membranes on astrocytic differentiation

Composites of polymer and graphene-based nanomaterials (GBNs) combine easy processing onto porous 3D membrane geometries due to the polymer and cellular differentiation stimuli due to GBNs fillers. Aiming to step forward to the clinical application of polymer/GBNs composites, this study performs a systematic and detailed comparative analysis of the influence of the properties of four different GBNs: (i) graphene oxide obtained from graphite chemically processes (GO); (ii) reduced graphene oxide (rGO); (iii) multilayered graphene produced by mechanical exfoliation method (Gmec); and (iv) low-oxidized graphene via anodic exfoliation (Ganodic); dispersed in polycaprolactone (PCL) porous membranes to induce astrocytic differentiation. PCL/GBN flat membranes were fabricated by phase inversion technique and broadly characterized in morphology and topography, chemical structure, hydrophilicity, protein adsorption, and electrical properties. Cellular assays with rat C6 glioma cells, as model for cell-specific astrocytes, were performed. Remarkably, low GBN loading (0.67 wt%) caused an important difference in the response of the C6 differentiation among PCL/GBN membranes. PCL/rGO and PCL/GO membranes presented the highest biomolecule markers for astrocyte differentiation. Our results pointed to the chemical structural defects in rGO and GO nanomaterials and the protein adsorption mechanisms as the most plausible cause conferring distinctive properties to PCL/GBN membranes for the promotion of astrocytic differentiation. Overall, our systematic comparative study provides generalizable conclusions and new evidences to discern the role of GBNs features for future research on 3D PCL/graphene composite hollow fiber membranes for in vitro neural models.

Novel read-through fusion transcript Bcl2l2-Pabpn1 in glioblastoma cells.

Read‐through fusion transcripts have recently been identified as chimeric RNAs and have since been linked to tumour growth in some cases. Many fusion genes generated by chromosomal rearrangements have been described in glioblastoma. However, read‐through fusion transcripts between neighbouring genes in glioblastoma remain unexplored. We performed paired‐end RNA‐seq of rat C6 glioma cells and normal cells and discovered a read‐through fusion transcript Bcl2l2‐Pabpn1 in which exon 3 of Bcl‐2‐like protein 2 (Bcl2l2) fused to exon 2 of Polyadenylate‐binding protein 1 (Pabpn1). This fusion transcript was found in both human glioblastoma and normal cells. Unlike other fusions reported in glioblastoma, Bcl2l2‐Pabpn1 appeared to result from RNA processing rather than genomic rearrangement. Bcl2l2‐Pabpn1 fusion transcript encoded a fusion protein with BH4, BCL and RRM domains. Functionally, Bcl2l2‐Pabpn1 knockdown by targeting its fusion junction decreased its expression, and suppressed cell proliferation, migration and invasion in vitro. Mechanistically, Bcl2l2‐Pabpn1 blocked Bax activity and activated PI3K/AKT pathway to promote glioblastoma progression. Together, our work characterized a glioblastoma‐associated Bcl2l2‐Pabpn1 fusion transcript shared by humans and rats.

Antitumor activity of irinotecan with ellagic acid in C6 glioma cells.

SUMMARYOBJECTIVE:Irinotecan-based combination chemotherapies in malignant gliomas need to be examined. The aim of this study was to investigate the synergetic effect of ellagic acid, a natural polyphenolic antioxidant compound, with irinotecan, an inhibitor of topoisomerase I enzyme, on the growth, cadherin switch, and angiogenic processes of a glioma cell line.METHODS:A combination of 100 μM ellagic acid and 100 μM irinotecan was applied to rat C6 glioma cells for 24th, 48th, and 72nd h. The cell proliferation was evaluated by 5-bromo-2′-deoxyuridine immunocytochemistry. The expression levels of vascular endothelial growth factor, E-cadherin, and N-cadherin were measured using real-time polymerase chain reaction and their immunoreactivities using immunocytochemistry.RESULTS:The treatment of irinotecan with combining ellagic acid enhanced antitumor activity and the synergistic effect of these reduced the cell proliferation of C6 glioma by inhibiting the cadherin switch and promoting the antiangiogenic processes.CONCLUSIONS:Further research is required to prove a negative relationship between C6 glial cell proliferation and irinotecan with ellagic acid application. Our preliminary data suggest that even with the extremely short-term application, irinotecan with ellagic acid may affect glioma cells at the level of gene and protein expression.

Investigation of the Anticancer Effect of S-Allyl-L Cysteine on the C6 Rat Glioma Cell Line in vitro in 2D- and 3D-Cell Culture Models.

In our study, we aimed to investigate the effects of SAC on C6 glioblastoma cells and its antiproliferative and apoptotic effects on two- and three-dimensional cell culture systems. The rat glioma cell line C6 was separated into 2D-Control, 2D-SAC, 3D-CMC-Control and 3D-CMC-SAC groups. While control cells were incubated under standard culture conditions, cells in the SAC groups were incubated with the IC50 dose (50 µM for both the 2D-SAC C6 and 3D-CMC-SAC groups) of SAC in culture medium for 24 and 48 hours. Thereafter, all groups were stained with antibodies recognizing NOTCH1 and JAGGED1, and the mRNA expression levels of NOTCH1 and JAGGED1 were evaluated by qRT-PCR. Increasing doses of SAC were administered for 24 hours in the C6 glioma cell line. The concentration of 50 μM was chosen as the most suitable dose for administration. The gene expression profiles were different in the two forms of culture. NOTCH1 receptor mRNA expression levels were significantly decreased in cells treated with 50 μM SAC for 24 hours compared to control cells in both the two-dimensional (2D) and three-dimensional (3D) cell cultures. The immunoreactivities of both of these markers (JAGGED1 and NOTCH1) were significantly decreased in the glioma cells in the SAC group compared to the control group. This study shows that SAC has potential as a drug for human use, as indicated by its nontoxic nature. The present study confirmed the anticancer activity of SAC by modulating NOTCH1 and JAGGED1 signaling in glioma cancer cells.

In silico analysis of expression and DNA methylation profiles of NLRP13 inflammasome in tumor cells

BackgroundNOD-like receptor proteins (NLRs) are essential innate pattern recognition receptors that play fundamental roles in inflammation, and they have been associated with many cancers. NLRP13 (NLR Family Pyrin Domain Containing 13) belongs to the NLRP protein family, but its biological function is not as well defined as other related proteins in this family. Methods and resultsThe expression profile and CpG-aggregated methylation status of the NLRP13 gene were determined using GENT2 and TCGA online platforms. Samples from patients with different tumor stages were used to evaluate the promoter methylation status of NLRP13 in cancer versus normal tissues in a tumor stage-dependent manner by using UALCAN server. We further studied NLRP13 expression in response to inflammatory and hypomethylating agents in two distinct cancer cell lines, HT-29 (human colon adenocarcinoma) and C6 (rat glioma cells), following the treatment with LPS and/or a hypomethylating agent, decitabine, to reveal NLRP13 methylation profiles in these cell lines. ConclusionsUsing bioinformatics tools, we found that NLRP13 expression is higher in healthy breast, head and neck, prostate tissues compared to tumor tissues, whereas its expression is significantly higher in colorectal adenocarcinoma tissue than in normal colon tissue. We also found that NLRP13 gene is mostly hypomethylated in cancer tissues compared to normal tissues, and its high expression is linked with worse overall survival depending on cancer type. Based on data from UALCAN database, NLRP13 promoter methylation is similarly decreased in many cancer patients depending on individual cancer stages. Protein-protein interactions (PPIs) data indicated that NLRP13 showed a confidence interaction with MEFV, NLRC4 and CASP1 proteins, and gene ontology terms associated with functional partners of NLRP13 were also identified in the current study. Our results further showed that NLRP13 expression is regulated by DNA methylation, and treatment with decitabine might effect NLRP13-mediated inflammatory responses in cancer since LPS-induced pro-inflammatory responses specified by IL-1β, IL-18 and caspase-1 levels change in certain cases in cancer cell lines following the pretreatment with this epigenetic modulator. Our study is the first to provide evidence for the differential expression and epigenetic regulation of the NLPR13 in multiple cancer types.

Methylmalonic Acid Impairs Cell Respiration and Glutamate Uptake in C6 Rat Glioma Cells: Implications for Methylmalonic Acidemia.

Methylmalonic acidemia is an organic acidemia caused by deficient activity of L-methylmalonyl-CoA mutase or its cofactor cyanocobalamin and it is biochemically characterized by an accumulation of methylmalonic acid (MMA) in tissue and body fluids of patients. The main clinical manifestations of this disease are neurological and observable symptoms during metabolic decompensation are encephalopathy, cerebral atrophy, coma, and seizures, which commonly appear in newborns. This study aimed to investigate the toxic effects of MMA in a glial cell line presenting astrocytic features. Astroglial C6 cells were exposed to MMA (0.1-10mM) for 24 or 48h and cell metabolic viability, glucose consumption, and oxygen consumption rate, as well as glutamate uptake and ATP content were analyzed. The possible preventive effects of bezafibrate were also evaluated. MMA significantly reduced cell metabolic viability after 48-h period and increased glucose consumption during the same period of incubation. Regarding the energy homeostasis, MMA significantly reduced respiratory parameters of cells after 48-h exposure, indicating that cell metabolism is compromised at resting and reserve capacity state, which might influence the cell capacity to meet energetic demands. Glutamate uptake and ATP content were also compromised after exposure to MMA, which can be influenced energy metabolism impairment, affecting the functionality of the astroglial cells. Our findings suggest that these effects could be involved in the pathophysiology of neurological dysfunction of this disease. Methylmalonic acid compromises mitochondrial functioning leading to reduced ATP production and reduces glutamate uptake by C6 astroglial cells.

Targeted gene delivery to the brain using CDX-modified chitosan nanoparticles.

Introduction: Blood-brain barrier with strictly controlled activity participates in a coordinated transfer of bioactive molecules from the blood to the brain. Among different delivery approaches, gene delivery is touted as a promising strategy for the treatment of several nervous system disorders. The transfer of exogenous genetic elements is limited by the paucity of suitable carriers. As a correlate, designing high-efficiency biocarriers for gene delivery is challenging. This study aimed to deliver pEGFP-N1 plasmid into the brain parenchyma using CDX-modified chitosan (CS) nanoparticles (NPs). Methods: Herein, we attached CDX, a 16 amino acids peptide, to the CS polymer using bifunctional polyethylene glycol (PEG) formulated with sodium tripolyphosphate (TPP), by ionic gelation method. Developed NPs and their nanocomplexes with pEGFP-N1 (CS-PEG-CDX/pEGFP) were characterized using DLS, NMR, FTIR, and TEM analyses. For in vitro assays, a rat C6 glioma cell line was used for cell internalization efficiency. The biodistribution and brain localization of nanocomplexes were studied in a mouse model after intraperitoneal injection using in vivo imaging and fluorescent microscopy. Results: Our results showed that CS-PEG-CDX/pEGFP NPs were uptaken by glioma cells in a dose-dependent manner. In vivo imaging revealed successful entry into the brain parenchyma indicated with the expression of green fluorescent protein (GFP) as a reporter protein. However, the biodistribution of developed NPs was also evident in other organs especially the spleen, liver, heart, and kidneys. Conclusion: Based on our results, CS-PEG-CDX NPs can provide a safe and effective nanocarrier for brain gene delivery into the central nervous system (CNS).

Apoptotic Effect of Novel Benzimidazole Derivatives Bearing Pyridyl/Pyrimidinyl Piperazine Moiety.

Benzimidazole derivatives bearing pyridyl/pyrimidinyl piperazine moiety has attracted attention in medicinal chemistry and modern drug discovery since it exhibited a variety of biological activities, including anticancer activity. In this study, we designed and synthesized novel 1-[2-oxo-2-(4-substituted phenyl)ethyl]benzimidazol-2- yl)methyl 4-(2-pyridyl/pyrimidin-2-yl)piperazine-1-carbodithioate derivatives (2a-m). We also investigated their anticancer activities against A549 lung adenocarcinoma and C6 rat glioma cell lines. We further studied the selectivity of the compounds against the NIH/3T3 mouse embryonic fibroblast cell line. Cholinesterase inhibition effects of these compounds were also investigated to measure the relationship between anticancer activity and cholinesterases. The cytotoxic activities of these acquired thirteen final compounds were screened using MTT assay on A549, C6, and NIH/3T3 cell lines. Cell proliferation ELISA, BRDU (colorimetric) assay was used to measure the proliferation in replicative cells in which DNA synthesis occurs. Flow cytometric analysis was used to measure apoptotic cell percentages, caspase 3 activity, and mitochondrial membrane depolarised cell percentages. Compounds 2e, 2f, and 2k were shown to be the most active antitumor agents with selective cytotoxicities (the results for A549 were 76.58±6.43, 55.13±5.75, and 32.94±3.02 μM, respectively; and for C6 they were 86.48±3.60, 97.12±30.21, and 59.29±3.95 μM, respectively), high DNA synthesis inhibition rates and high apoptotic cell percentages on both cell lines. The results showed that compounds 2e, 2f, and 2k have potential anticancer activity against A549 and C6 cell lines.

Glioma facilitates the epileptic and tumor-suppressive gene expressions in the surrounding region

Patients with glioma often demonstrate epilepsy. We previously found burst discharges in the peritumoral area in patients with malignant brain tumors during biopsy. Therefore, we hypothesized that the peritumoral area may possess an epileptic focus and that biological alterations in the peritumoral area may cause epileptic symptoms in patients with glioma. To test our hypothesis, we developed a rat model of glioma and characterized it at the cellular and molecular levels. We first labeled rat C6 glioma cells with tdTomato, a red fluorescent protein (C6-tdTomato), and implanted them into the somatosensory cortex of VGAT-Venus rats, which specifically expressed Venus, a yellow fluorescent protein in GABAergic neurons. We observed that the density of GABAergic neurons was significantly decreased in the peritumoral area of rats with glioma compared with the contralateral healthy side. By using a combination technique of laser capture microdissection and RNA sequencing (LCM-seq) of paraformaldehyde-fixed brain sections, we demonstrated that 19 genes were differentially expressed in the peritumoral area and that five of them were associated with epilepsy and neurodevelopmental disorders. In addition, the canonical pathways actively altered in the peritumoral area were predicted to cause a reduction in GABAergic neurons. These results suggest that biological alterations in the peritumoral area may be a cause of glioma-related epilepsy.