C6 Glioma Cells Research Articles

Iboga Inspired N-Indolylethyl-Substituted Isoquinuclidines as a Bioactive Scaffold: Chemoenzymatic Synthesis and Characterization as GDNF Releasers and Antitrypanosoma Agents.

The first stage of the drug discovery process involves the identification of small compounds with biological activity. Iboga alkaloids are monoterpene indole alkaloids (MIAs) containing a fused isoquinuclidine-tetrahydroazepine ring. Both the natural products and the iboga-inspired synthetic analogs have shown a wide variety of biological activities. Herein, we describe the chemoenzymatic preparation of a small library of novel N-indolylethyl-substituted isoquinuclidines as iboga-inspired compounds, using toluene as a starting material and an imine Diels–Alder reaction as the key step in the synthesis. The new iboga series was investigated for its potential to promote the release of glial cell line-derived neurotrophic factor (GDNF) by C6 glioma cells, and to inhibit the growth of infective trypanosomes. GDNF is a neurotrophic factor widely recognized by its crucial role in development, survival, maintenance, and protection of dopaminergic neuronal circuitries affected in several neurological and psychiatric pathologies. Four compounds of the series showed promising activity as GDNF releasers, and a leading structure (compound 11) was identified for further studies. The same four compounds impaired the growth of bloodstream Trypanosoma brucei brucei (EC50 1–8 μM) and two of them (compounds 6 and 14) showed a good selectivity index.

Heparan Sulfate Proteoglycans (HSPGs) Serve as the Mediator Between Monomeric Tau and Its Subsequent Intracellular ERK1/2 Pathway Activation.

The conversion of soluble tau protein to insoluble, hyperphosphorylated neurofibrillary tangles (NFTs) is a major hallmark leading to neuronal death observed in neurodegenerative tauopathies. Unlike NFTs, the involvement of monomeric tau in the progression of tau pathology has been less investigated. Using live-cell confocal microscopy and flow cytometry, we demonstrate that soluble 0N4R monomers were rapidly endocytosed by SH-SY5Y and C6 glioma cells via actin-dependent macropinocytosis. Further, cellular endocytosis of monomeric tau has been demonstrated to be HSPG-dependent, as shown in C6 glial cells with genetic knockouts of xylosyltransferase-1—a key enzyme in HSPG synthesis—with a reduced level of tau uptake. Tau internalization subsequently triggers ERK1/2 activation and therefore, the upregulation of IL-6 and IL-1β. The role of ERK1/2 in regulating the levels of pro-inflammatory gene transcripts was confirmed by inhibiting the MEK-ERK1/2 signaling pathway, which led to the attenuated IL-6 and IL-1β expressions but not that of TNF-α. Moreover, as a key regulator of tau internalization, LRP1 (low-density lipoprotein receptor-related protein 1) levels were downregulated in response to monomeric tau added to C6 cells, while it was upregulated in HSPG-deficient cells, suggesting that the involvement of LRP1 in tau uptake depends on the presence of HSPGs on the cell surface. The subsequent LRP1 knockdown experiment we performed shows that LRP1 deficiency leads to an attenuated propensity for tau uptake and further elevated IL-6 gene expression. Collectively, our data suggest that tau has multiple extracellular binding partners that mediate its internalization through distinct mechanisms. Additionally, this study demonstrates the important role of both HSPGs and LRP1 in regulating cellular immune responses to tau protein monomers, providing a novel target for alleviating the neuroinflammatory environment before the formation of neurofibrillary tangles.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12031-021-01943-2.

In Vitro Evaluation of the Cytotoxic Effect of Streptococcus pyogenes Strains, Protegrin PG-1, Cathelicidin LL-37, Nerve Growth Factor and Chemotherapy on the C6 Glioma Cell Line.

Brain cancer treatment, where glioblastoma represents up to 50% of all CNS malignancies, is one of the most challenging calls for neurooncologists. The major driver of this study was a search for new approaches for the treatment of glioblastoma. We tested live S. pyogenes, cathelicidin family peptides and NGF, assessing the oncolytic activity of these compounds as monotherapy or in combination with chemotherapeutics. For cytotoxicity evaluation, we used the MTT assay, trypan blue assay and the xCELLigence system. To evaluate the safety of the studied therapeutic approaches, we performed experiments on normal human fibroblasts. Streptococci and peptides demonstrated high antitumor efficiency against glioma C6 cells in all assays applied, surpassing the effect of chemotherapeutics (doxorubicin, carboplatin, cisplatin, etoposide). A real-time cytotoxicity analysis showed that the cell viability index dropped to 21% 2–5 h after S. pyogenes strain exposure. It was shown that LL-37, PG-1 and NGF also exhibited strong antitumor effects on C6 glioma cells when applied at less than 10−4 M. Synergistic effects for combinations of PG-1 with carboplatin and LL-37 with etoposide were shown. Combinations of S. pyogenes strain #7 with NGF or LL-37 demonstrated a cytotoxic effect (56.7% and 57.3%, accordingly) on C6 glioma cells after 3 h of exposure.

Exploring the efficacy of ethylene glycol dimethacrylate crosslinked cationised pullulan for gene delivery in cancer cells

The balance between cytotoxicity and transfection efficiency is essential for any cationic polymers intended for gene delivery. However, increase in transfection efficiency of these cationic polymers is usually associated with an increase in the toxicity owing to the high positive surface charge. In this study pullulan was cationised with polyethyleneimine (pullulan-PEI) and was cross-linked with ethylene glycol dimethacrylate (EGDMA). Crosslinked pullulan –PEI (PPE) was found to be cytocompatible and formed nanoplexes of size of about 150 nm and 9.96 mV. The presence of EGDMA groups reduced the plasma protein interactions as well. The nanoplex uptake and transfection efficacy studies of PPE were done in C6 and HeLa cells. The interesting observation was that these nanoplexes exhibited differential uptake in these cell lines. The cellular uptake was very high in C6 glioma cells where as it was very low in HeLa and consequently gene expression was observed in the former cells only. Transfection efficacy of PPE derivatives was evaluated using p53 plasmid whose expression induces apoptosis in the cancerous cells. Immunostaining experiments and flow cytometry analysis following Annexin V staining confirmed the expression of p53 protein and apoptosis respectively in the PPEI/p53 poyplex transfected C6 cells.

Synergistic therapy for orthotopic gliomas via biomimetic nanosonosensitizer-mediated sonodynamic therapy and ferroptosis.

Ferroptosis is an emerging form of programmed cell death, and its combination with sonodynamic therapy (SDT) for anti-tumor activity is gradually attracting attention. However, their application against gliomas has not been studied. Herein, multifunctional cancer homologous targeting biomimetic nanoparticles (PIOC@CM NPs) encapsulating both Fe3O4 and Ce6 were constructed as a nanosonosensitizer. Based on focused ultrasound (US) combined with circulating microbubbles (MBs) to open the blood-brain barrier (BBB) in a safe and transient manner, the development of a therapeutic strategy to integrate the biomimetic nanosonosensitizer-mediated SDT and ferroptosis could achieve synergistic therapeutic effects against gliomas. We demonstrated that the glioma C6 cell membrane (CM) on the surface of the NPs allowed the nanosonosensitizer to accumulate selectively in tumors through homologous targeting in vitro. After efficient internalization in C6 cells, the PIOC@CM NPs could significantly increase the level of reactive oxygen species (ROS) and deplete glutathione (GSH) upon ultrasonic irradiation, resulting in the loss of glutathione peroxidase-4 (GPX4) activity, which facilitated SDT and ferroptosis to kill glioma C6 cells. Furthermore, the PIOC@CM NPs were intravenously injected after noninvasively opening the BBB via US-MBs, which enhanced the accumulation of the nanosonosensitizer in tumor tissues. Crucially, an attractive phenomenon of the significant reduction in orthotopic gliomas after the second US pulse-triggered SDT and ferroptosis was observed. Taken together, this study presents a novel combinatorial glioma therapeutic strategy based on noninvasive BBB opening with a biomimetic sonotheranostic system-mediated SDT and ferroptosis.

Preparation of resveratrol containing poly (lactic-co-glycolic acid) nanoparticles and investigation of its cytotoxic effect in C6 glioma cells

Preparation of resveratrol containing poly (lactic-co-glycolic acid) nanoparticles and investigation of its cytotoxic effect in C6 glioma cells

Angiopep-2-decorated titanium-alloy core-shell magnetic nanoparticles for nanotheranostics and medical imaging.

The poor permeability of therapeutic agents across the blood-brain barrier and blood-tumor barrier is a significant barrier in glioma treatment. Low-density lipoprotein receptor-related protein (LRP-1) recognises a dual-targeting ligand, angiopep-2, which is overexpressed in the BBB and gliomas. Here, we have synthesized Ti@FeAu core-shell nanoparticles conjugated with angiopep-2 (Ti@FeAu-Ang nanoparticles) to target glioma cells and treat brain cancer via hyperthermia produced by a magnetic field. Our results confirmed that Ti@FeAu core-shell nanoparticles were superparamagnetic, improved the negative contrast effect on glioma, and exhibited a temperature elevation of 12° C upon magnetic stimulation, which implies potential applications in magnetic resonance imaging (MRI) and hyperthermia-based cancer therapy. Angiopep-2-decorated nanoparticles exhibited higher cellular uptake by C6 glioma cells than by L929 fibroblasts, demonstrating selective glioma targeting and improved cytotoxicity up to 85% owing to hyperthermia produced by a magnetic field. The in vivo findings demonstrated that intravenous injection of Ti@FeAu-Ang nanoparticles exhibited a 10-fold decrement in tumor volume compared to the control group. Furthermore, immunohistochemical analysis of Ti@FeAu-Ang nanoparticles showed that coagulative necrosis of tumor tissues and preliminary safety analysis highlighted no toxicity to the haematological system, after Ti@FeAu-Ang nanoparticle-induced hyperthermia treatment.

Organophosphates modulate tissue transglutaminase activity in differentiated C6 neural cells.

The organophosphate compounds chlorpyrifos (O, O-diethyl O-[3,5,6-trichloro-2-pyridinyl] phosphorothioate, CPF) and phenyl saligenin phosphate (PSP) have been widely implicated in developmental neurotoxicity and neurodegeneration. However, the underlying mechanism remains unclear. Transglutaminase (TG)2 is a calcium ion (Ca2+)-dependent enzyme with an important role in neuronal cell outgrowth and differentiation and in neurotoxin activity and is modulated by organophosphates. We studied TG2 activity modulation by CPO and PSP during differentiation in C6 glioma cells. We studied the effects of CPO or PSP treatment with or without the TG2 inhibitor Z-DON and identified potential TG2 protein substrates via mass spectrometry. PSP and CPO did not affect cell viability but affected TG2 activity in differentiating cells. Our results indicate that the organophosphate-induced amine incorporation activity of TG2 may have a direct effect on neuronal outgrowth, differentiation, and cell survival by modifying several essential microtubule proteins, including tubulin. Inhibiting TG2 reduced neurite length but not cell survival. TG2 inhibitors can protect against organophosphate-induced neuropathy and could be used for developing novel therapeutic strategies for treating brain cancer and neurodegenerative disorders.

Chemical engineering methods in analyses of 3D cancer cell cultures: Hydrodinamic and mass transport considerations

A multidisciplinary approach based on experiments and mathematical modeling was used in biomimetic system development for three-dimensional (3D) cultures of cancer cells. Specifically, two cancer cell lines, human embryonic teratocarcinoma NT2/D1 and rat glioma C6, were immobilized in alginate microbeads and microfibers, respectively, and cultured under static and flow conditions in perfusion bioreactors. At the same time, chemical engineering methods were applied to explain the obtained results. The superficial medium velocity of 80 ?m s-1 induced lower viability of NT2/D1 cells in superficial microbead zones, implying adverse effects of fluid shear stresses estimated as ?67 mPa. On the contrary, similar velocity (100 ?m s-1) enhanced the proliferation of C6 glioma cells within microfibers compared to static controls. An additional study of silver release from nanocomposite Ag/honey/alginate microfibers under perfusion indicated that the medium partially flows through the hydrogel (interstitial velocity of ?10 nm s-1). Thus, a diffusion-advection-reaction model described the mass transport to immobilized cells within microfibers. Substances with diffusion coefficients of ?10-9-10-11 m2 s-1 are sufficiently supplied by diffusion only, while those with significantly lower diffusivities (?10-19 m2 s-1) require additional convective transport. The present study demonstrates the selection and contribution of chemical engineering methods in tumor model system development.

Lomustine Incorporated Lipid Nanostructures Demonstrated Preferential Anticancer Properties in C6 Glioma Cell Lines with Enhanced Pharmacokinetic Profile in Mice.

Effective treatment of glioma still stands as a challenge in medical science. The work aims for the fabrication and evaluation of lipid based nanostructures for improved delivery of lomustine to brain tumor cells. Experimental formulations (LNLs) were developed by modified lipid layer hydration technique and evaluated for different in vitro characteristics like particle size analysis, surface charge, surface morphology, internal structure, in vitro drug loading, drug release profile etc. Anticancer potential of selected LNLs was tested in vitro on C6 glioma cell line. Electron microscopic study depicted a size of less than 50 nm for the selected LNLs along 8.8% drug loading with a sustained drug release tendency over 48 h study period. Confocal microscopy revealed extensive internalization of the selected LNL in C6 cells. LNLs were found more cytotoxic than free drug and blank nanocarriers as depicted from MTT assay. The selected LNL showed improved pharmacokinetic profile both in blood and brain in the experimental mice models along with negligible hemolysis in mice blood cells. Further studies are warranted for the future translation of LNLs at clinics.

Suppression of PI3K/Akt/mTOR pathway in chrysoeriol-induced apoptosis of rat C6 glioma cells.

Chrysoeriol, a dietary methoxyflavonoid which is found in tropical medicinal plants, has been shown to have antioxidant, anti-inflammatory, and antineoplastic properties. The present study aimed to investigate the effects of chrysoeriol and its related mechanisms in rat C6 glioma cells. Cell viability in rat C6 glioma cells were measured by MTT assay. The protein expression levels of cleaved caspase-3, caspase-3, pro-apoptotic (Bax), anti-apoptotic protein (Bcl-2), and Annexin V were detected by Western blot analysis and immunocytochemical staining. Results showed that chrysoeriol significantly decreased cell viability and induced apoptosis in rat C6 glioma cells. Chrysoeriol significantly increased the levels of Bax/Bcl-2 ratio and cleaved caspase-3/caspase-3 ratio. Moreover, treatment with chrysoeriol significantly reduced the phosphorylation of PI3K, Akt, and mTOR expression in ratios. These results suggest that chrysoeriol promote apoptosis in rat C6 glioma cells via suppression of the PI3K/Akt/mTOR signaling pathway, thereby demonstrating the potential antineoplastic effects of chrysoeriol on glioma cells.

Structural Design, Synthesis and Antioxidant, Antileishmania, Anti-Inflammatory and Anticancer Activities of a Novel Quercetin Acetylated Derivative.

Quercetin (Q) is a bioflavonoid with biological potential; however, poor solubility in water, extensive enzymatic metabolism and a reduced bioavailability limit its biopharmacological use. The aim of this study was to perform structural modification in Q by acetylation, thus, obtaining the quercetin pentaacetate (Q5) analogue, in order to investigate the biological potentials (antioxidant, antileishmania, anti-inflammatory and cytotoxicity activities) in cell cultures. Q5 was characterized by FTIR, 1H and 13C NMR spectra. The antioxidant potential was evaluated against the radical ABTS•+. The anti-inflammatory potential was evaluated by measuring the pro-inflammatory cytokine tumor necrosis factor (TNF) and the production of nitric oxide (NO) in peritoneal macrophages from BALB/c mice. Cytotoxicity tests were performed using the AlamarBlue method in cancer cells HepG2 (human hepatocarcinoma), HL-60 (promyelocytic leukemia) and MCR-5 (healthy human lung fibroblasts) as well as the MTT method for C6 cell cultures (rat glioma). Q and Q5 showed antioxidant activity of 29% and 18%, respectively, which is justified by the replacement of hydroxyls by acetyl groups. Q and Q5 showed concentration-dependent reductions in NO and TNF production (p < 0.05); Q and Q5 showed higher activity at concentrations > 40µM when compared to dexamethasone (20 µM). For the HL-60 lineage, Q5 demonstrated selectivity, inducing death in cancer cells, when compared to the healthy cell line MRC-5 (IC50 > 80 µM). Finally, the cytotoxic superiority of Q5 was verified (IC50 = 11 µM), which, at 50 µM for 24 h, induced changes in the morphology of C6 glioma cells characterized by a round body shape (not yet reported in the literature). The analogue Q5 had potential biological effects and may be promising for further investigations against other cell cultures, particularly neural ones.

Abstract 12472: Development of an in vitro System to Study Mechanisms of Ultrasound-Targeted Microbubble Cavitation-Mediated Blood Brain Barrier Opening

Introduction: Ultrasound-targeted microbubble cavitation ( UTMC ) is being explored as a method to transiently open the blood brain barrier ( BBB ). While UTMC-mediated BBB opening is a promising avenue for increasing drug delivery to the central nervous system, its underlying mechanisms are incompletely understood. We sought to develop an in vitro system simulating cerebral endothelial barrier function, to facilitate studies aimed at exploring mechanisms of UTMC-mediated BBB opening. Methods: We developed a transwell model with murine brain microvasculature endothelial cells (bEnd.3) and rat C6 glioma cells on opposite sides of a support membrance. Three concentrations of lipid microbubbles ( MB ) were added before applying pulsed ultrasound (1 MHz, 10 μs pulse duration, 10 ms pulse interval) at 3 peak negative pressures ( PNP ) for 20 s. Endothelial barrier function was assessed using transendothelial electrical resistance ( TEER ) and permeability using 10 kDa Texas Red dextran. Cells were stained with Hoechst (nucleus), CellMask Deep Red (cell coverage), calcein-AM (viability), and propidium iodide ( PI ; sonoporation) Differences between experimental conditions were compared using unpaired 2-tailed t-test. Significance was defined as p &lt;0.05. Results: At a given MB:cell ratio, higher PNPs increased the percent of viably sonoporated cells ( Fig 1A ), but caused increasing cell loss from the transwell (decrease in % area coverage) at PNP greater than 250kPa and MB:cell ratio higher than 3:1 ( Fig 1B ). Therefore, 250kPa ultrasound and 3:1 MB ratio were used in the permeability studies: BBB permeability increased by 15 min after UTMC as measured by TEER ( Fig 1C ) and dextran flux ( Fig 1D ), but recovered 1 hour later. Conclusions: We developed a co-culture transwell model of the BBB to simulate UTMC-induced reversible endothelial barrier hyperpermeability. This model will uniquely allow systematic investigation of mechanisms of UTMC-mediated BBB opening.

Selective in vitro anticancer effect of blueberry extract (Vaccinium virgatum) against C6 rat glioma: exploring their redox status.

The aim of this study was to investigate the anticancer potential of blueberry extract (Vaccinium virgatum) against a C6 rat glioma lineage. Cultures of the C6 cells were exposed to blueberry extract at concentrations of 50 to 600µg/mL for 12, 24, 48, or 72h and then evaluated for cell viability, proliferation, migration, colony formation and oxidative stress. We also evaluated the effects of blueberry extract on primary rat cortical astrocytes. Our results show that treatment with blueberry extract did not alter the viability or proliferation of normal primary astrocytes but it did significantly reduce the viability in 21.54 % after 48h and proliferation in 8.59 % after 24h of C6 cells at 200µg/mL. We also observed a reduction in the size of the colonies of 29.99 % at 100µg/mL when compared to the control cells and cell migration was also reduced at 50µg/mL. After 72h, there was a reduction in the reactive oxygen species levels ranging from 46.26 to 34.73 %, in addition to a 380.2 % increase in total thiol content. Superoxide dismutase, catalase, and glutathione S-transferase activities were also enhanced when compared to the control. Taken together this data suggests that blueberry extract exerts some selective anticancer activity in C6 glioma cells.

Miniatured Fluidics-Mediated Modular Self-Assembly of Anticancer Drug–Amino Acid Composite Microbowls for Combined Chemo-Photodynamic Therapy in Glioma

A particulate carrier with the ability to load a combination of therapeutic molecules acting via diverse modes to initiate cancer cell ablation would help heighten anticancer therapeutic outcomes and mitigate harmful side effects due to high doses of mono drug therapy. Moving a step closer, herein, we have developed doxorubicin-curcumin-amino acid-based composite microbowls (CMBs) following miniaturized fluid flow-based self-assembly. The CMBs were further exploited as dual chemo-photodynamic therapeutic agents in C6 glioma cells cultured in both two-dimensional (2D) monolayer and as three-dimensional (3D) spheroids. These CMBs showed synergistic and visible (blue)-light-sensitive cell-killing effects in both C6 cells and 3D spheroids. Furthermore, these bowl-shaped structures also demonstrated good stability and excellent in vitro cytocompatibility in C6 glioma cells. Our results indicated that CMBs with asymmetric cavities could potentially be used as a combinatorial drug carrier enabling simultaneous chemo- and phototherapy for effective cancer treatment. The use of blue light, from the visible part of the electromagnetic system, to generate the phototherapeutic effect further advocates for the ease and widespread applicability of the systems.

MiR-221/222 suppression induced by activation of the cAMP/PKA/CREB1 pathway is required for cAMP-induced bidirectional differentiation of glioma cells.

Factors that increase cAMP levels can induce lineage-specific differentiation of glioma cells into astrocyte-like cells. However, the differentiation pattern and underlying mechanisms remain unclear. Here, we find that cAMP/protein kinase A (PKA)/cAMP responsive element binding protein 1 (CREB1)-induced miR-221/222 suppression contributes to the neuron-like differentiation of gliomas. cAMP agonists selectively induced neuron- and astrocyte-like but not oligodendrocyte-like differentiation of C6 glioma cells. PKA inhibitors and CREB1 knockout blocked neuron-like differentiation of glioma cells. cAMP inhibited miR-221/222 in a PKA/CREB1-dependent manner. Importantly, both in vitro and in vivo assays demonstrated that transcriptional suppression of miR-221/222 is required for neuronal differentiation of glioma cells. Our findings suggest that increasing cAMP levels can induce bidirectional differentiation of glioma cells. Furthermore, the miR-221/222 cluster acts as an epigenetic brake during glioma differentiation.

Synergism of Temozolomide, Metformin, and Epigallocatechin Gallate Promotes Oxidative Stress-Induced Apoptosis in Glioma Cells

Aim: This study aimed to study the synergistic anti-glioma efficacies of Temozolomide, Metformin, and Epigallocatechin Gallate in U87MG and C6 glioma cells. Background: Glioblastoma (GBM) is the most malignant and invasive tumor of the central nervous system. The current standard treatment comprises surgical resection, followed by adjuvant radiotherapy and chemotherapy employing temozolomide (TMZ). Yet the survival rates for GBM patients are very low. Hence there is a need for new treatment regimes. Objective: This study aimed to unravel the synergistic anti-tumor potential of a biguanide drug, Metformin (MET) and a polyphenol, Epigallocatechin gallate (EGCG) to enhance the anti-GBM efficacy of the standard drug. Methods: The anti-proliferative effect of TMZ, MET, and EGCG, individually and in combination was elucidated in U87MG (human) and C6 (rat) glioma cells using MTT assay, and combination index was used to determine synergism. Cytotoxicity of the drugs was carried out in HEK293T noncancerous cells. Apoptotic morphological changes in the cells were observed by AO/EtBr staining. Furthermore, the effects of drugs on antioxidant and apoptotic genes (SOD, CAT, Nrf-2, Caspase- 9, and Bcl-2) were evaluated using qRT-PCR, and the protein levels of Nrf-2 and Caspase-9 were evaluated using ELISA. Results: The triple-drug combination (TMZ+ MET+ EGCG) synergistically inhibited the proliferation of U87MG and C6 glioma cells in a dose-dependent manner and promoted the apoptosis of glioma cells. The triple-drug combination significantly up-regulated the expression of antioxidant and apoptotic genes and induced oxidative stress, suggesting a shift in equilibrium towards apoptosis. Conclusion: MET and EGCG in combination with TMZ synergistically promoted oxidative stressinduced apoptosis in glioma cells. Hence, the combination of TMZ, MET, and EGCG may be therapeutically exploited for improving the clinical outcomes of patients with GBM.

The Nuclear Function of IL-33 in Desensitization to DNA Damaging Agent and Change of Glioma Nuclear Structure.

Glioma, the most common subtype of primary brain tumor, is an aggressive and highly invasive neurologically tumor among human cancers. Interleukin-33 (IL-33) is considered as a dual functional cytokine, an alarmin upon tissue damage and a nuclear chromatin-associated protein. Despite that, IL-33 is known to foster the formation of the inflammatory tumor microenvironment and facilitate glioma progression, evidence showing nuclear IL-33 function is still poor. In this study using lentivirus-mediated IL-33 gene knockdown (IL33KD) and IL-33 overexpression (IL33oe) in rat C6 glioma cells and human glioma cell lines (U251MG and U87MG), we found that IL33oe-glioma cells had resistance to the insults of the alkylating agent, temozolomide (TMZ), possibly because of the increased expression of DNA repair genes (i.e., BRCA1, BRCA2, Rad51, FANCB, and FANCD) in IL33oe-glioma cells. Alternatively, examination of glioma nuclear shape from transmission electron microscopy (TEM) imaging analysis and immunofluorescence for histone protein H2A staining showed that IL33KD attenuated the abnormal cancerous nuclear characteristic, such as indentation, long clefts, and multiple nucleoids. Yet, IL33oe promoted the changes in glioma nuclear shapes, such as the formation of multiple lobes. We further found that histone proteins, H2A and H3, were reduced in IL33KD glioma cells. The non-histone DNA-binding nucleoproteins, the high mobility group A1 (HMGA1) and HMGA2, were also downregulated by IL33KD. In contrast, IL33oe increased H2A and H3 proteins and HMGA1 and HMGA2 in glioma cells. Altogether, the upregulation of nuclear IL-33 expression was along with an increase in the expression of DNA repair genes, contributing to the desensitization of glioma cells to DNA damaging agents. Moreover, nuclear IL-33 proteins in cooperation with chromatin-associated proteins regulate glioma nuclear structure, which might be crucial for glioma progression and malignancy.

Design, synthesis and biological evaluation of N-anthraniloyl tryptamine derivatives as pleiotropic molecules for the therapy of malignant glioma.

COX-2 and STAT3 are two key culprits in the glioma microenvironment. Herein, to inhibit COX-2 and block STAT3 signaling, we disclosed 27N-anthraniloyl tryptamine compounds based on the combination of melatonin derivatives and N-substituted anthranilic acid derivatives. Among them, NP16 showed the best antiproliferative activity and moderate COX-2 inhibition. Of note, NP16 decreased the level of p-JAK2 and p-STAT3, and blocked the nuclear translocation of STAT3 in GBM cell lines. Moreover, NP16 downregulated the MMP-9 expression of BV2 cells in a co-culture system of BV2 and C6 glioma cells, abrogated the proliferative/invasive/migratory abilities of GBM cells, induced apoptosis by ROS and the Bcl-2-regulated apoptotic pathway, and induced obvious G2/M arrest in glioma cells invitro. Furthermore, NP16 displayed favorable pharmacokinetic profiles covering long half-life (11.43±0.43h) and high blood-brain barrier permeability. Finally, NP16 effectively inhibited tumor growth, promoted the survival rate, increased the expression of E-cadherin and reduced overproduction of PGE2, MMP-9, VEGF-A and the level of p-STAT3 in tumor tissue, and improved the anxiety-like behavior in C6 glioma model. All these evidences demonstrated N-anthraniloyl tryptamine derivatives as multifunctional anti-glioma agents with high potency could drain the swamp to beat glioma.

Synthesis and Characterization of a Series of Temozolomide Esters and Its Anti-glioma Study.

Temozolomide is a first-line therapeutic drug for glioblastoma (GBM), and it has a low solubility, short biological half-life, and resistance to drug limits in clinical applications. Therefore, it is necessary to find more effective anti-tumor drugs to overcome drug resistance and enhance its anti-glioma activity. We therefore used n-butanol, n-hexanol, n-octanol, 1-dodecanol and 1-hexadecanol to synthesize a series of temozolomide ester compounds (TMZEs) and then investigated their physicochemical properties and anti-glioma efficacy. Our results showed that TMZEs had a higher lipophilicity compared to TMZ and could stably exist in plasma and brain homogenates. TMZEs had significantly increased cytotoxicity and cellular uptake in C6 glioma cells as chain lengths increased. Additionally, the IC50 of TMZ-16E towards TMZ-resistant cells (T98G) was 85.9-fold lower than that of TMZ (p<0.001), and Western blot results demonstrated that TMZ-16E could significantly reduce the expression of O6-methylguanine-DNA-methyltransferase (MGMT). The in vivo anti-glioma efficacy of TMZ-16E were then investigated in orthotopic and subcutaneous GBM models. TMZ-16E prolonged the survival time to 35 days in orthotopic glioma bearing rats, which was 1.94-fold longer than the survival time of rats treated with TMZ, and TMZ-16E increased tumor cell apoptosis based on TUNEL staining. Moreover, TMZ-16E (50mg/kg) noticeably slowed the growth of T98G subcutaneous tumors by down-modulating MGMT expression in subcutaneous GBM-bearing mice, indicating that TMZ-16E could effectively reverse drug resistance. In conclusion, TMZEs improved the lipophilicity and stability of these drugs. Especially, TMZ-16E could reverse drug resistance and improve therapeutic effects of TMZ, which has clinical application potential for GBM treatment.