Rat C6 Glioma Cells Research Articles

DDDR-14. IMPROVED ANTI-GLIOBLASTOMA EFFICACY BY BRG399, A NOVEL ORAL MICROTUBULE BINDING AGENT

Abstract Glioblastoma (GBM) is a highly malignant form of brain tumor with a poor prognosis despite advances in treatments. Therapeutic resistance/relapse remains a significant challenge necessitating the development of novel approaches. BRG399 is a brain permeant, non P-gp substrate, microtubule-targeting agent that displays anti-cancer activity in vitro and in vivo. BRG399 was well tolerated in rat and dog toxicology studies. Anti-cancer activity (IC50) of BRG399 in in vitro models of GBM was assessed to have potency ranging from 42nm to 89nM in the 3 cell lines tested. Here, in vivo tumor activity of BRG399 was assessed in Sprague Dawley rats implanted with 3x105 C6 rat glioma cells into the right entorhinal cortex/subiculum region. 14 days post successful implantation tumors were assessed by MRI, rats were randomized to receive vehicle (2% Labrasol) or BRG399 (5, 10, 20 mg/kg groups) twice daily by oral gavage for 16 days, and MRIs were performed on survivors on days 31 and 45 post implantation. BRG399 administration resulted in a marked improvement in survival (from 7.7 ± 2.6 days to 25.5 ± 15.6 days, log-rank test, p:0.007) compared to controls. Six animals receiving BRG399 remained alive beyond day 60 of post-treatment initiation. To assess biodistribution of BRG399 in the brain, rats were implanted with C6 cells and tumor establishment assessed 12 days later by MRI. Animals were treated with vehicle or BRG399 (10, 20, 25 mg/kg) and plasma and brains were collected for bioanalytical assessment. Tissue single cell biodistribution was performed using TIMSTOF FLEX spatial OMICS. These results demonstrate a therapeutic effect of BRG399 on intracranial glioma-bearing rats with survival of the BRG399 treatment groups of strong statistical significance over the control group. Analysis of drug levels in plasma and brain will serve as guidance for dose and scheduling decisions for further therapeutic development.

Perfluorooctane sulfonate (PFOS) and benzo[a]pyrene (BaP) synergistically induce neurotoxicity in C6 rat glioma cells via the activation of neurotransmitter and Cyp1a1-mediated steroid hormone synthesis pathways

Humans are often exposed to complex mixtures of multiple pollutants rather than a single pollutant. However, the combined toxic effects and the molecular mechanism of PFOS and BaP remain poorly understood. In this study, two typical environmental pollutants, perfluorooctane sulfonate acid (PFOS) and benzo[a]pyrene (BaP), were selected to investigate their combined neurotoxic effects on rat C6 glioma cells at environmentally relevant concentrations. The results showed that coexposure to low-dose PFOS and BaP induced greater toxicity (synergistic effect) than did single exposure. PFOS-BaP coexposure had stronger toxic effects on inducing oxidative stress and promoting early apoptosis. Targeted metabolomics confirmed that increased levels of the neurotransmitters 5-hydroxytryptophan, dopamine, tryptophan and serotonin disturb the phenylalanine, tyrosine and tryptophan biosynthesis pathways. Mechanistically, exposure to a low-dose PFOS-BaP binary mixture induces steroid hormone synthesis disorder through the activation of Cyp1a1 and Hsd17b8 (steroid hormone synthesis genes) and Dhcr24 and Dhcr7 (cholesterol synthesis genes). These findings are useful for comprehensively and systematically elucidating the biological safety of PFOS-BaP and its potential threats to human health.

Uncovering naringin’s anticancer mechanisms in glioblastoma via molecular docking and network pharmacology approaches

Naringin, a flavonoid, exhibits diverse therapeutic properties and has been proven to exert cytotoxic effects on cancer cells. Nevertheless, the precise mechanism of naringin maintaining its cytotoxic effect on glioblastoma (GBM) remains unknown. Thus, the current study aimed to establish a plausible cellular mechanism for Naringin’s inhibition of GBM. We employed various system biology techniques to forecast the primary targets, including gene ontology and cluster analysis, KEGG enrichment pathway estimation, molecular docking, MD (molecular dynamic) simulation and MMPBSA analysis. Glioblastoma target sequences were obtained via DisGeNet and Therapeutic Target Prediction, aligned with naringin targets, and analyzed for gene enrichment and ontology. Gene enrichment analysis identified the top ten hub genes. Further, molecular docking was conducted on all identified targets. For molecular dynamics modelling, we selected the two complexes that exhibited the most docking affinity and the two most prominent genes of the hub identified through analysis of the enrichment of genes. The PARP1 and ALB1 signalling pathways were found to be the main regulated routes. Naringin exhibited the highest binding potential of − 12.90 kcal/mol with PARP1 (4ZZZ), followed by ABL1 (2ABL), with naringin showing a − 8.4 kcal/mol binding score, as determined by molecular docking. The molecular dynamic approach and MM-PBSA investigation along with PCA study revealed that the complex of Naringin, with 4ZZZ (PARP1) and, 2ABL (ABL1), are highly stable compared to that of imatinib and talazoparib. Analyses of the signalling pathway suggested that naringin may have anticancer effects against GBM by influencing the protein PARP and ALB1 levels. Cytotoxicity assay was performed on two different glioblastoma cell lines C6 and U87MG cells. Naringin demonstrates a higher cytotoxic potency against U87MG human glioblastoma cells compared to C6 rat glioma cells.

Potential for boron neutron capture therapy (BNCT) with ASCT2-targeted boron agents.

233 Background: Boron Neutron Capture Therapy (BNCT) is a cutting-edge particle irradiation technique that utilizes the nuclear reaction triggered by the irradiation of non-radioactive boron-10 with thermal neutrons, selectively annihilating tumor cells that have incorporated boron. In the clinical arena, Boronophenylalanine (BPA), a phenylalanine compound targeting amino acid receptors, has been the cornerstone of BNCT. While BNCT has been effective against malignant gliomas, its dependency on BPA uptake has illuminated the existence of inherent resistance within specific cells, tissues, and cancer types. This study pivots towards the Alanine-serine-cysteine transporter 2 (ASCT2)—a transporter distinct from LAT1—investigating its potential as a gateway for the development of innovative boron carriers, with the aim of expanding the clinical applicability and effectiveness of BNCT. Methods: This research embarked on in vitro investigations to evaluate boron accumulation in F98 and C6 rat glioma cells, and 9L rat gliosarcoma, following 24 hours of exposure to BPA and GluB-2 (10 μg B/ml each). Complementary in vivo studies involved the intravenous administration of these compounds to an F98 rat brain tumor model, with the subsequent measurement of boron distribution at 2.5, 6, and 24 hours post-administration. The efficacy of these treatments was then ascertained through neutron irradiation experiments, gauging therapeutic outcomes via the survival periods of the subjects. Results: In vitro findings highlighted that GluB-2 facilitated a significantly higher intracellular boron concentration compared to BPA in F98 cells (p=0.02, Student's t-test). In vivo results indicated that the apex of boron biodistribution in tumors was achieved at 2.5 hours post-BPA treatment and at 6 hours post-GluB-2 treatment, with GluB-2 securing a superior maximum intratumor boron concentration. Survival analysis revealed a mean survival of 28.0±2.5 days for the neutron alone group, 37.7±5.0 days for the group receiving BPA IV + BNCT at 2.5 hours, 55.1±19.9 days for the group treated with GluB-2 IV + BNCT at 6 hours, and 25.3±1.4 days for the untreated group. Remarkably, the group treated with GluB-2 demonstrated significantly prolonged survival compared to the BPA-treated group (p<0.001, log-rank test). Conclusions: Combining BNCT with GluB-2 showed superior efficacy over BPA. Further research is needed to optimize boron concentration timing and ASCT2 expression levels, yet GluB-2 presents a promising advancement in BNCT, potentially transforming malignant brain tumor treatment.

Effect of Xanthohumol, a Bioactive Natural Compound from Hops, on Adenosine Pathway in Rat C6 Glioma and Human SH-SY5Y Neuroblastoma Cell Lines.

Xanthohumol (Xn) is an antioxidant flavonoid mainly extracted from hops (Humulus lupulus), one of the main ingredients of beer. As with other bioactive compounds, their therapeutic potential against different diseases has been tested, one of which is Alzheimer's disease (AD). Adenosine is a neuromodulatory nucleoside that acts through four different G protein-coupled receptors: A1 and A3, which inhibit the adenylyl cyclases (AC) pathway, and A2A and A2B, which stimulate this activity, causing either a decrease or an increase, respectively, in the release of excitatory neurotransmitters such as glutamate. This adenosinergic pathway, which is altered in AD, could be involved in the excitotoxicity process. Therefore, the aim of this work is to describe the effect of Xn on the adenosinergic pathway using cell lines. For this purpose, two different cellular models, rat glioma C6 and human neuroblastoma SH-SY5Y, were exposed to a non-cytotoxic 10 µM Xn concentration. Adenosine A1 and A2A, receptor levels, and activities related to the adenosine pathway, such as adenylate cyclase, protein kinase A, and 5'-nucleotidase, were analyzed. The adenosine A1 receptor was significantly increased after Xn exposure, while no changes in A2A receptor membrane levels or AC activity were reported. Regarding 5'-nucleotidases, modulation of their activity by Xn was noted since CD73, the extracellular membrane attached to 5'-nucleotidase, was significantly decreased in the C6 cell line. In conclusion, here we describe a novel pathway in which the bioactive flavonoid Xn could have potentially beneficial effects on AD as it increases membrane A1 receptors while modulating enzymes related to the adenosine pathway in cell cultures.

Synthesis of New Indole-Containing Benzo[f]coumarin Derivatives and Their Effect on the Proliferation and Redox State of Rat C6 Glioma Cells

Synthesis of New Indole-Containing Benzo[f]coumarin Derivatives and Their Effect on the Proliferation and Redox State of Rat C6 Glioma Cells

Cytotoxic effects of nerve growth factor and its combinations with chemotherapeutic drugs on anaplastoc astrocytoma, glioblastoma and medubloblastoma cells <i>in vitro</i>

BACKGROUND: Currently, the effectiveness of the treatment of malignant tumors using surgical resection, radiotherapy and chemotherapy is insufficient. Therefore, new research is needed to find alternative molecules with antitumor effects. It is known that nerve growth factor (NGF) inhibits invasion, migration, and angiogenesis of tumor cells. Studying the effects of NGF on brain tumors, as well as its combinations with chemotherapy drugs used in medicine, may contribute to the development of new treatment regimens for malignant neoplasms in the central nervous system. AIM: The purpose of this study is an exploration the molecular and cellular mechanisms of anticancer effects of individual and combined preparations of NGF and chemotherapeutic drugs on brain tumor cells (gliomas C6, U251, anaplastic astrocytoma, glioblastoma and medulloblastoma). MATERIALS AND METHODS: The study was performed on rat glioma C6, human U251 glioma cell lines, as well as on primary cells of anaplastic astrocytoma (n = 9), glioblastoma (n = 9) and medulloblastoma (n = 38) patients. The cytotoxicity of chemotherapeutic drugs, NGF and their combinations against tumor cells was assessed using the MTT assay. The expression of TrkA and p75 receptors on anaplastic astrocytoma, glioblastoma and medulloblastoma cells was assessed by immunofluorescence analysis using anti-TrkA and anti-p75 monoclonal antibodies. RESULTS: Nerve growth factor exhibits in vitro cytotoxic activity that exceeds the activity of chemotherapy drugs towards rat glioma C6, human U251, anaplastic astrocytoma (AA), glioblastoma (GBM) and medulloblastoma cells. The cytotoxic activity of NGF in combination with chemotherapy drugs is significantly higher than the activity of the individual NGF drug against medulloblastoma cells, while against anaplastic astrocytoma cells it is comparable to the indicators of the isolated action of NGF, and lower for glioblastoma cells. The effectiveness of the cytotoxic effect of the combinations NGF + cisplatin and NGF + temozolomide (TMZ) on AA and GBM cells correlates with both the expression of TrkA, p75 receptors, and their coexpression, indicating that expression indicators can be considered as markers of tumor cell sensitivity to NGF. CONCLUSIONS: The data obtained allow us to consider NGF as a potential anticancer drug for the treatment of brain tumors. Thus, NGF can act as a potential anticancer drug for the development of new therapeutic regimens for brain tumors.

In Vitro and In Silico Anti-Glioblastoma Activity of Hydroalcoholic Extracts of Artemisia annua L. and Artemisia vulgaris L.

Glioblastoma, the most aggressive and challenging brain tumor, is a key focus in neuro-oncology due to its rapid growth and poor prognosis. The C6 glioma cell line is often used as a glioblastoma model due to its close simulation of human glioma characteristics, including rapid expansion and invasiveness. Alongside, herbal medicine, particularly Artemisia spp., is gaining attention for its anticancer potential, offering mechanisms like apoptosis induction, cell cycle arrest, and the inhibition of angiogenesis. In this study, we optimized extraction conditions of polyphenols from Artemisia annua L. and Artemisia vulgaris L. herbs and investigated their anticancer effects in silico and in vitro. Molecular docking of the main phenolic compounds of A. annua and A. vulgaris and potential target proteins, including programmed cell death (apoptosis) pathway proteins proapoptotic Bax (PDB ID 6EB6), anti-apoptotic Bcl-2 (PDB ID G5M), and the necroptosis pathway protein (PDB ID 7MON), mixed lineage kinase domain-like protein (MLKL), in complex with receptor-interacting serine/threonine-protein kinase 3 (RIPK3), revealed the high probability of their interactions, highlighting the possible influence of chlorogenic acid in modulating necroptosis processes. The cell viability of rat C6 glioma cell line was assessed using a nuclear fluorescent double-staining assay with Hoechst 33342 and propidium iodide. The extracts from A. annua and A. vulgaris have demonstrated anticancer activity in the glioblastoma model, with the synergistic effects of their combined compounds surpassing the efficacy of any single compound. Our results suggest the potential of these extracts as a basis for developing more effective glioblastoma treatments, emphasizing the importance of further research into their mechanisms of action and therapeutic applications.

Effects of Glutamine Synthetase on Neovascularization in Glioma: In Vivo MR Vessel Size Imaging and Histology.

Glutamine Synthetase (GS) could induce vascular sprouting through the improvement of endothelial cell migration in inflammatory diseases. MR vessel-size imaging has been proposed as a valuable approach for visualizing the underlying angiogenic processes in the brain. This study aims to investigate the role of GS in the neovascularization of gliomas through the utilization of MR vessel-size imaging and histopathological techniques. In this exploratory animal study, we randomly divided the C6 glioma rat models into a control group and an L-methionine sulfoximine (MSO) treatment group. Daily intraperitoneal injections were administered for three consecutive days, starting from day 10 following the implantation of C6 glioma cells in rats. Subsequently, MR vessel size imaging was conducted using a BRUKER 7 T/200 mm MRI scanner, and the MRI results were validated through histopathological examination. A significant decrease in microvessel density was observed in both the tumor periphery and center areas in the MSO treatment group compared to that in the control group. The mean vessel diameter (mVD) and vessel size index (VSI) did not exhibit significant changes compared to the control group. Moreover, the staining intensity of platelet endothelial cell adhesion molecule-1 (CD31) and GS in the tumor periphery was significantly decreased in the MSO treatment group. Additionally, the MSO treatment demonstrated a substantial inhibition of tumor growth. GS inhibitors significantly reduced angiogenesis in the periphery area of C6 glioma, exerting an inhibitory effect on tumor progression. Thus, GS inhibitors could be potential therapeutic agents for treating glioma. Additionally, in vivo MR vessel size imaging detects changes in vascularrelated parameters after tumor treatment, making it a promising method for detecting neovascularization in glioma.</p>.

Morin Inhibits Cell Proliferation and Induces Caspase-mediated Apoptotic Cell Death in Glioma C6 Cell Line

Background Glioma is a recurrent form of primary malignant cancer that occurs in the brain and central nervous system. The adults are the major victims of gliomas, and the men are mostly affected. At present, gliomas are treated with surgical resection followed by radiation and the administration of chemodrugs such as temozolomide. The invasive and high recurrence rate of gliomas often reduces the effectiveness of treatment, which leads to poor recovery. Hence, more potent drugs without side effects on long-term treatment need to be discovered to treat gliomas. Morin is one such promising phytochemical that possesses immense pharmacological properties. It is proven to exert anti-inflammatory, antioxidant, anticancer, antibacterial, antidiabetic, and neuroprotective properties. The current research focuses on examining the growth inhibition and apoptosis-inducing potency of morin against glioma C6 cells. Materials and Methods Rat C6 glioma cells were treated with different concentrations of morin and analyzed for cytotoxicity using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. The intracellular reactive oxygen species (ROS) production and alteration of mitochondrial membrane potential (MMP) by morin in glioma cells were examined with dichlorodihydrofluorescein diacetate (DCHF-DA) and rhodamine 123 staining. The apoptotic induction in glioma cells was analyzed with dual staining and it was confirmed by quantifying the apoptotic protein using the enzyme-linked immunosorbent assay (ELISA) technique. The anti-inflammatory property of morin was assessed by the inflammatory cytokines tumor necrosis factor-α (TNF-α), nuclear factor-kappa B (NF-κB), cyclooxygenase-2 (COX-2), and interleukin-6 (IL-6) using the ELISA technique. Results Our MTT results show that morin treatment significantly reduced the cell viability of rat C6 glioma cells. It also significantly increased ROS generation and decreased MMP in rat C6 glioma cells. Morin treatment also effectively increased caspase-3 and caspase-9, the proapoptotic protein Bax, and decreased the antiapoptotic protein Bcl-2. The induction of apoptosis by morin in glioma cells was evident with our acridine orange/ethidium bromide (AO/EtBr) staining results. Morin also decreased the inflammatory cytokine levels in rat C6 glioma cells. Conclusion Our findings have proven that morin significantly induces apoptosis by increasing the levels of apoptotic protein via the generation of ROS. It also effectively reduced inflammatory cytokine levels, thereby exerting anticancer activity against glioma cells. Therefore, morin can be subjected to further research to be designed as a potent anticancer drug to treat gliomas.

Development of Hollow Gold Nanoparticles for Photothermal Therapy and Their Cytotoxic Effect on a Glioma Cell Line When Combined with Copper Diethyldithiocarbamate.

Gold-based nanoparticles hold promise as functional nanomedicines, including in combination with a photothermal effect for cancer therapy in conjunction with chemotherapy. Here, we synthesized hollow gold nanoparticles (HGNPs) exhibiting efficient light absorption in the near-IR (NIR) region. Several synthesis conditions were explored and provided monodisperse HGNPs approximately 95-135 nm in diameter with a light absorbance range of approximately 600-720 nm. The HGNPs were hollow and the surface had protruding structures when prepared using high concentrations of HAuCl4. The simultaneous nucleation of a sacrificial AgCl template and Au nanoparticles may affect the resulting HGNPs. Diethyldithiocarbamate (DDTC) is metabolized from disulfiram and is a repurposed drug currently attracting attention. The chelation of DDTC with copper ion (DDTC-Cu) has been investigated for treating glioma, and here we confirmed the cytotoxic effect of DDTC-Cu towards rat C6 glioma cells in vitro. HGNPs alone were biocompatible and showed little cytotoxicity, whereas a mixture of DDTC-Cu and HGNPs was cytotoxic in a dose dependent manner. The temperature of HGNPs was increased by NIR-laser irradiation. The photothermal effect on HGNPs under NIR-laser irradiation resulted in cytotoxicity towards C6 cells and was dependent on the irradiation time. Photothermal therapy by HGNPs combined and DDTC-Cu was highly effective, suggesting that this combination approach hold promise as a future glioma therapy.

Expression of brain-derived neurotrophic factor and formation of migrasome increases in the glioma cells induced by the adipokinetic hormone.

It has been previously shown that brain-derived neurotrophic factor is linked with various types of cancer. Brain-derived neurotrophic factor is found to be highly expressed in multiple human cancers and associated with tumor growth, invasion, and metastasis. Adipokinetic hormones are functionally related to the vertebrate glucagon, as they have similar functionalities that manage the nutrient-dependent secretion of these two hormones. Migrasomes are new organelles that contain numerous small vesicles, which aid in transmitting signals between the migrating cells. Therefore, the aim of this study was to investigate the effects of Anax imperator adipokinetic hormone on brain-derived neurotrophic factor expression and ultrastructure of cells in the C6 glioma cell line. The rat C6 glioma cells were treated with concentrations of 5 and 10 Anax imperator adipokinetic hormone for 24 h. The effects of the Anax imperator adipokinetic hormone on the migrasome formation and brain-derived neurotrophic factor expression were analyzed using immunocytochemistry and transmission electron microscope. The rat C6 glioma cells of the 5 and 10 μM Anax imperator adipokinetic hormone groups showed significantly high expressions of brain-derived neurotrophic factor and migrasomes numbers, compared with the control group. A positive correlation was found between the brain-derived neurotrophic factor expression level and the formation of migrasome, which indicates that the increased expression of brain-derived neurotrophic factor and the number of migrasomes may be involved to metastasis of the rat C6 glioma cell line induced by the Anax imperator adipokinetic hormone. Therefore, the expression of brain-derived neurotrophic factor and migrasome formation may be promising targets for preventing tumor proliferation, invasion, and metastasis in glioma.

Synthesis of Some New 1,3,4-Oxadiazole Derivatives and Evaluation of Their Anticancer Activity.

In this work, some new 2-[(5-((2-acetamidophenoxy)methyl)-1,3,4-oxadiazol-2-yl)thio]acetamide derivatives (4a-4l) were synthesized and studied for their anticancer activity. Twelve new compounds were tested on the A549 human lung cancer cell line, C6 rat glioma cell line, and L929 murine fibroblast cell line. Compounds 4f, 4i, 4k, and 4l (IC50: 1.59-7.48 μM), and especially 4h (IC50: <0.14 μM), exhibited excellent cytotoxic profile on A549 with selectivity. Compounds 4g and 4h showed remarkable antiproliferative activity on the C6 cell line with IC50 values of 8.16 and 13.04 μM, respectively. The compounds with the lowest IC50 value on the A549 cell line (4f, 4h, 4i, 4k, and 4l) were further studied to determine the mechanism of action. These compounds were found to induce apoptosis with a higher ratio (16.10-21.54%) than that of the standard drug cisplatin (10.07%). Compound 4f displayed mitochondrial membrane depolarization and caspase-3 activation at most, whereas compounds 4h (89.66%) and 4i (78.78%) had outstanding retention rates in the G0/G1phase of the cell cycle (cisplatin 74.75%). Compounds 4f, 4g, 4h, and 4l exhibited matrix metalloproteinase-9 (MMP-9) inhibition higher than 75% at 100 μg/mL; even IC50 values were found to be 1.65 and 2.55 μM for 4h and 4l. In addition, in silico physicochemical properties of the compounds and molecular docking interaction of compound 4h on the MMP-9 enzyme were evaluated; the desired and expected results were obtained.

Aquaporin 1 overexpression may enhance glioma tumorigenesis by interacting with the transcriptional regulation networks of Foxo4, Maz, and E2F families

BackgroundThe glioblastoma has served as a valuable experimental model system for investigating the growth and invasive properties of glioblastoma. Aquaporin-1 (AQP1) in facilitating cell migration and potentially contributing to tumor progression. In this study, we analyzed the role of AQP1 overexpression in glioblastoma and elucidated the main mechanisms involved.MethodsAQP1 overexpression recombinant vector was introduced into C6 rat glioma cells to construct an AQP1 overexpression C6 cell line, and its effect on cell viability and migration ability was detected by MTT and Transwell. RNA was extracted by Trizol method for gene sequencing and transcriptomics analysis, and the differentially expressed genes (DEGs) were enriched for up- and downregulated genes by Principal component analysis (PCA), and the molecular mechanism of AQP1 overexpression was analyzed in comparison with the control group using the NCBI GEO database. Statistical analysis was performed using Mann-Whitney paired two tailed t test.ResultsThe cell viability of AQP1-transfected cell lines increased by 23% and the mean distance traveled increased by 67% compared with the control group. Quantitative analysis of gene expression showed that there were 12,121 genes with an average transcripts per million (TPM) value greater than 1. DEGs accounted for 13% of the genes expressed, with the highest correlation with upregulated genes being FOXO4 and MAZ, and the highest with downregulated genes being E2F TFs.ConclusionsAQP1 may be implicated in glioma formation by interacting with the transcriptional regulation networks involving the FOXO4, MAZ, and E2F1/2. These findings shed light on the potential significance of AQP1 in glioma pathogenesis and warrant further investigations to unravel the underlying molecular mechanisms.

Inhibition of HDAC6 alleviates cancer‑induced bone pain by reducing the activation of NLRP3inflammasome.

Cancer‑induced bone pain (CIBP) is characterized as moderate to severe pain that negatively affects the daily functional status and quality of life of patients. When cancer cells metastasize and grow in bone marrow, this activates neuroinflammation in the spinal cord, which plays a vital role in the generation and persistence of chronic pain. In the present study, a model of CIBP was constructed by inoculating of MRMT‑1 rat breast carcinoma cells into the medullary cavity of the tibia in male Sprague‑Dawley rats. Following two weeks of surgery, CIBP rats exhibited damaged bone structure, increased pain sensitivity and impaired motor coordination. Neuroinflammation was activated in the spinal cords of CIBP rats, presenting with extensive leukocyte filtration, upregulated cytokine levels and activated astrocytes. Histone deacetylase6 (HDAC6) works as a therapeutic target for chronic pain. The intrathecal injection of the HDAC6 inhibitor tubastatin A (TSA) in the lumbar spinal cord resulted in decreased spinal inflammatory cytokine production, suppressed spinal astrocytes activation and reduced NOD‑like receptor pyrin domain containing 3 (NLRP3) inflammasome activity. Consequently, this effect alleviated spontaneous pain and mechanical hyperalgesia and recovered motor coordination in CIBP rats. It was demonstrated by immunoprecipitation assay that TSA treatment reduced the interaction between HDAC6 and NLRP3. Cell research on C6 rat glioma cells served to verify that TSA treatment reduced HDAC6 and NLRP3 expression. In summary, the findings of present study indicated that TSA treatment alleviated cancer‑induced bone pain through the inhibition of HDAC6/NLRP3inflammasome signaling in the spinal cord.

Toxicity of Carboplatin-Niosomal Nanoparticles in a Brain Cancer Cell Line.

Cancer poses a significant challenge in modern medicine, standing as the primary cause of death in many countries, second only to cardiovascular diseases. Among the various treatments available, carboplatin, a chemotherapy drug, is employed for specific cancer types, including brain carcinoma. The main objective of this investigation is to enhance the therapeutic efficacy of carboplatin by utilizing niosomal nanocarriers. We synthesized nanoniosomal carboplatin using the reverse-phase evaporation technique and conducted an assessment of its particle size, zeta potential, and drug-release properties. Subsequently, we evaluated the cytotoxicity of nanoniosomal carboplatin using the C6 rat glioma cell line. Our research revealed that these niosomal nanoparticles possessed a particle size of 290.5±5.5 nm and a zeta potential of -21.7±7.4 mV. The amount of encapsulated drug and drug loading level were found to be 60.2±2.3% and 2.5±1.1%, respectively. Importantly, the cytotoxic impact of these nanoniosomes on the C6 rat glioma cell line exhibited a significant increase compared to the free drug (P<0.05). Based on our discoveries, it is evident that carboplatin niosomal nanocarriers hold potential as an innovative approach to chemotherapy for brain cancer therapy.

αV-Integrin-Dependent Inhibition of Glioblastoma Cell Migration, Invasion and Vasculogenic Mimicry by the uPAcyclin Decapeptide.

Among the deadliest human cancers is glioblastoma (GBM) for which new treatment approaches are urgently needed. Here, the effects of the cyclic decapeptide, uPAcyclin, are investigated using the U87-MG, U251-MG, and U138-MG human GBM and C6 rat cell models. All GBM cells express the αV-integrin subunit, the target of uPAcyclin, and bind specifically to nanomolar concentrations of the decapeptide. Although peptide exposure affects neither viability nor cell proliferation rate, nanomolar concentrations of uPAcyclin markedly inhibit the directional migration and matrix invasion of all GBM cells, in a concentration- and αV-dependent manner. Moreover, wound healing rate closure of U87-MG and C6 rat glioma cells is reduced by 50% and time-lapse videomicroscopy studies show that the formation of vascular-like structures by U87-MG in three-dimensional matrix cultures is markedly inhibited by uPAcyclin. A strong reduction in the branching point numbers of the U87-MG, C6, and U251-MG cell lines undergoing vasculogenic mimicry, in the presence of nanomolar peptide concentrations, was observed. Lysates from matrix-recovered uPAcyclin-exposed cells exhibit a reduced expression of VE-cadherin, a prominent factor in the acquisition of vascular-like structures. In conclusion, these results indicate that uPAcyclin is a promising candidate to counteract the formation of new vessels in novel targeted anti-GBM therapies.

Effects of mitragynine on viability, proliferation, and migration of C6 rat glioma, SH-SY5Y human neuroblastoma, and HT22 immortalized mouse hippocampal neuron cell lines

Mitragynine (MG) is an indole alkaloid found in the extract of Mitragyna speciosa Korth native to Southeast Asia. Although MG is known for its pain-relieving and psychoactive effects, reports have suggested that it has therapeutic potential against neoplasms and psychiatric disorders. However, no evidence currently exists to support the effect of MG on brain tumors. This study aimed to investigate the antitumor effects of MG in C6 rat glioma and SH-SY5Y human neuroblastoma tumor cell lines compared with those in the non-tumor HT22 mouse hippocampal neuronal cell line. MTT assay for cell viability, clonogenic and wound healing assays for cell migration, Hoechst 33342/propidium iodide staining for nuclear morphology, and cell cycle distribution using flow cytometry were performed. MG at 125.47 μM (50 μg/ml) significantly reduced the viability of all cell lines, and the clonogenicity of C6 glioma cells began decreasing at 75.28 μM (30 μg/ml) of MG. Cell migration was inhibited in C6 and HT22 cells treated with 75.28 μM (30 μg/ml) of MG. Apoptotic nuclear condensation and fragmentation were observed in all cell lines treated with 125.47 μM (50 μg/ml) MG, whereas late-phase apoptotic cells were predominant in the group treated with 250.94 μM (100 μg/ml) of MG. The cell cycle assay results suggest that MG arrested the S phase in the C6 cell line and the G2/M phase in the HT22 cell lines. This study showed that MG induces cell death and cell cycle arrest, disrupting cell migration and reducing the clonogenicity of brain tumor cells.

GDNF triggers proliferation of rat C6 glioma cells via the NF-κB/CXCL1 signaling pathway.

Glioblastoma multiforme (GBM) is the most common primary malignant brain tumor that is characterized by its high proliferative and migratory potential, leading to a high invasiveness of this tumor type. However, the underlying mechanism of GBM proliferation and migration has not been fully elucidated. In this study, at first, we used RNA-seq together with bioinformatics technology to screen for C-X-C motif ligand 1 (CXCL1) as a proliferation-related gene. And exogenous glial cell line-derived neurotrophic factor (GDNF) induced proliferation and up-regulated the level of CXCL1 in rat C6 glioma cells determined by sqPCR and ELISA. Then, we manipulated the CXCL1 expression by using a lentiviral vector (CXCL1-RNAi) approach. By this, the proliferation of C6 cells was decreased, suggesting that CXCL1 plays a key role in proliferation in these cells. We hypothesized that exogenous GDNF promoted NF-κB nuclear translocation and therefore, analyzed the interaction of CXCL1 with NF-κB by Western Blot and immunofluorescence. Additionally, we used BAY 11-7082, a phosphorylation inhibitor of NF-κB, to elucidate NF-κB mediated the effect of GDNF on CXCL1. These results demonstrated that GDNF enhanced the proliferation of rat C6 glioma cells through activating the NF-κB/CXCL1 signaling pathway. In summary, these studies not only revealed the mechanism of action of exogenous GDNF in promoting the proliferation of C6 glioma cells but may also provide a new biological target for the treatment of malignant glioma.

The first evidence of biological activity for free Hypusine, an enigmatic amino acid discovered in the '70s.

Hypusine amino acid [Nε-(4-amino-2-hydroxybutyl)-lysine] was first isolated in 1971 from bovine brain extracts. Hypusine originates from a post-translational modification at the eukaryotic translation initiation factor 5A (eIF5A), a protein produced by archaebacteria and eukaryotes. The eIF5A protein is the only one described containing the hypusine residue, which is essential for its activity. Hypusine as a free amino acid is a consequence of proteolytic degradation of eIF5A. Herein, we showed, for the first time, evidence of biological activity for the free hypusine. C6 rat glioma cells were treated with hypusine, and different cellular parameters were evaluated. Hypusine treatment significantly reduced C6 cell proliferation and potently suppressed their clonogenic capacity without leading to apoptosis. Hypusine also decreased the Eif5A transcript content and the global protein synthesis profile that may occur due to negative feedback in response to high hypusine concentration, controlling the content of newly synthesized eIF5A, which can affect the translation process. Besides, hypusine treatment also altered cellular metabolism by changing the pathways for energy production, reducing cellular respiration coupled with oxidative phosphorylation, and increasing the anaerobic metabolism. These observed results and the relationship between eIF5A and tumor processes led us to test the combination of hypusine with the chemotherapeutic drug temozolomide. Combining temozolomide with hypusine reduced the MTT conversion to the same levels as those observed using double temozolomide dosage alone, demonstrating a synergetic action between the compounds. Thus, since 1971, this is the first study showing evidence of biological activity for hypusine not associated with being an essential component of the eiF5A protein. Finding out the molecular targets of hypusine are the following efforts to completely characterize its biological activity.