Rat Glioma Research Articles

Evaluation of HIF-1α Expression in a Rat Glioma Model Using Intravoxel Incoherent Motion and R2* Mapping.

Accurate evaluation of HIF-1α levels can facilitate the detection of hypoxia niches in glioma and treatment decisions. To investigate the feasibility of intravoxel incoherent motion (IVIM) and R2* Mapping for detecting HIF-1α expression levels, sixteen rats with intracranial C6 gliomas were subjected to IVIM and R2* Mapping using a 7 Tesla MRI scanner. For each model, the brain tissue on the HIF-1α-stained slices was subdivided into multiple square regions of interest (ROIs) with areas of 1 mm2, for which HIF-1α expression was assessed by HALO software to form a maps of HIF scores with a 0–300 range. The IVIM and R2* Mapping images were processed to create maps of the D, D*, f and R2* that were then paired with the corresponding HIF score maps. The average D, D*, f, perfusion (f × D*) and R2* values were calculated for the ROIs in the tumor and normal brain regions with different HIF-1α levels and used in further analysis. In this study, the average tumor size of sixteen C6 model rats was 458 ± 46.52 mm3, and the 482 included ROIs consisted of 280 tumoral and 202 normal ROIs. The average HIF score for the tumor regions was significantly higher than normal brain tissue (p < 0.001), and higher HIF scores were obtained for the central part of tumors than peripheral parts (p=0.03). Compared with normal brain tissues, elevated perfusion and f values were observed in tumor regions (p = 0.021, 0.004). In tumoral ROIs, the R2* values were higher in the group with high HIF-1α expression than in the group with low HIF-1α expression (p = 0.003). A correlation analysis revealed a positive correlation between the R2* value and HIF scores (r = 0.43, p < 0.001) and a negative correlation between D* and the HIF scores (r = -0.30, p = 0.001). Discrepancies in HIF-1α expression were found among different intratumoral areas, and IVIM and R2* Mapping were found to be promising means of noninvasive detection of the distribution and expression level of HIF-1α.

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.

Antitumor effect of partially purified venom protein from Conus virgo on rat glioma cell line

BackgroundSince many research works have been carried out to identify the pharmacological properties of conotoxins from many Conus sp. The pharmacological properties of higher molecular weight toxin from the venom of Conus sp. have been poorly elucidated. Therefore, the present investigation was made to isolate the higher molecular weight toxin from the venom of Conus virgo and to test its antitumor effect on rat glioma cell line.ResultsThe partially purified fraction 1 showed better activity (IC50 50 μg/ml) than fraction 2 (IC50 150 µg/ml) after 24 h incubation. The decreased elevation of creatine kinase was documented on fraction 1treated C6 cells. Vero cells showed minimal cell damage at 150 μg/ml 24 h as well. The nuclear morphology and nuclear fragmentation showed the presence of nuclear damage in C6 cells. Western blot analysis revealing the upregulation of the p53-mediated intrinsic apoptotic pathway was documented.ConclusionsThe present investigation emphasizes that the partially purified fraction 1 has triggered p53 mediated apoptosis by synergism of two higher molecular weight toxins in Rat glioma cell.

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.

Evaluation of Temozolomide Treatment for Glioblastoma Using Amide Proton Transfer Imaging and Diffusion MRI.

Simple SummaryGlioblastoma (GBM), the most frequent and malignant histological type of glioma, is associated with a very high mortality rate. MRI is a useful method for the evaluation of tumor growth. However, there are few studies that have quantitatively evaluated the changes in disease state after TMZ treatment against GBM, and it is not fully understood how the effects of treatment are reflected in the quantitative values measured on MRI. We used the C6 glioma rat model to evaluate the tumor changes due to chemotherapy at different doses of TMZ in terms of quantitative values measured by neurite orientation dispersion and density imaging (NODDI) and amide proton transfer (APT) imaging using 7T-MRI. These methods can evaluate the microstructural changes caused by TMZ-induced tumor growth inhibition.This study aimed to evaluate tumor changes due to chemotherapy with temozolomide (TMZ) in terms of quantitative values measured by APT imaging and NODDI. We performed TMZ treatment (administered orally by gavage to the TMZ-40 mg and TMZ-60 mg groups) on 7-week-old male Wistar rats with rat glioma C6 implanted in the right brain. T2WI, APT imaging, diffusion tensor imaging (DTI), and NODDI were performed on days 7 and 14 after implantation using 7T-MRI, and the calculated quantitative values were statistically compared. Then, HE staining was performed on brain tissue at day 7 and day 14 for each group to compare the results with the MR images. TMZ treatment inhibited tumor growth and necrotic area formation. The necrotic areas observed upon hematoxylin and eosin (HE) staining were consistent with the MTR low-signal areas observed upon APT imaging. The intracellular volume fraction (ICVF) map of the NODDI could best show the microstructure of the tumor, and its value could significantly highlight the difference in treatment effects at different TMZ doses. APT imaging and NODDI can be used to detect the microstructural changes caused by TMZ-induced tumor growth inhibition. The ICVF may be useful as a parameter for determining the effect of TMZ.

Microtubular Assessment of C6 Rat Glioma Cell Spheroids Developed in Transparent Liquid Marbles or Hanging Drops.

Simple SummaryDue to its high refractoriness to therapies, glioblastoma brain tumour is frequently used as a model to develop new therapeutic approaches. Many of these treatments may target the microtubular network of the cell, also considering that tubulin post-translational modifications (PTMs) are markers of tumour plasticity. The two-dimensional (2D) culture systems are now being replaced by three-dimensional (3D) systems capable of mimicking in vivo conditions. In this work, spheroids were developed from C6 rat glioma cells (RGCs) using two 3D systems: liquid marbles (LMs) or hanging drops (HD) and analysed in terms of the morphology and behaviour of the two main tubulin PTMs, tyrosinated α-tubulin (Tyr-T) and acetylated α-tubulin (Ac-T). RGCs spontaneously formed spheroids more rapidly in the LM than in the HD system. An increase in Tyr-T and Ac-T was observed in both the HD and LM system during IVC, with the highest values shown in LM spheroids. In conclusion, the present work shows that the LM 3D system boosts the induction and maintenance of a high plasticity state in glioma cells and could provide a novel approach to set up a biological system to evaluate new anticancer therapies and advance knowledge on glioblastoma.Glioblastoma is a brain tumour frequently used as an experimental model to exploit innovative therapeutic approaches due to its high lethality and refractoriness to therapies. Part of these innovative anticancer therapies address cytoskeletal microtubules (MTs) since specific tubulin post-translational modifications (PTMs) are considered markers of tumour plasticity. In vitro studies, which traditionally employ two-dimensional (2D) culture systems, are now being replaced by three-dimensional (3D) systems that more closely mimic in vivo physiological conditions and allow a better understanding of the signalling between cells. In this work, we compared 2 liquid base 3D methods for the generation of spheroids from C6 rat glioma cells (RGCs) using 30 µL of liquid marble (LM) or the hanging drops (HDs), which contained 2 different cell numbers (5000 or 15,000). After 24 or 48 h of in vitro culture (IVC), the morphology of the spheroids was observed and the behaviour of the two main tubulin PTMs, tyrosinated α-tubulin (Tyr-T) and acetylated α-tubulin (Ac-T), was evaluated by fluorescence and Western blot (WB). RGCs spontaneously formed spherical agglomerates more rapidly in the LM than in the HD system. Cell density influenced the size of the spheroids, which reached a larger size (> of 300 µm Ø), with 15,000 cells compared to 5000 cells (150 µm Ø). Moreover, an increase in Tyr-T and Ac-T was observed in both the HD and LM system from 24 to 48 h, with the highest values shown in the 48 h/LM spheroids of 5000 cells (p < 0.05). In conclusion, by comparing the morphology and microtubular architecture of spheroids from C6 rat glioma cells developed by LM or HD methodology, our findings demonstrate that the use of a fumed silica microbioreactor boosts the induction and maintenance of a high plasticity state in glioma cells. RGCs cultured in LM express levels of tubulin PTMs that can be used to evaluate the efficacy of new anticancer therapies.

Synthesis and in vitro evaluation of a novel thienopyrimidine with phototoxicity towards rat glioma F98 cells

Glioblastoma multiforme is one of the most aggressive cancer forms in humans, and has low recovery rates after surgery, ionizing radiation and chemotherapy. Therefore, there is a high interest in the development of new treatment methods, as for instance photodynamic therapy (PDT). It is here presented results of the cytotoxic properties of the novel compound N1-(4-(4-(benzyl(methyl)amino)thieno[2,3-d]pyrimidin-6-yl)benzyl)-N2,N2-dimethylethane-1,2-diamine (1), which was found ten-fold more active on the rat glioma cell model F98 than the reference drug Temozolomide (TMZ). Further cell survival studies showed a profound increase in F98 cell death on UVA-radiation (330 nm, 0.5 mW/cm2). Photochemical internalization induced delivery of compound 1, but in contrast to the cytostatic drug Bleomycin, a higher cytotoxicity was not observed. Localization studies using fluorescence microscopy revealed that compound 1 readily internalized into the cytosol but did not enter the cell nucleus. The compound was shown to be a relatively weak epidermal growth factor receptor inhibitor, which is not likely to explain its cytotoxicity. However, the quantum efficiency for generation of singlet oxygen was 23%, suggesting generation of reactive oxygen species as one possible mechanism. Although more studies are needed to reveal detailed mode of action, compound 1 is a promising photosensitizer candidate for further development in tests of animal models.

Antioxidant and antiplasmodial activities of Malus sikkimenmsis bioactives and their role in the suppression of LPS-induced neuroinflammation in glial cells

As a part of our continued effort to isolate and identify the pharmacological properties of polyphenols from Malus Sikkimensis, here in this work we have evaluated the neuroprotective effects of these polyphenols along with their antioxidant and antiplasmodial properties. The antioxidant potential of these bioactives was estimated using enzymatic and non-enzymatic methods of free radical generation, where phloretin (PT) displayed better antioxidant potential under both the conditions. Additionally, PT also exhibited significant antiplasmodial activity. Phloridzin (PZ) and phloretin (PT) were also studied for their effect on lipopolysaccharide (LPS) induced neuro-inflammation in rat (C6)glioma cells. Our results showed that pre-treatment of PZ and PT at 5 and 10 µg/ml concentrations significantly (p < 0.05) suppressed the release of pro-inflammatory cytokines (TNF-α and IL-6) in a dose dependent manner. Additionally, PT exhibited more potent anti-inflammatory activity than PZ without any cytotoxic effects, as estimated using MTT assay. These findings suggest that PT possessed promising anti-neuroinflammatory potential along with antioxidant and antiplasmodial activities, which may be enhanced further by structural modification and chemical derivatization.

The effects of thymoquinone and quercetin on the toxicity of acrylamide in rat glioma cells.

Acrylamide is a neurotoxic agent forming in foods. Thymoquinone and quercetin are plant-derived antioxidants in various foods with known benefits. C6 cells are glioblastoma cells. In this study, we aimed at preventing acrylamide toxicity by thymoquinone and quercetin in the C6 cell line. In our study, first, toxic doses of acrylamide, nontoxic doses of thymoquinone and quercetin in C6 cells for 24 h were determined by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) colorimetric test. After that, caspase 3/7 and annexin V tests were performed by flow cytometry to evaluate whether the apoptosis pathway was inducted. Furthermore, autophagy and oxidative stress were assessed by flow cytometry. The amount of Nrf2 (nuclear factor erythroid 2-related factor 2) was determined by immunocytochemistry. The morphological examination was performed by microscopic analyses. As a result, 4 mM of acrylamide was determined to be used to induce toxicity in C6 cells. The nontoxic doses of thymoquinone and quercetin were respectively determined as 3.9 and 2.0 µM. Thymoquinone and quercetin not only reduced acrylamide-induced apoptosis in annexin V and caspase 3/7 assays but also morphological deformations in microscopic examinations. In autophagy, it was revealed that acrylamide-induced autophagy was decreased by quercetin and thymoquinone pretreatments. As for Nrf2 expression, it was observed that acrylamide increased Nrf2 expression, and thymoquinone and quercetin pretreatments increased it even further. In conclusion, in the study, acrylamide demonstrated a damaging effect on C6 glioblastoma cells, and thymoquinone and quercetin pretreatments exerted a protective effect against acrylamide-induced damage in C6 cells.

Bioprospecting of new Antarctic seaweed selective antitumor molecules: chemical characterization and in vitro analysis

BackgroundThe Antarctic continent exposes seaweeds to extreme environmental conditions, which may facilitate the production of novel metabolites. Algae represent a highly diverse group of little explored organisms. Thus, marine biomass has emerged as a possible source of new biologically active molecules for the treatment of diseases requiring novel therapeutic options. PurposeThe ultrasonic and Soxhlet extractions were used to evaluate chemical profiles and antitumor activities of Desmarestia anceps, Iridaea cordata, and Pyropia endiviifolia extracts using solvents of different polarity. MethodsChemical characterization of the extracts was performed using gas chromatography. The anticancer activities of the extracts were evaluated by exposing rat glioma (C6), human glioblastoma (U87), and human lung adenocarcinoma epithelial (A549) cell lines to increasing extract concentrations (10–1000 µg mL−1) for 24 and 48 h. In parallel, rat astrocyte and human lung fibroblast (MRC-5) were treated under the same conditions to represent non-transformed cell controls, and permit tumor selectivity evaluations of the extracts. ResultsAn MTT assay was used to measure cell cytotoxicity. Hexane, chloroform, and ethanol extracts reduced glioma and lung cancer cell viability. The hexane and ethanol extracts of P. endiviifolia showed the best antiproliferative effect, decreasing glioma cell viability by 40% at 10 µg mL−1, and the chloroform extract reduced lung adenocarcinoma cell viability by 50% at 46 µg mL−1 after 24 h of treatment. None of the extracts affected the viability of non-transformed cells, as was observed in tumor cells. Fatty acids, fatty alcohols, and phthalate esters were the main compounds identified in the seaweed extracts. ConclusionsAntarctic seaweeds displayed selective antitumor activity against glioma and lung cancer cells, particularly the endemic red alga P. endiviifolia. Therefore, these algae could play an important role as novel prototype molecule source for oncology drug development.

Nose-to-brain delivery of simvastatin mediated by chitosan-coated lipid-core nanocapsules allows for the treatment of glioblastoma in vivo

Glioblastoma is the most common and lethal malignant brain tumor. Despite simvastatin (SVT) showing potential anticancer properties, its antitumoral effect against glioblastoma appears limited when the conventional oral administration route is selected. As a consequence, nose-to-brain delivery has been proposed as an alternative route to deliver SVT into the brain. This study aimed to prepare chitosan-coated simvastatin-loaded lipid-core nanocapsules (LNCSVT-chit) suitable for nose-to-brain delivery and capable of fostering antitumor effects against glioblastoma both in vitro and in vivo. Results showed that the nanocapsules present adequate particle size (mean diameter below 200 nm), narrow particle size distribution (PDI < 0.2), positive zeta potential and high encapsulation efficiency (nearly 100%). In vitro cytotoxicity of LNCSVT-chit was comparable to non-encapsulated SVT in C6 rat glioma cells, whereas LNCSVT-chit were more cytotoxic than non-encapsulated SVT after 72 h of incubation against U-138 MG human glioblastoma cell line. In studies carried out in rats, LNCSVT-chit significantly enhanced the amount of drug in rat brain tissue after intranasal administration (2.4-fold) when compared with free SVT. Moreover, LNCSVT-chit promoted a significant decrease in tumor growth and malignancy in glioma-bearing rats in comparison to control and free SVT groups. Additionally, LNCSVT-chit did not cause any toxicity in treated rats. Considered overall, the results demonstrated that the nose-to-brain administration of LNCSVT-chit represents a novel potential strategy for glioblastoma treatment.

Burrowing behaviour of rats: Strain differences and applicability as well-being parameter after intracranial surgery.

In mice, burrowing is considered a species-typical parameter for assessing well-being, while this is less clear in rats. This exploratory study evaluated burrowing behaviour in three rat strains during training and in the direct postoperative phase after complex intracranial surgery in different neuroscience rat models established at Hannover Medical School or Aachen University Hospital. Male Crl:CD (SD; n = 18), BDIX/UlmHanZtm (BDIX; n = 8) and RjHan:WI (Wistar; n = 35) rats were individually trained to burrow gravel out of a tube on four consecutive days. Thereafter, BDIX rats were subjected to intracranial injection of BT4Ca cells and tumour resection (rat glioma model), SD rats to injection of 6-hydroxydopamine (6-OHDA) or vehicle (rat Parkinson's disease model) and Wistar rats to endovascular perforation or sham surgery (rat subarachnoid haemorrhage (SAH) model). Burrowing was retested on the day after surgery. During training, BDIX rats burrowed large amounts (mean of 2370 g on the fourth day), while SD and Wistar rats burrowed less gravel (means of 846 and 520 g, respectively). Burrowing increased significantly during training only in Wistar rats. Complex surgery, that is, tumour resection (BDIX), 6-OHDA injection (SD) and endovascular perforation or sham surgery for SAH (Wistar) significantly reduced burrowing and body weight, while simple stereotactic injection of tumour cells or vehicle did not affect burrowing. Despite the training, burrowing differed between the strains. In the direct postoperative phase, burrowing was reduced after complex surgery, indicating reduced well-being. Reduced burrowing was accompanied with postoperative weight loss, a validated and recognised quantitative measure for severity assessment.

Cytotoxicity of a Cell Culture Medium Treated with a High-Voltage Pulse Using Stainless Steel Electrodes and the Role of Iron Ions.

High-voltage pulses applied to a cell suspension cause not only cell membrane permeabilization, but a variety of electrolysis reactions to also occur at the electrode–solution interfaces. Here, the cytotoxicity of a culture medium treated by a single electric pulse and the role of the iron ions in this cytotoxicity were studied in vitro. The experiments were carried out on mouse hepatoma MH-22A, rat glioma C6, and Chinese hamster ovary cells. The cell culture medium treated with a high-voltage pulse was highly cytotoxic. All cells died in the medium treated by a single electric pulse with a duration of 2 ms and an amplitude of just 0.2 kV/cm. The medium treated with a shorter pulse was less cytotoxic. The cell viability was inversely proportional to the amount of electric charge that flowed through the solution. The amount of iron ions released from the stainless steel anode (>0.5 mM) was enough to reduce cell viability. However, iron ions were not the sole reason of cell death. To kill all MH-22A and CHO cells, the concentration of Fe3+ ions in a medium of more than 2 mM was required.

RETRACTED ARTICLE: Cirsilineol inhibits cell growth and induces apoptosis in glioma C6 cells via inhibiting MAPK and PI3K/Akt/mTOR signaling pathways

BackgroundGlioma is a major type of brain cancer and responsible for high death rate worldwide due to the delayed diagnosis and chemo-resistance. It is responsible for nearly 16% of all brain cancer incidences with increased prevalence globally.ObjectiveThe present research study deals with discovering the in vitro anticancer potential of cirsilineol on the rat glioma C6 cells through the inhibition of PI3K/Akt/mTOR and MAPK signaling pathways.MethodologyThe in vitro antioxidant potential of cirsilineol was examined by different free radical scavenging analyses. The effects of cirsilineol on the cell viability of C6 and normal Vero cells were investigated by MTT assay. The intracellular ROS production and apoptotic cell death in the cirsilineol-administered C6 cells were scrutinized by fluorescent staining techniques. The contents of TBARS and GSH, SOD and caspase activity were examined using assay kits. The PI3K, mTOR, Akt, ERK1/2, JNK1/2, and p38 expressions in the cirsilineol-supplemented C6 cells were examined by RT-PCR study.ResultsThe cirsilineol treatment appreciably scavenged the DPPH, superoxide, and peroxyl radicals. The cell viability of C6 cells was markedly reduced by the cirsilineol; however, it does not affected the Vero cell viability. The cirsilineol treatment demonstrated the augmented ROS production and apoptosis in the C6 cells. The TBARS level and caspase activity were improved, and GSH level and SOD activity were depleted by the cirsilineol. Cirsilineol effectively inhibited PI3K, mTOR, Akt, ERK1/2, JNK1/2, and p38 expressions in the C6 cells.ConclusionOur findings confirmed that the cirsilineol inhibited cell growth and induced apoptosis in C6 cells by inhibiting the PI3K/Akt/mTOR and MAPK signaling cascades. Hence, it can be a potential candidate to treat the glioma in the future.

Application of Intravoxel Incoherent Motion Diffusion-Weighted Imaging in Predicting and Monitoring Early Efficacy of Anti-Angiogenic Therapy in the C6 Glioma Rat Model.

ObjectiveTo investigate the feasibility of intravoxel incoherent motion (IVIM) diffusion-weighted imaging (DWI) in evaluating early effects of anti-angiogenic therapy in the C6 glioma rat model.MethodsTwenty-six rats of the C6 glioma model were randomly divided into a treatment group (received bevacizumab) and a control group (physiological saline). IVIM-DWI was performed on days 0, 1, 3, 5, and 7 after anti-angiogenic therapy and tumor growth and IVIM-DWI parameters were dynamically observed. Hematoxylin and eosin, CD34 microvessel density (MVD), proliferation of cell nuclear antigen (PCNA), and Hif-α staining were performed on day 7. One-way ANOVA was used to compare intra-group differences and an independent-samples t-test was used to compare inter-group differences of MRI parameters. Correlations between IVIM-DWI parameters, tumor size, and pathological results were analyzed.ResultsThe relative change in tumor volume (ΔVolume) in the two groups differed significantly on days 5 and 7 (p = 0.038 and p < 0.001). The perfusion-related parameters D*- and f-values decreased in the treatment group and demonstrated significant differences compared with the control group on days 3, 5, and 7 (p = 0.033, p < 0.001, and p < 0.001, respectively). The diffusion-related parameters ADC and D-values increased in the treatment group and were found to be significantly differently different from the control group on days 5 and 7 (both p < 0.001). The initial D-value showed a negative correlation with ΔVolume (γ = −0.744, p < 0.001), whereas the initial D*-value and relative change of D-value had a positive correlation with ΔVolume (γ = 0.718, p < 0.001 and γ = 0.800, p < 0.001, respectively). MVD was strongly positively correlated with D*-value (r = 0.886, p = 0.019), PCNA was negatively correlated with ADC- and D-values (r = −0.848, p = 0.033; and r = −0.928 p = 0.008, respectively), and Hif-1α was strongly negatively correlated with D*-value (r = −0.879, p = 0.010).ConclusionIVIM-DWI was sensitive and accurate in predicting and monitoring the effects of early anti-angiogenesis therapy in a C6 glioma rat model.

Sacsin Deletion Induces Aggregation of Glial Intermediate Filaments.

Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is a neurodegenerative disorder commonly diagnosed in infants and characterized by progressive cerebellar ataxia, spasticity, motor sensory neuropathy and axonal demyelination. ARSACS is caused by mutations in the SACS gene that lead to truncated or defective forms of the 520 kDa multidomain protein, sacsin. Sacsin function is exclusively studied on neuronal cells, where it regulates mitochondrial network organization and facilitates the normal polymerization of neuronal intermediate filaments (i.e., neurofilaments and vimentin). Here, we show that sacsin is also highly expressed in astrocytes, C6 rat glioma cells and N9 mouse microglia. Sacsin knockout in C6 cells (C6Sacs−/−) induced the accumulation of the glial intermediate filaments glial fibrillary acidic protein (GFAP), nestin and vimentin in the juxtanuclear area, and a concomitant depletion of mitochondria. C6Sacs−/− cells showed impaired responses to oxidative challenges (Rotenone) and inflammatory stimuli (Interleukin-6). GFAP aggregation is also associated with other neurodegenerative conditions diagnosed in infants, such as Alexander disease or Giant Axonal Neuropathy. Our results, and the similarities between these disorders, reinforce the possible connection between ARSACS and intermediate filament-associated diseases and point to a potential role of glia in ARSACS pathology.

Influence of N-alkynylaminosteroids on mitochondria function and autophagy in glioma cells

In this work we examined the synthesized N-alkynyl-17-aminosteroids and N-alkynyl-20-aminosteroids (based on dehydroepiandrosterone and pregnenolone, respectively) for their effect on C6 rat glioma cell functions. At 10 μM, the compounds had an insignificant effect on C6 glioma mitochondrial membrane potential, but increased cell autophagy by 70-90%, comparable to the known autophagy inducer dexamethasone. Docking simulations predict a potential high-affinity interaction between N-alkynylaminosteroids and Keap1 and the Hedgehog pathway protein, Smoothened, which are involved in autophagy regulation. The possible mechanisms of observed processes are discussed.

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.