Cultured C6 Glioma Cells Research Articles

Changes in intracellular water diffusion and energetic metabolism in response to ischemia in perfused C6 rat glioma cells

This work reports results of experiments in hollow-fiber bioreactor C6 glioma cell cultures where the apparent diffusion coefficient (ADC) of intracellular water (iADC) was measured at diffusion times between 0.83 and 40 ms. The experiments were carried out before and after the onset of permanent ischemia. The changes in iADC following ischemia were dependent on the diffusion time employed in the experiment. An ischemia-induced decrease in the iADC was measured at short diffusion times, while at long diffusion times the iADC increased. Decreases in the iADC measured at short diffusion times are interpreted to be a result of a decrease in the intrinsic diffusivity of intracellular water due to energy failure. Increases in iADC measured at long diffusion times, are interpreted to result from cell swelling.

Blood-Brain Barrier Disruption with Focused Ultrasound Enhances Delivery of Chemotherapeutic Drugs for Glioblastoma Treatment

To demonstrate the feasibility of using focused ultrasound to enhance delivery of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) to glioblastomas in rats with induced tumors and determine if such an approach increases treatment efficacy. All animal experiments were approved by the animal committee and adhered to the experimental animal care guidelines. A 400-kHz focused ultrasound generator was used to transcranially disrupt the blood-brain barrier (BBB) in rat brains by delivering burst-tone ultrasound energy in the presence of microbubbles. The process was monitored in vivo by using magnetic resonance (MR) imaging. Cultured C6 glioma cells implanted in Sprague-Dawley rats were used as the tumor model. BCNU (13.5 mg/kg) was administered intravenously and its concentration in brains was quantified by using high-performance liquid chromatography. MR imaging was used to evaluate the effect of treatments longitudinally, including analysis of tumor progression and animal survival, and brain tissues were histologically examined. Methods including the two-tailed unpaired t test and the Mantel-Cox test were used for statistical analyses, with a significance level of .05. Focused ultrasound significantly enhanced the penetration of BCNU through the BBB in normal (by 340%) and tumor-implanted (by 202%) brains without causing hemorrhaging. Treatment of tumor-implanted rats with focused ultrasound alone had no beneficial effect on tumor progression or on animal survival up to 60 days. Administration of BCNU only transiently controlled tumor progression; nevertheless, relative to untreated controls, animal survival was improved by treatment with BCNU alone (increase in median survival time [IST(median)], 15.7%, P = .023). Treatment with focused ultrasound before BCNU administration controlled tumor progression (day 31: 0.05 cm(3) + or - 0.1 [standard deviation] vs 0.28 cm(3) + or - 0.1) and improved animal survival relative to untreated controls (IST(median), 85.9%, P = .0015). This study demonstrates a means of increasing localized chemotherapeutic drug delivery for brain tumor treatment and strongly supports the feasibility of this treatment in a clinical setting.

Microgravity-induced apoptosis in cultured glial cells.

Apoptosis is a form of naturally occurring cell death that plays fundamental roles during embryonic developement. In adults, it neatly disposes of cells damaged by injuries provoked by external causes such as UV radiation, ionisation and heat shock. Alteration of the gravity vector may be one of the external apoptosis inducers. Neurophysiological impairment signs were seen during space flights in astronauts, but very few studies were carried out on the nervous system and none at the cellular level. In this study, we submitted cultured C6 glioma cells to microgravity (0xg) of varying duration, obtained by clinorotation in a Fokker three-dimensional clinostat for 15 min, 30 min, 1h, 20h or 32h. After 30 min at 0xg, numerous nuclei underwent the classical morphological alterations (chromatin condensation, nuclear fragmentation, apoptotic bodies) that lead to the programmed cell death. After 30 min at 0xg, immunostaining for the enzyme caspase-7 was present in the cytoplasm of many cells concurrently with DNA fragmentation identified by the TUNEL method. At 32h, the number of apoptotic nuclei was much reduced indicating the ability of glial cells to adapt to altered gravity.

Cytotoxic, cytoprotective and antioxidant activities of resveratrol and analogues in C6 astroglioma cells in vitro

Resveratrol (3,4′,5- trans-trihydroxystilbene) and other hydroxystilbenes exhibit in vitro antioxidant as well as prooxidant effects. The antioxidant properties are assumed to enable these compounds to protect cells from oxidative damage. The prooxidant effects are held likely to be responsible for their cytotoxic, anti-proliferative or pro-apoptotic effects observed in vitro. Regarding antioxidant/prooxidant activities in the past various studies were performed aiming at defining structure–activity relationships for hydroxystilbenes using cell-free systems. In the present study cultured C6 glioma cells were used in order to investigate the relationship between the antioxidant, cytoprotective and cytotoxic activities of resveratrol and selected analogues, e.g., 3,3′,4′,5- trans-tetrahydroxystilbene (piceatannol), 3,3′,5,5′- trans-tetrahydroxystilbene (3,3′,5,5′-THS) and 3,3′,4′,5,5′- trans-pentahydroxystilbene (3,3′,4′,5,5′-PHS). All these compounds were cytotoxic to growth-arrested C6 cells, with EC 50-values between 20 and 85 μM. A higher cytotoxic potency in proliferating cells indicated a specific cytostatic activity of resveratrol and 3,3′,4′,5,5′-PHS. All hydroxystilbenes studied inhibited cellular radical generation induced by cumene hydroperoxide (CHP). The rank order of antioxidant potency was resveratrol > piceatannol > 3,3′,5,5′-THS > 3,3′,4′,5,5′-PHS. However, only resveratrol and piceatannol inhibited cellular radical generation at lower than cytotoxic concentrations. At subcytotoxic concentrations only piceatannol was able to protect the cells from damage caused by CHP. Taken together, these results show that neither the cytotoxic or cytostatic activities of hydroxystilbenes nor their cytoprotective and antioxidant activities in living cells can be predicted from their antioxidant and prooxidant activity, respectively, in cell-free systems.

Selective COX-2 Inhibitor (Rofecoxib) Inhibits Vinorelbine Cytotoxicity in C6 Glioma Cells In Vitro

We examined the effects of gemcitabine, a novel nucleotide analogue, and vinorelbine, a semi-synthetic vinca alcaloid, in C6 glioma cell culture. We simultaneously monitored the modulation of its activity in combination with the te- lomerase inhibitory agent dimetilsulfoxid and with a specific cyclooxygenase-2 inhibitory agent Rofecoxib. The effects of the gemcitabine/ vinorelbine combination were observed over a 96 hour period. Plating, S-phase (bromodeoxyuridine- labelling index) and ultrastructure were selected as the evaluation parameters of drug interactions. Gemcitabine (10� g/ml) was found to possess significant blocking activity of C6 glioma plating and cells' S-phase. Vi- norelbine (10� g/ml) also demonstrated significant inhibitory activity. Vinorelbine reduced BrdU-LI but was not as effec- tive as gemcitabine. Rofecoxib (10� g/ml) showed no synergism on cell inhibition either with gemcitabine or vinorelbine and protected against the S-phase depleting activity of vinorelbine. Dimetilsulfoxid (20� g/ml) reduced the effect of vinorelbine in spheroid culture. But at the final 96 th hour tie point dimetil- sulfoxid reduced the increasing cell numbers which were previously seen in the gemcitabine group. In conclusion, both gemcitabine and vinorelbine effectively reduced both the monolayer and spheroid growth of C6 glioma cells. At the single cell level, only dimetilsulfoxid could sensitise for gemcitabine but not vinorelbine. Despite re- ducing spheroid growth, treatment using cyclooxygenase-2 inhibitors with microtubule inhibitors should be avoided.

Transcriptional regulation of PSA-NCAM expression by NMDA receptor activation in RA-differentiated C6 glioma cultures

N-Methyl- d-aspartate (NMDA) receptors exhibit a dichotomy of signaling with both toxic and plastic responses. Recent reports have shown that exposure to subtoxic concentration of NMDA results in a neuroprotective state that was measured when these neurons were subsequently challenged with toxic doses of glutamate or kainate. Control of polysialylated neural cell adhesion molecule (PSA-NCAM) expression by NMDA receptor activation has been described in several systems, suggesting a functional link between these two proteins. The perception of glial role in CNS function has changed dramatically over the past few years from simple trophic functions to that of cells with important roles in development and maintenance of CNS in cooperation with neurons. We report here the transcriptional regulation of PSA-NCAM expression by subtoxic dose of NMDA in retinoic acid differentiated C6 glioma cell cultures. C6 glioma cell cultures differentiated with retinoic acid (10 μM) were exposed to NMDA (100 μM) or to antagonist MK-801 (200 nM) prior to treatment with NMDA and cells were harvested after 24 h of treatment to study the expression of total NCAM, PSA-NCAM, nuclear factor kappaB (NF-κB) and activator protein-1 (AP-1) by Western blotting and dual immunocytofluorescence and expression of PST mRNA by fluorescent in situ hybridization (FISH). Significant increase in the levels of PSA-NCAM, NF-κB, AP-1 and PST mRNA was observed in NMDA treated cultures. Treatment of cultures with MK-801, a non-competitive NMDA receptor antagonist, prior to NMDA exposure prevented the NMDA-mediated changes indicating the involvement of NMDA receptor activation. The results elucidate the possible cellular and molecular mechanisms of regulation of PSA-NCAM expression in astroglial cultures by extracellular signals.

The effect of photodynamic therapy on cultured rat C6 glioma cells under different conditions

Objective To evaluate the effect of hepatoprophyrin derivative (HpD) mediated photodynamic therapy (PDT) on cultured C6 glioma cells. Methods Cell survival rate examination after HpD-PDT treatment: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyhertrazolium bromide (MTr) assay was used to determine eell survival rate 24 hrs after irradiation by Lumaeare-051 as follows: (1) C6 cells were irradiated at 628 nm, 20 mW/cm2 for 5 rain afrer being incubated with different concentration of HpD (0, 2, 5, 10, 20, 30 and 40 mg/L) respectively; (2) Cells were irradiated at 628 nm, 20 mW/cm2 for 5 min after being ineubated with HpD for different time periods (0. 5, 1, 3, 6, 12 and 24 hrs); (3) After being incubated with HpD, Cells were irradiated at 628 nm for 5 min at different light output power (10, 20 and 30 mW/cm2). Cell survival rate was calculated as follows: Survival(%)=(mean O. D. value of treated cell/mean O. D. value of control cell) × 100%. Results The effect of HpD-PDT on C6 cells differed under different conditions: No cell death was seen in low HpD concentration (2 mg/L) or short time incubation (< 3 h) groups; Cell survival rate decreased as the concentraion of HpD, incubation time with HpD and light output power increased. Conclusions The effect of HpD-PDT differes under different conditions, which is correlated to the concentration of HpD, incubation time of C6 cells with HpD and also the light output power. Key words: Glioma; Photodynamic therapy (PDT); C6 glioma cell; Hematoporphyrin derivative(HpD)

Morphine enhances purine nucleotide catabolism in vivo and in vitro

To investigate the effect and mechanism of morphine on purine nucleotide catabolism. The rat model of morphine dependence and withdrawal and rat C6 glioma cells in culture were used. Concentrations of uric acid in the plasma were measured by the uricase-rap method, adenosine deaminase (ADA) and xanthine oxidase (XO) in the plasma and tissues were measured by the ADA and XO test kit. RT-PCR and RT-PCR-Southern blotting were used to examine the relative amount of ADA and XO gene transcripts in tissues and C6 cells. (i) the concentration of plasma uric acid in the morphine-administered group was significantly higher (P<0.05) than the control group; (ii) during morphine administration and withdrawal periods, the ADA and XO concentrations in the plasma increased significantly (P<0.05); (iii) the amount of ADA and XO in the parietal lobe, liver, small intestine, and skeletal muscles of the morphine-administered groups increased, while the level of ADA and XO in those tissues of the withdrawal groups decreased; (iv) the transcripts of the ADA and XO genes in the parietal lobe, liver, small intestine, and skeletal muscles were higher in the morphine-administered group. The expression of the ADA and XO genes in those tissues returned to the control level during morphine withdrawal, with the exception of the skeletal muscles; and (v) the upregulation of the expression of the ADA and XO genes induced by morphine treatment could be reversed by naloxone. The effects of morphine on purine nucleotide metabolism might be an important, new biochemical pharmacological mechanism of morphine action.

Effects of Gamma Knife surgery on C6 glioma in combination with adenoviral p53 in vitro and in vivo

The authors sought to study the combined potential of wild-type p53 gene transfer and Gamma Knife surgery (GKS) for the treatment of glioblastomas multiforme. Modification of the radiation response in C6 glioma cells in vitro and in vivo by the wild-type p53 gene was investigated. Stable expression of wild-type p53 in C6 cells was achieved by transduction of the cells with adenoviral p53. Two days later, some cells were treated with GKS. Forty-eight hours after irradiation, the comparative survival rate was assessed by monotetrazolium (MTT) assays. Treated and control C6 glioma cells (4 x 10(3) per well) were plated into a 96-well plate in octuplicate and tested every 24 hours. Meanwhile, immunohistopathological examination of proliferating cell nuclear antigen (PCNA) and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (TUNEL) assays were performed. The MTT assays indicated the p53, GKS, and combined treated cells proliferated at a significantly lower rate than those of the control group (p < 0.01, Days 2-6) and the positive fraction of PCNA in p53-treated group and GKS-treated group was 70.18 +/- 3.61 and 50.71 +/- 2.61, respectively, whereas the percentage in the combined group was 30.68 +/- 1.49 (p < 0.01). Fifty-six male Sprague-Dawley rats were anesthetized and inoculated with 10(6) cultured C6 glioma cells into the cerebrum. Forty-eight hours after transduction with adenoviral p53, some rats underwent GKS. A margin dose of 15 Gy was delivered to the 50% isodose line. Two days later, six rats in each group were killed. Their brains were removed and paraffin-embedded section were prepared for immunohistopathological examination and TUNEL assays. The remaining rats were observed for the duration of the survival period. The survival curve indicated that a modest but significant enhancement of survival duration was seen in the p53-treated or GKS alone groups, whereas a more marked and highly significant enhancement of survival duration was achieved when these two treatment modalities were combined. When PCNA expression was downregulated, apoptotic cells become obvious after TUNEL staining. The findings of this study suggest that p53-based gene therapy in combination with GKS may be superior to single-modality treatment of C6 glioma.

A possible cellular explanation for the NMR-visible mobile lipid (ML) changes in cultured C6 glioma cells with growth

The NMR-visible mobile lipid (ML) signals of C6 glioma cells have been monitored at 9.4 and 11.7 T (single pulse and 136 ms echo time) from cell pellets by 1H NMR spectroscopy. A reproducible behavior with growth has been found. ML signals increase from log phase (4 days of culture) to postconfluence (7 days of culture). This ML behavior is paralleled by the percentage of cells containing epifluorescence detectable Nile Red stained cytosolic droplets (range 23%–60% of cells). The number of positive cells increases after seeding (days 0–1), decreases at log phase (days 2–4), increases again at confluence (day 5) and even further at post-confluence (day 7). C6 cells proliferation arrest induced by growth factors deprivation induces an even higher accumulation of cytosolic droplets (up to 100% of cells) and a large ML increase (up to 21-fold with respect to 4-day log phase cells). When neutral lipid content is quantified by thin-layer chromatography (TLC) on total lipid extracts of C6 cells, no statistically significant change can be detected (in μg/10 8 cells) with growth or growth arrest in major neutral lipid containing species (triacylglycerol, TAG, diacylglycerol, DAG, cholesteryl esters, ChoEst) except for DAG, which decreased in post-confluent, 7-day cells. The apparent discrepancy between NMR, optical microscopy and TLC results can be reconciled if possible biophysical changes in the neutral lipid pool with growth are taken into account. A cellular explanation for the observed results is proposed: the TAG-droplet-size-change hypothesis.

In vitro evidence for an antioxidant role of 3-hydroxykynurenine and 3-hydroxyanthranilic acid in the brain

We investigated the in vitro effect of 3-hydroxykynurenine (3HKyn), 3-hydroxyanthranilic acid (3HAA), kynurenine (Kyn) and anthranilic acid (AA) on various parameters of oxidative stress in rat cerebral cortex and in cultured C6 glioma cells. It was demonstrated that 3HKyn and 3HAA significantly reduced the thiobarbituric acid-reactive substances (TBA-RS) and chemiluminescence measurements in rat cerebral cortex, indicating that these metabolites prevent lipid peroxidation in the brain. In addition, GSH spontaneous oxidation was significantly prevented by 3HAA, but not by the other kynurenines in cerebral cortex. We also verified that 3HKyn and 3HAA significantly decreased the peroxyl radicals induced by the thermolysis of 2,2′-azo-bis-(2-amidinopropane)-derived peroxyl radicals, and to a higher degree than the classical peroxyl scavenger trolox. 2-Deoxy- d-ribose degradation was also significantly prevented by 3HKyn, implying that this metabolite was able to scavenge hydroxyl radicals. Furthermore, the total antioxidant reactivity of C6 glioma cells was significantly increased when these cells were exposed from 1 to 48 h to 3HKyn, being the effect more prominent at shorter incubation times. TBA-RS values in C6 cells were significantly reduced by 3HKyn when exposed from 1 to 6 h with this kynurenine. However, C6 cell morphology was not altered by 3HKyn. Finally, we tested whether 3HKyn could prevent the increased free radical production induced by glutaric acid (GA), the major metabolite accumulating in glutaric acidemia type I, by evaluating the isolated and combined effects of these compounds on TBA-RS levels and 2′,7′-dihydrodichlorofluorescein (DCFH) oxidation in rat brain. GA provoked a significant increase of TBA-RS values and of DCFH oxidation, effects that were attenuated and fully prevented, respectively, by 3HKyn. The results strongly indicate that 3HKyn and 3HAA behave as antioxidants in cerebral cortex and C6 glioma cells from rats.

Regulatory Effect of Ginkgo Biboba Extract on the Expression of NF-κB and Nitroxide Production in Glioma Cells

Objective To study the effects of Ginkgo biloba extract(EGb761) on the expression of nuclear factor-kappa B(NF-κB), contents of nitric oxide(NO) and inducible nitric oxide synthase(iNOS) in C6 glioma cells. Methods Upon stimulation with lipopolysaccharide(LPS) and phorbol-1,2-myristate-1,3-acetate (PMA), iNOS gene was expressed in cultured C6 glioma cells, which caused the consequent generation of high concentration of nitric oxide. The effects of EGb761 on the intracellular NO concentration and the expression of iNOS gene in stimulated C6 glioma cells was studied by fluorescence probe and laser scanning confocal system, Western blot analysis and reverse transcription polymerase chain reaction techniques. Results The results showed that EGb761 decreased the expression of iNOS at either the protein or mRNA level. Further investigation showed that EGb761 significantly inhibited the degradation of IκB-α, and blocked the translocation of p65/RelA into the nuclei. Conclusion The results suggest that EGb761 inhibits the expression of iNOS by modulating the NF-κB pathway.

Amantadine and memantine induce the expression of the glial cell line-derived neurotrophic factor in C6 glioma cells

Aminoadamantanes are commonly used in the treatment of Parkinson's and Alzheimer's diseases. While these drugs are shown to antagonise ionotropic glutamate receptors on neuronal cells, additional mechanisms could contribute to their neuroprotective properties. The aim of the present study was to investigate the effect of aminoadamantanes on the production of the glial cell line-derived neurotrophic factor (GDNF) in glial cells. For this purpose, we measured the modulation of GDNF release in C6 glioma cell cultures treated for 24 h with amantadine and memantine. Both drugs dose-dependently increased GDNF level in the culture medium with similar potency (submicromolar range) and efficacy (three to four-fold induction). RT-PCR studies revealed that both compounds also increased GDNF mRNA levels and their influence on the GDNF gene transcription was further evidenced using a rat GDNF promoter luciferase reporter assay. Together, these results demonstrate that the neuroprotective effect of amantadine and memantine could involve the regulation of GDNF production by glial cells.

HSP70 induction and oxidative stress protection mediated by a subtoxic dose of NMDA in the retinoic acid-differentiated C6 glioma cell line

NMDA class of glutamate receptors plays an important role in regulating toxic and plastic responses in CNS. Astrocytes are the predominant cell type in the adult CNS and recent studies have suggested their role in many aspects of CNS function and dysfunction. We report here the protective effect of a subtoxic dose of NMDA in retinoic acid differentiated C6 glioma cell cultures. C6 glioma cell cultures differentiated with retinoic acid (10 μM) were exposed to NMDA (100 μM) or to antagonist MK-801 (200 nM) alone as well as with NMDA and cells were harvested after 24 h of treatment to study the expression of HSP70 and for biochemical assay of free radical scavenger system. The protection imparted by a subtoxic dose of NMDA was checked by challenging the differentiated controls as well as NMDA treated and MK-801 treated cultures with a toxic dose of glutamate and subsequently estimating the free radical scavenger system profile. Biochemical analysis revealed a significant increase in the activities of glutathione peroxidase (GPx), copper zinc-superoxide dismutase (CuZnSOD) and reduced glutathione (GSH) content upon exposure to NMDA. No significant change was observed in the level of lipid peroxidation (LPx). A significant increase was observed in HSP70 expression as seen by Western blotting and immunocytofluorescent studies in NMDA treated cultures. Treatment of cultures with MK-801 alone, a non-competitive NMDA receptor antagonist, or pretreatment with MK-801 prior to NMDA exposure prevented the NMDA mediated changes indicating the involvement of NMDA receptors mediated mechanism. The results illustrate the protective effect of a subtoxic dose of NMDA in RA differentiated C6 glioma cell line.

Blockage of angiotensin II type I receptor decreases the synthesis of growth factors and induces apoptosis in C6 cultured cells and C6 rat glioma

Angiotensin II (Ang II) is a main effector peptide in the renin–angiotensin system and participates in the regulation of vascular tone. It also has a role in the expression of growth factors that induce neovascularisation which is closely associated to the growth of malignant gliomas. We have shown that the selective blockage of the AT1 receptor of angiotensin inhibites tumour growth, cell proliferation and angiogenesis of C6 rat glioma. The aim of this study was to study the effects of the blockage of AT1 receptor on the synthesis of growth factors, and in the genesis of apoptosis in cultured C6 glioma cells and in rats with C6 glioma. Administration of losartan at doses of 40 or 80 mg kg−1 to rats with C6 glioma significantly decreased tumoral volume and production of platelet-derived growth factor, vascular endothelial growth factor and basic fibroblast growth factor. It also induced apoptosis in a dose-dependent manner. Administration of Ang II increased cell proliferation of cultured C6 cells which decreased by the administration of losartan. Our results suggest that the selective blockage of AT1 diminishes tumoral growth through inhibition of growth factors and promotion of apoptosis.

Tamoxifen-induced cell death and expression of neurotrophic factors in cultured C6 glioma cells

The effects of ( Z)-2[ p -(1,2-diphenyl-1-butenyl)phenoxy]-N ,N -dimethylamine citrate (tamoxifen) on cell survival and the expression of neurotrophic factors (NTF) were investigated in rat C6 glioma cells (C6). C6 cells do not express the estrogen receptor. Cytotoxic effect was detected from 24 h after the treatment with 10 microM tamoxifen and increased with time in a dose-dependent manner. C6 cells treated with tamoxifen also displayed various morphological types such as elliptical, round and aggregated form. As the treatment time increased, the proliferation of C6 cells was reduced remarkably and most of them became the round or aggregated form. To examine the relationship of the expression of NTF and the cytotoxicity of tamoxifen, the mRNA level of brain-derived neurotrophic factor (BDNF), glial-derived neurotrophic factor (GDNF), and basic fibroblast growth factor (bFGF) was measured after 24 h treatment with tamoxifen by RT-PCR. The expression of mRNA of BDNF or GDNF in C6 cells treated with various concentrations of tamoxifen was comparable to controls. The expression of bFGF mRNA was significantly reduced in C6 cells treated with 10 or 15 microM tamoxifen. The results suggest that tamoxifen exerts cytotoxic effect on estrogen receptor-negative C6 cells through the inhibition of the transcription of bFGF.

Therapeutic Effects of Holmium-166 Chitosan Complex in Rat Brain Tumor Model

This study examined the effectiveness of Holmium-166 (Ho-166) chitosan complex therapy for a malignant glioma. Cultured C6 glioma cells (100,000 in 5 µl) were injected into the caudate/putamen of 200 - 250 gram Wistar rats. Five days later, a Ho-166 chitosan complex was injected into the same site of the glioma injection. Four injection doses were administered: the control group received PBS 10 µl, group 1 received an injection of 100 µCi (10 µl), group 2 received an injection of 50 µCi (5 µl), and group 3 received an injection of 10 µCi (1 µl). The average tumor volume for each group was 1.385 mm3 for the control group, 0.036 mm3 for group 1, 0.104 mm3 for group 2, and 0.111 mm3 for group 3. Compared with the control group, the size of the tumors in groups 1, 2 and 3 was reduced by an average of 97.4%, 92.5% and 91.9%, respectively. The Kaplan-Meier survival curve of group 2 was the longest, followed by groups 3, group 1 and the control. The mean survival was 22.8, 59, 60, and 44.6 days for the control group and groups 3, 2 and 1, respectively. H-E staining revealed that group 2 yielded the best results in the destruction of the malignant glioma. TUNEL staining and immunohistochemical studies indicated apoptotic features. The Ho-166 chitosan complex proved to be effective in destroying the malignant glioma.

Δ9-tetrahydrocannabinol increases C6 glioma cell death produced by oxidative stress

(−)Δ9-Tetrahydrocannabinol is a scavenger of free radicals. However, the activation of the CB1 receptor in cultured C6 glioma cells by (−)Δ9-tetrahydrocannabinol in the presence of reagents generating reactive oxygen species leads to amplification of the cellular damage from oxidative stress. This was evident by increased loss of cell wall integrity, impaired mitochondrial function and reduction of glucose uptake. In addition, (−)Δ9-tetrahydrocannabinol treatment was also found to be deleterious to the cells under conditions of glucose starvation. Free radicals have been implicated in various conditions leading to cell death and, as a routine, the Fenton reaction is utilized for modeling reactive oxygen species production. Our study was performed using a cell permeating Fe(III) chelating quinone that provides more physiological conditions for mimicking the naturally occurring oxidative stress within the cell and thus serves as a better model for natural reactive oxygen species formation.

Inhibition of cell cycle progression by penta-acetyl geniposide in rat C6 glioma cells

Penta-acetyl geniposide, (Ac) 5-GP, the acetylated compound of geniposide, is able to inhibit the growth of rat C6 glioma cells in culture and in the bearing rats. Our recent data indicated that the induction of cell apoptosis and cell cycle arrest at G 0/gap phase 1 (G 1) by (Ac) 5-GP might be associated with the induction of p53 and c-Myc, and mediated via the apoptosis-related bcl-2 family proteins. In this report, we further investigated the mechanism involved in the cell cycle arrest induced by (Ac) 5-GP in C6 glioma cells. The inhibitory effect of (Ac) 5-GP on the cell cycle progression of C6 glioma cells which arrested cells at the G 0/G 1 phase was associated with a marked decrease in the protein expression of cyclin D 1, and an induction in the content of cyclin-dependent kinase (cdk) inhibitor p21 protein. This effect was correlated with the elevation in p53 levels. Further immunoprecipitation studies found that, in response to the treatment, the formation of cyclin D 1/cdk 4 complex declined, preventing the phosphorylation of retinoblastoma (Rb) and the subsequent dissociation of Rb/E2F complex. These results illustrated that the apoptotic effect of (Ac) 5-GP, arresting cells at the G 0/G 1 phase, was exerted by inducing the expression of p21 that, in turn, repressed the activity of cyclin D 1/cdk 4 and the phosphorylation of Rb.

Isoflurane enhances the expression and activity of glutamate transporter type 3 in C6 glioma cells.

Glutamate transporters play an important role in maintaining extracellular glutamate homeostasis. Volatile anesthetics have been shown to affect glutamate transporter activity acutely (within minutes after the exposure). It is not known whether volatile anesthetics affect the expression of glutamate transporters. Rat cultured C6 glioma cells that express excitatory amino acid transporter type 3 (EAAT3) were exposed to isoflurane at various concentrations (0.5-4.0%) or for different periods (1-24 h) at 37 degrees C. EAAT3 mRNA, proteins, and activity were quantified. Isoflurane induced a time- and concentration-dependent increase in the mRNA and protein levels of EAAT3 in C6 cells. The maximal increase was induced by 2% isoflurane, and the cells incubated with 2% isoflurane for 3 and 7 h expressed the highest levels of EAAT3 mRNA and proteins, respectively. Similarly, glutamate uptake was higher in C6 cells exposed to 2% isoflurane for 7 h than in control cells. Actinomycin D and cycloheximide, inhibitors for mRNA and protein synthesis, respectively, did not affect the isoflurane-induced increase in EAAT3 mRNA and protein expression. Phorbol 12-myristate 13-acetate, a protein kinase C activator, also enhanced EAAT3 expression. The combination of 2% isoflurane and phorbol 12-myristate 13-acetate caused a higher level of EAAT3 expression than that induced by 2% isoflurane alone. Neither staurosporine, a protein kinase C inhibitor, nor wortmannin, a phosphatidylinositol 3 kinase inhibitor, inhibited the isoflurane-induced increase in EAAT3 expression. The results of this study suggest that isoflurane increases the expression and activity of EAAT3 by stabilizing EAAT3 mRNA and proteins via protein kinase C- and phosphatidylinositol 3 kinase-independent pathways.