Rat C6 Glioma Cells Research Articles

Enhanced cell growth and tumorigenicity of rat glioma cells by stable expression of human CD133 through multiple molecular actions

CD133 (Prominin-1/AC133) is generally treated as a cell surface marker found on multipotent stem cells and tumor stem-like cells, and its biological function remains debated. Genetically modified rat glioma cell lines were generated by lentiviral gene delivery of human CD133 into rat C6 glioma cells (hCD133(+) -C6) or by infection of C6 cells with control lentivirus (mock-C6). Stable hCD133 expression promoted the self-renewal ability of C6-formed spheres with an increase in the expression of the stemness markers, Bmi-1 and SOX2. Akt phosphorylation, Notch-1 activation, and Notch-1 target gene expression (Hes-1, Hey1 and Hey2) were increased in hCD133(+) -C6 when compared to mock-C6. The inhibition of Akt phosphorylation, Notch-1 activation, and Hes-1 in hCD133(+) -C6 cells effectively suppressed their clonogenic ability, indicating that these factors are involved in expanding the growth of hCD133(+) -C6. An elevated expression of GTPase-activating protein 27 (Arhgap27) was detected in hCD133(+) -C6. A decline in the invasion of hCD133(+) -C6 by knockdown of Arhgap27 expression indicated the critical role of Arhgap27 in promoting cell migration of hCD133(+) -C6. In vivo study further showed that hCD133(+) -C6 formed aggressive tumors in vivo compared to mock-C6. Exposure of hCD133(+) -C6 to arsenic trioxide not only reduced Akt phosphorylation, Notch-1 activation and Hes-1 expression in vitro, but also inhibited their tumorigenicity in vivo. The results show that C6 glioma cells with stable hCD133 expression enhanced their stemness properties with increased Notch-1/Hes-1 signaling, Akt activation, and Arhgap27 action, which contribute to increased cell proliferation and migration of hCD133(+) -C6 in vitro, as well as progressive tumor formation in vivo.

Shikonin Kills Glioma Cells through Necroptosis Mediated by RIP-1

Background and PurposeShikonin was reported to induce necroptosis in leukemia cells, but apoptosis in glioma cell lines. Thus, it is needed to clarify whether shikonin could cause necroptosis in glioma cells and investigate its underlying mechanisms.MethodsShikonin and rat C6 glioma cell line and Human U87 glioma cell line were used in this study. The cellular viability was assayed by MTT. Flow cytometry with annexin V-FITC and PI double staining was used to analyze cellular death modes. Morphological alterations in C6 glioma cells treated with shikoinin were evaluated by electronic transmission microscopy and fluorescence microscopy with Hoechst 33342 and PI double staining. The level of reactive oxygen species was assessed by using redox-sensitive dye DCFH-DA. The expressional level of necroptosis associated protein RIP-1 was analyzed by western blotting.ResultsShikonin induced cell death in C6 and U87 glioma cells in a dose and time dependent manner. The cell death in C6 and U87 glioma cells could be inhibited by necroptosis inhibitor necrotatin-1, not by pan-caspase inhibitor z-VAD-fmk. Shikonin treated C6 glioma cells presented electron-lucent cytoplasm, loss of plasma membrane integrity and intact nuclear membrane in morphology. The increased ROS level caused by shikonin was attenuated by necrostatin-1 and blocking ROS by anti-oxidant NAC rescued shikonin-induced cell death in both C6 and U87 glioma cells. Moreover, the expressional level of RIP-1 was up-regulated by shikonin in a dose and time dependent manner as well, but NAC suppressed RIP-1 expression.ConclusionsWe demonstrated that the cell death caused by shikonin in C6 and U87 glioma cells was mainly via necroptosis. Moreover, not only RIP-1 pathway, but also oxidative stress participated in the activation of shikonin induced necroptosis.

Glutamine synthetase functions as a negative growth regulator in glioma

Our recent study demonstrated that glutamine synthetase (GS) may not only serve as a glutamate-converting enzyme in glial cells, but may also function as a regulator of astrocyte migration after injury. In this report, we showed that GS expression increased in cultured rat C6 glioma cells that underwent long-term serially propagation. The stable overexpression of GS in C6 glioma cells resulted in growth arrest and motility suppression; however the stable knockdown of GS resulted in motility enhancement. In correlation with cell aggregation, N-cadherin levels increased at sites of cell-cell contact in C6 cells overexpressing GS, and decreased in C6 cells with stable GS knockdown; total N-cadherin expression levels remained unchanged in these cells. In addition, levels of p21, a potent cyclin-dependent kinase inhibitor, increased, while cyclin D1 levels decreased in C6 cells overexpressing GS. Our additional studies showed that N-cadherin-mediated cell-cell contacts were implicated in GS-induced cell growth arrest and impairment of cell migration, as evidenced by the inhibition of GS on cell growth and motility by the neutralizing anti-N-cadherin monoclonal antibody (GC-4 mAb). Collectively, these observations suggest a novel mechanism of growth regulation by GS that involves N-cadherin mediated cell-cell contact.

Inhibitory effect of pseudolaric acid B on proliferation of rat glioma C6 cells

Objective To observe the inhibitory effects of pseudolaric acid B(PAB) on the proliferation of rat glioma C6 cells in vivo and in vitro.Methods MTT assay was used to examine the inhibitory effect of 0,0.1,0.2,0.4,0.8,1.6,8,and 16 μg/ml PAB on C6 cells;the distribution of cell cycle was determined by flow cytometry.C6 cells were injected subcutaneously to establish glioma-bearing rat model and the tumor volume was measured.The expression of proliferating cell nuclear antigen(PCNA) was detected immunohistochemically in the implanted glioma.Results In vitro results showed that PAB inhibited C6 cell growth in a dose-dependent manner,with the IC50 being 0.03 μg/ml.It also arrested the cells in G2/M phase.In vivo results showed that the tumor size of PAB group was decreased compared with the control group;moreover,PCNA expression was also decreased by PAB.Conclusion PAB can inhibit rat glioma C6 cell growth,which might be associated with cell arrest in G2/M phase and down-regulation of PCNA expression.

Enhancement Effect of Cytotoxicity Response of Silver Nanoparticles Combined with Thermotherapy on C6 Rat Glioma Cells

The present studies reveal that silver nanoparticles (AgNPs) can induce apoptosis and enhance radio-sensitivity on cancer cells. In this paper, we mainly investigated the effect of AgNPs on rat glioma C6 cells upon the combination treatment of hyperthermia treatment (HTT). AgNPs were synthesized by a polyol process and the mean size was 15 nm. The particles showed dose-dependent cytotoxicity on C6 cells from the experimental data. Besides, we found that heating cells could enhance the contents of cell uptake of AgNPs. From the survival curves, AgNPs showed the ability to enhance thermo-sensitivity on C6 cells. Our results revealed that AgNPs could have a potential application in enhancing effect on HTT induced killing of glioma cells.

Comparative study on methyl- and ethylmercury-induced toxicity in C6 glioma cells and the potential role of LAT-1 in mediating mercurial-thiol complexes uptake

Various forms of mercury possess different rates of absorption, metabolism and excretion, and consequently, toxicity. Methylmercury (MeHg) is a highly neurotoxic organic mercurial. Human exposure is mostly due to ingestion of contaminated fish. Ethylmercury (EtHg), another organic mercury compound, has received significant toxicological attention due to its presence in thimerosal-containing vaccines. This study was designed to compare the toxicities induced by MeHg and EtHg, as well as by their complexes with cysteine (MeHg-S-Cys and EtHg-S-Cys) in the C6 rat glioma cell line. MeHg and EtHg caused significant (p<0.0001) decreases in cellular viability when cells were treated during 30min with each mercurial following by a washing period of 24h (EC50 values of 4.83 and 5.05μM, respectively). Significant cytotoxicity (p<0.0001) was also observed when cells were treated under the same conditions with MeHg-S-Cys and EtHg-S-Cys, but the respective EC50 values were significantly increased (11.2 and 9.37μM). l-Methionine, a substrate for the l-type neutral amino acid carrier transport (LAT) system, significantly protected against the toxicities induced by both complexes (MeHg-S-Cys and EtHg-S-Cys). However, no protective effects of l-methionine were observed against MeHg and EtHg toxicities. Corroborating these findings, l-methionine significantly decreased mercurial uptake when cells were exposed to MeHg-S-Cys (p=0.028) and EtHg-S-Cys (p=0.023), but not to MeHg and EtHg. These results indicate that the uptake of MeHg-S-Cys and EtHg-S-Cys into C6 cells is mediated, at least in part, through the LAT system, but MeHg and EtHg enter C6 cells by mechanisms other than LAT system.

Glioma therapy using tumor homing and penetrating peptide-functionalized PEG–PLA nanoparticles loaded with paclitaxel

By taking advantage of the excessively upregulated expression of neuropilin (NRP) on the surface of both glioma cells and endothelial cells of angiogenic blood vessels, the ligand of NRP with high affinity – tLyp-1 peptide, which also contains a CendR motif ((R/K)XX(R/K)), was functionalized to the surface of PEG–PLA nanoparticles (tLyp-1-NP) to mediate its tumor homing, vascular extravasation and deep penetration into the glioma parenchyma. The tLyp-1-NP was prepared via a maleimide-thiol coupling reaction with uniformly spherical shape under TEM and particle size of 111.30 ± 15.64 nm. tLyp-1-NP exhibited enhanced cellular uptake in both human umbilical vein endothelial cells and Rat C6 glioma cells, increased cytotoxicity of the loaded PTX, and improved penetration and growth inhibition in avascular C6 glioma spheroids. Selective accumulation and deep penetration of tLyp-1-NP at the glioma site was confirmed by in vivo imaging and glioma distribution analysis. The longest survival was achieved by those mice bearing intracranial C6 glioma treated with PTX-loaded tLyp-1-NP. The findings here strongly indicate that tLyp-1 peptide-functionalized nanoparticulate DDS could significantly improve the efficacy of paclitaxel glioma therapy.

High-Confidence de Novo Peptide Sequencing Using Positive Charge Derivatization and Tandem MS Spectra Merging

De novo peptide sequencing holds great promise in discovering new protein sequences and modifications but has often been hindered by low success rate of mass spectra interpretation, mainly due to the diversity of fragment ion types and insufficient information for each ion series. Here, we describe a novel methodology that combines highly efficient on-tip charge derivatization and tandem MS spectra merging, which greatly boosts the performance of interpretation. TMPP-Ac-OSu (succinimidyloxycarbonylmethyl tris(2,4,6-trimethoxyphenyl)phosphonium bromide) was used to derivatize peptides at N-termini on tips to reduce mass spectra complexity. Then, a novel approach of spectra merging was adopted to combine the benefits of collision-induced dissociation (CID) and electron transfer dissociation (ETD) fragmentation. We applied this methodology to rat C6 glioma cells and the Cyprinus carpio and searched the resulting peptide sequences against the protein database. Then, we achieved thousands of high-confidence peptide sequences, a level that conventional de novo sequencing methods could not reach. Next, we identified dozens of novel peptide sequences by homology searching of sequences that were fully backbone covered but unmatched during the database search. Furthermore, we randomly chose 34 sequences discovered in rat C6 cells and verified them. Finally, we conclude that this novel methodology that combines on-tip positive charge derivatization and tandem MS spectra merging will greatly facilitate the discovery of novel proteins and the proteome analysis of nonmodel organisms.

Synergistic cytotoxicity induced by α-solanine and α-chaconine

α-Solanine and α-chaconine are well-known potato toxins, but the mechanism of the synergistic cytotoxic effect of these alkaloids has been little clarified. This study confirmed their synergistic cytotoxic effects on C6 rat glioma cells by three different cell viability tests, namely WST-1 (water-soluble tetrazolium) assay sensitive to intracellular NADH concentration, menadione-catalysed chemiluminescent assay depending on both NAD(P)H concentration and NAD(P)H:quinone reductase activity, and LDH (lactate dehydrogenase) assay sensitive to the release of LDH from damaged cells. The maximum cytotoxic effect was observed at a ratio of 1:1 between α-solanine and α-chaconine at micromolar concentrations. The cytotoxic effects of these alkaloids were observed immediately after incubation and were constant after 30min, suggesting that rapid damage of plasma membrane causes the lethal disorder of metabolism.

Abstract 1023: Regulation of cell proliferation by GPR55/cannabinoid receptors using (R,R’)-4’-methoxy-1-naphthylfenoterol in rat C6 glioma cell line.

Abstract (R,R’)-4’-Methoxy-1-naphthylfenoterol (MNF) is a potent inhibitor of the proliferation of rat C6 glioma cells with an IC(50) of 0.45 nM and initial in vivo data indicate that the compound may be potentially useful in the treatment of malignant gliomas. MNF is an agonist of the β(2)-adrenergic receptor and an antagonist of the GPR55 receptor. Previous studies have demonstrated that MNF inhibits proliferation in 1321N1 astrocytoma cells via the β(2)-adrenergic receptor and in HepG2 hetapocallular carcinoma cells via GPR55. In this study, the mechanism of the observed MNF effect in C6 cells was investigated using [(3)H]-thymidine incorporation, cell motility assay, and internalization of the fluorescent GPR55 ligand, Tocrifluor 1117 (T1117). The anti-mitogenic response of MNF was not affected by the β(2)-AR antagonist 3-(isopropylamino)-1-[(7-methyl-4-indanyl)oxy]butan-2-ol (ICI 118,551). Cell treatment with synthetic agonists of cannabinoid receptors 1 and 2 (WIN-55212,2 and CP55940) led to growth inhibition, whereas pretreatment with AM251 (0.1-5 μM) dose-dependently blocked the anti-mitogenic responses of MNF. Both AM251 and the atypical cannabinoid O-1602 increased cell motility, which was blocked by MNF. Finally, there was marked inhibition in the cellular accumulation of T1117 in MNF-treated C6 glioma cells. These results indicate that MNF inhibits proliferation of C6 cells via GPR55/cannabinoid receptors and not via the β(2)-adrenergic receptor. This work was supported entirely by the Intramural Research Program of the NIA, NIH. Citation Format: Rajib K. Paul, Artur Wnorowski, Fred E. Indig, Michel Bernier, Irving W. Wainer. Regulation of cell proliferation by GPR55/cannabinoid receptors using (R,R’)-4’-methoxy-1-naphthylfenoterol in rat C6 glioma cell line. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1023. doi:10.1158/1538-7445.AM2013-1023

Abstract 268: Circadian rhythms of glioma stem cells and progenitor cells.

Abstract The circadian timing system generates and maintains daily rhythms in physiological processes throughout the body. Disruption of circadian timing is associated with higher cancer incidence and more malignant cancers, although the interactions between cancer and circadian rhythms remain unclear. Embryonic stem cells appear to lack endogenous circadian rhythms until they differentiate. We asked whether cancer stem cells (CSCs) also lack persistent circadian oscillations at the molecular level. An inability to generate circadian rhythms may be linked to the undifferentiated state of CSCs, and treatments that can induce clock gene expression might help to initiate rhythms and promote differentiation. Because CSCs appear to evade standard cancer therapies that are effective against non-CSCs new methods to force their differentiation would be useful. To examine the circadian properties of CSCs, we selected the C6 rat glioma cell line because it expresses a strong circadian rhythm in gene expression. C6 CSCs have been characterized through flow cytometry by the side-population cell sorting method that relies on the ability of CSCs to efflux the fluorescent dye Hoechst 33342. We developed a Hoechst dye-exclusion method to identify putative CSCs in C6 monolayer cultures. This cell subpopulation failed to show a circadian rhythm in nuclear transport of a fusion protein of GFP and the circadian clock component mPER2, while the remaining cells were rhythmic. Evidence that these non-rhythmic cells are CSCs was shown by their proliferation in a medium that promotes stem cell growth and their prevalence in neurospheres grown from the C6 line that are masses of CSCs and partially differentiated cells including progenitor cells. The spheres showed immunostaining for the stem cell surface marker CD-133 and they could be induced to form astrocyte and neuron-like cells according to the cell-specific markers GFAP and beta III-tubulin, respectively. To determine whether the neurospheres are capable of generating circadian rhythms, C6 cells expressing a firefly luciferase and mPER2 fusion protein under control by the mPer2 gene promoter were imaged after they formed neurospheres. Circadian rhythms in bioluminescence persisted when the cells were synchronized with a 2-hour pulse of forskolin that induces clock genes but not when the spheres were imaged without forskolin treatment. We interpret these results as indicating a lack of circadian clocks in CSCs but circadian clocks functioning in progenitor cells of neurospheres that require synchronization for coordinated circadian activity. Because CSCs in the microenvironment of tumors may also be arrhythmic, efforts to manipulate circadian clock genes should be examined when treating gliomas and possibly other cancers. Citation Format: Vishal P. Sharma, Michael E. Geusz. Circadian rhythms of glioma stem cells and progenitor cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 268. doi:10.1158/1538-7445.AM2013-268

Abstract 5514: Antitumor activity of (R,R’)-4′-methoxy-1-naphthylfenoterol in a rat C6 glioma xenograft model in the mouse.

Abstract (R,R’)-4’-methoxy-1-naphthylfenoterol (MNF) inhibits in vitro proliferation of several types of cancer cell lines. In this study, the in vivo antitumor effects of MNF were evaluated using rat C6 glioma cells implanted subcutaneously into the lower flank of 5 week-old NMRI/Nude female Swiss mice (0.5 x 10(6) cells, 100μl). Three days after the inoculation, the mice were subjected to intraperitoneal injections of saline or MNF (10μl/g, 2mg/kg of body weight) for 5 days per week for 2 weeks. Tumor volumes were measured everyday using slide calipers. Significant reductions in mean tumor volumes were observed in mice receiving MNF when compared with the saline-treated group (p &amp;lt; 0.001, n=17-19). At the end of the study, animals were sacrificed and tumors were collected for cDNA microarray, quantitative PCR and immunoblot analyses. Comparison of cDNA microarray analysis of tumors from mice treated with MNF vs. saline demonstrated a significant decrease in expression of genes involved in cellular proliferation, including mitotic checkpoint kinase MAD3L (BUB1B), cyclin-dependent kinase inhibitor 3 (CDKN3) and cyclin A2 (CCNA2), as well as molecular markers for glioblastoma, such as oligodendrocyte transcription factor 1 (OLIG1) and SRY-box 4 (SOX4). Target gene validation is currently in progress. Our results show that MNF reduces growth of flank xenografted C6 glioma tumors and expression of genes related to tumor proliferation and suggest that this compound may be useful in cancer treatment. This work was supported in part by the Intramural Research Program of the NIA/ NIH. Citation Format: Rajib K. Paul, Anuradha Ramamoorthy, Yongqing Zhang, Kevin G. Becker, Arnaud Paris, Jean-Francois Cloix, Michel Bernier, Irving W. Wainer. Antitumor activity of (R,R’)-4′-methoxy-1-naphthylfenoterol in a rat C6 glioma xenograft model in the mouse. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5514. doi:10.1158/1538-7445.AM2013-5514

Antitumor activity of methyl gallate by inhibition of focal adhesion formation and Akt phosphorylation in glioma cells

BackgroundMethyl gallate (MG) possesses a wide range of biological properties that include anti-oxidant, anti-inflammatory, and anti-microbial activities. However, its anti-tumor activity has not been extensively examined in cancer cells. Thus, we examined the effect of MG in both glutamate-induced rat C6 and human U373 glioma cell proliferation and migration. MethodsMG was isolated from the stem bark of Acer barbinerve. Cell viability and migration were analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and scratch wound-healing assay, respectively. Focal adhesion formation was detected with immunofluorescence. ResultsTreatment of C6 and U373 glioma cells with MG significantly reduced cell viability, migration, and Akt phosphorylation level. Glutamate stimulation markedly increased the level of ERK1/2 phosphorylation. However, cells treated with MG displayed decreased ERK1/2 phosphorylation. Inhibition of ERK1/2 by MG or MEK1/2 inhibitor significantly inhibited paxillin phosphorylation at Ser83 and focal adhesion turn-over produced inefficient glioma cell migration. In addition, activation of Akt and ERK1/2 upon glutamate stimulation was independently regulated by Ca2+ and protein kinase C activity, respectively, via the α-amino-3-hydroxy-5-methy-4-isoxazolepropionate acid glutamate receptor and metabotropic glutamate receptor. General significanceOur results clearly indicate that MG has a strong anti-tumor effect through the down-regulation of the Akt and ERK1/2 signaling pathways. Thus, methyl gallate is a potent anti-tumor and novel therapeutic agent for glioma.

Water soluble cinnamon extract reverses the hydrogen peroxide‐induced down regulation of S100 β secretion and sirtuin protein expression in C6 rat glioma cells

Dietary polyphenols exert neuroprotective effects in a variety of brain disorders, including Alzheimer's disease, ischemia/stroke, and Parkinson's disease. The brain is particularly susceptible to oxidative damage due to its high consumption of oxygen and oxidative stress contributes to such damage in these neural disorders. The objective of this study was to investigate the effects of a water soluble extract of cinnamon, high in type A polyphenols (CP), on oxidative stress induced by hydrogen peroxide in rat C6 glioma cells. After 4 hr of H2O2 (100μM) incubation, the secretion and intracellular expression of the Ca2+‐binding protein, S100β, were significantly decreased as determined by immunoprecitation/immunoblotting and immunofluorescence imaging. CP counteracted the down regulation of S100 β by H2O2. CP also reversed H2O2 reduction of sirtuin 1, 2, and 3, deacetylases important in cell survival. In addition, H2O2 induced the expression of Bcl‐xl, an antiapoptotic member of the Bcl‐2 family, TNF‐α, and phospho‐Nf‐kB p65. CP suppressed the H2O2‐induced expression of these proteins. In summary, our results suggest a protective role of CP on glioma cells during oxidative stress. The water soluble extract of cinnamon, containing a minimum of 3% type A polyphenols, may exert neuroprotective effects in astroglial cells by upregulating pro‐survival proteins and suppressing pro‐inflammatory proteins.

In vivo dynamic monitoring of the biological behavior of labeled C6 glioma by MRI

Gliomas are the most common type of intracranial tumor and have the highest rate of mortality. The aims of this study were to investigate the long-term course and biological behavior of orthotopically implanted C6 gliomas and to dynamically monitor the distribution of superparamagnetic iron oxide(SPIO) nanocomposite-labeled C6 glioma cells in rats using 7.0TMRI. We observed that in the MRI of the rats implanted with SPIO-labeled cells, there were pronounced hypointense signal bands, which faded over time, but remained visible up to day27 after implantation. We observed that the first tumors were detected as early as 2days after implantation, presenting as slightly hyperintense regions with indefinite boundaries in the T1-weighted images (T1WIs). On the 9thday, thick tumor feeder vessels, ~0.2mm in diameter, were observed and these increased rapidly over time. Edema was observed in the labeled and unlabeled groups in the T2WIs. Both the central hypointense signal area and the peripheral cogwheel-shaped hypointense signal band in the tumor were observed on the post-contrast T1WIs, in accordance with the necrosis observed in the photomicrographs following hematoxylin and eosin (HE) staining. In conclusion, labeling tumor cells with SPIO and performing an MRI scan dynamically monitors the development and biological behavior of glioma at a very early stage.

The prospective application of a hypoxic radiosensitizer, doranidazole to rat intracranial glioblastoma with blood brain barrier disruption

BackgroundGlioblastoma is one of the intractable cancers and is highly resistant to ionizing radiation. This radioresistance is partly due to the presence of a hypoxic region which is widely found in advanced malignant gliomas. In the present study, we evaluated the effectiveness of the hypoxic cell sensitizer doranidazole (PR-350) using the C6 rat glioblastoma model, focusing on the status of blood brain barrier (BBB).MethodsReproductive cell death in the rat C6 glioma cell line was determined by means of clonogenic assay. An intracranial C6 glioma model was established for the in vivo experiments. To investigate the status of the BBB in C6 glioma bearing brain, we performed the Evans blue extravasation test. Autoradiography with [14C]-doranidazole was performed to examine the distribution of doranidazole in the glioma tumor. T2-weighted MRI was employed to examine the effects of X-irradiation and/or doranidazole on tumor growth.ResultsDoranidazole significantly enhanced radiation-induced reproductive cell death in vitro under hypoxia, but not under normoxia. The BBB in C6-bearing brain was completely disrupted and [14C]-doranidazole specifically penetrated the tumor regions. Combined treatment with X-irradiation and doranidazole significantly inhibited the growth of C6 gliomas.ConclusionsOur results revealed that BBB disruption in glioma enables BBB-impermeable radiosensitizers to penetrate and distribute in the target region. This study is the first to propose that in malignant glioma the administration of hydrophilic hypoxic radiosensitizers could be a potent strategy for improving the clinical outcome of radiotherapy without side effects.

Adenovirus-mediated expression of BmK CT suppresses growth and invasion of rat C6 glioma cells

BmK CT, one of the key toxins in the venom of the scorpion, Buthus martensii Karsch, can interact specifically with glioma cells as a chloride channel blocker and inhibit the invasion and migration of those cells via MMP-2. A recombinant adenovirus, Ad-BmK CT, was constructed and characterized by in vitro and in vivo studies, using MTT cytotoxicity assay and the glioma C6/RFP (red fluorescence protein)/BALB/c allogeneic athymic nude mice model, respectively. The adenovirus-mediated expression of BmK CT displayed a high activity in suppressing rat C6 glioma cells growth and invasion thereby suggesting that this recombinant adenovirus may be a powerful method for treating glioblastoma.

Effects of Pregnane Glycosides on Food Intake Depend on Stimulation of the Melanocortin Pathway and BDNF in an Animal Model

Pregnane glycosides appear to modulate food intake by possibly affecting the hypothalamic feeding circuits; however, the mechanisms of the appetite-regulating effect of pregnane glycosides remain obscure. Here, we show that pregnane glycoside-enriched extracts from swamp milkweed Asclepias incarnata at 25-100 mg/kg daily attenuated food intake (up to 47.1 ± 8.5% less than controls) and body weight gain in rats (10% for males and 9% for females, respectively) by activating melanocortin signaling and inhibiting gastric emptying. The major milkweed pregnane glycoside, ikemagenin, exerted its appetite-regulating effect by decreasing levels of agouti-related protein (0.6-fold) but not NPY satiety peptides. Ikemagenin treatment also increased secretion of brain-derived neurotropic factor (BDNF) downstream of melanocortin receptors in the hypothalamus (1.4-fold) and in the C6 rat glioma cell culture in vitro (up to 6-fold). These results support the multimodal effects of pregnane glycosides on feeding regulation, which depends on the activity of the melanocortin signaling pathway and BDNF.

The proteasome inhibitor lactacystin exerts its therapeutic effects on glioma via apoptosis: An in vitro and in vivo study

To examine the effect and underlying mechanism of action of the proteasome inhibitor lactacystin on glioma, in vitro and in vivo. Rat C6 glioma cells were cultured with or without lactacystin. Cell proliferation, apoptosis and mitochondrial membrane potential were determined. A glioma xenograft model was established in mice and animals were treated with 0, 1 or 5 µg/20 g body weight lactacystin for 7 days. Animals were sacrificed on day 17 after completion of treatment. Apoptosis in tumour tissue was examined by terminal deoxynucleotidyl transferase dUTP nick end labeling staining. Levels of B cell lymphoma 2 (Bcl-2), and Bcl2-associated X protein (Bax) protein and mRNA, were determined in C6 cells and tumour tissues. Lactacystin significantly inhibited the proliferation of C6 cells, increased apoptosis and reduced mitochondrial membrane potential in vitro, and suppressed tumour growth in vivo. Lactacystin increased the ratio of Bax to Bcl-2 at the mRNA and protein levels, both in vitro and in vivo. The effects of lactacystin are associated with apoptosis induction. Proteasome inhibition may represent an effective treatment option for glioma.

Radiotracers for cardiac sympathetic innervation: Transport kinetics and binding affinities for the human norepinephrine transporter

Most radiotracers for imaging of cardiac sympathetic innervation are substrates of the norepinephrine transporter (NET). The goal of this study was to characterize the NET transport kinetics and binding affinities of several sympathetic nerve radiotracers, including [(11)C]-(-)-meta-hydroxyephedrine, [(11)C]-(-)-epinephrine, and a series of [(11)C]-labeled phenethylguanidines under development in our laboratory. For comparison, the NET transport kinetics and binding affinities of some [(3)H]-labeled biogenic amines were also determined. Transport kinetics studies were performed using rat C6 glioma cells stably transfected with the human norepinephrine transporter (C6-hNET cells). For each radiolabeled NET substrate, saturation transport assays with C6-hNET cells measured the Michaelis-Menten transport constants Km and Vmax for NET transport. Competitive inhibition binding assays with homogenized C6-hNET cells and [(3)H]mazindol provided estimates of binding affinities (KI) for NET. Km, Vmax and KI values were determined for each NET substrate with a high degree of reproducibility. Interestingly, C6-hNET transport rates for 'tracer concentrations' of substrate, given by the ratio Vmax/Km, were found to be highly correlated with neuronal transport rates measured previously in isolated rat hearts (r(2)=0.96). This suggests that the transport constants Km and Vmax measured using the C6-hNET cells accurately reflect in vivo transport kinetics. The results of these studies show how structural changes in NET substrates influence NET binding and transport constants, providing valuable insights that can be used in the design of new tracers with more optimal kinetics for quantifying regional sympathetic nerve density.