Heat shock induction of a 65 kDa ATP-binding proteinase in rat C6 glioma cells

The 45, 55, 65 and 100 kDa ATP-binding proteinases (ATP-BPases) of the heat-shocked (44 ℃ for 30 min, recovery for 12h) rat C6 glioma cells were purified by DEAE-ionexchange and ATP-affinity chromatography. Their molecular masses, isoelectric points (pI), pH-optima and other properties were analyzed by native proteinase gels.It was shown that the 65 kDa ATP-BPase is specifically induced by heat shock and not detectable in control cells.Its N-terminal 1-9 amino acid sequence was determined by Edman degradation, but no homologies to other proteins in the protein data bases were found. 30 and 31 kDa proteinases can be cleaved from the 45, 55 and 65 kDa proteinases to which they are linked. A possible relationship of the heat-induced 65 kDa ATP-BPase with the ATP-dependent proteinases (ATP-DPases) in prokaryotes and eukaryotes is discussed.

Lipid-albumin nanoassemblies co-loaded with borneol and paclitaxel for intracellular drug delivery to C6 glioma cells with P-gp inhibition and its tumor targeting

Successful chemotherapy with paclitaxel(PTX)is impeded by multidrug resistance(MDR)in tumor cells.In this study,lipid-albumin nanoassemblies co-loaded with borneol and paclitaxel(BOR/PTX LANs)were prepared to circumvent MDR in C6 glioma cells.The physiochemical properties including particle size,encapsulation efficiency and morphology were evaluated in vitro.Quantitative and qualitative investigations of cellular uptake were carried out in C6 glioma cells.The cytotoxicity of the BOR/PTX LANs was determined by MTT assay.After that,the tumor targeting was also evaluated in C6 glioma bearing mice by in vivo imaging analysis.BOR/PTX LANs have a higher entrapment efficiency(90.4±1.2%),small particle size(107.5±3.2 nm),narrow distribution(P.I.=0.171±0.02).The cellular uptake of PTX was significantly increased by BOR/PTX LANs compared with paclitaxel loaded lipidalbumin nanoassemblies(PTX LANs)in quantitative research.The result was further confirmed by confocal laser scanning microscopy qualitatively.The cellular uptake was energy-,timeand concentration-dependent,and clathrin-and endosome/lysosome-associated pathways were involved.The BOR/PTX LANs displayed a higher cytotoxicity agaist C6 glioma cells in comparion with PTX LANs and Taxol.Moreover,the encapsulation of BOR in LANs obviously increased the accumulation of the drug in tumor tissues,demonstrating the tumor targeted ability of BOR/PTX LANs.These results indicated that BOR/PTX LANs could overcome MDR by combination of drug delivery systems and P-gp inhibition,and shown the potential for treatment of gliomas.

Inhibitory effects of lapachol on rat C6 glioma in vitro and in vivo by targeting DNA topoisomerase Ⅰ and topoisomerase Ⅱ

OBJECTIVE The aim of this study is to investigate the inhibitory effects of lapachol on rat C6 glioma both in vitro and in vivo,as well as the potential mechanisms.METHODS First,the model of C6 glioma in Wistar rats was established and verified by hemotoxylin and eosin staining,immunohistochemical staining and magnetic resonance imaging(MRI).Then different doses of lapachol were gavaged and tumor volumes of the C6 glioma were detected by MRI.The effects of lapachol on C6 cell proliferation,apoptosis and DNA damage were detected by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium(MTS)/phen-azinemethosulfate(PMS)assay,Hoechst33358 staining,AnnexinⅤ-FITC/PI staining,and comet assay.Effects of lapachol on topoisomeraseⅠ(TOPⅠ)and topoisomeraseⅡ(TOPⅡ)activities were detected by TOPⅠand TOPⅡmediated supercoiled p BR322 DNA relaxation assay.Molecular docking was used to predict the interaction of lapachol-TOPⅠand lapachol-TOPⅡ.TOP I and TOPⅡexpression levels in C6 cells were determined by Enzymelinked immunosorbent assay kits and real-time polymerase chain reaction(RT-PCR).RESULTS The rat C6 glioma model was successfully established.High dose lapachol showed significant inhibitory effect on the C6 glioma in Wistar rats(P<0.05).MTS/PMS assay,Hoechst 33258 staining,AnnexinⅤ-FITC/PI staining,and comet assay showed that lapachol could inhibit proliferation,induce apoptosis and DNA damage of C6 cells in dose dependent manners.Lapachol could inhibit the activities of both TOPⅠandⅡ.Molecular docking showed that lapachol-TOPⅠshowed relatively stronger interaction than that of lapachol-TOPⅡ.Enzyme-linked immunosorbent assay and RT-PCR showed that lapachol could inhibit TOPⅡexpression levels,but not TOPⅠexpression levels.CONCLUSION These results showed that lapachol could significantly inhibit C6 glioma both in vivo and in vitro,which might be related with inhibiting TOPⅠand TOPⅡactivities,as wel as TOPⅡexpression.

Effects of antigliomatin from the scorpion venom of Buthus martensii Karsch on chloride channels on C6 glioma cells

Using whole-cell patch-clamp recordings, the effects of antigliomatin were observed on chloride channels on C6 glioma cells cultured in vitro. Antigliomatin was extracted from the venom of the scorpion Buthus martensii Karsch. Chloride channels are closed under normal osmotic pressure. When osmotic pressure was reduced to 120, 110 and 100 mV, the cell volume enlarged, chloride channels opened, and the chloride channel current increased. Three minutes after antigliomatin treatment, the chloride channel current decreased in a dose-dependent manner. These results show that antigliomatin extracted from the venom of the scorpion Buthus martensii Karsch diminishes chloride channel currents on C6 glioma cells.

Bone marrow stromal cell versus neural stem cell transplantation in a C6 glioma rat model

BACKGROUND： Embryonic neural stem cells （NSCs） have provided positive effects for the treatment of glioma. However, the source for embryonic NSCs remains limited and high amplification conditions are required. Bone marrow stromal cells （BMSCs） have been proposed for the treatment of glioma. OBJECTIVE： To investigate biological changes in NSCs and BMSCs following transplantation into rat models of glioma. DESIGN, TIME AND SETTING： A randomized, controlled, animal experiment was performed at the Embryonic Stem Cell Research Laboratory of Yunyang Medical College from February 2006 to August 2008. MATERIALS： The rat C6 glioma cell line was purchased from Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences; mouse anti-bromodeoxyuridine （BrdU） monoclonal antibody and Cy3-1abeled goat anti-mouse IgG antibody was purchased from Upstate, USA. METHODS： A total of 95 Sprag6ue Dawley rats were randomly assigned to three groups： NSC （n = 35）, transplanted with 〉 6 × 10^6 NSCs via left medial hind limb; BMSC （n = 35）, transplanted with 〉 1 × 10^6 BMSCs via left medial hind limb; model group （n = 25）, injected with the same volume of 0.1 mmol/L phosphate buffered saline. MAIN OUTCOME MEASURES： Gliomal growth and size were assessed by nuclear magnetic resonance, and glioma morphological features were observed following hematoxylin-eosin staining and BrdU immunohistochemistry 3 and 4 weeks following transplantation. RESULTS： The average survival of rats in the BMSC, NSC, and model groups was 4.03, 4.28, and 3.88 weeks. At 3 weeks, there was no significant difference in the average glioma diameter between the BMSC and model groups （P 〉 0.05）. However, gliomal diameter was significantly decreased in the NSC group compared with the model group （P 〈 0.05）. At 4 weeks, there was no statistical difference between the groups （P 〉 0.05）. BrdU immunohistochemistry revealed that BMSCs and NSCs appeared to migrate to the gliomas. CONCLUSION： NSCs inhibited glioma cell growth and prolonged rat survival. BMSCs did not significantly suppress glioma cell growth.

Inhibitory effects of Pseudomonas aeruginosa mannose-sensitive hemagglutinin on rat C6 glioma cell proliferation

BACKGROUND： Pseudomonas aeruginosa mannose-sensitive hemagglutinin （PA-MSHA） parenteral injection is used as a broad-spectrum immunomodulator. It remains unclear whether PA-MSHA exhibits inhibitory effects on tumor cell growth. OBJECTIVE： To investigate inhibitory mechanisms of PA-MSHA-induced proliferation in rat C6 glioma cells in vitro. DESIGN, TIME AND SETTING： Comparative observation and in vitro experiments were performed at the Key Laboratory of Natural Medicine, Kunming Medical College, China from July 2008 to April 2009. MATERIALS： Rat C6 glioma cell line （Shanghai Institute of Cell Biology, Chinese Academy of Sciences, China） and PA-MSHA parenteral injection （Beijing Wanteer Bio-Pharmaceutical, China） were used in the present study. METHODS： Rat C6 glioma cells in logarithmic growth phase were harvested in vitro. Adherent monolayer cells were respectively treated with PA-MSHA at final colony-forming units （cfu） of 1 ×10^8 cfu/mL, 2 × 10^8 cfu/mL, 4 × 10^8 cfu/mL, 6 × 10^8 cfu/mL, and 8 ×10^8 cfu/mL following 24 hours of conventional culture. MAIN OUTCOME MEASURES： MTT colorimetric assay was utilized to determine the inhibitory rate of C6 glioma cells following treatment with various concentrations of PA-MSHA at different times. Cell apoptosis was detected by fluorescent microscopy following Hoechst 33258 staining. Flow cytometry was used to measure PA-MSHA effects on C6 cell cycle. RESULTS： Inhibitory rate of C6 glioma cells increased with prolonged time and increased dose. Hoechst 33258 staining revealed obvious morphological changes in apoptotic C6 glioma cells. Flow cytometry revealed hypodiploid peaks, Le., apoptotic peak, and the apoptotic rate in cells during S-phase significantly increased with increased concentrations in the experimental groups. CONCLUSION： With in vitro experiments, PA-MSHA preparations inhibited C6 glioma cell proliferation in a time- and dose-dependent manner. These mechanisms are likely associated with cell apoptosis induction and inhibition of the S phase.

Effects of selective cyclooxygenase-2 inhibitor on C6 glioma cell proliferation and apoptosis

BACKGROUND： Studies have shown that cyclooxygenase-2 is associated with proliferation and apoptosis of glioma cells. OBJECTIVE： To investigate the effects of selective cyclooxygenase-2 inhibitor celecoxib on proliferation and apoptosis of C6 glioma cells in vitro. DESIGN, TIME AND SETTING： A cellular, molecular, controlled study was performed at the Central Laboratory and Room of Electron Microscope, Medical School, Xi＇an Jiaotong University, China from March 2007 to March 2008. MATERIALS： C6 glioma cells during in vitro log phase were assigned to control and experimental groups. Celecoxib （Pfizer, USA）, dimethyl sulfoxide （Sigma, USA）, and MTT （Sigma, USA） were used for this study. METHODS： The control group was subdivided into blank control and dimethyl sulfoxide control groups. C6 glioma cells in the blank control and dimethyl sulfoxide control groups were incubated in Dulbecco＇s modified Eagle＇s medium supplemented with 10% calf serum and 0.3% dimethyl sulfoxide respectively. C6 glioma cells in the experimental group were separately treated with 60, 80 and 100 μmol/L celecoxib. MAIN OUTCOME MEASURES： Activity of C6 glioma cells was examined by MTT assay. C6 glioma cell cycle and apoptosis were determined by annexin V-fluorescein isothiocyanate/propidium iodide double-staining, followed by flow cytometry. Morphology and ultrastructure of C6 glioma cells were observed with an inverted microscope and a transmission electron microscope, respectively. RESULTS： Compared with the blank control group, cell density was reduced, adherence ability weakened, and irregular nuclei were visible, with the presence of chromatin condensation, margination, and some apoptotic bodies in the experimental group. Activity of C6 glioma cells was significantly decreased （P 〈 0.05）, cell number was reduced during S phase, cell number was significantly increased during G2/M phase （P 〈 0.01 ）, and the apoptotic rate was significantly increased （P 〈 0.05） in the experimental group. These results were displayed in a dose- and time-dependent fashion. The outcomes were obvious in the 100 IJmol/L celecoxib group following 72 hours of treatment. CONCLUSION： Celecoxib blocked proliferation and induced apoptosis of C6 glioma cells in a dose- and time-dependent fashion.

Inhibitory effects of lapachol on rat C6 glioma in vitro and in vivo by targeting DNA topoisomeraseⅠ and topoisomeraseⅡ

OBJECTIVE Lapachol is a natural naphthoquinone compound that possesses extensive biological activities.The aim of this study is to investigate the inhibitory effects of lapachol on rat C6 glioma both in vitro and in vivo,as well as the potential mechanisms.METHODS The antitumor effect of lapachol was firstly evaluated in the C6 glioma model in Wistar rats.The effects of lapachol on C6 cell proliferation,apoptosis and DNA damage were detected by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium(MTS)/phenazinemethosulfate(PMS)assay,hoechst 33358 staining,annexinⅤ-FITC/PI staining,and comet assay.Effects of lapachol on topoisomerase I(TOP I)and topoisomeraseⅡ(TOPⅡ)activities were detected by TOPⅠand TOPⅡmediated supercoiled p BR322DNA relaxation assays and molecular docking.TOPⅠand TOPⅡexpression levels in C6 cells were also determined.RESULTS High dose lapachol showed significant inhibitory effect on the C6 glioma in Wistar rats(P<0.05).It was showed that lapachol could inhibit proliferation,induce apoptosis and DNA damage of C6 cel s in dose dependent manners.Lapachol could inhibit the activities of both TOPⅠ and Ⅱ.Lapachol-TOPⅠshowed relatively stronger interaction than that of lapachol-TOPⅡin molecular docking study.Also,lapachol could inhibit TOPⅡexpression levels,but not TOPⅠexpression levels.CONCLUSION These results showed that lapachol could significantly inhibit C6 glioma both in vivo and in vitro,which might be related with inhibiting TOPⅠ and TOPⅡ activities,as wel as TOPⅡ expression.

RNA interference affects tumorigenicity and expression of insulin-like growth factor-1,insulin-like growth factor-1 receptor,and basic fibroblast growth factor-2 in rat C6 glioma cells

BACKGROUND： Human gliomas are more likely to express basic fibroblast growth factor-2 （FGF-2） insulin-like growth factor-1（IGF-1）, and IGF-1 receptor （IGF-1R） than normal brain tissue. These factors activate signal transduction systems of Ras/MAPK and PI3K/Akl, which promote glioma growth. OBJECTIVE： To utilize RNA interference （RNAi） technique to down-regulate FGF-2, IGF-1, and IGF-1R gene expression, and to investigate the effects of these genes on rat C6 glioma cells, as well as the feasibility of RNAi for treating glioma. DESIGN, TIME AND SETTING： This neurooncological, randomized, controlled, in vivo and in vitro experiment, which used RNAi methodology, was performed at the Laboratory of Molecular Biology, Institute of Biochemistry, Chinese Academy of Sciences between August 2005 and February 2008. MATERIALS： Rat C6 cell lines were purchased from Shanghai Institute of Cellular Biology Affiliated to Chinese Academy of Sciences. Small interfering RNA （siRNA） was synthesized by Shanghai GenePharma. Anti-IGF-1, anti-IGF-1R, anti-FGF-2, anti-mouse and anti-rabbit IgG G1-HRP antibodies were provided by Santa Cruz Biotechnology, USA. Four to six week-old BALB/c nude mice were purchased from the Laboratory Animal Center, Chinese Academy of Sciences. METHODS： C6 glioma cells were transfected with siRNA, which was chemically synthesized in vitro to correspond to endogenous FGF-2, IGF-1, and IGF-1R genes. The inhibition ratio of targeting mRNA expression was detected by semiquantitative RT-PCR, and protein expression was determined by Western blot analysis. C6 glioma cell proliferation was observed using a growth curve C6 glioma cell apoptosis rate and cell cycle were detected by flow cytometry. C6 glioma cell growth regression was observed by transwell migration assay. In addition, nude mouse subcutaneous tumor models were used in this study. For studying the anti-tumor effects of IGF-1 and IGF-1R siRNA, two blank control groups, with six mice each, were set up： A （2.5 μg siRNA was injected one week after C6 cells were inoculated, Le., when tumor volume reached 8 mm × 8 mm） and B （siRNA was injected at the same time with C6 cells were inoculated. To study the effects of FGF-2 siRNA, the groups consisted of a blank control group, negative control group, 2.6 μg siRNA group, 4 μg siRNA group, and 5.3 μg siRNA group, with six mice each. MAIN OUTCOME MEASURES： mRNA and protein inhibition ratio of FGF-2, IGF-1, and IGF-1 R; C6 glioma cell proliferation, apoptosis, and cycle growth arrest; C6 glioma cell growth regression and subcutaneous tumorigenicity rates. RESULTS： All siRNA constructs proved to be effective. After 48 hours, transfection of 200 nmol/L siRNA resulted in a FGF-2 or IGF-1R gene inhibition ratio 〉 80% and an IGF-1 gene inhibition ratio of approximately 70%. Protein expression levels for FGF-2, IGF-1, and IGF-1R decreased in a dose-dependent manner following siRNA transfection, with an inhibition rate 〉 85%, 60%, and 50%, respectively. C6 glioma cell proliferation and apoptosis rates increased in proportion to siRNA. The apoptosis rate of C6 glioma cells induced by FGF-2, IGF-1, and IGF-1R siRNA was 39.96%, 15.07% and 22.47%, respectively （P 〈 0.01）. Transfection of 200 nmol/L IGF or IGF-1R siRNA for 48 hours suppressed C6 glioma cell migration. At 30 days after intratumoral injection of 2.6, 4, and 5.3 tJg FGF-2 siRNA, tumor growth regression rate of FGF-2 siRNA was 56%, 67%, and 86%, respectively. The tumor growth regression rate was 71.88% and 45.71%, respectively, when IGF-1 or IGF-1R siRNA was intratumorally injected 1 week after C6 glioma cell transplantation. When IGF-1 or IGF-1 R siRNA was intratumorally injected during C6 glioma cell transplantation, the tumor growth regression rate was 78.13% and 74.29%, respectively. CONCLUSION： siRNA transfection downregulated gene expression of FGF-2, IGF-1, and IGF-1R In addition, siRNA treatment markedly suppressed glioma cell proliferation, growth, and migration, and concomitantly reduced subcutaneous tumorigenicity.

Induction of apoptosis and inhibition of proliferation of C6 glioma cells in vitro by tamoxifen

Objective To investigate the anti-tumor effect and mechanism of tamoxifen on rat C6 glioma cells. Methods C6 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 3% fetal calf serum (FCS), and treated with tamoxifen of different concentrations, i.e. group A (1.25μmol/L), group B (2.50 μmol/L), group C (5.00 μmol/L), group D (10.00 μmol/L), group E (20.00 μmol/L) and control group (0.00 μmol/L). Morphological changes, MTT assay and 5-bromo-2’-deoxyuriding labeling ratio were assessed. Apoptosis was observed by flow cytometry. Results C6 cells treated with different doses of tamoxifen for 24, 48, and 72 hours became irregular in shape, while cells treated with vehicle grew normally. MTT assay showed that tamoxifen did not suppress C6 cell growth until 72 hours after treatment. Seventy-two hours after treatment, there were significant differences in cell viable rate between group A versus groups C, D and E; so did group B versus group D as well as group E (P<0.05). BrdU incorporation assay indicated significant difference of BrdU labbled index (BrdU LI) among groups A, C, E and control group 48 hours after treatment (P<0.05). And the BrdU LI decreased with the increased concentration of tamoxifen. Flow cytometry (FCM) showed significant difference between treated group and control group at 24, 48, and 72 hours after treatment (P<0.05). Conclusion Tamoxifen significantly suppresses the growth of C6 glioma cells in a time-and dose-dependent manner. The mechanism of tamoxifen suppressing C6 glioma cells may be inhibiting proliferation and inducing apoptosis. Therefore, tamoxifen can be a candidate as a chemotherapy agent for glioma.

IN VIVO GROWTH OF CEREBRAL C6 GLIOMA CELLS TRANSFECTED AND TREATED WITH CX43 GENE

Objective: To study the role of connexin gene (Cx 43) on the development of glioma and the feasibility of using Cx43cDNA as a target of gene therapy of gliomas. Methods: Parental rat C6 cells and C6 cells transfected with Cx43cDNA were implanted into right caudate nucleus of SD rats as control and transfected group. Rats bearing cerebral C6 gliomas were treated with Cx43cDNA and empty vector as treated group and empty vector group. The general manifestation, survival time, MRI dynamic scanning and histopathological changes of all rats were observed. In situ hybridization and immunohisto- chemistry were used for examination of Cx43mRNA and its protein in gliomas. Average number of AgNOR staining was used for detection of cell proliferation activity, and TUNEL method for determination of cell apoptosis. Results: All rats in control and empty vector group died of cerebral gliomas within 3 weeks after implantation of C6 cells. Six out of nine rats in the transfected group and eight out of ten rats in treated group kept alive beyond 120 days with totally disappearing of the tumor foci, except one treated rat having a little residue of tumor. In gliomas of transfected and treated groups Cx43 gene expression was upregulated, proliferation activity was lowered, However, the apoptotic cells did not increase. Conclusion: The present study indicates that Cx43 gene is of crucial importance in the development of malignant glioma. It can be an effective target for gene therapy of gliomas.

Polymer Delivery of Hydroxycamptothecin against C6 Glioma

Hydroxycamptothecin is a potent antineoplastic agent that has shown efficacy against multiple tumor lines in vitro. This is the first study to investigate the release, distribution, and efficacy of hydroxycamptothecin which was incorporated into the biodegradable polymer Polylactic Acid (PLA), and implant into brain directly. In vitro release curve generated showed that a large initial release occurred over the first three days and was followed by a steady, but considerably slower rate of release over the next 25 days. After implanting the discs into 40 male SD rats, the animals were followed up to 28 days, where the concentration in brain tissue was far higher than that in peripheral blood at the each of the eight time-points evaluated, and it was also within the therapeutic range for C6 cells tested in vitro. The in vivoefficacy of the discs was evaluated with rats inoculated intracranially with C6 glioma and treated with hydroxycamptothecin polymer compared to intravenous as well as intratumoral injections;the median survival is 21.1, 13.9, 14.9 days, respectively. Given these data, we conclude that the biodegradable polymer PLA releases hydroxycamptothecin, producing tumoricidal levels in brain tissues and prolonging survival in a rat glioma model.

Ethanol Extract of Lycopodium serratum Thunb. Attenuates Lipopolysaccharide-Induced C6 Glioma Cells Migration via Matrix Metalloproteinase-9 Expression

Objectives： To elucidate how ethanol extract of L. serratum（ELS） could exert anti-migratory effects on glioma with the suppression of nuclear factor kappa B（NF-κB） downstream pathway. Methods： Cell viability of ELS on C6 glioma was detected by 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide（MTT） assay. Nitric oxide（NO） assay and 2＇,7＇-dichlorofluorescin diacetate（DCFH-DA） assay were applied to measure NO production and reactive oxygen species（ROS） generation on lipopolysaccharide（LPS）-induced C6 glioma cells. NF-κB, mitogen-activated protein kinase（MAPK）, inducible nictric oxide synthase（i NOS） and cyclooxygenase-2（COX-2） protein were determined by Western blot. Wound healing assay was used to investigate the inhibitory effect of ELS on fetal bovine serum（FBS）-induced migration and matrix metalloproteinase（MMP）-9 and-2 activity was examined by zymography. Results： ELS suppressed LPS-induced phosphorylation of extracellular signal-regulated kinase（ERK）, c-Jun N-terminal kinase（JNK）, and p38 through inhibiting the expression of chemokine CCL2（or monocyte chemoattractant protein-1, MCP-1）. In addition, ELS inhibited the expression of i NOS, COX-2, and the production of NO by LPS in C6 glioma cells. ELS also significantly decreased serum-induced migration of C6 glioma cells in scratch wound healing in a dose-dependent manner（P〈0.01）. The activity of MMP-9 and-2 were also significantly attenuated by ELS with LPS treatment（P〈0.01）. Conclusion： Our results suggest that downregulation of MMP-9 gene expression might be involved in the anti-migration effect of ELS against LPS-induced C6 glioma cells.

Characterization of Acidic Glycosphingolipid Changes in C6 Glioma Rats Treated With Temozolomide Using Ultra-High-Performance Liquid Chromatography Coupled With Quadrupole Time-of-Flight Mass Spectrometry

Ganglioside and sulfatide are primary components of acidic glycosphingolipids(AGSLs),which are abundant in the brain tissue.AGSLs are potential tumor markers.In this paper,we use ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry to generate a first-ever comprehensive profile of AGSLs in brain and plasma of C6 glioma rats treated with temozolomide(TMZ).The AGSLs detected consisted mainly of C18-/C20-sphingosine.12,20,19,14 and 12 species were identified in GM1,GD1a,GD1b,GM3 and GT1b ganglioside groups which were abundant in rat brain,respectively.These five groups accounted for 88-89%of total ganglioside content.However,no AGSLs were detected in rat plasma.Possible biomarkers for abnormal changes in the glioma model and the protective effect of TMZ were mainly found in these ganglioside groups and sulfatide.The main lipid component of central and peripheral nervous system myelin sheathes is sulfatide,which is upregulated in many tumors.Antitumor properties of TMZ are due to modulation of sulfatide levels in tumor tissues.

Intramedullary Spinal Cord Glioma Following Microinjection of Glioblastoma Cell Line C6 in Rats

Background: This paper describes the establishment of a rat intramedullary spinal cord tumor (IMSCT) model and histopathological characterization of the tumor model. Methods: Fourteen male Wistar rats were randomized into two groups. The rats in group 1 (control group, n = 7) received a 5 μl intramedullary injection of serum physiologic (SF). Those in group 2 (experimental group, n= 7) received a 5 μl intramedullary implantation of media containing 5 × 105 C6 glioma cells. The animals were sacrificed for histopathological examination at 21 days. Results: The control group showed normal functional and histopathological findings. The group 2 rats implanted with C6 glioblastoma cells developed hind-limb paraplegia. Pathological sections confirmed intramedullary C6 glioblastoma invading the spinal cord. Conclusions: A rat C6 IMSCT model was successfully established. This model may be useful in increasing understanding of intramedullary spinal cord gliomas in humans.

鲜壁虎冻干粉诱导C6胶质瘤细胞凋亡的血清药理学研究

目的探讨鲜壁虎冻干粉对Ｃ6胶质瘤细胞凋亡的影响。方法将30只小鼠随机分为3组,分别给予顺铂腹腔注射,口服鲜壁虎冻干粉及口服等量蒸馏水。20天后,取小鼠血清体外处理Ｃ6胶质瘤细胞。通过形态学分析、流式细胞仪及ＴＵＮＥＬ法,观察细胞凋亡情况;Ｓ Ｐ免疫组化法检测细胞凋亡相关基因ｂｃｌ 2、ｂａｘ的表达水平。结果形态学变化、流式细胞仪分析和ＴＵＮＥＬ法结果均证实含鲜壁虎血清在体外可诱导Ｃ6胶质瘤细胞凋亡;鲜壁虎组与空白对照组比较,细胞内的ｂｃｌ 2基因表达无明显变化,ｂａｘ基因表达升高。结论含鲜壁虎血清能够诱导细胞凋亡,其机制可能与上调ｂａｘ基因有关。

苦参碱对C6胶质瘤细胞的增殖和原癌基因表达的影响

目的 :本研究拟探讨苦参碱对胶质瘤细胞株 (C6细胞株 )的抑制作用及其作用机理。方法 :应用MTT比色法检测不同浓度苦参碱对C6细胞的增殖抑制 ,应用流式细胞仪观察苦参碱对C6细胞周期的影响 ,应用RT PCR观察原癌基因C myc表达的变化。结果 :苦参碱对C6细胞增殖抑制作用呈剂量依赖性 ,当浓度为 0 2 0g/L时 ,对C6细胞增殖的抑制率达到 ( 5 3 8± 5 6 ) %。流式细胞仪分析显示 ,用 0 2 0g/L苦参碱培养 3d ,细胞周期中G0 /G1期所占比例增加 ,S期占细胞周期的比例降低。RT PCR结果显示 ,随着苦参碱作用剂量的增加 ,C myc基因的表达被明显抑制。结论 :苦参碱对大鼠胶质瘤细胞系C6的增殖具有明显的抑制作用 ,并对原癌基因C

苦参碱引起C6胶质瘤细胞凋亡及其对TRADD表达的影响

目的:应用一系列分子生物学检测技术来检测苦参碱对胶质瘤细胞中TRADD表达的影响,以进一步推测其诱导胶质瘤细胞凋亡的作用机制。方法:采用MTT法测定不同浓度苦参碱作用于C6胶质瘤细胞后的吸光值以计算其抑制率;采用Annex- in/FITC染色进行流式细胞术检测来测定不同浓度苦参碱诱导C6胶质瘤细胞的凋亡率;采用ICC(免疫细胞化学)、Western-blot- ting及Real-time PCR array(实时定量PCR芯片)方法来检测苦参碱对胶质瘤细胞TRADD表达的影响。结果:MTT结果显示细胞抑制率随着药物剂量的增加而增加;Annexin/FITC染色进行流式细胞术的检测结果显示胶质瘤细胞的凋亡率随着药物剂量的增加而增加;ICC、Western-bloning及Real-time PCR array结果显示苦参碱可诱导胶质瘤细胞TRADD基因及蛋白表达的上调。结论:苦参碱可以诱导胶质瘤细胞凋亡,其诱导胶质瘤细胞凋亡可能是通过上调TRADD的表达,以死亡受体途径来实现的。

异甘草素对大鼠C6胶质瘤细胞增殖和分化的影响

目的研究异甘草素对大鼠C6胶质瘤细胞增殖和分化的影响。方法采用磺酰罗丹明B(SRB)比色法和克隆形成实验考察异甘草素对C6胶质瘤细胞增殖抑制作用;Giemsa染色观察细胞形态学变化;半定量RT-PCR、Western blot鉴定细胞分化相关基因及蛋白表达水平。结果大鼠C6胶质瘤细胞经异甘草素诱导后,以浓度依赖性抑制细胞增殖(P<0.01);光镜观察到异甘草素诱导前的C6胶质瘤细胞呈两极长梭形或多角形,胞质多,胞突短;而诱导后细胞突起数目不同程度增多,细胞形态变细变长。诱导前克隆形成出现早,数量多,直径大,克隆形成率高;异甘草素处理组克隆形成率降低,与对照组相比差异有统计学意义(P<0.01)。RT-PCR、Western blot结果显示,异甘草素诱导后C6细胞GFAP mRNA和蛋白表达均明显上升(P<0.01)。结论异甘草素能抑制大鼠C6胶质瘤细胞的增殖,并能诱导C6细胞分化。