The effects of tibolone on the human primary glioblastoma multiforme cell culture and the rat C6 glioma model

Abstract

Objectives: In the light of recent advances in tumor biology and genetics, we hypothesized that tibolone, an estrogen receptor agonist, may have antiproliferative effects on primary human glioblastoma cells and rat C6 malignant glioma cell lines. We thought that tibolone should exert its antiproliferative effects by augmenting glial cell differentiation through the naive, nonhypermethylated estrogen receptors in the glioma cells.

Methods: Human primary glioblastoma multiforme (GBM) cells were acquired perioperatively from ten patients aged between 45 and 69 years, diagnosed clinically and radiologically with GBM. The diagnosis was confirmed using immunohistochemical assays. Human GBM and rat C6 malignant glioma cells were cultivated in vitro to obtain monolayer cell cultures. Tibolone was then applied to these cultures in wells, each containing 500,000 tumor cells.

Results: Tibolone significantly decreased the number of human GBM cells at the concentrations of 10 and 100 mg/ml. For tibolone, a strong dose-dependent correlation in tumor inhibition was found (p=0.001). This antiproliferative effect of tibolone in human GBM cells was not observed in rat C6 malignant glioma cells. Tibolone demonstrated differential effects on human GBM and rat C6 glioma cells.

Discussion: In vitro antiproliferative effects of tibolone on human GBM need to be investigated further in in vivo works.     

Intra-vital ultrasonographic monitoring of intra-cerebral tumor growth in a rat glioma model: technical note

Abstract

The assessment of therapeutic effects in rodent glioma models by comparison of post mortem tumor sizes has to deal with differing individual growth kinetics and the possibility of spontaneous tumor regression. This technical note describes the intravital ultrasonographical monitoring of cerebral tumor growth in individual animals. In the experiments C6 lacZ glioma cells were injected intracerebrally into female Wistar rats. Extended craniectomy allowed for transcutaneous sonographic examination of the tumor growth. Four animals were followed ultrasonographically, the volumes of the tumors were calculated and plotted graphically, and on day 21 histological evaluation was performed. Our results show that ultrasonography is an easy and reliable imaging modality for frequent assessment of tumor growth kinetics in the intra-cerebral rat glioma model. It allows for the intravital monitoring of treatment with new therapeutic strategies and increases the reliability of the model by visualization of the tumor size before initiation of treatment.     

C6大鼠胶质瘤模型及其在脑胶质瘤治疗研究中应用的缺陷

C6细胞是大鼠经化学致癌剂N-亚硝基甲脲体内诱发产生的脑胶质瘤细胞。具有较典型的人脑胶质瘤组织学特点。大鼠脑内立体定向接种C6细胞建立的动物模型。因成瘤高，动物手术死亡率低。所以广泛应用于脑胶质瘤治疗的研究中。国外研究发现，来源于非纯种大鼠的C6细胞具有有强的免疫原性；其主要组织相容性基因复合体中存在与大鼠是异源性的基因；C6细胞接种于大鼠体内引发强烈的免疫排斥反应可使肿瘤不能持续生长甚至自行消退，因此混淆了治疗所产生的效应。尤其当用于免疫和基因免疫治疗的研究时，存在较大缺陷。     

注射维生素C治疗脑胶质瘤的实验研究

目的探讨维生素C治疗脑胶质瘤的作用机制,为胶质瘤的临床治疗提供实验室依据。方法制作大鼠C6脑胶质瘤模型。荷瘤7d后将Wistar大鼠随机分为5组,每组10只,即对照组;维生素C低剂量组（2．0g／kg）;维生素C中剂量组（4．0g／kg）;维生素C高剂量组（8．0g／kg）;5-Fu组（20mg／kg）。于用药结束后次日处死各组小鼠,计算肿瘤体积和抑瘤率;流式细胞术检测各组肿瘤组织细胞凋亡率;免疫组化法检测各组肿瘤组织Ki-67蛋白表达情况。结果维生素C可抑制大鼠肿瘤生长,且呈剂量依赖性,实验组肿瘤体积与对照组相比有显著差异（P〈0.05）,维生素C高剂量组与5--Fu组肿瘤体积比较无统计学差异（P〉0.05）。维生素C可诱导大鼠肿瘤细胞凋亡,维生素C各组与对照组相比均有极显著性差异（P〈0.01）,维生素C高剂量组凋亡率高于5-Fu组（P〈0.05）。免疫组化法检测肿瘤组织细胞Ki-67蛋白表达中,维生素C各组与对照组相比均有极显著性差异（P〈0.01）,维生素C高剂量组与5-Fu组细胞凋亡率比较无统计学差异（P〉0.05）。结论维生素C在-定剂量范围内可抑制C6大鼠脑胶质瘤的生长,诱导肿瘤细胞发生凋亡是其机制之一;药理剂量的维生素C能降低C6大鼠脑胶质瘤细胞增殖活性,具有-定的抗肿瘤作用。     

Signaling effects of α-thrombin and SFLLRN in rat glioma C6 cells

Effects of thrombin on brain cells, including change of neurite outgrowth and astrocyte shape, are described, but the molecular mechanisms are unclear. We investigated the effects of human α-thrombin and a six amino acid thrombin receptor activating peptide (TRAP-6, SFLLRN) on [Ca²⁺]₁, phosphoinositide hydrolysis, and protein kinase C in rat glioma C₆ cells. Stimulation of C₆ cells with both α-thrombin and TRAP-6 resulted in [Ca²⁺]₁ mobilization, [³H]Inositol phosphate response, and enhanced immunoreactivity of the protein kinase C (PKC) isoenzymes α, β, γ, δ, and ϵ. Results suggest that α-thrombin and TRAP-6 activate at least partially the same intracellular signaling pathways in rat glioma C₆ cells, which is evidence for involvement of “tethered ligand” receptor in thrombin induced signaling in glioma C₆ cells. © 1996 Wiley-Liss, Inc.     

精神分裂症患者血浆白介素6、10、12水平研究

目的　探讨白介素 (IL) 6、1 0、1 2在精神分裂症的病因与发病机制中的作用。方法　采用酶联免疫吸附法 (ELSIA)检测 57例精神分裂症和 2 9名健康对照的血浆IL 6、IL 1 0、IL 1 2水平。结果　精神分裂症患者组血浆IL 1 2水平明显高于对照组 (P <0 0 5) ,而IL 6、IL 1 0水平两组间无显著性差异 ;女性患者的血浆IL 1 0水平明显低于女性对照组 (P <0 0 5) ,其IL 1 2则高于对照组 (P <0 0 5) ;Ⅱ型精神分裂症患者的血浆IL 6水平明显高于Ⅰ型(P <0 0 5) ,家族性亚型患者的IL 6水平明显高于散发性亚型 (P <0 0 1 ) ;患者组IL 6与IL 1 0呈正相关 (P <0 0 0 1 )。结论　精神分裂症患者存在IL 1 2介导的免疫功能异常 ,血浆IL 6水平升高可能是其某些临床亚型的特征性免疫学指标之一 ,细胞因子间的相互关系在精神病理状态下可能发生改变     

槲皮素对大鼠脑胶质瘤抑瘤作用的体内实验研究

目的观察槲皮素及顺铂对大鼠脑胶质瘤生长的抑制作用,初步探讨其作用机制。方法将30只颅内接种C6胶质瘤细胞的SD大鼠随机分为3组:分别给予顺铂、槲皮素及对照治疗7 d,观察各组肿瘤生长情况,于接种后17 d处死各组大鼠,收集肿瘤标本进行肿瘤大小、HE染色及电镜观察,并通过免疫组化检测肿瘤组织增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)的表达。结果顺铂组[(66.2±5.9)mm3]、槲皮素组[(59.6±5.4)mm3]肿瘤体积低于对照组[(101.1±9.6)mm3]槲皮素组优于顺铂组(t=10.61,P<0.01),槲皮素组、顺铂组肿瘤抑制率分别为41.3%、34.8%。光镜观察槲皮素组、顺铂组肿瘤细胞密度降低,电镜显示有细胞凋亡。PCNA免疫组化结果显示槲皮素组(42.2%±5.1%)、顺铂组(50.0%±6.7%)肿瘤PCNA表达减少,与对照组(79.6%±8.6%)相比,具有统计学差异(F=416.12,P<0.01),两两比较,槲皮素组治疗效果优于顺铂组(q=55.25,P<0.01)。结论槲皮素和顺铂均可显著抑制大鼠颅内C6胶质瘤细胞生长,其机制可能与诱导肿瘤细胞凋亡和抑制肿瘤细胞增殖有关。     

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 × 108 cfu/mL, 2 × 108 cfu/mL, 4 × 108 cfu/mL, 6 × 108 cfu/mL, and 8 × 108 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, i.e., 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.     

Cerebrospinal fluid levels of interleukin-6 and interleukin-12 in children with meningitis

Purpose  Certain cytokines play important roles in the pathophysiology of meningitis. The main purpose of this study was to investigate if the levels of interleukin-6 (IL-6) and interleukin-12 (IL-12) in cerebrospinal fluid (CSF) could be diagnostic predictors of bacterial meningitis in children. Methods  CSF was obtained from 95 patients suspected with meningitis. These cases were classified to the bacterial meningitis (n = 12), aseptic meningitis (n = 41), and nonmeningitis (n = 42) groups. The levels of IL-6 and IL-12 in CSF were measured using the enzyme-linked immmunosorbent assays test. Results  The CSF IL-6 levels in the bacterial meningitis group (45.2 ± 50.0 pg/ml) were significantly higher than those in the aseptic meningitis group (12.9 ± 10.2 pg/ml) and the nonmeningitis group (6.5 ± 7.8 pg/ml; p < 0.05). The CSF IL-12 levels in the bacterial meningitis group (69.8 ± 67.1 pg/ml) were significantly higher than those in the aseptic meningitis group (22.9 ± 10.8 pg/ml) and the nonmeningitis group (15.3 ± 11.2 pg/ml; p < 0.05). With regard to diagnosis, the measurement of CSF IL-6 and IL-12 levels showed sensitivities of 96% and 96%, respectively, and specificities of 51% and 75%, respectively. Conclusion  It is suggested that the CSF IL-6 and IL-12 levels are useful markers for distinguishing bacterial meningitis from aseptic meningitis.     

白介素-6、10、12与精神分裂症临床特征的关系

目的 检测精神分裂症患者血浆IL-6、IL-10和IL-12水平,探讨其与精神分裂症临床特征的关系。方法 对57例精神分裂症患者和29例健康人,采用酶联免疫吸附法(ELSIA)检测其血浆IL-6、IL-10、IL-12水平,采用阳性和阴性症状评定量表(PANSS)评定患者的症状特征。结果 患者组血浆IL-12水平高于正常对照组(P<0.05),且与疾病的严重程度皇正相关;阴性症状组患者IL-6水平明显高于阳性症状组和正常对照组,而其IL-10水平则低于阳性症状组;急性或亚急性起病者血浆IL-10水平明显低于慢性起病组。 结论 精神分裂症存在细胞免疫异常,IL-12在精神分裂症的发病机制中起一定作用,IL-6和IL-10则与其部分临床特征有关。     

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-labeled goat anti-mouse IgG antibody was purchased from Upstate, USA. METHODS:A total of 95 Sprague Dawley rats were randomly assigned to three groups:NSC (n = 35), transplanted with > 6 × 106 NSCs via left medial hind limb; BMSC (n = 35), transplanted with > 1 × 106 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.     

Morphogenesis of measles virus on C6 rat glioma cells

Rat glioma cells (C₆) persistently infected with measles virus show a locally dissociated distribution of budding processes at the cell surface.     

顺铂缓释剂瘤内治疗大鼠C6胶质瘤实验观察

观察顺铂缓释剂瘤内用药治疗大鼠Ｃ６胶质瘤的生物学效应。方法用几丁质做载体,与顺铂制成缓释剂型,进行瘤内植入治疗大鼠脑胶质瘤。结果瘤内给予１ｍｇ／ｍ＾２顺铂缓释剂无毒性,瘤内铂浓度比全身用药高２－５０倍,维持时间达１月以上,血铂浓度比全身用药低１０－６０倍,荷瘤鼠生存时间较单纯顺铂液瘤内或全身用药明显延长。     

Identification of caveolae and caveolin in C6 glioma cells

Caveolae (CAV) constitute a novel subcellular transport vesicle that has received special attention based on its proven and postulated participation in transcytosis, potocytosis, and in cell signaling events. One of the principal components of CAV are caveolin protein isoforms. Here, we have undertaken the immunochemical identification of CAV and the known caveolin isoforms (1alpha, 1beta, 2 and 3) in cultured rat C6 glioma cells. Immunoblot analysis revealed that particulate fractions from rat C6 glioma cells express caveolin-1 and caveolin-2. The relative detergent-insolubility of these caveolin isoforms was also determined by Western blot analysis. Indirect immunofluorescence analysis with caveolin-1 and -2 antibodies revealed staining patterns typical of CAV's known subcellular distribution and localization. For both caveolin isoforms immunocytochemical staining was characterized by intensely fluorescent puncta throughout the cytoplasm and diffuse micropatches at the level of the plasmalemma. Perinuclear staining was also detected, consistent and suggestive of caveolin's localization in the trans Golgi region. The caveolin-1 and -2 immunoreactivity seen in Western blots and immunocytochemically is related to structurally relevant CAV as supported by the isolation of caveolin-enriched membrane complexes using two different methods. Light-density, Triton X-100-insoluble caveolin-1- and caveolin-2-enriched fractions were obtained after fractionation of rat C6 glioma cells and their separation over 5-40% discontinuous sucrose-density gradients. Similar fractions were obtained using a detergent-free, sodium carbonate-based fractionation method. These results further support the localization of CAV and caveolins in glial cells. In addition, they demonstrate that cultured C6 glioma cells can be useful as a model system to study the role of CAV and caveolins in subcellular transport and signal transduction events in glial cells and the brain.     

Regulation of c-fos gene expression by lipopolysaccharide and cycloheximide in C6 rat glioma cells

The effect of lipopolysaccharide (LPS) on the expression of immediate early genes, such as c-fos and c-jun, was examined in C6 rat glioma cells. LPS (1 microg/ml) alone did not affect c-fos mRNA level. LPS, however, transiently increased c-jun mRNA level. Cycloheximide (CHX, 20 microM), a protein synthesis inhibitor, alone caused increases of c-fos and c-jun mRNA levels. LPS showed a potentiating effect in the regulation of c-fos mRNA level, whereas LPS showed an additive action for the regulation of CHX-induced c-jun mRNA expression. To determine if CREB and mitogen-activated protein kinases (MAPKs) are involved in the regulation of c-fos mRNA expression by LPS and CHX, Western blot was carried out using the phosphorylated form of antibodies against ERK, JNK, p38, and CREB. LPS transiently increased the phosphorylation of p38-MAPK and CREB. In addition, LPS alone elevated phosphorylation of ERK (p44/p42) MAPK in a time-dependent manner. Furthermore, LPS plus CHX enhanced phosphorylation of ERK, p38, and CREB in a synergistic manner. Our results suggest that the phosphorylation of ERK, p38, and CREB may be involved in the regulation of synergistic c-fos mRNA expression induced by LPS plus CHX in C6 rat glioma cells.     

雷公藤红素对C6胶质瘤细胞凋亡及细胞周期阻滞的影响

目的 探讨雷公藤红素对体外C6胶质瘤细胞凋亡及细胞周期阻滞的影响及机制.方法 雷公藤红素处理体外培养的C6胶质瘤细胞,MTT法检测细胞增殖,Hochest33342染色荧光显微镜观察以及锇铀铅染色透射电镜观察胶质瘤细胞形态变化,流式细胞术分析细胞凋亡率及细胞周期改变.蛋白印迹分析检测凋亡相关蛋白及细胞周期调控蛋白的表达变化.结果 雷公藤红素对C6胶质瘤细胞的增殖抑制作用呈浓度和时间依赖性;2.56 μmol/L雷公藤红素处理C6胶质瘤细胞24h后,荧光显微镜、透射电镜下均可见凋亡形态学改变.流式细胞术检查显示,凋亡细胞比例由1.32％升高到19.34％;并且,处于G0/G1期的细胞比例由76.42％降低到54.52％,G2/M期细胞比例由9.29％升高到30.50％.蛋白印迹分析显示,雷公藤红素降低了Bcl -2及XIAP蛋白表达,促进了Bax及Caspase -3的表达以及PARP的剪切;雷公藤红素在诱导细胞周期调控蛋白p21、p27及cyclin B1蛋白表达增加的同时抑制了cdk2蛋白的表达.结论 雷公藤红素能够通过调节凋亡相关蛋白及细胞周期相关因子的表达影响C6胶质瘤细胞的凋亡及细胞周期阻滞.     

Dehydroepiandrosterone inhibits the death of immunostimulated rat C6 glioma cells deprived of glucose

Pretreatment of interferon-gamma and lipopolysaccharides made C6 glioma cells highly vulnerable to glucose deprivation. Neither 12 h of glucose deprivation nor 2-day treatment with interferon-gamma (100 U/ml) and lipopolysaccharides (1 microg/ml) altered the viability of C6 glioma cells. However, significant death of immunostimulated C6 glioma cells was observed after 5 h of glucose deprivation. The augmented death was prevented by dehydroepiandrosterone (DHEA) treatment during immunostimulation, but not by DHEA treatment during glucose deprivation. DHEA reduced the rise in nitrotyrosine immunoreactivity, a marker of peroxynitrite, and superoxide production in glucose-deprived immunostimulated C6 glioma cells. DHEA, however, did not protect glucose-deprived C6 glioma cells from the exogenously produced peroxynitrite by 3-morpholinosydnonimine. Further, DHEA did not alter the production of total reactive oxygen species and nitric oxide in immunostimulated C6 glioma cells. Superoxide dismutase (SOD) and the synthetic SOD mimetic Mn(III)tetrakis (4-benzoic acid) porphyrin inhibited the death of glucose-deprived immunostimulated C6 glioma cells. In addition, a superoxide anion generator paraquat reversed the protective effect of DHEA on the augmented death. The data indicate that DHEA prevents the glucose deprivation-evoked augmented death by inhibiting the production of superoxide anion in immunostimulated C6 glioma cells.