Stimulation of cell growth and DNA synthesis by peripheral benzodiazepine

The effects of peripheral benzodiazepine receptor ligands on cell proliferation were evaluated. PK11195 increased the growth rate of C6 glioma cells by 20-30% in the nanomolar range in serum free medium. [3H]thymidine incorporation into C6 glioma cells also were increased 22% and 25% after treatment by PK11195 and Ro5-4864, respectively. The effect of PK11195 as a mitogenic agent was estimated by mitogenic agent was estimated by [3H]thymidine incorporation using Swiss 3T3 cells. PK11195 increased DNA synthesis 170% over control at 10 nM. Higher concentrations of benzodiazepines showed inhibition of the DNA synthesis. Peripheral benzodiazepine binding sites underwent downregulation after exposure to serum free medium or to 10 nM PK11195. These findings suggest that peripheral benzodiazepines may be involved in the regulation of cell proliferation as a growth factor in lower concentration and as a antiproliferative agent in higher concentration.     

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 inhibits 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.     

The effect of calphostin C,a potent photodependent protein kinase C inhibitor,on the proliferation of glioma cells in vitro

Recent studies have suggested that the proliferation of malignant gliomas may result from activation of protein kinase C (PKC)-mediated pathways; conversely, inhibitionof PKC may provide a strategy for blocking tumor growth.In the current studies, we examined the effect of a novelPKC inhibitor, calphostin C, which is a selective, highlypotent, photo-activatable inhibitor of the PKC regulatorydomain, on the proliferation and viability of three established and three low-passage malignant glioma cell lines, four low-passage low-grade glioma cell lines, and in adult human and neonatal rat non-neoplastic astrocyte cell lines in vitro. Under light-treated conditions, calphostin C consistently inhibited cell proliferation in each of the tumor cell lines and in the neonatal rat astrocyte cell line with a 50% effective concentration of 30 to 50 ng/ml (40 to 60 nm), which was comparable to the previously reported median inhibitory concentration (IC₅₀) for PKC inhibition by calphostin C. Complete elimination of proliferation was achieved atconcentrations of 50 to 100 ng/ml (60 to 125 nM). Cell viability decreased sharply with calphostin C concentrations of 100 to 300 ng/ml (125 to 380 nM). In contrast, under light-shielded conditions, calphostin C had a comparatively modest effect on cell proliferation and viability, with a median effective concentration of approximately 300 ng/ml. No significant inhibition of proliferationwas noted in the non-neoplastic adult astrocyte cell line under either light-treated or light-shielded conditions. These findings provide further evidence that PKC may play an essential role in mediating the proliferation of both benign and malignant glioma cells in vitro and may also contribute to the proliferation of non-neoplastic immature astrocytes. Light-sensitive inhibition of proliferation and viability by agents such as calphostin C may provide a novel strategy for applying photodynamic therapy to the treatment of neoplastic glial cells.     

The role of thrombin in the neo-vascularization of malignant gliomas: an intrinsic modulator for the up-regulation of vascular endothelial growth factor

Thrombin is a key enzyme in the blood coagulation system where it converts fibrinogen to fibrin. It participates in a variety of biological processes such as the induction of mitogenesis and of morphological changes, the production of cytokines and growth factors, and apoptosis. To clarify the role of thrombin in the proliferation of human malignant gliomas, we investigated its effect on the expression of vascular endothelial growth factor (VEGF) in vitro and determined its intrinsic expression in human glioma tissues. In 3 human glioma cell lines tested, U-87 MG, U-251 MG, and U-105 MG, thrombin induced the VEGF mRNA expression and protein in a dose- and time-dependent manner. The thrombin receptor expression was detectable by RT-PCR and immunoblot. The secretion of VEGF protein in glioma cells was stimulated by the thrombin receptor agonist peptide and the induction of VEGF was significantly blocked by the thrombin inhibitor hirudin, indicating that the up-regulation of VEGF was mediated by the thrombin/thrombin receptor pathway. Immunoblot analysis demonstrated that prothrombin, the precursor of thrombin, was distributed in all 10 glioma tissues examined. In situ hybridization and immunohistochemical analysis revealed the co-localization of prothrombin mRNA-positive and GFAP-positive cells in the glioma tissues. Although various factors may be involved in the up-regulation of VEGF, our results suggest that human gliomas per se express prothrombin, and that thrombin, converted from prothrombin in glioma tissues, substantially stimulates angiogenesis in an autocrine fashion.     

全反式维甲酸诱导大鼠C6脑胶质瘤细胞凋亡的实验研究

目的: 探讨全反式维甲酸对大鼠C6脑胶质瘤细胞的增殖抑制及其分子机制。 方法: MTT法检测全反式维甲酸作用于大鼠C6脑胶质瘤细胞后,观察其对细胞增殖抑制率的影响。流式细胞仪观察肿瘤细胞周期及凋亡率的变化。电镜观察C6细胞超微结构变化。Western blot法在不同时间点对凋亡相关基因caspase-3活性蛋白产物的表达进行了检测。 结果: MTT结果表明ATRA对C6细胞的抑制作用具有时间和浓度依赖性。流式细胞仪检测证明与对照组相比,处理组C6细胞发生G₁期阻滞;S、G₂期细胞比例下降;细胞出现亚二倍峰,凋亡比例明显增加。电镜下全反式维甲酸作用72h后处理组C6细胞呈凋亡改变:如核固缩、染色质趋边凝聚。Western blot检测发现,处理组出现了caspase-3蛋白活性裂解片段。 结论: 全反式维甲酸抑制C6脑胶质瘤细胞生长,全反式维甲酸抑制脑胶质瘤的作用机理可能至少通过改变细胞周期分布、诱导凋亡来实现。     

Induced interleukin-33 expression enhances the tumorigenic activity of rat glioma cells

Background

Glioma development is a multistep process associated with progressive genetic alterations but also regulated by cellular and noncellular components in a tumor-associated niche.

Methods

Using 2 rat C6 glioma cell clones with different tumorigenesis, named C6-1 and C6-2, this study characterized genes associated with enhanced tumorigenic features of glioma cells by comparative cDNA microarray analysis combined with Q-PCR. Neurospehere formation and clonogenicity were examined to determine the growth of tumorigenic C6 glioma cells. The lentivirus-mediated gene knockdown approach was conducted to determine the role of interleukin-33 (IL-33) in glioma cell proliferation and migration. Transwell cell invasion assay was used to examine microglia migration induced by tumorigenic C6 cells.

Results

The functional analysis of gene ontology (GO) biological processes shows that the upregulated genes found in tumorigenic C6 (C6-1) cells are closely related to cell proliferation. Tumorigenic C6 cells expressed cytokines and chemokines abundantly. Among these genes, IL-33 was profoundly induced in tumorigenic C6 cells with the expression of IL-33 receptor ST2. Furthermore, the growth rate and colony formation of tumorigenic C6 cells were attenuated by the inhibition of IL-33 and ST2 gene expression. Moreover, IL-33 was involved in tumorigenic glioma cell migration and regulation of the expression of several glioma-associated growth factors and chemokines in tumorigenic C6 cells.

Conclusion

Accordingly, we concluded that glioma cells with abundant production of IL-33 grow rapidly; moreover, the interactions of multiple cytokines/chemokines induced by glioma cells may develop a microenvironment that facilitates microglia/macrophage infiltration and fosters glioma growth in the brain.     

VEGF反义寡核苷酸对C_6胶质瘤细胞VEGF表达和内皮细胞生长的抑制作用

目的　探讨VEGF反义寡核苷酸作为抗肿瘤血管生成治疗新药的可能性。方法　观察VEGF反义、正义、错义寡核苷酸制备的条件培养基对牛血管内皮细胞生长的抑制作用。应用免疫荧光流式细胞技术观察三者分别对C6胶质瘤细胞VEGF表达的影响。结果　反义寡核苷酸对C6胶质瘤细胞VEGF的表达和内皮细胞生长有明显抑制作用 ,而且抑制作用存在浓度依赖性。结论　反义VEGF寡核苷酸通过抑制C6胶质瘤细胞VEGF的表达 ,进而抑制内皮细胞的生长。     

Thalidomide and a Thalidomide Analogue Inhibit Endothelial Cell Proliferation in vitro

Angiogenesis is a crucial process in inflammatory reactions as well as in tumor implantation and growth. Tumors with high rates of invasion and recurrence such as gliomas, are specially dependent on neovascularization. This suggests that inhibition of angiogenesis might reduce the growth of these tumors. Thalidomide has been previously shown to inhibit angiogenesis induced by basic fibroblast growth factor in vivo, using the rabbit corneal micropocket assay. Therefore, the effect of thalidomide and a thalidomide analogue (cc-1069) on the proliferation in vitro of endothelial and glioma cells was tested. We observed a decrease in endothelial cell proliferation in cultures treated with thalidomide or the thalidomide analogue cc-1069. The analogue inhibited endothelial cell proliferation more efficiently than thalidomide. The inhibition occurred in association with a marked decrease in the activity of the nuclear factor SP1 and a moderate inhibition of NF-B activation in nuclear extracts of endothelial cells. The drugs did not impair cell viability. There was no effect of thalidomide or the thalidomide analogue on the proliferation of the glioma cell line (U251) in vitro.     

Transforming growth factor gene expression in human endometrial adenocarcinoma cells: regulation by progestins.

In an attempt to understand the antiproliferative effects of progestins in endometrial cancer, we have examined the effects of the potent progestin, medroxyprogesterone acetate (MPA), on the cell proliferation and the expression of transforming growth factor (TGF) alpha and beta genes in human endometrial adenocarcinoma cell lines. The two cell lines used were Ishikawa, var 1, and HEC-50. In addition, the effects of exogenous TGF-alpha and anti-epidermal growth factor (EGF) receptor monoclonal antibody on cell proliferation were determined. Incubation of both cell lines with MPA resulted in a time- and dose-dependent inhibition of cell proliferation. Half-maximal growth inhibition was observed at 0.6 nM. In Ishikawa cells, the relative abundance of TGF-alpha was significantly reduced by MPA. A significant decrease in TGF-alpha mRNA was apparent 6 h after exposure to MPA and a further decrease was seen 12-24 h after addition of the progestin. The concentration of TGF-alpha immunoreactivity in conditioned medium of MPA-treated cells was also significantly reduced compared to control cultures. MPA had no effect on TGF-alpha expression by HEC-50 cells. EGF mRNA was not detected by Northern blot analysis in either cell type. MPA had no significant effect on EGF receptor mRNA abundance but resulted in a small increase in EGF receptor number in Ishikawa cells. Anti-EGF receptor monoclonal antibody (0.6-6 nM) inhibited Ishikawa cell growth but had no effect on HEC-50 cell proliferation. Exogenous TGF-alpha stimulated proliferation of both cell lines, but Ishikawa cells were significantly more sensitive to exogenous TGF-alpha than HEC-50 cells. Furthermore, TGF-alpha could reverse the growth inhibitory effects of MPA on Ishikawa cells. A decrease in TGF-beta mRNA abundance was also observed in MPA-treated Ishikawa and HEC-50 cells. This effect was of small magnitude, variable, and only observed after prolonged exposure to MPA. These observations are consistent with the hypothesis that the antiproliferative effects of progestins on Ishikawa cells are mediated by decreased expression and autocrine action of TGF-alpha. Since similar growth inhibition is also seen in the HEC-50 cells in which progestins have no effect on TGF-alpha expression, additional mechanisms are likely to be involved in the antiproliferative effects of progestins in human endometrial cancer.     

VEGF抗体治疗胶质瘤的实验研究

目的 以血管内皮生长因子（ｖａｓｃｕｌａｒ ｅｎｄｏｔｈｅｌｉａｌ ｇｒｏｗｔｈｆａｃｔｏｒ,ＶＥＧＦ）抗体进行抗血管生成治疗胶原瘤的研究。方法 应用ＶＥＧＦ抗体分别作用于Ｃ６胶原瘤细胞系的体外培养细胞和动物模型,计算细胞存活率及抑溜率,ＡＢＣ免疫组化技术检测体内生长胶质瘤中微血管密度。结果 ＶＥＧＦ抗体不抑制体外培养细胞生长,细胞存活率为１００％。对体内生长的胶质瘤则有显著抑制作用且呈剂量依赖关     

Simvastatin, an inhibitor of cholesterol biosynthesis, shows a synergistic effect with N,N'-bis(2-chloroethyl)-N-nitrosourea and beta-interferon on human glioma cells.

The effect of simvastatin, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, on human glioma cell growth was investigated. When incubated with simvastatin, cell proliferation decreased in a concentration-dependent fashion, as measured by cell number and [3H]-thymidine incorporation into DNA (concentration producing 50% inhibition, 60 nM). The effect was detectable 12 h after cells were exposed to the drug and persisted for 2 days. Addition of mevalonate to cells exposed effect of simvastatin in combination with beta-interferon and N,N'-bis(2-chloroethyl)-N-nitrosourea, both antitumoral drugs, was also evaluated by cell growth inhibition assay. The concentration producing 50% inhibition for each of these drugs was 650 units/ml and 50 nM, respectively. Subliminal concentrations of beta-interferon or N,N'-bis(2-chloroethyl)-N-nitrosourea were incubated together with 1 nM simvastatin. The data were analyzed with the aid of an isobologram using the concept of an envelope of additivity. Simultaneous cell exposure to simvastatin with either N,N'-bis(2-chloroethyl)-N-nitrosourea or beta-interferon produced a strong synergistic inhibitory effect on cell proliferation. These data provide in vitro support for the possibility that 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, utilized as plasma cholesterol-lowering agents, could potentiate the effect of antiblastic drugs on tumor growth.     

Targeting hyaluronan interactions in malignant gliomas and their drug-resistant multipotent progenitors.

PURPOSE: To determine if hyaluronan oligomers (o-HA) antagonize the malignant properties of glioma cells and treatment-resistant glioma side population (SP) cells in vitro and in vivo. EXPERIMENTAL DESIGN: A single intratumoral injection of o-HA was given to rats bearing spinal cord gliomas 7 days after engraftment of C6 glioma cells. At 14 days, spinal cords were evaluated for tumor size, invasive patterns, proliferation, apoptosis, activation of Akt, and BCRP expression. C6SP were isolated by fluorescence-activated cell sorting and tested for the effects of o-HA on BCRP expression, activation of Akt and epidermal growth factor receptor, drug resistance, and glioma growth in vivo. RESULTS: o-HA treatment decreased tumor cell proliferation, increased apoptosis, and down-regulated activation of Akt and the expression of BCRP. o-HA treatment of C6SP inhibited activation of epidermal growth factor receptor and Akt, decreased BCRP expression, and increased methotrexate cytotoxicity. In vivo, o-HA also suppressed the growth of gliomas that formed after engraftment of C6 or BCRP+ C6SP cells, although most C6SP cells lost their expression of BCRP when grown in vivo. Interestingly, the spinal cord gliomas contained many BCRP+ cells that were not C6 or C6SP cells but that expressed nestin and/or CD45; o-HA treatment significantly decreased the recruitment of these BCRP+ progenitor cells into the engrafted gliomas. CONCLUSIONS: o-HA suppress glioma growth in vivo by enhancing apoptosis, down-regulating key cell survival mechanisms, and possibly by decreasing recruitment of host-derived BCRP+ progenitor cells. Thus, o-HA hold promise as a new biological therapy to inhibit HA-mediated malignant mechanisms in glioma cells and treatment-resistant glioma stem cells.     

全反式维甲酸与单纯疱疹病毒胸苷激酶基因对胶质瘤的协同杀伤作用

背景与目的：细胞间缝隙连接通讯（gap junctional intercellular communication,GJIC)是介导单纯疱疹病毒胸苷激酶（herpes simplx virus thymidine kinase,HSV-tk)基因治疗中旁观者效应的重要机制。全反式维甲酸(all-trans-retinotic acid,ATRA)或能通过上调胶质细胞GJIC和抑制肿瘤细胞生长的双重作用,促进HSV-tk基因治疗的疗效。本研究的目的在于证实HSV-tk和ATRA联合应用对胶质瘤细胞的协同杀伤作用。方法：分别以1μmol/L、10μmol/L、100μmol/L3种浓度的ARTA作用于培养的大鼠C6胶质瘤细胞,并研究ATRA对C6胶质瘤细胞分化、增殖、GJIC及connexin43(Cx43)基因转录等方面的影响。将C6细胞与稳定表达HSV-tk基因的C6tk细胞按不同比例混合,分别在含GCV和不同浓度ATRA或仅含GCV的培养基中培养,并于药物作用7天后用MTT法检测旁观者效应。结果：经3种浓度的ARTA作用后,C6胶质瘤细胞均显示出细胞分化的形态学特征。C6细胞的增殖也明显受到ATRA的抑制,绝大多数的活细胞都静止于G1期,100μmol/L ATRA对C6细胞增殖的抑制尤其明显,并诱导细胞凋亡。经3种浓度的ATRA分别作用后,C6细胞的GJIC也明显提高,而Cx43基因的转录未受明显影响。旁观者效应实验的结果显示,3种浓度的ATRA均可明显增强旁观者效应。结论：联合应用ATRA与HSV-TK基因治疗可以产生显著的杀伤胶质瘤细胞的作用。     

Antiglioma effects of N6‐isopentenyladenosine,an endogenous isoprenoid end product,through the downregulation of epidermal growth factor receptor

Malignant gliomas are highly dependent on the isoprenoid pathway for the synthesis of lipid moieties critical for cell proliferation. The isoprenoid derivative N6‐isopentenyladenosine (iPA) displays pleiotropic biological effects, including a direct anti‐tumor activity in several tumor models. The antiglioma effects of iPA was then explored in U87MG cells both in vitro and grafted in mice and the related molecular mechanism confirmed in primary derived patients' glioma cells. iPA powerfully inhibited tumor cell growth and induced caspase‐dependent apoptosis through a mechanism involving a marked accumulation of the pro‐apoptotic BIM protein and inhibition of EGFR. Indeed, activating AMPK following conversion into its iPAMP active form, iPA stimulated EGFR phosphorylation and ubiquitination along a proteasome‐mediated pathway which was responsible for receptor degradation and its downstream signaling pathways inhibition, including the STAT3, ERK and AKT cascade. The inhibition of AMPK by compound C prevented iPA‐mediated phosphorylation of EGFR, known to precede receptor loss. As expected the block of EGFR degradation, by exposure to the proteasome inhibitor MG132, significantly reduced iPA‐induced cell death. Given the importance of receptor degradation in iPA‐mediated cytotoxicity, we also documented that the EGFR expression levels in a panel of primary glioma cells confers them a high sensitivity to iPA treatment. In conclusion our study provides the first evidence of iPA antiglioma effect. Indeed, as glioma is driven by aberrant signaling of growth factor receptors, particularly the EGFR, iPA, alone or in association with EGFR targeted therapies, might be a promising therapeutic tool to achieve a potent anti‐tumoral effect.     

In vitro and in vivo potentiating the cytotoxic effect of radiation on human U251 gliomas by the c-Met antisense oligodeoxynucleotides

C-Met, a receptor tyrosine kinase, and its ligand, hepatocyte growth factor (HGF), are critical in cellular proliferation, motility, and invasion, and are known to be overexpressed in gliomas, which are related to the repair of damaged DNA. In this study, we investigated both in vitro and in vivo whether inhibition of the c-Met gene by antisense oligonucleotides (ODNs) enhances the cytotoxic effect of radiation on human U251 gliomas. A volume of 100 nM of c-Met antisense ODNs inhibited the level of mRNA by more than 95% and reduced the protein expression by about 70%. Treatment of human U251 glioma cells with 100 nM of c-Met antisense ODNs significantly enhanced the radiation-induced cell kill compared to control cells, and cells treated with nonsense ODNs. When the glioma cells were implanted in the cisterna magna of nude mice followed by treatment with c-Met antisense ODNs, the survival time of the nude mice was markedly prolonged compared to that of the untreated group (P < 0.001, logrank test). In addition, the combination of antisense ODNs and irradiation extended the survival time of the glioma-bearing nude mice much longer than could be achieved with radiation alone (P < 0.0001, logrank test). These results suggest that inhibition of c-Met can be expected to serve as a novel potentiator for radiation therapy in human U251 gliomas.     

IL-24基因对大鼠胶质瘤细胞生长状况的影响

目的　探讨IL 2 4基因对C6大鼠胶质瘤细胞生长状况的影响。方法　应用逆转录病毒载体 ,将IL 2 4基因导入C6细胞 ,经G4 18筛选后获得表达IL 2 4分子的阳性细胞克隆C6 /IL 2 4 ;用RT PCR方法检测目的基因表达 ;四甲基偶氮唑蓝 (MTT)法检测细胞体外增殖状况 ,流式细胞技术检测细胞的增殖活性 ,并制作荷瘤动物模型 ,观察C6 /IL 2 4和C6细胞的体内致瘤性。结果　RT PCR检测表明 ,外源IL 2 4基因于mRNA水平在C6 /IL 2 4细胞已获得稳定表达。C6 /IL 2 4细胞系的体外增殖性较亲代C6细胞明显下降 ,流式细胞术检测其细胞增殖指数 (PI)为 (2 9.71± 0 .89) %。 9只接种C6 /IL 2 4细胞的实验组大鼠中 ,6只颅内成瘤 ,肿瘤体积为 (14 .0 8± 9.81)mm3 ,明显小于接种C6细胞大鼠的肿瘤体积 (P <0 .0 5 )。结论　外源性IL 2 4基因可部分抑制胶质瘤细胞异常增殖的肿瘤特性。     

In vitro growth suppression of human glioma cells by a 16-mer oligopeptide: a potential new treatment modality for malignant glioma

Fibroblast growth factor-2 (FGF-2) is involved as an autocrine growth factor in the autonomous proliferation of glioma cells. To develop a new strategy for treating patients with glioma, we studied the effect on human glioma cells of a 16-mer oligopeptide with conformational similarity to the putative receptor-binding domain of FGF-2. A synthesized oligonucleotide was assessed its receptor-binding activity by BIAcore instrument. Its biological effect on glioma cell lines was examined in vitro by MTT assay. The peptide suppressed the in vitro growth of human glioma cells U87MG, T98G and U251MG cells, but not of A431 cells whose growth is not dependent on FGF-2. Apoptotic bodies were noted after 24-h incubation in the presence of the peptide; Ac-YVAD-CHO, a caspase-3 inhibitor, suppressed apoptosis. Furthermore, we examined the modulation of the cytotoxic effect of anticancer drugs by the oligopeptide. The addition of this oligopeptide to the chemotherapeutic agents CDDP, ACNU and VP16 had additive effects in vitro. These results suggest that the pathway of the FGF-2 autocrine loop through the FGF receptor plays an important role in the proliferation of glioma cells. New drugs targeting this loop may be highly effective in treating FGF-2-dependent tumors. Our results suggest that its addition to the therapeutic arsenal may lead to improved treatment regimens for patients with FGF-2-dependent tumors.     

Protamine inhibits angiogenesis and growth of C6 rat glioma; a synergistic effect when combined with carmustine

Protamine inhibits angiogenesis and blocks endothelial, fibroblast and platelet growth factors. Human and experimental gliomas spread and grow in response to both paracrine and autocrine release of these factors. Our objective was to study the effect of protamine administration on cell proliferation, angiogenesis and tumoral growth of C6 glioma. Additionally, we compared the antitumoral effect of protamine with that of another inhibitor of angiogenesis, suramin, and investigated a potential synergistic antitumoral action of low doses of protamine combined with the antineoplastic carmustine. C6 glioma cells were implanted subcutaneously in Wistar rats. A highly malignant glioma developed in 80% of animals; when the tumour reached a diameter of 1.5 cm, either protamine, suramin, carmustine or protamine plus carmustine were administered in various doses. Tumour parameters were measured and compared between groups. In a dose-dependent manner, protamine reduced tumour volume (P < 0.001), mitotic index (P < 0.05), vascular density (P < 0.05) and cell viability (P < 0.005) of C6 glioma. An ultrastructural study demonstrated membranous inclusions in the cytoplasm of 28% of tumoral and endothelial cells of tumours from animals treated with protamine. The inhibition of tumoral growth produced by moderate doses of protamine was similar to that produced by toxic doses of suramin. The combination of protamine and carmustine had a synergistic curtailing effect on tumoral growth (P < 0.001). Our results indicate that protamine is an effective agent against glioblastoma; in non-toxic doses it could potentiate the antineoplastic effect of nitrosoureas for the treatment of glial tumours.     

Nerve Growth Factor Plays a Divergent Role in Mediating Growth of Rat C6 Glioma Cells via Binding to the P75 Neurotrophin Receptor

Dysregulation of proliferation, differentiation and cell death play a major role in glial tumors, and there is evidence for regulatory mechanisms involving nerve growth factor (NGF) and its receptors in various CNS-derived tumor cell lines. The aim of our study was to observe the effect of exogenous recombinant NGF on C6 rat glioma growth, to characterize the role of endogenous NGF and the p75 neurotrophin receptor (p75) and to rule out whether p75 is necessary to mediate the effect of exogenous NGF. Recombinant exogenous NGF (1–100ng/ml) was applied under different serum conditions (0%, 1%, 5%) and knockdown of endogenous NGF and p75 was achieved by lipid-mediated antisense oligonucleotide treatment. In presence of serum, NGF had a positive whereas in absence of serum NGF produced a negative effect on C6 cell number. A knockdown of NGF or p75 increased cell numbers and enhanced BrdU incorporation. In p75-knocked down cells NGF did not enhance C6 glioma growth in presence of serum. We conclude that (1) exogenous recombinant NGF enhances C6 glioma growth under serum conditions but decreases cell number in absence of serum, that (2) the effect of exogenous NGF is mediated by p75 alone or by heterodimers containing p75 and that (3) either basal levels of endogenous NGF or basal levels of p75 receptor moderate C6 glioma growth and represent an autoregulatory potential of C6 glioma cells.