Somatostatin suppression of meningioma cell proliferation in vitro

Considering the presence of a stereospecific receptor for somatostatin (SST) in human meningioma cells and the possible involvement of this neuropeptide in the growth control of certain meningioma cell lines, the effects of SST on the proliferation of human meningioma cells in vitro was investigated. Tumour tissues for primary cell cultures were obtained surgically from 2 women with histopathological diagnosis of meningothelial meningioma. The incorporation of [3H]-thymidine into meningioma cells DNA was measured as an index of the cells proliferation. It was shown that SST (10(-7)-10(-5) M) significantly inhibited the [3H]-thymidine incorporation. The results have indicated that SST may have an antiproliferative effects on the meningioma tumour cells in vitro.     

Inhibitory effect of calcium channel blockers on proliferation of human glioma cells in vitro

The effects of 2 specific calcium channel blockers, verapamil and nimodipine, on the proliferation of human glioma tumour cells were investigated in vitro. Tumour tissues for primary cell cultures were obtained bioptically from 3 patients with the histopathological diagnosis of glioblastoma. The [3H]-thymidine incorporation into glioma tumour cells DNA was used as a sensitive index of the cell proliferation. It was found that verapamil (10(-4)-10(-5) M) and nimodipine (10(-4)-10(-6) M) significantly inhibited the [3H]-thymidine uptake in a dose-related manner. The inhibitory effect of both calcium channel antagonists was reversed by simultaneous addition of calcium chloride (5 x 10(-3) M). These results indicate that verapamil and nimodipine may exert an antiproliferative effect on glioma cells growth acting through a blockade of specific voltage-dependent calcium channels.     

逆转录酶抑制剂AZT对胶质瘤干细胞端粒酶活性和细胞增殖的影响

目的 探讨逆转录酶抑制剂3-叠氮-3-脱氧胸腺核苷(AZT)对脑胶质瘤干细胞增殖的影响及相关机制.方法 原代分离培养脑胶质瘤干细胞和脑胶质瘤细胞并鉴定,两种细胞同时设立为实验组(0.125 mot/L、0.250 mol/L、0.500 mol/L AZT)和对照组.MTT法检测AZT对两种细胞增殖的影响;流式细胞仪检测细胞凋亡和细胞周期的改变;端粒重复序列扩增技术-酶联免疫吸附试验(TRAP-ELISA)检测端粒酶活性的变化.结果 AZT对两组细胞的生长抑制呈浓度-时间依赖性(均P<0.05),在同一浓度和时间点,AZT对脑胶质瘤干细胞的生长抑制作用弱于脑胶质瘤细胞(均P<0.05).0.250 mol/L、0.500 mol/L AZT作用72 h后,脑胶质瘤干细胞的凋亡率分别为(4.21±1.53)%、(10.60±0.38)%,而脑胶质瘤细胞的凋亡率分别为(6.75±1.25)%,(14.30±2,59)%,明显高于胶质瘤干细胞(均P<0.05).AZT对两组细胞端粒酶活性的抑制呈浓度-时间依赖性(均P<0.05),且在同一浓度和时间点对脑胶质瘤细胞的作用明显强于脑胶质瘤干细胞(均P<0.05).结论 逆转录酶抑制剂AZT对脑胶质瘤干细胞和脑胶质瘤细胞均有明显的生长抑制作用,可能机制是通过抑制端粒酶活性、调控细胞周期和诱导细胞凋亡实现.脑胶质瘤干细胞的耐药性强于胶质瘤细胞,其机制有待进一步研究.     

In vitro analysis of BCNU-sensitivity in human malignant gliomas

Like all chloroethyl-nitrosoureas of major clinical use, 1,3 bis-(2-chloroethyl)-1-nitrosourea (BCNU) - which is one of the most effective chemotherapeutic agents for CNS malignancies - biologically degrades into active alkylating and carbamoylating moieties. Using a human brain tumor stem cell assay, we analyzed a series of anaplastic astrocytomas of pediatric age, characterized by different degrees of BCNU-resistance. Early (2-4) passage cultures from these tumors were treated in vitro with model drugs for alkylation (BCNU, CHLZ (2-[3-(2-chloroethyl)-3-nitrosoureido]-2-deoxy-D-glucopyranose), ENU (N-ethyl-N-nitrosourea), cross-linking (BCNU, CHLZ) and carbamoylation BHCNU (1,3 bis (trans-4-hydrocyclohexyl)-1-nitrosourea): dose-schedules were compatible with clinically achievable levels. Results of chemosensitivity tests confirmed that - as previously reported in malignant gliomas of the adult - cellular resistance to BCNU was closely related to the cross-linking activity of alkylating species. However, in pediatric gliomas the levels of cell kill after treatment with the purely carbamoylating agent BHCNU, even at the highest doses tested, were lower than expected.     

In vitro analysis of BCNU-sensitivity in human malignant gliomas

With the use of the Human Brain Tumor Stem Cell Assay (HBTSCA) in a cross-resistance study, four early (3-4) culture passages of human malignant gliomas (glioblastoma multiforme) were tested for in vitro chemosensitivity with three of the most effective single agents for brain tumor chemotherapy: BCNU, CCNU and cisplatinum (DDP). The shapes of the dose-response curves indicated complete cross-resistance between BCNU and CCNU, i.e. two chloroethyl-nitrosoureas sharing a common alkylating-carbamoylating activity, with no evident cross-resistance between the two nitrosoureas and the DDP, a DNA binder with a putatively different antitumor action. Probably because of differences in drug delivery kinetics or in the cytotoxic mechanism, DDP might play a role in the treatment of nitrosourea-resistant gliomas.     

miR‐193b promotes cell proliferation by targeting Smad3 in human glioma

Studies have shown that several miRNAs play important roles in regulating a variety of cellular processes in gliomas. In these reports, upregulation of miR‐193b has been found to be associated with a poor prognosis for glioma, but its functional mechanism in glioma remains unclear. This study investigates the roles of miR‐193b in glioma tumor growth. We first showed that the expression of miR‐193b was elevated in both glioma samples and glioma cells. Furthermore, downregulation of miR‐193b by inhibitors was statistically correlated with a decrease in cell growth and a restored G1 accumulation. Luciferase assay and Western blot analysis revealed that Smad3 is a direct target of miR‐193b. To prove that miR‐193b regulated cell growth through the transforming growth factor‐β (TGF‐β) pathway in glioma cells by regulating Smad3, we tested endogenous targets of the TGF‐β pathway by measuring the accumulation of p21 mRNAs after downregulation of miR‐193b. The results confirmed that induction of p21 was promoted by miR‐193b inhibitors in glioma cells, although this induction disappeared when Smad3 was knocked down with siRNA. Moreover, downregulation of Smad3 mitigates the miR‐193b suppression of glioma proliferation. In conclusion, these results suggest that miR‐193b regulated cell growth in glioma through the TGF‐β pathway by regulating Smad3. Thus, our study indicates that miR‐193b promotes cell proliferation by targeting Smad3 in human glioma, which may serve as a potentially useful target for development of miRNA‐based therapies in the future. © 2014 Wiley Periodicals, Inc.     

端粒酶基因hTERT在脑胶质瘤的表达及与肿瘤增殖相关性的研究

目的研究人类端粒酶催化亚单位(hTERT)mRNA在胶质瘤的表达情况,探讨hTERT基因表达与胶质瘤增殖的关系.方法应用原位杂交方法检测45例胶质瘤标本、8例正常脑组织标本中hTERTmRNA的表达,采用免疫组织化学法测定增殖指数(PCNA LI).结果hTERTmRNA总的阳性表达率为60.0%(27/45例),低度恶性组为31.3%(5/16例),高度恶性组为75.9%(22/29例);8例正常脑组织均为阴性.高度恶性组与低度恶性组、正常组间相比较均有显著性差异(P＜0.05)hTERT阴性组PCNA LI均低于hTERT阳性组,胶质瘤hTERT mRNA表达率与肿瘤增殖指数呈正相关(r=0.727,P＜0.05).结论端粒酶hTERT在正常脑组织中未见表达,而在胶质瘤标本呈不同程度的表达,阳性表达率与肿瘤病理分级呈正相关,与肿瘤增殖程度一致.表明端粒酶hTERT的激活可能发生在胶质瘤的癌变过程中,并起关键性作用,有可能成为靶向基因治疗的候选基因.     

吴茱萸碱对人胶质瘤U251细胞增殖及凋亡影响的研究

目的研究吴茱萸碱(evodiamine)对人胶质瘤U251细胞增殖和凋亡的影响及其可能的作用机制。方法体外培养人胶质瘤U251细胞,并将其分为空白对照组及25、50、100μg/mL吴茱萸碱4组。应用MTT法检测吴茱萸碱对U251细胞的增殖抑制作用;Hoechst33258荧光染色法检测吴茱萸碱诱导胶质瘤U251细胞凋亡;采用Annexin V-FITC/PI双染法检测各组早期凋亡率;Western blot法分析凋亡相关蛋白的变化。结果与空白对照组同期比较,25、50、100μg/mL吴茱萸碱组生长抑制率在24、48、72 h均增加,差异有统计学意义。Hoechst 33258荧光染色显示吴茱萸碱作用24 h后U251细胞出现典型的细胞凋亡特征,各处理组均可见凋亡小体。与空白对照组自发早期凋亡率3.12%比较,25、50、100μg/mL吴茱萸碱组早期凋亡率分别为8.65%、19.47%及28.97%,差异均有统计学意义。Western blot实验显示,与空白对照组同期比较,25、50、100μg/mL吴茱萸碱上调了FAS、FADD、Caspase-8及Caspase-3蛋白表达,Bcl-2蛋白表达明显下降,Bax蛋白表达明显上升,差异均有统计学意义。上述指标均呈时间和剂量依赖性。结论吴茱萸碱对U251细胞具有明显的抑制细胞增殖和促进细胞凋亡的作用,其机制可能与上调Fas途径和下调Bcl-2/Bax有关。     

Inhibition of glial proliferation in vitro by serum from patients with multiple sclerosis

Primary cell cultures from fetal rat CNS have been employed to evaluate the effects caused by the addition of serum from patients affected by multiple sclerosis (MS). MS-serum supplemented media caused a decrease in [3H]-thymidine incorporation into the cultures, thus indicating an inhibitory effect on proliferating glial cells. Sera from patients in remission stage of the disease showed an inhibitory effect not significatively lower than those from patients in acute stage. These results suggest that glial cells may be a target of circulating factors present in MS.     

GM3 as a novel growth regulator for human gliomas

The simple ganglioside GM3 inhibits proliferation and induces apoptosis in proliferating immature rodent CNS cells. To determine whether GM3 influenced the expansion of human neural tumors the effects of GM3 treatment on primary human brain tumors were assayed. Here we demonstrate that GM3 treatment dramatically reduces cell numbers in primary cultures of high-grade human glioblastoma multiforme (GBM) tumors and the rat 9L cell gliosarcoma cell line. By contrast, GM3 treatment had little effect on cell number in cultures of normal human brain. A single injection of GM3 3 days after intracranial implantation of 9L tumor cells in a murine xenograft model system resulted in a significant increase in the symptom-free survival period of host animals. The effects of GM3 were not restricted to GBMs and 9L cells. Cultures of high-grade ependymomas, mixed gliomas, astrocytomas, oligodendrogliomas, and gangliogliomas were all susceptible to GM3 treatment. These results suggest that GM3 may have considerable value as a selectively toxic chemotherapeutic agent for human high-grade gliomas.     

Biologic parameters that correlate with the prognosis of human gliomas

Much clinical and biologic data have been processed in the search for useful objective parameters to predict brain tumor behavior. Seventy cases of astrocytic glioma collected by a single clinical team were studied using a full complement of clinical procedures: follow up (7 years), histologic analysis, DNA content estimation, and cell kinetics by flow cytometry. Proliferating cell nuclear antigen (PCNA) was determined by immunocytochemical‐coupling flow cytometry (PFC) and also by counting under light microscopy (PIHC). A statistical evaluation was carried out to establish the usefulness of several parameters for glioma prognosis. The cases were histologically classified as 14 low‐grade astrocytomas, 20 anaplastic astrocytomas, and 36 glioblastomas multiforme. The survival curve showed significant differences between his‐tologic groups. Diploid populations were more frequent in low‐grade astrocytomas, and aneuploid tumors often had increased S‐phase and proliferative fractions. The PCNA‐labeled index (PCNA‐LI) increased with malignancy and correlated with histologic grading (P = 0.01). The PCNA‐LI and age segregated low‐ from high‐grade astrocytomas (including anaplastic astrocytoma and glioblastoma multiforme), but none of the variables considered differentiated anaplastic astrocytoma from glioblastoma multiforme. The Cox regression test displayed significant values for age, histologic diagnosis, and PCNA determinations when considered in tandem. Discriminant analysis obtained a function integrating age and specifically PIHC‐LI to help in the prognosis of doubtful cases. The results emphasize the importance of parameters integrating different variables in an attempt to provide an accurate prognosis, the most significant being age, histopathologic diagnosis, and the proliferative fraction determined by PCNA.     

miR-137调控人脑胶质瘤细胞增殖侵袭能力的体内外研究

目的探讨miR-137调控人脑胶质瘤细胞的增殖侵袭性生长能力及其机制。方法采用实时PCR分析miR-137在不同品系胶质瘤细胞及不同级别胶质瘤样本中的表达;脂质体介导miR-137模拟物转染胶质瘤细胞,实时PCR检测转染后miR-137的表达;应用MTT法、流式细胞术评价细胞生长和增殖的生物学特征变化;划痕实验、transwell细胞体外迁移实验检测肿瘤细胞迁移、侵袭能力;动物实验评价体内条件下肿瘤生长能力变化;Western blot、免疫组织化学染色检测肿瘤细胞Ki-67、MMP9表达水平。结果实时PCR分析显示：miR-137在胶质瘤中低表达。miR-137模拟物转染LN229和U87细胞后,实时PCR显示：miR-137表达上调;MTT法及流式细胞术显示细胞生长受抑,出现G0/G1期阻滞;划痕实验及transwell实验证实：细胞迁移侵袭能力下降;进一步Western blot、免疫组织化学染色显示：增殖侵袭相关蛋白Ki-67、MMP9表达降低;动物实验反映：肿瘤细胞生长受抑制。结论 miR-137高表达可抑制胶质瘤细胞生长和侵袭能力,提示miR-137可作为基因治疗脑胶质瘤的候选靶点。     

Inhibition of cold-induced TSH release by benzodiazepines

Diazepam (1–5 mg/kg, i.p.) did not alter basal thyroid-stimulating hormone (TSH) release in rats; however, both diazepam (2 mg/kg, i.p.) and clonazepam (1 mg/kg, i.p.) blocked the 3–5-fold increase in TSH induced by cold exposure. The benzodiazepine antagonist, Ro15-1788 (10 mg/kg, i.p.) was effective in reversing clonazepam inhibition of cold-induced TSH release, but when administered alone had no effect on either basal TSH levels or the rise in TSH following cold exposure. Diazepam did not affect the ability of TRH to increase TSH secretion.     

Secretion of cathepsin B by human gliomas in vitro

There is evidence from investigations of non-CNS neoplasms that secreted proteolytic enzymes may facilitate tumour invasion by partially degrading extracellular matrix (ECM). Among the enzymes which may be involved are members of the cysteine proteinase superfamily and especially cathepsin B (CB). In the present investigation we have studied CB in human gliomas in vitro , concentrating particularly on CB secretion, as extracellular enzyme is of prime importance in this context. We have found that CB is secreted by gliomas in vitro as a latent zymogen, requiring activation. This has been confirmed by gel chromatography which indicated that CB is secreted as a 42 kDa proenzyme which may be proteolytically processed to an enzymatically active 29 kDa molecule. The inactive, high molecular weight, latent CB is stable at extracellular pH in contrast to the activated low molecular weight form which rapidly loses activity at this pH. We have also measured secretion of cysteine proteinase inhibitors (CPI), as their presence would have a direct influence on the effective activity of CB, and found that all of the gliomas secreted significant amounts of a CPI as assessed by papain inhibition. Our experiments suggest that a number of factors are involved in the regulation of extracellular glioma-derived CB activity. These include: rate of secretion of pro-CB, rate of CB activation, destabilization of CB at neutral pH and the presence of cysteine proteinase inhibitors.     

Diffuse intrinsic pontine gliomas: Diagnostic approach and treatment strategies

Diffuse intrinsic pontine gliomas (DIPG) are high grade gliomas of the brainstem with fatal outcomes. Radiation is known to be partially effective to control the immediate flare but relapse is frequent. There has been ongoing research to study the role of molecular subgroups and identification of specific targets but this is not possible with histopathological diagnosis alone. The authors’ objective is to highlight the need for and discuss ongoing molecular research. There is an inherent need for the availability of tumor tissue to be able to conduct research studies. The authors advocate the use of neuronavigation assisted stereotactic technique for tumor biopsy. The technique is feasible with a predefined surgical trajectory. After obtaining tissue diagnosis further work can be performed to isolate and identify histone protein genetic mutations and methylation changes responsible for DIPG molecular subgrouping. Moreover, convection enhanced delivery of therapeutic agents is being developed for better instillation of future drug agents. Despite identification of genetic/epigenetic mutations, growth factors, receptors, and tissue biomarkers, the oncogenesis of DIPG remains elusive. The authors’ effort to provide a comprehensive review on DIPG to better understand the disease, need for tissue diagnosis, described surgical technique, and need for pre-clinical and clinical future research is novel.     

Effect of benzodiazepines on the proliferation of mouse spleen lymphocytes in vitro

Summary The effect of several benzodiazepines (clonazepam, diazepam, Ro 5-4864, Ro 15-1788) and two pineal gland indoleamines (N-acetylserotonin, melatonin) on the spontaneous proliferation of mouse spleen lymphocytes was estimated in vitro by the 3 H-thymidine uptake assay. It was found that diazepam and Ro 5-4864 (a selective peripheral-type benzodiazepine receptor ligand) produced the concentration-dependent inhibition of 3 H-thymidine incorporation into the DNA of these cells. Ro 15-1788, a specific central-type receptor ligand, evoked a slight inhibitory effect in a high concentration (10^–4M), whereas clonazepam did not produce any significant inhibition. When Ro 5-4864 was tested in combination with diazepam, the inhibition of lymphocyte proliferation did not exceed the effect of diazepam given alone. Ro 15-1788 was unable to reverse the inhibitory action of diazepam in the same experimental conditions. Melatonin and its precursor N-actetylserotonin tested in the concentration range of 10^–4–10^–8 M had no significant influence on the spleen lymphocyte DNA replication in our assay system.These data suggest that diazepam inhibition of lymphocyte proliferation is mediated by peripheral-type sites. Additionally, the fact that melatonin and N-acetylserotonin were unable to affect 3 h-thymidine incorporation argues against any benzodiazepine receptor mediated effect of pineal indoleamines on a cellular proliferation.     

Expression of cell cycle inhibitors p21, p27, p14 and p16 in gliomas. Correlation with classic prognostic factors and patients' outcome

Gliomas are among the most aggressive and treatment‐refractory of all human tumors. The aim of the present study is to evaluate the role of the expression of cell cycle molecules as prognostic indicators in gliomas. We immunohistochemically analyzed the expression of p21, p27, p14, p16, p53 and proliferation marker Ki67, in 67 low and high grade astrocytic tumors. High grade tumors exhibited higher labeling indices for Ki67 (P = 0.004), p53 (P = 0.039) and slightly higher index for p21 (P = 0.07) compared to low grade tumors. p14 and p16 were more frequently present in low grade tumors (P = 0.001 and P = 0.052, respectively). Worse survival was correlated with high grade tumors (P < 0.0001) and higher Ki67 index (P < 0.0001). Cox regression analysis revealed that only age, grade and marginally Ki67 index were independent prognostic factors. Cell cycle alterations are involved in the malignant progression of astrocytomas, but only age, tumor grade and proliferating index can predict the outcome of the patients with glioma.     

人脑胶质瘤干细胞的分离、增殖和分化的研究

目的从人胶质瘤中分离并鉴定肿瘤干细胞。方法用神经干细胞无血清培养方法从胶质瘤中分离出具有单细胞克隆能力的细胞球,通过核型分析证明其肿瘤特性,用MTT法检测单克隆细胞增殖能力,应用免疫组织化学的方法检测克隆细胞nestin抗原和分化后相关神经细胞特异性抗原的表达。结果所获得的细胞球具有连续克隆的能力,nestin抗原阳性,并具有肿瘤核型,分化后的细胞表达星形胶质细胞和神经元特异性抗原。结论成功培养出脑胶质瘤肿瘤干细胞,该细胞具有自我更新、多向分化和很强的增殖能力等干细胞的特征。     

Neurotrophin gene expression by cell lines derived from human gliomas

The expression of neurotrophin (NGF, BDNF, and NT-3) mRNAs in 24 cell lines derived from human malignant gliomas was studied by Northern analysis. Widespread expression of neurotrophin genes was found with BDNF being the most abundantly expressed. Nearly all cell lines expressed BDNF, and about two-thirds of the cell lines expressed NGF and NT-3. Half of the cell lines analyzed expressed all three neurotrophins. Secretion of NGF into the medium of several cell lines could be detected by ELISA and a PC12 neurite outgrowth assay. Immuno- and bioactive NGF was isolated from conditioned medium of one cell line. No evidence of expression of the neurotrophin receptors trk and trkB by Northern analysis was found. Receptor crosslinking with radiolabeled cognate ligands failed to detect functional receptors in all but one cell line. In this cell line a receptor complex for BDNF was found that corresponded to truncated trkB receptors that lack the signal transducing tyrosine kinase domain. Neurotrophins did not stimulate mitosis of the glioma cultures. The findings suggest that production of neurotrophins by glioma cells is a general phenomenon, although neurotrophins made by gliomas lacking their receptors may not play an autocrine but rather a paracrine role. © 1993 Wiley-Liss, Inc.