Therapeutic potential of chlorotoxin-like neurotoxin from the Chinese scorpion for human gliomas

Chlorotoxin, one of the key toxins in scorpion Leiurus quinquestriatus venom, has been shown to bind specifically to glioma cell surface as a specific chloride channel blocker. In this study, a purified, recombinant chlorotoxin-like peptide from the scorpion Buthus martensii Karsch (named rBmK CTa) was characterized by in vivo and in vitro studies. The results from cell proliferation assay with human glioma (SHG-44) cells showed that rBmK CTa inhibits the growth of glioma cells in a dose-dependent manner, with an IC₅₀ value of approximately 0.28 μM. Under the same conditions, the IC₅₀ value for normal astrocytes increased to 8 μM. This clearly indicated that rBmK CTa had specific toxicity against glioma cells but not astrocytes. Results from whole-cell patch-clamp recording showed that chloride current in SHG-44 was inhibited by rBmK CTa in a voltage-dependent manner and percent inhibitions for the blocking action of rBmK CTa (0.07 and 0.14 μM) on I_Cl was 17.64 ± 3.06% and 55.86 ± 2.83%, respectively. Histological analysis of rBmK CTa treated mice showed that brain, leg muscle and cardiac muscle were the target organs of this toxin. These results suggest that rBmK CTa may have potential therapeutic application in clinical treatment of human glioma. It represents an approach for developing a novel therapeutic agent.     

人脑胶质瘤侵袭微生态系统的形态学观察

目的　探讨人脑胶质瘤侵袭微生态系统的形态学特征。　方法　利用光镜及透射电镜观察 12例手术切除的脑胶质瘤标本中微血管内皮细胞和基底膜及其与肿瘤细胞的相互关系。　结果　光镜下可见侵袭性生长的瘤细胞可在血管周形成巢状结构。电镜下不同胶质细胞起源的胶质瘤中的微血管内皮细胞基本上为无孔型 ,偶见窗孔形成。内皮细胞下基质完整 ,基底膜呈局限性或广泛性增厚 ,有的伴有多层基板样结构形成。基底膜靠近瘤细胞侧常出现大小不等、数量不一的虫蚀样空洞 ,含有较大空洞的基底膜常突向瘤细胞侧。有的血管外腔扩张 ,腔内可见瘤细胞以单个形式侵袭 ,少见瘤细胞伸出伪足突入基底膜及穿透内皮细胞间隙现象 ,瘤细胞与基底膜相互接触处呈光滑平整的界面。　结论　无孔型内皮细胞、肿瘤微血管基底膜结构的局限性或广泛性增厚和虫蚀样空洞的改变 ,及少见瘤细胞伸出伪足突入其间 ,可能是不同胶质细胞起源的人脑胶质瘤侵袭微生态系统形态学上共有的特征     

Fuzzy rules to predict degree of malignancy in brain glioma

The current pre-operative assessment of the degree of malignancy in brain glioma is based on magnetic resonance imaging (MRI) findings and clinical data. 280 cases were studied, of which 111 were high-grade malignancies and 169 were low-grade, so that regular and interpretable patterns of the relationships between glioma MRI features and the degree of malignancy could be acquired. However, as uncertainties in the data and missing values existed, a fuzzy rule extraction algorithm based on a fuzzy min-max neural network (FMMNN) was used. The performance of a multi-layer perceptron network (MLP) trained with the error back-propagation algorithm (BP), the decision tree algorithm ID3, nearest neighbour and the original fuzzy min-max neural network were also evaluated. The results showed that two fuzzy decision rules on only six features achieved an accuracy of 84.6% (89.9% for low-grade and 76.6% for high-grade cases). Investigations with the proposed algorithm revealed that age, mass effect, oedema, post-contrast enhancement, blood supply, calcification, haemorrhage and the signal intensity of the T1-weighted image were important diagnostic factors.     

miR-107抑制胶质瘤细胞的体外增殖、迁移和侵袭

目的探讨miR-107对胶质瘤细胞增殖、迁移和侵袭的调控机制。方法运用RT-qPCR检测人正常的星形胶质细胞系NHA、神经胶质瘤细胞系U87、A172、U251中miR-107和FOXK1的表达;将细胞分为miR-NC组(转染miR-NC)、miR-107组(转染miR-107 mimics)、si-NC组(转染si-NC)、si-FOXK1组(转染si-FOXK1)、miR-107+pcDNA3.1组(共转染miR-107 mimics和pcDNA3.1)和miR-107+pcDNA3.1-FOXK1组(共转染miR-107 mimics和pcDNA3.1-FOXK1);用脂质体法分别转染至U87细胞;CCK-8法检测细胞的增殖;Transwell小室实验检测细胞的迁移和侵袭;Western blot检测细胞中FOXK1的蛋白表达;双荧光素酶报告基因检测实验检测细胞的荧光活性。结果与正常的星形胶质细胞NHA相比,神经胶质瘤细胞U87、A172、U251中miR-107表达明显下调,FOXK1表达明显上调(P<0.05);过表达miR-107、敲减FOXK1均可抑制U87细胞的增殖、迁移和侵袭;miR-107可抑制野生型FOXK1的细胞荧光活性,并负向调控FOXK1的表达;过表达FOXK1可逆转miR-107对U87细胞增殖迁移侵袭的抑制作用。结论 miR-107抑制胶质瘤细胞增殖、迁移和侵袭的作用机制可能与靶向负调控FOXK1有关,将可为胶质瘤的诊断和治疗提供靶向治疗的依据。     

The expression of Rho proteins decreases with human brain tumor progression: Potential tumor markers

Astrocytic tumors are the most common human brain tumors. Establishment of tumor grade is a key determinant both in the choice of a therapeutic approach and in the prognosis. The diagnosis of astrocytic tumors is currently determined following histopathological analysis. The identification of molecular markers would offer a complementary tool for characterizing tumors with respect to their clinical behavior. In this study we determined the expression levels of 3 small GTP binding proteins (RhoA, RhoB and Rac1), of their inhibitor RhoGDI and of caveolin-1 in 24 human astrocytic tumors of grades I to IV. Our results demonstrated that the expression of RhoA and RhoB decreased significantly in all brain tumors studied and was inversely related with tumor of grade II to IV malignancy. The amount of caveolin-1 immunodetected was not significantly different from normal brain samples while the Rac1 expression level was diminished in astrocytic tumors of grades III and IV. Our finding that RhoA and RhoB expression levels are correlated to tumor malignancy suggests that they may serve as novel and efficient diagnostic markers for astrocytic brain tumors of histological grade II to IV and complement currently applied histopathological analysis.     

Radiotherapy and combination chemotherapy with carmustine,vincristine, and procarbazine (BVP) in primary brain tumors

Summary 26 patients with astrocytoma grade 11–111, and 36 with malignant glioma (astrocytoma grade IV or glioblastoma) were submitted three days after surgery to a cycle of combination chemotherapy, including BCNU, VCR, PCZ (BVP). Eighteen days after surgery, patients received 40 Gy (astrocytoma grade 11–111) or 45 Gy (malignant glioma) of megavoltage whole-brain irradiation, with an additional boost to the tumor bed of 20 Gy, delivered in 6 weeks. Vincristine was injected weekly during radiotherapy. At the end of radiotherapy, patients received BVP every 6 weeks for at least 8 cycles or until a recurrence or progressive disease. Performance status of grade 1 or 2 was achieved in 15 (60%) and in 5 (20%), respectively, of patients with astrocytoma grade 11–111 after 6 months, and in 6 ps. (29%) and in 9 ps. (42%) after 12 months of follow-up. Only 2 (5.5%) and 18 (64%) patients with malignant glioma achieved a performance status of grade 1 or 2 after 6 months, and these proportions are 6% and 35%, respectively, after 12 months. After a 5-year follow-up, 59% of patients with astrocytoma are still alive, with a median survival time of 60+ months, whereas only 4% of patients with malignant glioma are alive, with a median of 11.2 months.     

Growth inhibitory effect of recombinant α and β interferon on human glioma cells

Summary Growth inhibitory activity of recombinant and interferon on two human glioma cell lines, EFC-2 and KE cells, was determined by two different growth assays. Recombinant interferon showed slight growth inhibitory effect on EFC-2 cells at day 3, and maximum inhibition was seen on day 6 with an ID50 of 50 U/ml. Recombinant interferon showed no significant growth inhibition at any concentration. KE cells were resistant to both recombinant and interferon. The growth inhibitory activity of recombinant interferon on EFC-2 cells was not blocked by recombinant interferon, although recombinant and interferons shared same receptors on EFC-2 cells. Addition of DFMO (-difluoromethylornithine) to interferon in the media showed additive effect rather than synergistic effect in growth inhibition of glioma cells. Out of 7 glioma cell lines tested, 4 showed heterogeneous sensitivity to recombinant interferon, and all were resistant to recombinant interferon. These results suggest differential sensitivity of EFC-2 cells to recombinant interferon, as well as a heterogeneous sensitivity to recombinant interferon among different glioma cell lines.     

Effects of sodium and GTP on the binding kinetics of [3H]diprenorphine in NG 108-15 cell membranes

Summary Equilibrium binding isotherms of [³H]diprenorphine in membranes from NG 108-15 cells are consistent with a homogenous population of binding sites. Upon addition of Na⁺, Mg²⁺ and GTP, only a 2-fold reduction in affinity with a minor decrease in the number of sites is observed. Dissociation curves of [³H]diprenorphine, however, are clearly biphasic: in the absence of Na⁺, Mg²⁺ and GTP, 80% of the bound ligand dissociates slowly with at _1/2 of 100 min, and only 20% rapidly (t _1/2 4.5 min). In the presence of Mg²⁺, nearly all the binding is found in the slowly dissociating form. Upon the addition of either Na⁺ or GTP, 20–30% of the binding dissociates more rapidly. The rate constant of the rapidly dissociating form generated by Na⁺, however, is 2.5 times greater than that induced by the presence of GTP. Thus, the addition of both, Na⁺ and GTP, converts about 80% of the receptor into a very fast dissociating form (t _1/2 1.7 min).Exposure of intact cells to pertussis toxin (10 ng/ml) or treatment of membranes with N-ethyl maleimide (500 M), strongly reduces the proportion of the slowly dissociating component. Following these treatments, the effect of GTP is reduced or abolished, but that of Na⁺ remains unaffected.We conclude from these data that the effects of Na⁺ and GTP are not only distinct in site but also in mechanism of action and that there are three forms of opioid receptors that can be differentiated by their kinetic properties. The slowly dissociating receptor form requires a functional N unit.     

长链非编码RNA AGAP2-AS1在胶质瘤增殖过程中作用机制的研究

目的探究长链非编码RNA AGAP2-AS1在胶质瘤增殖过程中的作用机制。方法选取2018年12月-2019年11月期间的30例胶质瘤患者为观察组,同时期的30例脑外伤患者为对照组。比较两组的组织AGAP2-AS1 mRNA表达量、Ras及ERK1/2蛋白表达情况,并比较观察组中不同分级胶质瘤的AGAP2-AS1 mRNA表达量、Ras及ERK1/2蛋白表达情况,比较转染siRNA AGAP2-AS1与siRNANC的U87及U251细胞克隆数。结果观察组的RNA AGAP2-AS1 mRNA表达量、Ras及ERK1/2蛋白表达显著高于对照组,不同分级胶质瘤的RNA AGAP2-AS1 mRNA表达量、Ras及ERK1/2蛋白表达比较,差异有统计学意义(P<0.05),转染siRNA AGAP2-AS1的U87及U251细胞克隆数显著低于siRNA-NC,差异有统计学意义(P<0.05)。结论长链非编码RNA AGAP2-AS1可通过调控ERK/MAPK信号通路影响胶质瘤的增殖,下调AGAP2-AS1可显著影响胶质瘤细胞的克隆形成能力。     

木香内酯诱导人脑星形胶质母细胞瘤细胞凋亡

[目的] 探究木香内酯诱导人脑星形胶质母细胞瘤细胞凋亡的作用机制。[方法] 通过 CCK8 法检测木香内酯对 U87 细胞增殖作用的影响; 采用 Annexin V-FITC/PI 双染法检测木香内酯对 U87 细胞凋亡率的影响, 采用 JC-1染色法检测木香内酯对 U87 细胞线粒体膜电位的影响; 采用 DCFH-DA 探针检测木香内酯对 U87 细胞中 ROS 含量的影响;采用生化法检测木香内酯对 U87 细胞氧化应激指标丙二醛（MDA）含量、超氧化物歧化酶（SOD）活性、微量还原型谷胱甘肽（GSH）和一氧化氮（NO）含量的影响; 采用实时荧光定量聚合酶链式反应（RT-qPCR）检测木香内酯对U87 细胞 Caspase-3、Bax、Bcl-2 的 mRNA 表达水平的影响; 采用分子对接寻找木香内酯与 Caspase-3、Bax、Bcl-2 的具体结合位点。[结果] 木香内酯呈剂量依赖性抑制 U87 细胞的生长, 细胞半抑制浓度 IC₅₀ 为 11.27 μmol/L;细胞凋亡水平检测结果显示木香内酯 20 μmol/L 组 U87 细胞凋亡率增加, 差异具有统计学意义（P<0.001）; 线粒体膜电位水平检测结果显示木香内酯 5 μmol/L 组、10 μmol/L 组、20 μmol/L 组 U87 细胞线粒体膜电位降低, 差异具有统计学意义（P<0.001）;氧化应激检测水平显示, 木香内酯呈剂量依赖性上调 U87 细胞中 ROS、MDA、NO 水平; 下调 SOD、GSH 水平, 差异具有统计学意义（P<0.05）; 木香内酯 20 μmol/L 组 U87 细胞 Caspase-3、Bax mRNA 表达水平均上调, 差异具有统计学意义（P<0.01）, 而 Bcl-2 mRNA 表达水平下调, 差异具有统计学意义（P<0.05）。分子对接结果显示木香内酯分别与 Caspase-3、Bcl-2、Bax 氨基酸残基 ARG-164, LEU-168, ARG-66 形成稳定的结合能。[结论] 木香内酯能明显抑制 U87 细胞生长和介导细胞高氧化应激水平, 降低线粒体膜电位并促进细胞凋亡。