Endogenous Expression of Adenosine A<Subscript>1</Subscript>, A<Subscript>2 </Subscript>and A<Subscript>3</Subscript> Receptors in Rat C6 Glioma Cells

Inhibitory and stimulatory adenosine receptors have been identified and characterized in both membranes and intact rat C6 glioma cells. In membranes, saturation experiment performed with [³H]DPCPX, selective A₁R antagonist, revealed a single binding site with a K _D = 9.4 ± 1.4 nM and B _max = 62.7 ± 8.6 fmol/mg protein. Binding of [³H]DPCPX in intact cell revealed a K _D = 17.7 ± 1.3 nM and B _max = 567.1 ± 26.5 fmol/mg protein. On the other hand, [³H]ZM241385 binding experiments revealed a single binding site population of receptors with K _D = 16.5 ± 1.3 nM and B _max = 358.9 ± 52.4 fmol/mg protein in intact cells, and K _D = 4.7 ± 0.6 nM and B _max = 74.3 ± 7.9 fmol/mg protein in plasma membranes, suggesting the presence of A_2A receptor in C6 cells. A₁, A_2A, A_2B and A₃ adenosine receptors were detected by Western-blotting and immunocytochemistry, and their mRNAs quantified by real time PCR assays. Giα and Gsα proteins were also detected by Western-blotting and RT-PCR assays. Furthermore, selective A₁R agonists inhibited forskolin- and GTP-stimulated adenylyl cyclase activity and CGS 21680 and NECA stimulated this enzymatic activity in C6 cells. These results suggest that C6 glioma cells endogenously express A₁ and A₂ receptors functionally coupled to adenylyl cyclase inhibition and stimulation, respectively, and suggest these cells as a model to study the role of adenosine receptors in tumoral cells.     

OKN-007 decreases free radical levels in a preclinical F98 rat glioma model

Free radicals are associated with glioma tumors. Here, we report on the ability of an anticancer nitrone compound, OKN-007 [Oklahoma Nitrone 007; a disulfonyl derivative of α-phenyl-tert-butyl nitrone (PBN)] to decrease free radical levels in F98 rat gliomas using combined molecular magnetic resonance imaging (mMRI) and immunospin-trapping (IST) methodologies. Free radicals are trapped with the spin-trapping agent, 5,5-dimethyl-1-pyrroline N-oxide (DMPO), to form DMPO macromolecule radical adducts, and then further tagged by immunospin trapping by an antibody against DMPO adducts. In this study, we combined mMRI with a biotin–Gd-DTPA–albumin-based contrast agent for signal detection with the specificity of an antibody for DMPO nitrone adducts (anti-DMPO probe), to detect in vivo free radicals in OKN-007-treated rat F98 gliomas. OKN-007 was found to significantly decrease (P < 0.05) free radical levels detected with an anti-DMPO probe in treated animals compared to untreated rats. Immunoelectron microscopy was used with gold-labeled antibiotin to detect the anti-DMPO probe within the plasma membrane of F98 tumor cells from rats administered anti-DMPO in vivo. OKN-007 was also found to decrease nuclear factor erythroid 2-related factor 2, inducible nitric oxide synthase, 3-nitrotyrosine, and malondialdehyde in ex vivo F98 glioma tissues via immunohistochemistry, as well as decrease 3-nitrotyrosine and malondialdehyde adducts in vitro in F98 cells via ELISA. The results indicate that OKN-007 effectively decreases free radicals associated with glioma tumor growth. Furthermore, this method can potentially be applied toward other types of cancers for the in vivo detection of macromolecular free radicals and the assessment of antioxidants.     

贝伐单抗对ALA—PDT诱导的新生血管形成和胶质瘤生长的影响

探讨贝伐单抗对低剂量5-氨基乙酰丙酸（5-Aminolevulinicacid,ALA）介导的光动力学疗法（photodynamic therapy,PDT）诱导的脑组织新生血管形成和U87脑胶质瘤生长的影响。通过将裸小鼠随机分为对照组、ALA．PDT预处置组（ALA：2．3×10-3mol／kg,能量密度：10J／cm2）、贝伐单抗预处置组（1．6×10-5mol／kg）和联合预处置组（ALA—PDT＋贝伐单抗）,并接受相应处置。第10d,检测PDT照射及相应区域内新生血管形成和VEGF表达并种植U87脑胶质瘤细胞,21d后观察肿瘤体积。与对照组比较,低剂量ALA-PDT预处置后新生血管增多、VEGF表达增高,肿瘤体积增大,这些变化在联合预处置组被抑制;贝伐单抗预处置组的血管形态和VEGF表达虽无明显变化,但肿瘤体积减小。研究结果表明低剂量ALA-PDT可通过刺激VEGF表达诱导照射区新生血管形成,这种微环境的改变有利于U87胶质瘤细胞生长,但这些作用可被贝伐单抗所抑制。     

Acidic stress promotes a glioma stem cell phenotype

Malignant gliomas are lethal cancers that display cellular hierarchies with cancer stem cells at the apex. Glioma stem cells (GSCs) are not uniformly distributed, but rather located in specialized niches, suggesting that the cancer stem cell phenotype is regulated by the tumor microenvironment. Indeed, recent studies show that hypoxia and its molecular responses regulate cancer stem cell maintenance. We now demonstrate that acidic conditions, independent of restricted oxygen, promote the expression of GSC markers, self-renewal and tumor growth. GSCs exert paracrine effects on tumor growth through elaboration of angiogenic factors, and low pH conditions augment this expression associated with induction of hypoxia inducible factor 2α (HIF2α), a GSC-specific regulator. Induction of HIF2α and other GSC markers by acidic stress can be reverted by elevating pH in vitro, suggesting that raising intratumoral pH may be beneficial for targeting the GSC phenotype. Together, our results suggest that exposure to low pH promotes malignancy through the induction of a cancer stem cell phenotype, and that culturing cancer cells at lower pH reflective of endogenous tumor conditions may better retain the cellular heterogeneity found in tumors.     

MicroRNA与胶质瘤的分子病理特征研究进展

MicroRNAs（miRNAs）是一类非编码的内源性小RNA分子,通过调节mRNA的稳定性及蛋白翻译过程控制基因表达,从而发挥促癌或抑癌作用;研究表明,在胶质瘤的发生、进展、侵袭过程中,伴随发生了许多分子病理特征的改变,这一过程中miRNAs发挥着重要作用,本文就此方面研究进展进行综述。     

Method for Novel Anti-Cancer Drug Development using Tumor Explants of Surgical Specimens

The current therapies for malignant glioma have only palliative effect. For therapeutic development, one hurdle is the discrepancy of efficacy determined by current drug efficacy tests and the efficacy on patients. Thus, novel and reliable methods for evaluating drug efficacy are warranted in pre-clinical phase. In vitro culture of tumor tissues, including cell lines, has substantial phenotypic, genetic, and epigenetic alterations of cancer cells caused by artificial environment of cell culture, which may not reflect the biology of original tumors in situ. Xenograft models with the immunodeficient mice also have limitations, i.e., the lack of immune system and interspecies genetic and epigenetic discrepancies in microenvironment. Here, we demonstrate a novel method using the surgical specimens of malignant glioma as undissociated tumor blocks to evaluate treatment effects. To validate this method, data with the current first-line chemotherapeutic agent, temozolomide (TMZ), are described. We used the freshly-removed surgical specimen of malignant glioma for our experiments. We performed intratumoral injection of TMZ or other drug candidates, followed by incubation and analysis on surgical specimens. Here, we sought to establish a tumor tissue explant method as a platform to determine the efficacy of novel anti-cancer therapies so that we may be able to overcome, at least, some of the current limitations and fill the existing gap between the current experimental data and the efficacy on an actual patient''s tumor. This method may have the potential to accelerate identifying novel chemotherapeutic agents for solid cancer treatment.     

Evidence that protein kinase Calpha interacts with and regulates the glial glutamate transporter GLT-1

Many of the sodium‐dependent neurotransmitter transporters are rapidly (within minutes) regulated by protein kinase C (PKC), with changes in activity being correlated with changes in transporter trafficking to or from the plasma membrane. Our recent studies suggest that one of the classical subtypes of PKC, PKCα, may selectively mediate redistribution of the neuronal glutamate transporter, excitatory amino acid carrier (EAAC)1, and show that PKCα can be co‐immunoprecipitated with EAAC1. When the glial glutamate transporter GLT‐1a is transfected into C6 glioma cells, this transporter is internalized in response to activation of PKC, but the PKC subtype involved in this regulation is unknown. In the present study, expression of the phorbol ester‐activated subtypes of PKC was examined in C6 glioma transfected with GLT‐1. Of the classical subtypes, only PKCα was detected, and of the non‐classical subtypes, PKCδ and PKCε were detected. In this system, phorbol ester‐dependent internalization of GLT‐1 was blocked by a general inhibitor of PKCs (bisindolylmaleimide II) and by concentrations of Gö6976 that selectively block classical PKCs, but not by an inhibitor of PKCδ (rottlerin). PKCα immunoreactivity was found in GLT‐1 immunoprecipitates obtained from transfected C6 cells and from crude rat brain synaptosomes, a milieu that better mimics in vivo conditions. The amount of PKCα in both types of immunoprecipitate was modestly increased by phorbol ester, and this increase was blocked by a PKC antagonist. These studies suggest that PKCα may be required for the regulated redistribution of GLT‐1.     

Characterization of antiproliferative and cytotoxic properties of the HSV-1 immediate-early ICPo protein

BACKGROUND: The identification of novel proteins displaying cytostatic and/or cytotoxic actions that could eventually be used for gene therapy is a major issue in cancer research. Data from the literature suggested that the immediate-early ICP0 protein from herpes simplex virus type 1 (HSV-1) could fulfill these properties as it had been observed that this protein is involved in arrest of cell growth at the G1/S and G2/M phases of the cell cycle and that deletion of ICP0 from HSV-1 or HSV-1-recombinant vectors significantly reduced their cytotoxicity. The molecular basis of its action is likely related to the ability of ICP0, which possesses E3-ubiquitin ligase activity, to target destruction of key cellular proteins, including centromeric proteins, resulting in abnormal chromosome segregation, unusual cytokinesis, and emergence of nuclear morphological aberrations. However, neither the gene therapy potential of ICP0 on its own nor its action on primary quiescent cells has been assessed to date. The aim of this work was therefore to evaluate the antiproliferative and cytotoxic properties of ICP0 on a human glioblastoma cell line and on quiescent primary cells, and to explore whether this protein has a potential for gene therapy of cancer. METHODS: HSV-1-based amplicon particles were generated following a recently described method that produces relatively high titers of vector stocks that are essentially free of helper virus. These vectors express either wild-type ICP0 or FXE, a RING finger mutated inactive form of ICP0, together with reporter green fluorescent protein (GFP). The vectors were used to infect Gli36 cells, which derive from a human glioblastome, and cultured rat primary cardiomyocytes and brain cells, two well-established models of non-dividing cells. Expression and localization of ICP0 and FXE, as well as their action on centromeres and nuclear morphology, were evaluated by Western blotting, indirect immune fluorescence, and confocal microscopy using specific antibodies and DAPI labeling. The impact of ICP0 on cell growth, toxicity, and viability was evaluated in the different cells using a variety of methods, including FACS analysis after propidium iodide and AnnexinV staining, crystal violet staining, clonogenic capability, caspase 3 activation, MTT tests, and release of lactate dehydrogenase, after infection with the different vectors. RESULTS: The three cell types under study showed high levels of transduction by amplicons and strong expression of GFP, ICP0, and FXE transgenic proteins. Wild-type ICP0, but not FXE, induced centromeric disruption, appearance of micronuclei, arrest of proliferation, and significant cell death in glioblastoma Gli36 cells. In contrast, neither micronuclei formation nor any other sign of cell toxicity could be observed in cultured primary cardiomyocytes or brain cells, as evaluated by MTT tests and crystal violet staining. Furthermore, in the case of cardiomyocytes, expression of ICP0 did not interfere with beating as cells continued to beat at the same frequency as non-infected cells for several days post-infection. Neither AnnexinV early staining nor caspase 3 activation was observed in dying infected Gli36 cells, suggesting that these cells were not entering apoptosis. In contrast, release of lactate dehydrogenase by infected Gli36 cells suggested a necrotic way of death. CONCLUSIONS: ICP0 induced a strong cytostatic action followed by significant cell death on the glioblastoma Gli36 cell line. In contrast, neither cell death nor any other evidence of ICP0-induced toxicity affecting major physiological parameters was observed in primary cultured cardiomyoctes and brain cells, two models of primary non-cycling cells. These results suggest that ICP0 has gene therapy potential and could represent the first member of a novel family of directly acting proteins that could be used to treat cancers.