Identification of the angiogenesis signaling domain in pleiotrophin defines a mechanism of the angiogenic switch

Neoplasms progress through genetic and epigenetic mutations that deregulate pathways in the malignant cell that stimulate more aggressive growth of the malignant cell itself and/or remodel the tumor microenvironment to support the developing tumor mass. The appearance of new blood vessels in malignant tumors is known as the "angiogenic switch." The angiogenic switch triggers a stage of rapid tumor growth supported by extensive tumor angiogenesis and a more aggressive tumor phenotype and its onset is a poor prognostic indicator for host survival. Identification of the factors that stimulate the angiogenic switch thus is of high importance. Pleiotrophin (PTN the protein, Ptn the gene) is an angiogenic factor and the Ptn gene has been found to be constitutively expressed in many human tumors of different cell types. These studies use a nude mouse model to test if Ptn constitutively expressed in premalignant cells is sufficient to trigger an angiogenic switch in vivo. We introduced an ectopic Ptn gene into "premalignant" SW-13 cells and analyzed the phenotype of SW-13 Ptn cell tumor implants in the flanks of nude mice. SW-13 Ptn cell subcutaneous tumor implants grew very rapidly and had a striking increase in the density of new blood vessels compared to the SW-13 cell tumor implants, suggesting that constitutive PTN signaling in the premalignant SW-13 cell implants in the nude mouse recapitulates fully the angiogenic switch. It was found also that ectopic expression of the C-terminal domain of PTN in SW-13 cell implants was equally effective in initiating an angiogenic switch as the full-length PTN whereas implants of SW-13 cells in nude mice that express the N-terminal domain of PTN grew rapidly but failed to develop tumor angiogenesis. The data suggest the possibility that mutations that activate Ptn in premalignant cells are sufficient to stimulate an angiogenic switch in vivo and, since these mutations are frequently found in human malignancies, that constitutive PTN signaling may be an important contributor to progression of human tumors. The data also suggest that the C-terminal and the N-terminal domains of PTN equally initiate switches in premalignant cells to cells of a more aggressive tumor phenotype but the separate domains of PTN signal different mechanisms and perhaps signal through activation of a separate receptor-like protein.     

Anaplastic lymphoma kinase is expressed in different subtypes of human breast cancer

Pleiotrophin (PTN, Ptn) is an 18kDa cytokine expressed in human breast cancers. Since inappropriate expression of Ptn stimulates progression of breast cancer in transgenic mice and a dominant negative PTN reverses the transformed phenotype of human breast cancer cells that inappropriately express Ptn, it is suggested that constitutive PTN signaling in breast cancer cells that inappropriately express Ptn activates pathways that promote a more aggressive breast cancer phenotype. Pleiotrophin signals by inactivating its receptor, the receptor protein tyrosine phosphatase (RPTP)beta/zeta, and, recently, PTN was found to activate anaplastic lymphoma kinase (ALK) through the PTN/RPTPbeta/zeta signaling pathway in PTN-stimulated cells, not through a direct interaction of PTN with ALK and thus not through the PTN-enforced dimerization of ALK. Since full-length ALK is activated in different malignant cancers and activated ALK is a potent oncogenic protein, we examined human breast cancers to test the possibility that ALK may be expressed in breast cancers and potentially activated through the PTN/RPTPbeta/zeta signaling pathway; we now demonstrate that ALK is strongly expressed in different histological subtypes of human breast cancer; furthermore, ALK is expressed in both nuclei and cytoplasm and, in the ;;dotted" pattern characteristic of ALK fusion proteins in anaplastic large cell lymphoma. This study thus supports the possibility that activated ALK may be important in human breast cancers and potentially activated either through the PTN/RPTPbeta/zeta signaling pathway, or, alternatively, as an activated fusion protein to stimulate progression of breast cancer in humans.     

Tubeimoside V (1), a new cyclic bisdesmoside from tubers of Bolbostemma paniculatum, functions by inducing apoptosis in human glioblastoma U87MG cells

Malignant glioblastoma is one of the most common malignant tumors in the neurological system. Tubeimoside V (1), a new cyclic bisdesmoside from tubers of Bolbostemma paniculatum, appears to exhibit various biological activities, including antitumor effect, but the function and mechanism of this new agent on glioblastoma cells has not previously been determined. In the present study, we investigated the proliferation change of human glioblastoma U87MG cells exposured to different concentrations (0.9-14.8 microM) of Tubeimoside V (1) for a certain time. The results showed that Tubeimoside V (1) significantly suppressed U87MG cell proliferation in a time- and dose-dependent manner (IC(50) = 3.6 microM). Flow cytometric analysis of DNA in U87MG cells showed that Tubeimoside V (1) induces the prominent appearance of a sub-G1 peak in the cell cycle suggestive of apoptosis. Furthermore, U87MG cells' treatment with Tubeimoside V (1) resulted in nuclear condensation with apoptotic bodies observed by both fluorescence and electron microscopy. The result of annexin V/PI assay showed that phosphatidylserine externalization began after treatment, and then increased in the following 24h. Molecular changes explored through Western-blot staining showed Tubeimoside V (1) decreased the expression levels of Bcl-2 protein and increased the expression levels of Bax protein. The novel findings suggest that the cytotoxic actions of Tubeimoside V (1) toward U87MG cells result from the induction of cell apoptosis. Overall, our data demonstrate that Tubeimoside V (1) is an efficient apoptotic killing agent of glioblastoma cells and suggest that this mechanism may play a critical role in anti-tumor chemotherapy.     

UMSCs对U87MG细胞增殖的影响

目的：通过对研究脐带间充质干细胞（Umbilical cord mesenchymalstellcells,UCMSCs）与人恶性胶质母细胞瘤细胞U87MG细胞（U87 Malignant glioma cells）体外共培养,模拟肿瘤生长的内环境,以及其对U87MG细胞增值作用的影响及肿瘤细胞与间充质干细胞的共培养方法。方法：提取人脐带间充质干细胞进行体外培养、扩增,用MTT法测定uMSCS上清液对U87MG的影响,用瑞士染色法检测U87MG形态学变化。结果：MTT比色法结果显示UMSCS对U87MG有抑制作用。96小时培养后1：8、1：4、1：2及未稀释的UMSCs上清液对u87MG的抑制率分别为17％,24％,37．2％及46．4％,u87MG细胞形态亦随着培养时间的延长由多角形变为梭形,突起消失,细胞间骨架结构断裂。结论：通过对共培养前后U87MG与UMSCs共培养后形态学变化、生长曲线变化及对生长周期的影响作用的观察分析,得出UMSCs及其上清液对U87MG有抑制作用,而且呈时间及浓度依赖性。     

The receptor protein tyrosine phosphatase (RPTP)beta/zeta is expressed in different subtypes of human breast cancer

Increasing evidence suggests mutations in human breast cancer cells that induce inappropriate expression of the 18-kDa cytokine pleiotrophin (PTN, Ptn) initiate progression of breast cancers to a more malignant phenotype. Pleiotrophin signals through inactivating its receptor, the receptor protein tyrosine phosphatase (RPTP)beta/zeta, leading to increased tyrosine phosphorylation of different substrate proteins of RPTPbeta/zeta, including beta-catenin, beta-adducin, Fyn, GIT1/Cat-1, and P190RhoGAP. PTN signaling thus has wide impact on different important cellular systems. Recently, PTN was found to activate anaplastic lymphoma kinase (ALK) through the PTN/RPTPbeta/zeta signaling pathway; this discovery potentially is very important, since constitutive ALK activity of nucleophosmin (NPM)-ALK fusion protein is causative of anaplastic large cell lymphomas, and, activated ALK is found in other malignant cancers. Recently ALK was identified in each of 63 human breast cancers from 22 subjects. We now demonstrate that RPTPbeta/zeta is expressed in each of these same 63 human breast cancers that previously were found to express ALK and in 10 additional samples of human breast cancer. RPTPbeta/zeta furthermore was localized not only in its normal association with the cell membrane but also scattered in cytoplasm and in nuclei in different breast cancer cells and, in the case of infiltrating ductal carcinomas, the distribution of RPTPbeta/zeta changes as the breast cancer become more malignant. The data suggest that the PTN/RPTPbeta/zeta signaling pathway may be constitutively activated and potentially function to constitutively activate ALK in human breast cancer.     

Acridine yellow G blocks glioblastoma growth via dual inhibition of epidermal growth factor receptor and protein kinase C kinases

Amplification of the epidermal growth factor receptor (EGFR), frequently expressed as a constitutively active deletion mutant (EGFRvIII), occurs commonly in glioblastoma multiformes (GBM). However, blockade of EGFR is therapeutically disappointing for gliomas with PTEN deletion. To search for small molecules treating this aggressive cancer, we have established a cell-based screening and successfully identified acridine yellow G that preferentially blocks cell proliferation of the most malignant U87MG/EGFRvIII cells over the less malignant U87MG/PTEN cells. Oral administration of this compound markedly diminishes the brain tumor volumes in both subcutaneous and intracranial models. It directly inhibits EGFR and PKCs with IC(50) values of ~7.5 and 5 μM, respectively. It dually inhibits EGFR and PKCs, resulting in a blockade of mammalian target of rapamycin signaling and cell cycle arrest in the G(1) phase, which leads to activation of apoptosis in the tumors. Hence, combinatorial inhibition of EGFR and PKCs might provide proof of concept in developing therapeutic agents for treating malignant glioma and other human cancers.     

The lipid phosphatase activity of PTEN is critical for stabilizing intercellular junctions and reverting invasiveness.

To analyze the implication of PTEN in the control of tumor cell invasiveness, the canine kidney epithelial cell lines MDCKras-f and MDCKts-src, expressing activated Ras and a temperature-sensitive v-Src tyrosine kinase, respectively, were transfected with PTEN expression vectors. Likewise, the human PTEN-defective glioblastoma cell lines U87MG and U373MG, the melanoma cell line FM-45, and the prostate carcinoma cell line PC-3 were transfected. We demonstrate that ectopic expression of wild-type PTEN in MDCKts-src cells, but not expression of PTEN mutants deficient in either the lipid or both the lipid and protein phosphatase activities, reverted the morphological transformation, induced cell-cell aggregation, and suppressed the invasive phenotype in an E-cadherin-dependent manner. In contrast, overexpression of wild-type PTEN did not counteract Ras-induced invasiveness of MDCKras-f cells expressing low levels of E-cadherin. PTEN effects were not associated with marked changes in accumulation or phosphorylation levels of E-cadherin and associated catenins. Wild-type, but not mutant, PTEN also reverted the invasive phenotype of U87MG, U373MG, PC-3, and FM-45 cells. Interestingly, PTEN effects were mimicked by N-cadherin-neutralizing antibody in the glioblastoma cell lines. Our data confirm the differential activities of E- and N-cadherin on invasiveness and suggest that the lipid phosphatase activity of PTEN exerts a critical role in stabilizing junctional complexes and restraining invasiveness.     

Pleiotrophin stimulates tyrosine phosphorylation of beta-adducin through inactivation of the transmembrane receptor protein tyrosine phosphatase beta/zeta

Pleiotrophin (PTN the protein, Ptn the gene) signals through a unique mechanism; it inactivates the tyrosine phosphatase activity of its receptor, the transmembrane receptor protein tyrosine phosphatase (RPTP)beta/zeta, and increases tyrosine phosphorylation of the substrates of RPTPbeta/zeta through the continued activity of a yet to be described protein tyrosine kinase(s) in PTN-stimulated cells. We have now found that the cytoskeletal protein beta-adducin interacts with the intracellular domain of RPTPbeta/zeta in a yeast two-hybrid system, that beta-adducin is a substrate of RPTPbeta/zeta, that beta-adducin is phosphorylated in tyrosine in cells not stimulated by PTN, and that tyrosine phosphorylation of beta-adducin is sharply increased in PTN-stimulated cells, suggesting that beta-adducin is a downstream target of and regulated by the PTN/RPTPbeta/zeta signaling pathway. beta-Catenin was the first downstream target of the PTN/RPTPbeta/zeta signaling pathway to be identified; these data thus also suggest that PTN coordinately regulates steady state levels of tyrosine phosphorylation of the important cytoskeletal proteins beta-adducin and beta-catenin and, through PTN-stimulated tyrosine phosphorylation, beta-adducin may contribute to the disruption of cytoskeletal structure, increased plasticity, and loss of homophilic cell-cell adhesion that are the consequences of PTN stimulation of cells and a characteristic feature of different malignant cells with mutations that activate constitutive expression of the endogenous Ptn gene.     

Modulation of Sonic hedgehog signaling and WW domain containing oxidoreductase WOX1 expression enhances radiosensitivity of human glioblastoma cells

WW domain containing oxidoreductase, designated WWOX, FOR or WOX1, is a known pro-apoptotic factor when ectopically expressed in various types of cancer cells, including glioblastoma multiforme (GBM). The activation of sonic hedgehog (Shh) signaling, especially paracrine Shh secretion in response to radiation, is associated with impairing the effective irradiation of cancer cells. Here, we examined the role of Shh signaling and WOX1 overexpression in the radiosensitivity of human GBM cells. Our results showed that ionizing irradiation (IR) increased the cytoplasmic Shh and nuclear Gli-1 content in GBM U373MG and U87MG cells. GBM cells with exogenous Shh treatment exhibited similar results. Pretreatment with Shh peptides protected U373MG and U87MG cells against IR in a dose-dependent manner. Cyclopamine, a Hedgehog/Smoothened (SMO) inhibitor, reversed the protective effect of Shh in U87MG cells. Cyclopamine increased Shh plus IR-induced H2AX, a marker of DNA double-strand breaks, in these cells. To verify the role of Shh signaling in the radiosensitivity of GBM cells, we tested the effect of the Gli family zinc finger 1 (Gli-1) inhibitor zerumbone and found that it could sensitize GBM cells to IR. We next examined the role of WOX1 in radiosensitivity. Overexpression of WOX1 enhanced the radiosensitivity of U87MG (possessing wild type p53 or WTp53) but not U373MG (harboring mutant p53 or MTp53) cells. Pretreatment with Shh peptides protected both WOX1-overexpressed U373MG and U87MG cells against IR and increased the cytoplasmic Shh and nuclear Gli-1 content. Zerumbone enhanced the radiosensitivity of WOX1-overexpressed U373MG and U87MG cells. In conclusion, overexpression of WOX1 preferentially sensitized human GBM cells possessing wild type p53 to radiation therapy. Blocking of Shh signaling may enhance radiosensitivity independently of the expression of p53 and WOX1. The crosstalk between Shh signaling and WOX1 expression in human glioblastoma warrants further investigation.     

MicroRNA-330 Is an Oncogenic Factor in Glioblastoma Cells by Regulating SH3GL2 Gene

MicroRNAs have recently emerged as key regulators of cancers. This study was therefore conducted to investigate the role of miR-330 in biological behaviors of human glioblastoma U87 and U251 cell lines and its molecular mechanism. SH3GL2 gene was identified as the target of miR-330. MiR-330 overexpression was established by transfecting miR-330 precursor into U87 and U251 cells, and its effects on proliferation, migration, invasion, cell cycle and apoptosis were studied. Overexpression of miR-330 can enhance cellular proliferation, promote migration and invasion, activate cell cycle and also inhibit apoptosis in U87 and U251 cells. Collectively, these above-mentioned results suggest that miRNA-330 plays an oncogenic role in human glioblastoma by regulating SH3GL2 gene and might be a new therapeutic target of human glioblastoma.     

Angiogenin Promotes U87MG Cell Proliferation by Activating NF-κB Signaling Pathway and Downregulating Its Binding Partner FHL3

Angiogenin (Ang) is known to induce cell proliferation and inhibit apoptosis by cellular signaling pathways and its direct nuclear functions, but the mechanism of action for Ang in astrocytoma is not yet clear. Astrocytoma is the most frequent one among various neurogliomas, of which a subtype known as glioblastoma multiforme (GBM) is the most malignant brain glioma and seriously influences the life quality of the patients. The expression of Ang and Bcl-xL were detected in 28 cases of various grades of astrocytoma and 6 cases of normal human tissues by quantitative real-time PCR. The results showed that the expression of Ang and Bcl-xL positively correlated with the malignant grades. Cytological experiments indicated that Ang facilitated human glioblastoma U87MG cell proliferation and knock-down of endogenous Ang promoted cell apoptosis. Furthermore, Ang activated NF-κB pathway and entered the U87MG cell nuclei, and blocking NF-κB pathway or inhibiting Ang nuclear translocation partially suppressed Ang-induced cell proliferation. The results suggested that Ang participated in the regulation of evolution process of astrocytoma by interfering NF-κB pathway and its nucleus function. In addition, four and a half LIM domains 3 (FHL3), a novel Ang binding partner, was required for Ang-mediated HeLa cell proliferation in our previous study. We also found that knockdown of FHL3 enhanced IκBα phosphorylation and overexpression of Ang inhibited FHL3 expression in U87MG cells. Together our findings suggested that Ang could activate NF-κB pathway by regulating the expression of FHL3. In conclusion, the present study established a link between Ang and FHL3 proteins and identifies a new pathway for regulating astrocytoma progression.     

M2 receptor activation inhibits cell cycle progression and survival in human glioblastoma cells

Muscarinic receptors, expressed in several primary and metastatic tumours, appear to be implicated in their growth and propagation. In this work we have demonstrated that M2 muscarinic receptors are expressed in glioblastoma human specimens and in glioblastoma cell lines. Moreover, we have characterized the effects of the M2 agonist arecaidine on cell growth and survival both in two different glioblastoma cell lines (U251MG and U87MG) and in primary cultures obtained from different human biopsies. Cell growth analysis has demonstrated that the M2 agonist arecaidine strongly decreased cell proliferation in both glioma cell lines and primary cultures. This effect was dose and time dependent. FACS analysis has confirmed cell cycle arrest at G1/S and at G2/M phase in U87 cells and U251 respectively. Cell viability analysis has also shown that arecaidine induced severe apoptosis, especially in U251 cells. Chemosensitivity assays have, moreover, shown arecaidine and temozolomide similar effects on glioma cell lines, although IC50 value for arecaidine was significantly lower than temozolomide. In conclusion, we report for the first time that M2 receptor activation has a relevant role in the inhibition of glioma cell growth and survival, suggesting that M2 may be a new interesting therapeutic target to investigate for glioblastoma therapy.     

Glioma inhibition by HGF/NK2, an antagonist of scatter factor/hepatocyte growth factor

Strategies that antagonize growth factor signaling are attractive candidates for the biological therapy of brain tumors. HGF/NK2 is a secreted truncated splicing variant and potential antagonist of scatter factor/hepatocyte growth factor (SF/HGF), a multifunctional cytokine involved in the malignant progression of solid tumors including glioblastoma. U87 human malignant glioma cells that express an autocrine SF/HGF stimulatory loop were transfected with the human HGF/NK2 cDNA and clonal cell lines that secrete high levels of HGF/NK2 protein (U87-NK2) were isolated. The effects of HGF/NK2 gene transfer on the U87 malignant phenotype were examined. HGF/NK2 gene transfer had no effect on 2-dimensional anchorage-dependent cell growth. In contrast, U87-NK2 cell lines were approximately 20-fold less clonogenic in soft agar and approximately 4-fold less migratory than control-transfected cell lines. Intracranial tumor xenografts derived from U87-NK2 cells grew much slower than controls. U87-NK2 tumors were approximately 50-fold smaller than controls at 21 days post-implantation and HGF/NK2 gene transfer resulted in a trend toward diminished tumorigenicity. This report shows that the predominant effect of transgenic HGF/NK2 overexpression by glioma cells that are autocrine for SF/HGF stimulation is to inhibit their malignant phenotype.     

Continuous release of endostatin from microencapsulated engineered cells for tumor therapy

Research studies suggest that tumor-related angiogenesis contributes to the phenotype of malignant gliomas. We assessed the effect of local delivery of the angiogenesis inhibitor endostatin on human glioma cell line (U-87MG) xenografts. Baby hamster kidney (BHK) cells were stably transfected with a human endostatin (hES) expression vector and were encapsulated in alginate-poly L-lysine (PLL) microcapsules for long-term delivery of hES. The release of biologically active endostatin was confirmed using assays of bovine capillary endothelial (BCE) proliferation and of tube formation. Human endostatin released from the microcapsules brought about a 67. 2% inhibition of BCE proliferation. Furthermore, secreted hES was able to inhibit tube formation in KDR/PAE cells (porcine aortic endothelial cells stably transfected with KDR, a tyrosine kinase) treated with conditioned U-87MG medium. A single local injection of encapsulated endostatin-secreting cells in a nude mouse model resulted in a 72.3% reduction in subcutaneous U87 xenografts' weight 21 days post treatment. This inhibition was achieved by only 150.8 ng/ml human endostatin secreted from 2 x 10(5) encapsulated cells. Encapsulated endostatin-secreting cells are effective for the treatment of human glioblastoma xenografts. Continuous local delivery of endostatin may offer an effective therapeutic approach to the treatment of a variety of tumor types.     

NETRIN-4 Protects Glioblastoma Cells FROM Temozolomide Induced Senescence

Glioblastoma multiforme is the most common primary tumor of the central nervous system. The drug temozolomide (TMZ) prolongs lifespan in many glioblastoma patients. The sensitivity of glioblastoma cells to TMZ is interfered by many factors, such as the expression of O-6-methylguanine-DNA methyltransferase (MGMT) and activation of AKT signaling. We have recently identified the interaction between netrin-4 (NTN4) and integrin beta-4 (ITGB4), which promotes glioblastoma cell proliferation via activating AKT-mTOR signaling pathway. In the current work we have explored the effect of NTN4/ITGB4 interaction on TMZ induced glioblastoma cell senescence. We report here that the suppression of either ITGB4 or NTN4 in glioblastoma cell lines significantly enhances cellular senescence. The sensitivity of GBM cells to TMZ was primarily determined by the expression of MGMT. To omit the effect of MGMT, we concentrated on the cell lines devoid of expression of MGMT. NTN4 partially inhibited TMZ induced cell senescence and rescued AKT from dephosphorylation in U251MG cells, a cell line bearing decent levels of ITGB4. However, addition of exogenous NTN4 displayed no significant effect on TMZ induced senescence rescue or AKT activation in U87MG cells, which expressed ITGB4 at low levels. Furthermore, overexpression of ITGB4 combined with exogenous NTN4 significantly attenuated U87MG cell senescence induced by TMZ. These data suggest that NTN4 protects glioblastoma cells from TMZ induced senescence, probably via rescuing TMZ triggered ITGB4 dependent AKT dephosphorylation. This suggests that interfering the interaction between NTN4 and ITGB4 or concomitant use of the inhibitors of the AKT pathway may improve the therapeutic efficiency of TMZ.     

Up-regulation of Bak and Bim via JNK downstream pathway in the response to nitric oxide in human glioblastoma cells

Nitric oxide (NO) is a chemical messenger implicated in neuronal damage associated with ischemia neurodegenerative disease and excitotoxicity. In the present study, we examined the biological effects of NO and its mechanisms in human malignant glioblastoma cells. Addition of a NO donor, S-nitroso-N-acetyl-penicillamine (SNAP), induced apoptosis in U87MG human glioblastoma cells, accompanied by opening mitochondrial permeability transition pores, release of cytochrome c and AIF, and subsequently by caspase activation. NO-induced apoptosis occurred concurrently with significantly increased levels of the Bak and Bim. Treatment with SNAP resulted in sustained activation of JNK and its downstream pathway, c-Jun/AP-1. The expression of dominant-negative (DN)-JNK1 and DN-c-Jun suppressed the activation of AP-1, the induction of Bak and Bim, and the SNAP-induced apoptosis. In addition, de novo protein synthesis was required for the initiation of apoptosis in that the protein synthesis inhibitor, cycloheximide (CHX), inhibited NO-induced apoptotic cell death as well as up-regulation of Bak and Bim. These results suggest that NO activates an apoptotic cascade, involving sustained JNK activation, AP-1 DNA binding activity, and subsequent Bak and Bim induction, followed by cytochrome c and AIF releases and caspases cascade activation, resulting in human malignant brain tumor cell death.     

S1P(2) receptors mediate inhibition of glioma cell migration through Rho signaling pathways independent of PTEN

Sphingosine 1-phosphate (S1P) induced the inhibition of glioma cell migration. Here, we characterized the signaling mechanisms involved in the inhibitory action by S1P. In human GNS-3314 glioblastoma cells, the S1P-induced inhibition of cell migration was associated with activation of RhoA and suppression of Rac1. The inhibitory action of S1P was recovered by a small interference RNA specific to S1P₂ receptor, a carboxyl-terminal region of Gα12 or Gα13, an RGS domain of p115RhoGEF, and a dominant-negative mutant of RhoA. The inhibitory action of S1P through S1P₂ receptors was also observed in both U87MG glioblastoma and 1321N1 astrocytoma cells, which have no protein expression of a phosphatase and tensin homolog deleted on chromosome 10 (PTEN). These results suggest that S1P₂ receptors/G_12/13-proteins/Rho signaling pathways mediate S1P-induced inhibition of glioma cell migration. However, PTEN, recently postulated as an indispensable molecule for the inhibition of cell migration, may not be critical for the S1P₂ receptor-mediated action in glioma cells.     

Pleiotrophin: a cytokine with diverse functions and a novel signaling pathway.

Pleiotrophin (PTN the protein, Ptn the gene) is a 136 amino acid secreted heparin-binding cytokine that signals diverse functions, including lineage-specific differentiation of glial progenitor cells, neurite outgrowth, and angiogenesis. Pleiotrophin gene expression is found in cells in early differentiation during different development periods and upregulated in cells with an early differentiation phenotype in wound repair. The Ptn gene is a protooncogene. It is strongly expressed in different human tumor cells and expression of the Ptn gene in tumor cells in vivo accelerates growth and stimulates tumor angiogenesis. Separate independent domains have been identified in PTN to signal transformation and tumor angiogenesis. Pleiotrophin is the first ligand of any of the known transmembrane tyrosine phosphatases. Pleiotrophin inactivates the receptor protein tyrosine phosphatase (RPTP) beta/zeta. The interaction of PTN and RPTP beta/zeta increases steady-state tyrosine phosphorylation of beta-catenin. Pleiotrophin thus regulates both normal cell functions and different pathological conditions at many levels. It signals these functions through a transmembrane tyrosine phosphatase.     

Changes in the expression of proteins associated with aerobic glycolysis and cell migration are involved in tumorigenic ability of two glioma cell lines

ABSTRACT: BACKGROUND: The most frequent and malignant brain cancer is glioblastoma multiforme (GBM). In gliomas, tumor progression and poor prognosis are associated with the tumorigenic ability of the cells. U87MG cells (wild-type p53) are known to be tumorigenic in nude mice, but T98G cells (mutant p53) are not tumorigenic. We investigated the proteomic profiling of these two cell lines in order to gain new insights into the mechanisms that may be involved in tumorigenesis. RESULTS: We found 24 differentially expressed proteins between T98G and U87MG cells. Gene Ontology supports the notion that over-representation of differentially expressed proteins is involved in glycolysis, cell migration and stress oxidative response. Among those associated with the glycolysis pathway, TPIS and LDHB are up-regulated in U87MG cells. Measurement of glucose consumption and lactate production suggests that glycolysis is more effective in U87MG cells. On the other hand, G6PD expression was 3-fold higher in T98G cells and this may indicate a shift to the pentose-phosphate pathway. Moreover, GRP78 expression was also three-fold higher in T98G than in U87MG cells. Under thapsigargin treatment both cell lines showed increased GRP78 expression and the effect of this agent was inversely correlated to cell migration. Quantitative RT-PCR and immunohistochemistry of GRP78 in patient samples indicated a higher level of expression of GRP78 in grade IV tumors compared to grade I and non-neoplastic tissues, respectively. CONCLUSIONS: Taken together, these results suggest an important role of proteins involved in key functions such as glycolysis and cell migration that may explain the difference in tumorigenic ability between these two glioma cell lines and that may be extrapolated to the differential aggressiveness of glioma tumors.     

Saponin 1 Induces Apoptosis and Suppresses NF-κB-Mediated Survival Signaling in Glioblastoma Multiforme (GBM)

Saponin 1 is a triterpeniod saponin extracted from Anemone taipaiensis, a traditional Chinese medicine against rheumatism and phlebitis. It has also been shown to exhibit significant anti-tumor activity against human leukemia (HL-60 cells) and human hepatocellular carcinoma (Hep-G2 cells). Herein we investigated the effect of saponin 1 in human glioblastoma multiforme (GBM) U251MG and U87MG cells. Saponin 1 induced significant growth inhibition in both glioblastoma cell lines, with a 50% inhibitory concentration at 24 h of 7.4 µg/ml in U251MG cells and 8.6 µg/ml in U87MG cells, respectively. Nuclear fluorescent staining and electron microscopy showed that saponin 1 caused characteristic apoptotic morphological changes in the GBM cell lines. Saponin 1-induced apoptosis was also verified by DNA ladder electrophoresis and flow cytometry. Additionally, immunocytochemistry and western blotting analyses revealed a time-dependent decrease in the expression and nuclear location of NF-κB following saponin 1 treatment. Western blotting data indicated a significant decreased expression of inhibitors of apoptosis (IAP) family members,(e.g., survivin and XIAP) by saponin 1. Moreover, saponin 1 caused a decrease in the Bcl-2/Bax ratio and initiated apoptosis by activating caspase-9 and caspase-3 in the GBM cell lines. These findings indicate that saponin 1 inhibits cell growth of GBM cells at least partially by inducing apoptosis and inhibiting survival signaling mediated by NF-κB. In addition, in vivo study also demonstrated an obvious inhibition of saponin 1 treatment on the tumor growth of U251MG and U87MG cells-produced xenograft tumors in nude mice. Given the minimal toxicities of saponin 1 in non-neoplastic astrocytes, our results suggest that saponin 1 exhibits significant in vitro and in vivo anti-tumor efficacy and merits further investigation as a potential therapeutic agent for GBM.