SNB19 Cells Research Articles

The radiosensitization effect of titanate nanotubes as a new tool in radiation therapy for glioblastoma: A proof-of-concept

Background and purposeOne of the new challenges to improve radiotherapy is to increase the ionizing effect by using nanoparticles. The interest of titanate nanotubes (TiONts) associated with radiotherapy was evaluated in two human glioblastoma cell lines (SNB-19 and U87MG). Materials and methodsTitanate nanotubes were synthetized by the hydrothermal treatment of titanium dioxide powder in a strongly basic NaOH solution. The cytotoxicity of TiONts was evaluated on SNB-19 and U87MG cell lines by cell proliferation assay. The internalization of TiONts was studied using Transmission Electron Microscopy (TEM). Finally, the effect of TiONts on cell radiosensitivity was evaluated using clonogenic assay. Cell cycle distribution was evaluated by flow cytometry after DNA labeling. DNA double-stranded breaks were evaluated using γH2AX labeling. ResultsCells internalized TiONts through the possible combination of endocytosis and diffusion with no cytotoxicity. Clonogenic assays showed that cell lines incubated with TiONts were radiosensitized with a decrease in the SF2 parameter for both SNB-19 and U87MG cells. TiONts decreased DNA repair efficiency after irradiation and amplified G2/M cell-cycle arrest. ConclusionOur results indicated that further development of TiONts might provide a new useful tool for research and clinical therapy in the field of oncology.

Hsp90 inhibition by NVP-AUY922 and NVP-BEP800 decreases migration and invasion of irradiated normoxic and hypoxic tumor cell lines

This study explores the impact of Hsp90 inhibitors NVP-AUY922 and NVP-BEP800 in combination with ionizing radiation (IR) on the migration and invasion of lung carcinoma A549 and glioblastoma SNB19 cells, under normoxia or hypoxia. Independent of oxygen concentration, both drugs decreased the migration and invasion rates of non-irradiated tumor cells. Combined drug-IR treatment under hypoxia inhibited cell invasion to a greater extent than did each treatment alone. Decreased migration of cells correlated with altered expression of several matrix-associated proteins (FAK/p-FAK, Erk2, RhoA) and impaired F-actin modulation. The anti-metastatic efficacy of the Hsp90 inhibitors could be useful in combinational therapies of cancer.

Engineered Drug Resistant γδ T Cells Kill Glioblastoma Cell Lines during a Chemotherapy Challenge: A Strategy for Combining Chemo- and Immunotherapy

Classical approaches to immunotherapy that show promise in some malignancies have generally been disappointing when applied to high-grade brain tumors such as glioblastoma multiforme (GBM). We recently showed that ex vivo expanded/activated γδ T cells recognize NKG2D ligands expressed on malignant glioma and are cytotoxic to glioma cell lines and primary GBM explants. In addition, γδ T cells extend survival and slow tumor progression when administered to immunodeficient mice with intracranial human glioma xenografts. We now show that temozolomide (TMZ), a principal chemotherapeutic agent used to treat GBM, increases the expression of stress-associated NKG2D ligands on TMZ-resistant glioma cells, potentially rendering them vulnerable to γδ T cell recognition and lysis. TMZ is also highly toxic to γδ T cells, however, and to overcome this cytotoxic effect γδ T cells were genetically modified using a lentiviral vector encoding the DNA repair enzyme O(6)-alkylguanine DNA alkyltransferase (AGT) from the O(6)-methylguanine methyltransferase (MGMT) cDNA, which confers resistance to TMZ. Genetic modification of γδ T cells did not alter their phenotype or their cytotoxicity against GBM target cells. Importantly, gene modified γδ T cells showed greater cytotoxicity to two TMZ resistant GBM cell lines, U373TMZ-R and SNB-19TMZ-R cells, in the presence of TMZ than unmodified cells, suggesting that TMZ exposed more receptors for γδ T cell-targeted lysis. Therefore, TMZ resistant γδ T cells can be generated without impairing their anti-tumor functions in the presence of high concentrations of TMZ. These results provide a mechanistic basis for combining chemotherapy and γδ T cell-based drug resistant cellular immunotherapy to treat GBM.

Hsp90 Inhibitors NVP-AUY922 and NVP-BEP800 May Exert a Significant Radiosensitization on Tumor Cells along with a Cell Type-Specific Cytotoxicity

Targeting heat shock protein 90 (Hsp90) provides a promising therapeutic approach to enhance the sensitivity of tumor cells to ionizing radiation (IR). To explore the impact of scheduling drug-IR administration, in the present study, we analyzed the response of lung carcinoma A549 and glioblastoma SNB19 cells to simultaneous drug-IR treatment followed by a long-term drug administration. Cellular response was evaluated at different time intervals after IR-alone, drug-alone, or combined drug-IR treatments by colony counts and expression profiles of Hsp90 and its clients, along with several apoptotic markers and cell cycle-related proteins, as well as by IR-drug-induced cell cycle arrest, DNA damage, and repair. A short 30-minute exposure to either Hsp90 inhibitor did not affect the radiosensitivity of both tumor cell lines. Increasing the duration of post-IR-drug treatment progressively enhanced the sensitivity of SNB19 cells to IR. In contrast, the response of A549 cells to drug-IR combination was largely determined by the cytotoxic effects of both drugs without radiosensitization. Combined drug-IR treatment induced more severe DNA damage in both tumor cell lines than each treatment alone and also protracted the kinetics of DNA damage repair in SNB19 cells. In addition to large cell cycle disturbances, drug-IR treatment also caused depletion of the antiapoptotic proteins Akt and Raf-1 in both cell lines, along with a decrease of survivin in A549 cells in case of NVP-AUY922. The data show that simultaneous Hsp90 inhibition and irradiation may induce cell type-specific radiosensitization as well as cytotoxicity against tumor cells.

PARP-1 Inhibitors DPQ and PJ-34 Negatively Modulate Proinflammatory Commitment of Human Glioblastoma Cells

Poly(ADP-ribose) polymerases (PARPs) are recognized as key regulators of cell survival or death. PARP-1 is essential to the repair of DNA single-strand breaks via the base excision repair pathway. The enzyme may be overactivated in response to inflammatory cues, thus depleting cellular energy pools and eventually causing cell death. Accordingly, PARP-1 inhibitors, acting by competing with its physiological substrate NAD(+), have been proposed to play a protective role in a wide range of inflammatory and ischemia/reperfusion-associated diseases. Recently, it has also been reported that PARP-1 regulates proinflammatory mediators, including cytokines, chemokines, adhesion molecules, and enzymes (e.g., iNOS). Furthermore, PARP-1 has been shown to act as a coactivator of NF-κB- and other transcription factors implicated in stress/inflammation, as AP-1, Oct-1, SP-1, HIF, and Stat-1. To further substantiate this hypothesis, we tested the biomolecular effects of PARP-1 inhibitors DPQ and PJ-34 on human glioblastoma cells, induced to a proinflammatory state with lipopolysaccharide and Interferon-γ. PARP-1 expression was evaluated by laser scanning confocal microscopy immunofluorescence (LSM); nitrite production, LDH release and cell viability were also determined. LSM of A-172, SNB-19 and CAS-1 cells demonstrated that DPQ and PJ-34 downregulate PARP-1 expression; they also cause a decrease of LDH release and nitrite production, while increasing cell viability. Similar effects were caused in all three cell lines by N-mono-methyl-arginine, a well known iNOS inhibitor, and by L-carnosine and trehalose, two antioxidant molecules. These results demonstrate that, similar to other well characterized drugs, DPQ and PJ-34 reduce cell inflammation and damage that follow PARP-1 overexpression, while they increase cell survival: this suggests their potential exploitation in clinical Medicine.

Abstract 1767: Anthracenyl isoxazole amides (AIMs) as novel agents for brain tumors

Abstract In the United States, the average survival rate for malignant brain tumors is approximately one year. Despite recent advancements in treatment, there remains a need for the development of new therapeutic agents to target and treat malignant brain tumors to increase survival and quality of life. Due to its pivotal role in cancer cell proliferation, telomerase has become an attractive target in the development of new anticancer agents. The enzymatic activity of telomerase is responsible for telomere maintenance and adds the guanine-rich DNA sequence repeats to the 3′ end of DNA strands in the telomere regions. Human telomeric DNA has the potential to adopt G-quartet conformations, which in turn can stack in various arrangements to form G quadruplexes (G4). To this end, molecules that bind telomeric G4 DNA and inhibit telomerase have substantial anticancer potential. The objective of this study was to evaluate the cytotoxicity of a novel series of anthracenyl isoxazole amides (AIMs), capable of selectively binding to G4 telomeric DNA, and determine the ability of the AIMs to penetrate cell membranes and enter the nuclei of cancer cells. These AIMs have demonstrated significant activity against brain tumor cell lines in the DTP NCI-60 cell line panel, and COMPARE analysis indicated that these molecules exerted their cytotoxicity via a unique mechanism. Two AIMs, referred to here as AIM-dimer and AIM-double tail (AIM-dt), showed single digit micromolar IC50 values (4.75 +/− 0.27 and 3.13 +/− 0.45, respectively) by MTT assay when tested in the human glioblastoma cell line SNB-19. As a followup to the cytotoxicity studies, apoptosis was evaluated by flow cytometry (Annexin V). The results indicated that both AIMs induced apoptosis in SNB-19 cells at higher levels than untreated cells after a four-hour treatment. Since the AIMs are known to autofluoresce, confocal microscopy was performed to determine if they were able to penetrate cell membranes of the SNB-19 cells. Contrary to bioavailability calculations made with Symyx Draw v3.1, which suggested that only the AIM-dt had acceptable properties for cell penetration, both compounds were found to enter the cells. Perhaps more intriguing, however, are the results from the laser scanning cytometry (iCys) studies. SNB-19 cells were co-treated with either AIM-dimer or AIM-dt and the nuclear stain propidium iodide. Images taken of each AIM clearly indicated co-localization of the AIM at the nucleus, suggesting that these AIMs are able to penetrate cell membranes and reach the nuclei and therefore have potential to bind DNA. Further studies will be focused on elucidating the mechanism of action of the AIMs as well as confirming their interactions at the level of G4 telomeric DNA. Supported by NIH Grant P20RR017670 (HDB). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1767. doi:1538-7445.AM2012-1767

Structural and Functional Characterization of Monomeric EphrinA1 Binding Site to EphA2 Receptor

The EphA2 receptor is overexpressed in glioblastoma multiforme and has been to shown to contribute to cell transformation, tumor initiation, progression, and maintenance. EphrinA1 (eA1) is a preferred ligand for the receptor. Treatment with monomeric eA1, the form of eA1 found in the extracellular environment, causes receptor phosphorylation, internalization, and down-regulation with subsequent anti-tumor effects. Here, we investigated the structure-function relationship of a monomeric eA1 focusing on its G-H loop ((108)FQRFTPFTLGKEFKE(123)G), a highly conserved region among eAs that mediates binding to their receptors. Alanine substitution mutants of the G-H loop amino acids were transfected into U-251 MG glioblastoma multiforme cells, and functional activity of each mutant in conditioned media was assessed by EphA2 down-regulation, ERK and AKT activation and cellular response assays. Alanine substitutions at positions Pro-113 Thr-115, Gly-117, Glu-122, and also Gln-109 enhanced the EphA2 receptor down-regulation and decreased p-ERK and p-AKT. Substitution mutants of eA1 at positions Phe-108, Arg-110, Phe-111, Thr-112, Phe-114, Leu-116, Lys-118, Glu-119, and Phe-120 had a deleterious effect on EphA2 down-regulation when compared with eA1-WT. Mutants at positions Phe-108, Lys-18, Lys-121, Gly-123 retained similar properties to eA1-WT. Recombinant eA1-R110A, -T115A, -G117A, and -F120A have been found to exhibit the same characteristics as the ligands contained in the conditioned media mainly due to the differences in their binding to the receptor. Thus, we have identified variants of eA1 that possess either superagonistic or antagonistic properties. These new findings will be important in the understanding of the receptor/ligand interactions and in further design of anti-cancer therapies targeting the eA/EphA system.

Abstract LB-115: EphA2/ephrinA1 system may regulate invadopodia formation of glioblastoma multiforme (GBM) cells

Abstract Background. Grade IV astrocytoma, or glioblastoma multiforme (GBM), is the most common, most malignant primary brain tumor in humans. With a median survival of approximately14 months, GBMs are characterized for their aggressive and highly invasive nature. We have previously explored the function of the EphA2 receptor and its ligand ephrinA1 in GBM. EphA2 was found to be over-expressed in various GBM cells, but not normal brain. EphrinA1, a naturally occurring GPI-linked protein originally found on epithelial cells, plays a tumor suppressive function by binding to and down-regulating cell surface expression of EphA2, leading to a subsequent decrease in cell migration and invasion. Invadopodia are electron-dense, actin-rich protrusions that bind to and proteolytically degrade extracellular matrix (ECM) proteins, potentially allowing for increased invasion. We have thus begun exploring the role of the EphA2/ephrinA1 complex in invadopodia formation and matrix degradation. Methods. Invadopodia formation and matrix degradation were assessed by an in situ gelatin zymography assay combined with fluorescence microscopy. Expression of the EphA2 receptor was examined by immunoblot using the following GBM cell lines: parental, vector control and ephrinA1-expressing U-251 cells, A-172, G48a, and Snb-19. Invadopodia-mediated degradation of the gelatin matrix was examined by fluorescence microscopy at various time points for each cell line. Src-transformed NIH3T3 (Src3T3) cells were used as a positive control. Results. The Snb-19 cells showed the most degradation of gelatin matrix among the GBM cell lines tested, closely mimicking control Src3T3 cells. This punctate pattern of gelatin degradation started to form as early as 2 hr after cell plating and continued until at least 72 hr. Other GBM cell lines degraded the matrix in the following order: Snb-19 ≫ A-127 > U-251 ephrinA1 > U-251 vector > U-251 parental > G-48a. Unexpectedly, immunoblot analysis showed low levels of EphA2 among the cells that degraded the most ECM. In preliminary experiments, diminishing levels of EphA2, either by knockdown or ligand activation, evoked more prominent gelatin-degrading invadopodia activity. Conclusions. GBM cells form invadopodia-like structures that degrade the ECM, with the Snb-19 cell line exhibiting the greatest degradation activity among the tested cell lines. Our preliminary results suggest a significant involvement of the EphA2/ephrinA1 system in invadopodia function based on an inverse correlation between matrix degradation and EphA2 expression. These findings have potentially important implications concerning the role of the EphA2/ephrinA1 complex in brain tumor migration and invasion. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-115. doi:10.1158/1538-7445.AM2011-LB-115

C‐Cbl regulates glioma invasion through matrix metalloproteinase 2

c-Cbl, a multifunctional adaptor and an E3 ubiquitin ligase, plays a role in such cytoskeleton-mediated events as cell adhesion and migration. Invasiveness of human glioma is dependent on cell adhesion, migration, and degradation of extracellular matrix (ECM). However, the function of c-Cbl in glioma invasion has never been investigated. We report here, for the first time, that c-Cbl plays a positive role in the invasion of ECM by SNB19 glioma cells. RNAi-mediated depletion of c-Cbl decreases SNB19 cell invasion and expression of matrix metalloproteinase 2 (MMP2). Consistent with these findings, SNB19 cells expressing wild-type, but not mutant c-Cbl show increased invasion and MMP2 expression. We demonstrate that the observed role of c-Cbl in invasion of SNB19 cells is not mediated by the previously shown effects of c-Cbl on cell adhesion and migration or on EGFR signaling. Together, our results suggest that c-Cbl promotes glioma invasion through up-regulation of MMP2.

Suppression of uPA and uPAR Attenuates Angiogenin Mediated Angiogenesis in Endothelial and Glioblastoma Cell Lines

BackgroundIn our earlier reports, we showed that downregulation of uPA and uPAR inhibited glioma tumor angiogenesis in SNB19 cells, and intraperitoneal injection of a hairpin shRNA expressing plasmid targeting uPA and uPAR inhibited angiogenesis in nude mice. The exact mechanism by which inhibition of angiogenesis takes place is not clearly understood.Methodology/Principal FindingsIn the present study, we have attempted to investigate the mechanism by which uPA/uPAR downregulation by shRNA inhibits angiogenesis in endothelial and glioblastoma cell lines. uPA/uPAR downregulation by shRNA in U87 MG and U87 SPARC co-cultures with endothelial cells inhibited angiogenesis as assessed by in vitro angiogenesis assay and in vivo dorsal skin-fold chamber model in nude mice. Protein antibody array analysis of co-cultures of U87 and U87 SPARC cells with endothelial cells treated with pU2 (shRNA against uPA and uPAR) showed decreased angiogenin secretion and angiopoietin-1 as well as several other pro-angiogenic molecules. Therefore, we investigated the role of angiogenin and found that nuclear translocation, ribonucleolytic and 45S rRNA synthesis, which are all critical for angiogenic function of angiogenin, were significantly inhibited in endothelial cells transfected with uPA, uPAR and uPA/uPAR when compared with controls. Moreover, uPA and uPAR downregulation significantly inhibited the phosphorylation of Tie-2 receptor and also down regulated FKHR activation in the nucleus of endothelial cells via the GRB2/AKT/BAD pathway. Treatment of endothelial cells with ruPA increased angiogenin secretion and angiogenin expression as determined by ELISA and western blotting in a dose-dependent manner. The amino terminal fragment of uPA down regulated ruPA-induced angiogenin in endothelial cells, thereby suggesting that uPA plays a critical role in positively regulating angiogenin in glioblastoma cells.Conclusions/SignificanceTaken together, our results suggest that uPA/uPAR downregulation suppresses angiogenesis in endothelial cells induced by glioblastoma cell lines partially by downregulation of angiogenin and by inhibition of the angiopoietin-1/AKT/FKHR pathway.

Co-Depletion of Cathepsin B and uPAR Induces G0/G1 Arrest in Glioma via FOXO3a Mediated p27Kip1 Upregulation

BackgroundCathepsin B and urokinase plasminogen activator receptor (uPAR) are both known to be overexpressed in gliomas. Our previous work and that of others strongly suggest a relationship between the infiltrative phenotype of glioma and the expression of cathepsin B and uPAR. Though their role in migration and adhesion are well studied the effect of these molecules on cell cycle progression has not been thoroughly examined.Methodology/Principal FindingsCathespin B and uPAR single and bicistronic siRNA plasmids were used to downregulate these molecules in SNB19 and U251 glioma cells. FACS analysis and BrdU incorporation assay demonstrated G0/G1 arrest and decreased proliferation with the treatments, respectively. Immunoblot and immunocyto analysis demonstrated increased expression of p27Kip1 and its nuclear localization with the knockdown of cathepsin B and uPAR. These effects could be mediated by αVβ3/PI3K/AKT/FOXO pathway as observed by the decreased αVβ3 expression, PI3K and AKT phosphorylation accompanied by elevated FOXO3a levels. These results were further confirmed with the increased expression of p27Kip1 and FOXO3a when treated with Ly294002 (10 µM) and increased luciferase expression with the siRNA and Ly294002 treatments when the FOXO binding promoter region of p27Kip1 was used. Our treatment also reduced the expression of cyclin D1, cyclin D2, p-Rb and cyclin E while the expression of Cdk2 was unaffected. Of note, the Cdk2-cyclin E complex formation was reduced significantly.Conclusion/SignificanceOur study indicates that cathepsin B and uPAR knockdown induces G0/G1 arrest by modulating the PI3K/AKT signaling pathway and further increases expression of p27Kip1 accompanied by the binding of FOXO3a to its promoter. Taken together, our findings provide molecular mechanism for the G0/G1 arrest induced by the downregulation of cathepsin B and uPAR in SNB19 and U251 glioma cells.

Abstract 681: Knockdown of connective tissue growth factor and treatment with temozolomide inhibited invasion, angiogenesis, and tumorigenesis of human glioblastomas

Abstract Connective tissue growth factor (CTGF) is a putative proto-oncogene and plays a crucial role in endothelial cell migration and tumor angiogenesis. CTGF is highly upregulated in proliferating endothelial cells and glioblastoma cells. In this investigation, we examined whether knockdown of CTGF at the mRNA level and treatment with temozolomide (TMZ) could inhibit cell invasion, angiogenesis, and growth of human glioblastoma SNB-19 and T98G cells in vivo. The cells were stably transfected with a plasmid vector carrying the human CTGF siRNA cDNA and then treated with 10 μM TMZ for 48 h. Semiquantitative PCR, Western blotting, and immunohistochemical staining demonstrated 80% downregulation of CTGF both at mRNA and protein levels after stable transfection. Matrigel invasion, cell migration from spheroids, and cell proliferation studies demonstrated significant inhibition of cell invasion, migration, and proliferation, respectively, of both cell lines after downregulation of CTGF and treatment with TMZ. In vitro and in vivo angiogenesis assays demonstrated inhibition of network formation of endothelial cells and neovascularization under the dorsal skin of nude mice, respectively, after knockdown of CTGF and treatment with TMZ. Both subcutaneous and intracerebral tumorigenesis in nude mice was markedly reduced in CTGF downregulated cells after TMZ treatment. Mechanistic studies demonstrated significant reduction of PCNA, VEGF, c-Myc, CDK2, CDK4, and cyclin D1 and upregulation of the cell cycle inhibitors p21Waf1 and p27Kip1. Flow cytometric analysis showed cell cycle arrest at G2/M phase in both cell lines after CTGF knockdown and TMZ treatment. Taken together, our study indicates that the CTGF knockdown and TMZ treatment effectively prevents cell invasion, migration, angiogenesis, and growth of glioblastomas in vivo. Therefore, CTGF knockdown during TMZ treatment offers a novel and potential therapeutic strategy for controlling the growth of glioblastomas. This investigation was supported in part by the R01 grants (CA-91460 and NS-57811) from the National Institutes of Health (Bethesda, MD). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 681.

Doxorubicin-mediated apoptosis in glioma cells requires NFAT3.

Nuclear factor of activated T cells (NFAT), a family of transcription factors, has been implicated in many cellular processes, including some cancers. Here, we characterize, for the first time, the role of NFAT3 in doxorubicin (DOX)-mediated apoptosis, migration, and invasion in SNB19 and U87 glioma cells. This study demonstrates that the specific knockdown of NFAT3 results in a dramatic inhibition of the apoptotic effect induced by DOX and favors cell survival. Inhibition of NFAT3 activation by shNFAT3 (shNF3) significantly downregulated tumor necrosis factor (TNF)-alpha induction, its receptor TNFR1, caspase 10, caspase 3, and poly (ADP-ribose) polymerase, abrogating DOX-mediated apoptosis in glioma cells. DOX treatment resulted in NFAT3 translocation to the nucleus. Similarly, shNF3 treatment in SNB19 and U87 cells reversed DOX-induced inhibition of cell migration and invasion, as determined by wound healing and matrigel invasion assays. Taken together, these results indicate that NFAT3 is a prerequisite for the induction of DOX-mediated apoptosis in glioma cells.

N-(4-Hydroxyphenyl)retinamide induced differentiation with repression of telomerase and cell cycle to increase interferon-γ sensitivity for apoptosis in human glioblastoma cells

Glioblastoma is the most malignant and prevalent brain tumor in humans. It is composed of heterogenic abnormal astroglial cells that avoid differentiation, maintain proliferation, and hardly commit apoptosis. N-(4-Hydroxyphenyl)retinamide (4-HPR) induced astrocytic differentiation and increased sensitivity to interferon-γ (IFN-γ) for apoptosis in human glioblastoma A172, LN18, and SNB19 cells. Combination of 4-HPR and IFN-γ significantly inhibited human telomerase reverse transcriptase (hTERT), cyclin dependent kinase 2 (CDK2), and survivin to up-regulate caspase-8, caspase-9, and caspase-3 for increasing apoptosis in all glioblastoma cell lines. Hence, combination of 4-HPR and IFN-γ should be considered for controlling growth of different human glioblastoma cells.

Fatty acid synthase: a novel target for antiglioma therapy

High levels of fatty acid synthase (FAS) expression have been observed in several cancers, including breast, prostate, colon and lung carcinoma, compared with their respective normal tissue. We present data that show high levels of FAS protein in human and rat glioma cell lines and human glioma tissue samples, as compared to normal rat astrocytes and normal human brain. Incubating glioma cells with the FAS inhibitor cerulenin decreased endogenous fatty acid synthesis by approximately 50%. Cell cycle analysis demonstrated a time- and dose-dependent increase in S-phase cell arrest following cerulenin treatment for 24 h. Further, treatment with cerulenin resulted in time- and dose-dependent decreases in glioma cell viability, as well as reduced clonogenic survival. Increased apoptotic cell death and PARP cleavage were observed in U251 and SNB-19 cells treated with cerulenin, which was independent of the death receptor pathway. Overexpressing Bcl-2 inhibited cerulenin-mediated cell death. In contrast, primary rat astrocytes appeared unaffected. Finally, RNAi-mediated knockdown of FAS leading to reduced FAS enzymatic activity was associated with decreased glioma cell viability. These findings suggest that FAS might be a novel target for antiglioma therapy.

Regression of pre-established intracranial tumor growth by ON 01910.Na, a selective anticancer agent currently in phase I trials

1576 Background: Despite considerable progress in the treatment modalities used for brain tumors (surgery, radiation therapy and chemotherapy), local recurrence continues to be an important reason for treatment failure. Since multiple regulatory pathways are defective in these tumor cells, novel approaches must tackle more than one mechanism involved in the process of neoplasia. One approach is to effectively combine anti-tumor agents with anti-angiogenic assault. Onconova has developed agents that fit this profile. ON 01910.Na, an injectable product now undergoing Phase I studies at multiple centers, is a potent inhibitor of mitotic progression. It arrests cancer cells in mitosis by depleting cells of CDC25c and efficiently induces apoptosis in the irreversibly arrested cells. Methods: Here, we have evaluated the invasiveness, angiogenesis, and tumorigenicity of glioblastoma cells in both in vitro and in vivo models after treatment with ON 01910. Results: SNB19 cells treated with ON 01910 were significantly inhibited in migration, invasion, and angiogenesis in in vitro models compared to the control group. After treatment with ON 01910, SNB19 cells showed decreased levels of MMPs, uPA, pAKT and PI3 kinase levels compared to controls. Significantly increased levels of PARP, caspase 3 and caspase 9 were also shown. In addition, there was complete regression of pre-established intracranial tumor growth and angiogenesis in in vivo models. The effect was more significant in relation to invasion, angiogenesis and tumor growth when compared to other drugs such as BCNU and Gleevec. Toxicology studies, where ON 01910 was administered systemically or directly into the brain, demonstrate the safey and tolerability of this agent in animals and ongoing Phase I studies indicate that the agent is well-tolerated at dose levels generating pharmacologically relevant concentration of the drug in circulation. Conclusions: These studies validate the potential usefulness of this compound as a novel therapeutic agent for human glioblastoma. [Table: see text]

Specific Interference of Urokinase-type Plasminogen Activator Receptor and Matrix Metalloproteinase-9 Gene Expression Induced by Double-stranded RNA Results in Decreased Invasion, Tumor Growth, and Angiogenesis in Gliomas

We have previously demonstrated the effectiveness of adenovirus-mediated expression of antisense urokinase-type plasminogen activator receptor (uPAR) and matrix metalloproteinase-9 (MMP-9) in inhibiting tumor invasion in vitro and ex vivo. However, the therapeutic effect of the adenovirus-mediated antisense approach was shown to be transient and required potentially toxic, high viral doses. In contrast, RNA interference (RNAi)-mediated gene targeting may be superior to the traditional antisense approach, because the target mRNA is completely degraded and the molar ratio of siRNA required to degrade the target mRNA is very low. Here, we have examined the siRNA-mediated target RNA degradation of uPAR and MMP-9 in human glioma cell lines. Using RNAi directed toward uPAR and MMP-9, we achieved specific inhibition of uPAR and MMP-9. This bicistronic construct (pUM) inhibited the formation of capillary-like structures in both in vitro and in vivo models of angiogenesis. We demonstrated that blocking the expression of these genes results in significant inhibition of glioma tumor invasion in Matrigel and spheroid invasion assay models. RNAi for uPAR and MMP-9 inhibited cell proliferation, and significantly reduced the levels of phosphorylated forms of MAPK, ERK, and AKT signaling pathway molecules when compared with parental and empty vector/scrambled vector-transfected SNB19 cells. Furthermore, using RNAi to simultaneously target two proteases resulted in total regression of pre-established intracerebral tumor growth. Our results provide evidence that the use of hairpin siRNA expression vectors for uPAR and MMP-9 may provide an effective tool for cancer therapy.

In vivo and in vitro analysis of the human tissue‐type plasminogen activator gene promoter in neuroblastomal cell lines: evidence for a functional upstream κB element

Besides its well-established role in wound healing and fibrinolysis, tissue-type plasminogen activator (t-PA) has been shown to contribute to cognitive processes and memory formation within the central nervous system, and to promote glutamate receptor-mediated excitotoxicity. The t-PA gene is expressed and regulated in neuronal cells but the regulatory transcriptional processes directing this expression are still poorly characterized. We have used DNase I-hypersensitivity mapping and in vivo foot printing to identify putative regulatory elements and transcription factor binding sites in two human neuroblastomal (KELLY and SK-N-SH) and one human glioblastomal (SNB-19) cell lines. Hypersensitive sites were found in the proximal promoter region of all cell lines, and within the first exon for KELLY and SNB-19 cells. Mapping of methylation-protected residues in vivo detected a cluster of protected residues corresponding to a cAMP response element (CRE) and Sp1 sites in the proximal promoter previously shown to be essential for basal expression in other cell types. Protected residues were also found at other sites, notably a kappaB element at position bp -3081 to -3072 that was partly protected in KELLY and SNB-19 cells. Analysis of transfected reporter constructs in KELLY and SNB-19 cells confirmed that this particular element is functionally significant in the transactivation of the t-PA promoter in both cell types. This study defines, by in vivo and in vitro methods, a previously undescribed kappaB site in the t-PA gene promoter that influences t-PA expression in neuronal cells.

Recombinant adeno‐associated virus (rAAV) expressing TFPI‐2 inhibits invasion, angiogenesis and tumor growth in a human glioblastoma cell line

Recombinant adeno-associated viruses (rAAV) have become the vector of choice for many gene therapy protocols. rAAVs have a number of attractive features including long-term transgene expression and the ability to transduce both dividing and non-dividing cells. We have shown previously the anti-cancer role of tissue factor pathway inhibitor-2 (TFPI-2), a matrix-associated serine protease inhibitor, in human glioblastomas. As a result of our present study, in which 0.8-kb fragment of human TFPI-2 was cloned into the adeno-associated viral vectors (rAAA-TFPI-2), rAAV-TFPI-2 infection of SNB19 cells significantly increased TFPI-2 as determined by Western blotting. As assessed by spheroid and Matrigel assays, infection of SNB19 cells with rAAV-TFPI-2 significantly reduced migration and invasion in a dose-dependent manner. Tumor spheroids infected with rAAV-TFPI-2 and co-cultured with fetal rat brain aggregates did not invade rat brain aggregates, whereas 90-95% of the mock and AAV-CMV infected cells invaded rat brain aggregates. In vitro angiogenesis studies (tumor cells co-cultured with endothelial cells or endothelial cells seeded on matrigel) showed reduction of capillary-like structure formation in rAAV-TFPI-2-treated cells as compared to parental and mock-transfected cells. In in vivo angiogenesis results demonstrated the formation of microvessels in SNB19 parental cells and this formation was inhibited when the SNB19 cells were infected with rAAV-TFPI-2. Further, we observed a large reduction of tumor growth in SNB19 cells treated with rAAV-TFPI-2 virus injected intracerebrally when compared to controls. Our study demonstrates that rAAV-TFPI-2-mediated gene therapy offers a novel tool for the treatment of brain tumors.

Spotlight

Hasumi et al., 911–916 Prostate cancer is an extremely common form of cancer in older men. The disease has a complex etiology and many open questions also remain on its development and progression. Although hormone therapy is highly effective in shrinking tumor size, most patients become resistant to this treatment after several years. The biological mechanism underlying hormonal treatment resistance, known as recurrent hormone refractory prostate cancer (HRPC), is not clear. In this study, Hasumi et al., hunted for novel genes that may be differentially expressed in prostate cancer patients. Using GeneChip technology with both malignant and normal tissue samples taken from untreated prostate cancer patients, they identified an almost 3-fold increased expression of the protease inhibitor neuroserpin. Real-time quantitative PCR analysis identified the presence of neuroserpin in all prostate cancer (n = 45) and paired normal tissue (n = 45) samples and in patients with HRPC. However, neuroserpin levels were significantly higher in malignant than in normal tissue and higher again in HRPC samples. Importantly, neuroserpin expression had a significant negative correlation with recurrence-free and overall survival. How this protease inhibitor plays a role in prostate cancer disease progression remains to be seen, but in the interim its expression could be a very useful parameter to monitor disease recurrence and survival. Giwercman et al., 994–997 Giwercman et al. also look at prostate cancer, but from an epidemiological perspective. Although it is believed that prostate carcinogenesis is dependent on the presence of testosterone and related androgens, an association between androgen exposure and prostate cancer risk is inconclusive. Androgen status is not an absolute measure of, but is associated with, fertility. In this study, the authors postulate that male fertility—indirectly calculated by whether an individual has fathered offspring or not—is associated with an increased risk of prostate cancer. Using data from the Swedish National Cancer Registry, the authors analyzed nearly 49,000 diagnosed cases of prostate cancer matched by year of birth to healthy controls. The association between the number offspring and the risk of developing prostate cancer was estimated. The risk of developing prostate cancer is slightly lower in men who are childless (OR = 0.83; 95% confidence interval (CI) = 0.81 - 0.86) or who have only fathered one child (OR = 0.93; 95% CI = 0.90 - 0.96). The Odds Ratio for the association between the number of children and the risk of prostate cancer. The authors plainly admit that having fathered children does not take female fertility or the desire to have children into account and, therefore, is clearly not a perfect measurement. However, as these data are based on a huge cohort, the hypothesis that fertility is associated with a slightly increased prostate cancer risk should be further considered. Yanamandra et al., 998–1005 Glioblastoma is a highly invasive cancer with a high morbidity and mortality as neighboring tissue is invaded and destroyed by the malignant cells. Tissue factor pathway inhibitor (TFPI-2) is a serine protease inhibitor that is underexpressed in gliomas. The authors had previously demonstrated that overexpression of TFPI-2 in the human malignant glioma cell line SNB19 reduced invasiveness of tumor growth both in vitro and in vivo. In this study, Yanamandra et al., investigated whether TFPI-2 could be reliably expressed and display its anti-cancer effects when produced by a recombinant adeno-associated virus (rAAV). rAAV is an attractive gene therapy tool as it gives stable expression and can infect nondividing cells. SNB19 human glioma cells were grown as multi-cellular tumor spheroids and then infected with rAAV-TFPI-2. Both the migration rate and the invasiveness of SNB19 sharply declined when cells were treated with rAAV-TFPI-2 compared to control constructs. Neovascularization is also crucial to tumor progression and survival. rAAV-TFPI-2 infected SNB19 cells repressed capillary structure formation by 85% in vitro and angiogenesis by up to 100% in vivo. To test whether this gene therapy model could inhibit tumor growth in vivo, nude mice were injected intracerebrally with SNB19 cells infected with rAAV-TFPI-2. After 4 weeks, the mice that received SNB19 with the control vector had developed tumors, whereas mice receiving rAAV-TFPI-2 infected SNB19 had more than 85% reduction in tumor formation. The mechanism of how TFPI-2 causes these anti-cancer effects is not clear. Nonetheless, this comprehensive study gives very promising results for a novel gene therapy approach to treat malignant brain tumors and thus warrants further investigation.