D54-MG Cells Research Articles

Effects of Chemically-Functionalized Single-Walled Carbon Nanotubes on the Morphology and Vitality of D54MG Human Glioblastoma Cells.

The unique properties of single-walled carbon nanotubes (SWCNTs) have made them interesting candidates for applications in biomedicine. There are diverse chemical groups that can be attached to SWCNTs in order for these tiny tubes to gain various functionalities, for example, water solubility. Due to the availability of these “functionalization” approaches, SWCNTs are seen as agents for a potential anti-cancer therapy. In this context, we tested different chemically-functionalized forms of SWCNTs to determine which modifications make them better combatants against glioblastoma (astrocytoma grade IV), the deadliest brain cancer. We investigated the effects that two types of water soluble SWCNTs, functionalized with polyethylene glycol (SWCNT-PEG) or tetrahydrofurfuryl-terminated polyethylene glycol (SWCNT-PEG-THFF), have on the morphology and vitality, that is, cell adhesion, proliferation and death rate, of the D54MG human glioblastoma cells in culture. We found that SWCNT-PEG-THFF solute, when added to culture media, makes D54MG cells less round (measured as a significant decrease, by ~23%, in the form factor). This morphological change was induced by the PEG-THFF functional group, but not the SWCNT backbone itself. We also found that SWCNT-PEG-THFF solute reduces the proliferation rate of D54MG cells while increasing the rate of cell death. The functional groups PEG and PEG-THFF, on the other hand, reduce the cell death rate of D54MG human glioma cells. These data indicate that the process of functionalization of SWCNTs for potential use as glioma therapeutics may affect their biological effects.

CCAAT Box, Not CArG Motifs, Identify Surviving as a Highly Potent Radiation Inducible Promoter

Radiogenetic therapy is a novel approach in the treatment of cancer, which employs genetic modification to alter the sensitivity of tumor cells to the effect of applied radiation. Some previous studies concluded that CArG motifs are the elements responsible for radiation mediated transcription regulation, while other concluded that CCAAT box consensus are present in low frequencies in radioresponsive genes in comparison to normal genes. Using bioinformatics we aim to detect mechanism of radiation mediated transcription regulation in the context of glioblastoma adenoviral gene therapy. The effect of radiation was tested on 6 different in-house promoters (iNOS, FLT-1, DR5, Cox-2, VEGF, and survivin) where levels of mRNA expression of these promoters were assessed using Quantitative RTPCR in D54 MG cells before and after radiation exposure. Recombinant Ad/reporter genes driven by 7 different promoters; CMV, VEGF, FLT-1, DR5, iNOS, cox-2 and Survivin were constructed. Glioma cell lines were infected with different Multiplicity of infection of the (promoter) Ad or CMV Ad. Cells were then exposed to a range of radiation (0-12Gy) at single fraction. Fluorescent Microscopy, Luc assay and X-gal staining was used to detect the level of expression of related genes. A time course experiment was carried out to look for effect of time of radiation on gene expression. These promoters were screened for the presence CArG motifs and CCAAT box consensus using NCBI Blast bioinformatics software. Radiotherapy increase the expression of gene expression by1.75-fold in DR5, 2-fold in VEGF and 0 fold Flt-1 after 2 hr of radiation. Most importantly Cells treated with RT and Ad Luc driven by Survivin promoter showed 5-fold increase in expression after 2 Gy of radiation in compare to non-irradiated cells. RNA analysis was done and has shown increase copy no. of corresponding genes in level range from 2 fold for VEGF 3 fold for iNOS and as expected 30 fold more for Survivin. Using NCBI BLAST software, none of CArG motifs were found in the different 6 promoters. Survivin with the best response to radiation had the lowest number of CCAAT box (n=2) while iNOS with the lowest response to radiation had the highest number of CCAAT (n=14) with other promoters having different numbers of CCAAT ranging between these two values. Additionally, the initiator element consensus (YYANWYY) was not found in all the responsive promoters to radiation. Survivin as a radiation inducible promoter for glioblastoma viral gene therapy is independent of CArG motifs. Low frequency of CCAAT box could be responsible for this phenomena.

Abstract 2110: Anti-invasive and antimetastatic actions of omega-3 polyunsaturated fatty acids in glioblastoma cells

Abstract Glioblastoma (GBM) is the most aggressive and deadliest brain malignancy in adults. Despite of surgical techniques, radiotherapy and chemotherapy, GBM has remained an invariably lethal tumor with a median survival less than 15 months. Although the anticancer mechanisms of omega-3 polyunsaturated fatty acids (ω3-PUFAs) have been reported in several cancers, it is still unclear in brain cancer. Here, our data show the anti-invasive and anti-metastatic effects of ω3-PUFAs in GBM. Invasiveness using transwell chamber was inhibited by docosahexaenoic acid (DHA) treatment in D54MG and GL261 cells. In zymography, MMP-2 activity was suppressed by DHA in a dose dependent manner. MMP-2 promoter activity was also decreased. DHA inhibited both p-STAT3 and β-catenin levels that contribute to invasion by stimulating pro-invasion factors such as MMP-2. Additionally, fat-1 (ω3-desaturease) stable D54MG cell was established and the effect of the high level of ω3-PUFAs was investigated endogenously. The invasiveness were significantly inhibited in the fat-1 stable cells compared to control cell. Moreover, the metastasis in vivo was significantly reduced when GL261 mouse glioma cells were injected through tail vein into the fat-1 transgenic mice. In immunohistochemistry, intensity of p-STAT-3 and β-catenin were significantly weak in metastatic lung tumor from Fat-1 mice compared to wild type mice. These results indicate that anti-invasive and anti-metastatic actions of ω3-PUFA may be correlated with inhibition of MMP-2 through decrease of STAT3 and β-catenin in GBM cells. Thus, these findings provide important preclinical evidence and molecular insight for utilization of ω-3 PUFAs for the chemoprevention and treatment of human GBM. [This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (2007-0054932, NRF-2015R1D1A1A01056887) and by the framework of international cooperation program managed by National Research Foundation of Korea (2015K2A2A6002008)]. Citation Format: Soyeon Kim, Soyeon Shin, Soyeon Jeong, Seung-Hyeon Han, Yoon-Seon Yoo, Prashanta Silwal, Young-Joo Jeon, Jun-Young Heo, Gi-Ryang Kweon, Seung-Kiel Park, Jong-Il Park, Kyu Lim. Anti-invasive and antimetastatic actions of omega-3 polyunsaturated fatty acids in glioblastoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2110. doi:10.1158/1538-7445.AM2017-2110

Survivin a radiogenetic promoter for glioblastoma viral gene therapy independently from CArG motifs

BackgroundRadiogenetic therapy is a novel approach in the treatment of cancer, which employs genetic modification to alter the sensitivity of tumor cells to the effect of applied radiation.AimTo select a potent radiation inducible promoter in the context of brain tumors and to investigate if CArG radio responsive motifs or other elements in the promoter nucleotide sequences can correlate to its response to radiation.MethodsTo select initial candidates for promoter inducible elements, the levels of mRNA expression of six different promoters were assessed using Quantitative RTPCR in D54 MG cells before and after radiation exposure. Recombinant Ad/reporter genes driven by five different promoters; CMV, VEGF, FLT-1, DR5 and survivin were constructed. Glioma cell lines were infected with different multiplicity of infection of the (promoter) Ad or CMV Ad. Cells were then exposed to a range of radiation (0–12 Gy) at single fraction. Fluorescent microscopy, Luc assay and X-gal staining was used to detect the level of expression of related genes. Different glioma cell lines and normal astrocytes were infected with Ad survivin and exposed to radiation. The promoters were analyzed for presence of CArG radio-responsive motifs and CCAAT box consensus using NCBI blast bioinformatics software.ResultsRadiotherapy increases the expression of gene expression by 1.25–2.5 fold in different promoters other than survivin after 2 h of radiation. RNA analysis was done and has shown an increase in copy number of tenfold for survivin. Most importantly cells treated with RT and Ad Luc driven by survivin promoter showed a fivefold increase in expression after 2 Gy of radiation in comparison to non-irradiated cells. Presence or absence of CArG motifs did not correlate with promoter response to radiation. Survivin with the best response to radiation had the lowest number of CCAAT box.ConclusionSurvivin is a selective potent radiation inducible promoter for glioblastoma viral gene therapy and this response to radiation could be independent of CArG motifs.

Interferon regulatory factor 1 and histone H4 acetylation in systemic lupus erythematosus

Histone acetylation modulates gene expression and has been described as increased in systemic lupus erythematosus (SLE). We investigated interferon regulatory factor 1 (IRF1) interactions that influence H4 acetylation (H4ac) in SLE. Intracellular flow cytometry for H4 acetylated lysine (K) 5, K8, K12, and K16 was performed. Histone acetylation was defined in monocytes and T cells from controls and SLE patients. RNA-Seq studies were performed on monocytes to look for an imbalance in histone acetyltransferases and histone deacetylase enzyme expression. Expression levels were validated using real-time quantitative RT-PCR. IRF1 induction of H4ac was evaluated using D54MG cells overexpressing IRF1. IRF1 protein interactions were studied using co-immunoprecipitation assays. IRF1-dependent recruitment of histone acetyltransferases to target genes was examined by ChIP assays using p300 antibody. Flow cytometry data showed significantly increased H4K5, H4K8, H4K12, and H4K16 acetylation in SLE monocytes. HDAC3 and HDAC11 gene expression were decreased in SLE monocytes. PCAF showed significantly higher gene expression in SLE than controls. IRF1-overexpressing D54MG cells were associated with significantly increased H4K5, H4K8, and H4K12 acetylation compared to vector-control D54MG cells both globally and at specific target genes. Co-immunoprecipitation studies using D54MG cells revealed IRF1 protein-protein interactions with PCAF, P300, CBP, GCN5, ATF2, and HDAC3. ChIP experiments demonstrated increased p300 recruitment to known IRF1 targets in D54MG cells overexpressing IRF1. In contrast, p300 binding to IRF1 targets decreased in D54MG cells with IRF1 knockdown. SLE appears to be associated with an imbalance in histone acetyltransferases and histone deacetylase enzymes favoring pathologic H4 acetylation. Furthermore, IRF1 directly interacts with chromatin modifying enzymes, supporting a model where recruitment to specific target genes is mediated in part by IRF1.

Abstract 1809: Marizomib (NPI-0052) activity as a single agent in malignant glioma

Abstract Background: Glioblastoma multiforme (GBM) is a highly aggressive brain tumor, which displays innate resistance to multiple treatment modalities. Previous studies have shown that the proteasome plays a vital role in the physiology of GBM, and that proteasome inhibition can be used as a strategy for treating malignant gliomas. Marizomib (NPI-0052) is a second generation irreversible proteasome inhibitor, which has a more lipophilic structure and has a broader and more prolonged inhibition profile for 20S proteasome activities compared to bortezomib and carfilzomib, two proteasome inhibitors approved by FDA for treatment of multiple myeloma. While bortezomib and carfilzomib have only modest activity as a treatment of malignant gliomas, marizomib might potentially be a novel therapeutic strategy for primary brain tumors. Unfortunately, to date, the number of studies that have analyzed the effect of marizomib on glioma is limited. Methods: In these studies, we investigated the in vitro activities of marizomib in primary cell cultures derived from a multitude of human brain tumors (high-grade and low-grade gliomas and meningiomas), normal neural stem/progenitor cells (NSC) and as well as in the established human malignant glioma lines U251-MG and D54-MG. The effect of marizomib on cell proliferation, proteasome activity, motility, apoptosis and Reactive Oxygen Species (ROS) were evaluated in glioma cell lines. The inhibition of marizomib by the ROS quenching agent, N-acetyl cysteine (NAC) was also tested. Results: The sensitivities varied in function of the pathology of the tumor, with the malignant glioma stem-like cells being the most severely affected, in contrast with the low-grade glioma, meningioma and NSC-derived cultures. Marizomib inhibited the proliferation of U251-MG and D54-MG cell lines with a half maximal effective concentration (EC50) of 52nM and 20nM respectively, along with a significant decrease in cell migration and invasion. Treatment with marizomib at a concentration of 60nM for 4 hours inhibited proteasome chymotrypsin-like (CT-L, β5) activity by 85% in U251-MG and D54-MG cells. Marizomib treatment of human glioma cells was associated with increased free radical production and apoptosis, along with activation of caspase-3 and cleavage of PARP. Those effects of marizomib can be suppressed by exposure to the ROS quenching agent N-acetyl cysteine (NAC). Conclusion: These preclinical studies demonstrate a significant anti-tumor effect of marizomib in malignant glioma cells. Marizomib has relatively little effect on neural stem/progenitor cells suggesting minimal neurotoxicity, while severely affecting both malignant glioma stem cells and glioma cell lines. But importantly, unlike bortezomib and carfilzomib, marizomib can cross the blood brain barrier. Additional research into the use of marizomib as a potential treatment for malignant gliomas as a single agent or in combination with SOC therapies for glioma is warranted. Citation Format: Kaijun Di, Xing Gong, Dana M. Curticiu, Michael A. Palladino, Daniela A. Bota. Marizomib (NPI-0052) activity as a single agent in malignant glioma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1809. doi:10.1158/1538-7445.AM2014-1809

Proteasome inhibition with bortezomib induces cell death in GBM stem-like cells and temozolomide-resistant glioma cell lines, but stimulates GBM stem-like cells' VEGF production and angiogenesis

Recurrent malignant gliomas have inherent resistance to traditional chemotherapy. Novel therapies target specific molecular mechanisms involved in abnormal signaling and resistance to apoptosis. The proteasome is a key regulator of multiple cellular functions, and its inhibition in malignant astrocytic lines causes cell growth arrest and apoptotic cell death. The proteasome inhibitor bortezomib was reported to have very good in vitro activity against malignant glioma cell lines, with modest activity in animal models as well as in clinical trials as a single agent. In this paper, the authors describe the multiple effects of bortezomib in both in vitro and in vivo glioma models and offer a novel explanation for its seeming lack of activity. Glioma stem-like cells (GSCs) were obtained from resected glioblastomas (GBMs) at surgery and expanded in culture. Stable glioma cell lines (U21 and D54) as well as temozolomide (TMZ)-resistant glioma cells derived from U251 and D54-MG were also cultured. GSCs from 2 different tumors, as well as D54 and U251 cells, were treated with bortezomib, and the effect of the drug was measured using an XTT cell viability assay. The activity of bortezomib was then determined in D54-MG and/or U251 cells using apoptosis analysis as well as caspase-3 activity and proteasome activity measurements. Human glioma xenograft models were created in nude mice by subcutaneous injection. Bevacizumab was administered via intraperitoneal injection at a dose of 5 mg/kg daily. Bortezomib was administered by intraperitoneal injection 1 hour after bevacizumab administration in doses of at a dose of 0.35 mg/kg on days 1, 4, 8, and 11 every 21 days. Tumors were measured twice weekly. Bortezomib induced caspase-3 activation and apoptotic cell death in stable glioma cell lines and in glioma stem-like cells (GSCs) derived from malignant tumor specimens Furthermore, TMZ-resistant glioma cell lines retained susceptibility to the proteasome inhibition. The bortezomib activity was directly proportional with the cells' baseline proteasome activity. The proteasome inhibition stimulated both hypoxia-inducible factor (HIF)-1α and vascular endothelial growth factor (VEGF) production in malignant GSCs. As such, the VEGF produced by GSCs stimulated endothelial cell growth, an effect that could be prevented by the addition of bevacizumab (VEGF antibody) to the media. Similarly, administration of bortezomib and bevacizumab to athymic mice carrying subcutaneous malignant glioma xenografts resulted in greater tumor inhibition and greater improvement in survival than administration of either drug alone. These data indicate that simultaneous proteasome inhibition and VEGF blockade offer increased benefit as a strategy for malignant glioma therapy. The results of this study indicate that combination therapies based on bortezomib and bevacizumab might offer an increased benefit when the two agents are used in combination. These drugs have a complementary mechanism of action and therefore can be used together to treat TMZ-resistant malignant gliomas.

Sodium butyrate increases the effect of the photodynamic therapy: a mechanism that involves modulation of gene expression and differentiation in astrocytoma cells

In order to evaluate the improvement of the photodynamic therapy (PDT) due to sodium butyrate (NaBu), its effectiveness in U373-MG and D54-MG astrocytoma cell lines was evaluated. Cells were exposed to delta-aminolevulinic acid (δ-ALA) as a precursor to endogenous photosensitizer protoporphyrin IX (PpIX). In both astrocytoma cells, an important increase by ALA was observed in uroporphyrinogen synthetase gene expression: 1.8- and 52-fold for D54-MG and U373-MG cells, respectively. After irradiation, they showed 16.67 and 28.9% of mortality in U373-MG and D54-MG, respectively. These mortalities increased to 70.62 and 96.7% when U373-MG and D54-MG cells, respectively, were exposed 24 h to 8 mM NaBu, before to PpIX induction. NaBu induced expression of caspase-3, caspase-9, and Bcl-2 and increased Bax in U373-MG cells. ALA-induced morphological changes are compatible to differentiation. Genes and differentiation induced mainly by NaBu improve cell death performed by PDT in astrocytoma cells. These facts prove the synergistic effect of NaBu on cytotoxic damage induced by PDT.

Dynamics of signaling, cytoskeleton and cell cycle regulation proteins in glioblastoma cells after sub-lethal photodynamic treatment: Antibody microarray study

BackgroundPhotodynamic therapy (PDT) that induces oxidative stress and cell death is used for tumor destruction in oncology. To characterize early molecular events in photosensitized glioblastoma cells, we studied expression of 224 proteins after sublethal PDT that doesn't kill but wounds cells. MethodsCultured glioblastoma D54Mg cells were photosensitized with 5-aminolevulinic acid so that cell survival was 95–100%. At following 0.5–5.5h protein expression and phosphorylation was assayed using proteomic antibody microarrays. ResultsWithin the first post-treatment hour we observed phosphorylation of protein kinase Raf, adhesion-related kinases FAK and Pyk2, and microtubule-associated protein tau. Protein kinase Cγ and microtubule-associated protein MAP-1B were overexpressed. Dystrophin, calponin, and vinculin, components of the actin cytoskeleton scaffold, microtubule-associated proteins MAP2 and CNP, cytokeratins 4 and 7 were down-regulated that indicated changes in adhesion and cell shape. Down-regulation of cyclins A, D1 and D3, c-Myc, checkpoint proteins chk1/2 and up-regulation of Smad4 could arrest the cell cycle. Overexpression of Bcl-xL and down-regulation of caspase 9 demonstrated anti-apoptotic response. At 2h post-treatment protein expression changed lesser but at 5.5h levels of PKCγ and β-synuclein and phosphorylation of Raf, FAK, Pyk2, and tau increased again. ConclusionsSub-lethal PDT induces complex response of glioblastoma cells including changes in activity and expression of proteins involved in adhesion-mediated signaling, signal transduction, cytoskeleton remodeling, cell cycle regulation and anti-apoptotic processes. General significanceMultiple reactions of various cellular subsystems including adhesion, cytoskeleton, signal transduction, cell cycle, and apoptosis are integrated into the general cell response to a sublethal impact.

Glioma-specific Cation Conductance Regulates Migration and Cell Cycle Progression

In this study, we have investigated the role of a glioma-specific cation channel assembled from subunits of the Deg/epithelial sodium channel (ENaC) superfamily, in the regulation of migration and cell cycle progression in glioma cells. Channel inhibition by psalmotoxin-1 (PcTX-1) significantly inhibited migration and proliferation of D54-MG glioma cells. Both PcTX-1 and benzamil, an amiloride analog, caused cell cycle arrest of D54-MG cells in G(0)/G(1) phases (by 30 and 40%, respectively) and reduced cell accumulation in S and G(2)/M phases after 24 h of incubation. Both PcTX-1 and benzamil up-regulated expression of cyclin-dependent kinase inhibitor proteins p21(Cip1) and p27(Kip1). Similar results were obtained in U87MG and primary glioblastoma multiforme cells maintained in primary culture and following knockdown of one of the component subunits, ASIC1. In contrast, knocking down δENaC, which is not a component of the glioma cation channel complex, had no effect on cyclin-dependent kinase inhibitor expression. Phosphorylation of ERK1/2 was also inhibited by PcTX-1, benzamil, and knockdown of ASIC1 but not δENaC in D54MG cells. Our data suggest that a specific cation conductance composed of acid-sensing ion channels and ENaC subunits regulates migration and cell cycle progression in gliomas.

Abstract LB-334: Imaging caspase dependent cell death as a surrogate for efficacy of cancer therapeutics

Abstract Induction of cell death in malignant lesions still represents the major goal in the treatment of cancer patients. Imaging of apoptosis would enhance the development of optimal dosing, schedule and combination therapies. Here we used a luciferase biosensor (Promega) and developed a bioluminescent assay system with optimized signal to noise for the detection of cell death in vitro and in living animals. Using D54-MG cells stably expressing the reporter, we demonstrate that TRAIL treatment resulted in a 100–200 fold induction of bioluminescence activity which correlated with cell death as demonstrated by increased cleavage of Caspase-3. Furthermore inhibition of apoptosis using the pan caspase inhibitor Z-VAD-FMK resulted in inhibition of reporter activity. The utility of this cell death reporter was further demonstrated in human glioma xenografted tumors wherein induction of apoptosis was correlated with reporter activation. The high signal to noise and dynamic range of reporter activity provides for a sensitive and quantitative surrogate for evaluation of experimental therapeutics. In addition, the ability to image repeatedly provides a unique opportunity to understand the dynamics of cell death in response to specific drugs or combination therapies. We are also investigating the application of this technology in a high throughput screening of compound and targeted siRNA libraries in diverse cells and in pre-clinical cancer mouse models. 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-334. doi:10.1158/1538-7445.AM2011-LB-334

Regulation of Migration and Proliferation of Glioblastoma Cells by Modulating the Acid Sensitive Ion Channel‐1 (ASIC‐1) Activity

Inhibition of glioma cell specific cation channels is a promising opportunity for the therapy of glioblastoma multiforme. One of the major features of high grade glioma cells is their extreme ability to migrate and proliferate through brain parenchyma. The movement of cations through ion channels is an important requirement for the metastasis of glioma cells. We have previously reported that high grade malignant glioma cells express an amiloride-sensitive cation conductance which is not present in normal astrocytes or low grade glioma cells. In the present study, we have investigated the potential role of a component of this channel, Acid Sensitive Ion Channel-1 (ASIC-1) to regulate migration and the cell cycle process in glioma cells. We found that the blocking of ASIC-1 by Psalmotoxin-1 (PcTX-1) (100 nM), a tarantula spider toxin, significantly inhibits the migration and proliferation of D54MG and U251 glioma cells both in transwell migration and in-vitro scratch wound assays. Both PcTx-1 and benzamil (100mM), an amiloride analog, caused a significant cell cycle arrest of D54MG cells in G0/G1 phases (by 30% and 40%, respectively) and reduced accumulation in the S phase (by 50% and 86%, respectively), and G2/M phase (by 78% and 80%, respectively) (n=5, P<0.01) at 24h of incubation. An unfolded control peptide (100nM) had no significant effect compared to PcTX-1 and benzamil. PcTX-1 also induced an upregulation of cyclin dependent kinase inhibitor (CDKI) proteins p21Cip1/Waf1 and p27Kip1 in D54MG cells. We also found similar effects in D54MG cells by reducing the [Na+]O in the incubation medium. Knockdown of ASIC-1 expression had similar effects on p21Cip1/Waf1 and p27Kip1 expression and inhibited the cell cycle. Our data implicate the potentialrole of glioma specific cation conductance in migration and in progressionof the cell cycle in glioma cells. This study was supported by NIH grants CA101952 and DK037206.

Interaction of ASIC1 and ENaC subunits in human glioma cells and rat astrocytes.

Glioblastoma multiforme (GBM) is the most common and aggressive of the primary brain tumors. These tumors express multiple members of the epithelial sodium channel (ENaC)/degenerin (Deg) family and are associated with a basally active amiloride-sensitive cation current. We hypothesize that this glioma current is mediated by a hybrid channel composed of a mixture of ENaC and acid-sensing ion channel (ASIC) subunits. To test the hypothesis that ASIC1 interacts with αENaC and γENaC at the cellular level, we have used total internal reflection fluorescence microscopy (TIRFM) in live rat astrocytes transiently cotransfected with cDNAs for ASIC1-DsRed plus αENaC-yellow fluorescent protein (YFP) or ASIC1-DsRed plus γENaC-YFP. TIRFM images show colocalization of ASIC1 with both αENaC and γENaC. Furthermore, using TIRFM in stably transfected D54-MG cells, we also found that ASIC1 and αENaC both localize to a submembrane region following exposure to pH 6.0, similar to the acidic conditions found in the core of a glioblastoma lesion. Using high-resolution clear native gel electrophoresis, we found that ASIC1 forms a complex with ENaC subunits which migrates at ≈480 kDa in D54-MG glioma cells. These data suggest that different ENaC/Deg subunits interact and could combine to form a hybrid channel that likely underlies the amiloride-sensitive current seen in human glioma cells.

Recombinant single‐chain variable fragment antibodies against extracellular epitopes of human multidrug resistance protein MRP3 for targeting malignant gliomas

Multidrug resistance protein 3 (MRP3), a multidrug resistance protein identified by serial analysis of gene expression as a glioblastoma multiforme (GBM)-associated molecule, is highly expressed in GBM, but not in normal brain cells. Thus, MRP3 is a candidate for GBM immunotargeting, but to date, no monoclonal antibody has been isolated that can target an extracellular MRP3 epitope. By phage display, we have isolated 3 recombinant, fully human, single-chain Fv (scFv) antibodies, M25, M58 and M89, which specifically react with the extracellular N-terminus of human MRP3. In ELISA, these scFvs reacted only with the peptide used for screening and not with other MRP3-derived peptides. Flow cytometric analysis revealed that these scFv fragments bind specifically to viable human GBM cells displaying different MRP3 expression levels, but not to MRP3-null cells. Furthermore, these scFv antibodies failed to react with tumor cells overexpressing other MRP proteins, including MRP1, MRP2, MRP4 and MRP5. M25 and M58 also bound to viable neurospheres. Iodogen-labeled scFvs demonstrated a yield of 56-76%. The immunoreactive fractions of the radiolabeled M25, M58 and M89 scFvs were 32, 52 and 69%, respectively. M25 exhibited 20% internalization into D2159MG neurospheres, M58, 33% into D54MG cells and M89, 26% into D247MG. Immunohistochemical evaluation of human gliomas to determine the localization of MRP3 antigen using scFvs M25 and M58 showed a dense cytoplasmic and membranous staining pattern. These Fv-based recombinant antibodies, which possess superior tumor penetration capabilities and selectively target tumor cells that express MRP3, may potentially be used in immunotherapy and diagnosis for brain tumors and other cancers.

Regulation Of Migration and Proliferation of Glioma Cells by Modulating the Ion Channel Activity.

The movement of cations through ion channels is essential for the migration of glioma cells. We have previously reported that malignant glioma cells express an amiloride sensitive cation conductance that is not detected in normal astrocytes. In the present study, we investigate the potential role of this ion channel to regulate the migration and cell cycle processes of glioma cells. We found that the blocking of Acid Sensitive Ion Channel 1 by Psalmotoxin‐1 (40 amino acid synthetic peptide) significantly inhibited both migration and proliferation of D54MG and U251 glioma cells, using in vitro transwell migration and in‐vitro scratch wound assays. We also found that both PcTx‐1 and benzamil caused a significant cell cycle arrest of D54MG cells in G0/G1 (by 18% and 19%, respectively), and reduced accumulation in the S phase (by 61% and 49%, respectively), and G2/M phase (by 51% and 32%, respectively) (n=3–4; P&lt;0.01) at 24h of incubation. PcTX‐1 and benzamil showed the same effect after 48h of incubation time. An unfolded control peptide had no significant effect compared to PcTX‐1. PcTX‐1 also induced upregulation of cyclin dependent kinase inhibitor (CDKI) proteins (p21Cip1 and p27Kip1) in D54MG cells. Thus, our data implicate the potential role of this cation conductance in migration and in progression of the cell cycle in glioma cells. This study was supported by NIH grants CA101952 and DK037206.

An ASIC1 and ENaC hybrid channel is the amiloride sensitive channel in human glioma.

High grade gliomas express multiple members of the Epithelial Sodium Channel (ENaC)/Degenerin (Deg) family, displaying a basally active amiloride sensitive cation current. We hypothesize that this glioma current is mediated by a hybrid channel composed of a mixture of Epithelial Sodium Channel (ENaC) and Acid Sensing Ion Channel (ASIC) subunits. Using immunohistochemisty we show a higher expression level of ASIC1, αENaC, and γENaC in glioblastoma biopsy tissue compared with non‐neoplastic brain tissue. To test our hypothesis we made dominant negative (DN) cDNAs for ASIC1, αENaC, and γENaC. D54‐MG cells transfected with the DN constructs for ASIC1, αENaC or γENaC showed reduced protein expression and a significant reduction in the amiloride sensitive whole cell current as compared with untransfected D54‐MG cells. Using co‐immunoprecipitation we show interactions between ASIC1, αENaC, and γENaC at the plasma membrane of D54‐MG cells, consistent with these subunits interacting with each other to form an ion channel. Data using Total Internal Resolution Fluorescent Microscopy (TIRFM) also suggest that ASIC1 and ASIC2 interact with γENaC in CHO‐K1 cells. These data suggest that different ENaC/Deg subunits combine to form a hybrid channel that likely underlies the amiloride‐sensitive current in human gliomas. This study was supported by NIH Grants CA101952 and DK037206.

Annexin A2 Is a Molecular Target for TM601, a Peptide with Tumor-targeting and Anti-angiogenic Effects

TM601 is a synthetic form of chlorotoxin, a 36-amino acid peptide derived from the venom of the Israeli scorpion, Leirius quinquestriatus, initially found to specifically bind and inhibit the migration of glioma cells in culture. Subsequent studies demonstrated specific in vitro binding to additional tumor cell lines. Recently, we demonstrated that proliferating human vascular endothelial cells are the only normal cell line tested that exhibits specific binding to TM601. Here, we identify annexin A2 as a novel binding partner for TM601 in multiple human tumor cell lines and human umbilical vein endothelial cell (HUVEC). We demonstrate that the surface binding of TM601 to the pancreatic tumor cell line Panc-1 is dependent on the expression of annexin A2. Identification of annexin A2 as a binding partner for TM601 is also consistent with the anti-angiogenic effects of TM601. Annexin A2 functions in angiogenesis by binding to tissue plasminogen activator and regulating plasminogen activation on vascular endothelial cells. We demonstrate that in HUVECs, TM601 inhibits both vascular endothelial growth factor- and basic fibroblast growth factor-induced tissue plasminogen activator activation, which is required for activation of plasminogen to plasmin. Consistent with inhibition of cell surface protease activity, TM601 also inhibits platelet-derived growth factor-C induced trans-well migration of both HUVEC and U373-MG glioma cells.

Knockdown of ASIC1 and Epithelial Sodium Channel Subunits Inhibits Glioblastoma Whole Cell Current and Cell Migration

High grade gliomas such as glioblastoma multiforme express multiple members of the epithelial sodium channel (ENaC)/Degenerin family, characteristically displaying a basally active amiloride-sensitive cation current not seen in normal human astrocytes or lower grade gliomas. Using quantitative real time PCR, we have shown higher expression of ASIC1, alphaENaC, and gammaENaC in D54-MG human glioblastoma multiforme cells compared with primary human astrocytes. We hypothesize that this glioma current is mediated by a hybrid channel composed of a mixture of ENaC and acid-sensing ion channel (ASIC) subunits. To test this hypothesis we made dominant negative cDNAs for ASIC1, alphaENaC, gammaENaC, and deltaENaC. D54-MG cells transfected with the dominant negative constructs for ASIC1, alphaENaC, or gammaENaC showed reduced protein expression and a significant reduction in the amiloride-sensitive whole cell current as compared with untransfected D54-MG cells. Knocking down alphaENaC or gammaENaC also abolished the high P(K)(+)/P(Na)(+) of D54-MG cells. Knocking down deltaENaC in D54-MG cells reduced deltaENaC protein expression but had no effect on either the whole cell current or K(+) permeability. Using co-immunoprecipitation we show interactions between ASIC1, alphaENaC, and gammaENaC, consistent with these subunits interacting with each other to form an ion channel in glioma cells. We also found a significant inhibition of D54-MG cell migration after ASIC1, alphaENaC, or gammaENaC knockdown, consistent with the hypothesis that ENaC/Degenerin subunits play an important role in glioma cell biology.

Effects of Sodium Butyrate on Cell Death Induced by Photodynamic Therapy in U373‐MG and D54‐MG Astrocytoma Cell Lines

The damage induced by end products of photodynamic therapy (PDT) in astrocytoma tumors leads to cytotoxicity and cell death. Chromatin modifiers such as sodium butyrate (NaB) induce several genes involved in apoptosis, among others. The PDT improvement was evaluated by the measurement of its effectiveness in the treatment of U373-MG and D54-MG astrocytoma cell lines exposed to NaB. Cells exposed to 80 microg mL(-1) of delta-aminolevulinic acid (ALA) as precursor of endogenous photosensitizer (PS), protoporphyrin IX (PpIX), induced 16.67% and 28.9% of mortality in U373-MG and D54-MG, respectively. The mortality increased to 70.62% and 96.7%, respectively, when U373-MG and D54-MG cells were exposed for 24 h to 8 mm NaB prior to ALA-induction. In this condition, re-expression of some genes related to apoptosis in U373-MG, and differentiation in D54-MG were induced. PpIX accumulation was higher than ALA-induction and the acetylation of histone H4 induced by NaB was verified by immunocytochemistry in both cells. It can be concluded that modified chromatin and genes induced by NaB increment the cellular death induced by PDT in astrocytoma cells using PpIX as endogenous PS.

Epithelial Sodium Channel and Acid Sensing Ion Channel Subunits Interact With Each Other To Play A Role In Glioblastoma Whole Cell Current

High grade gliomas like glioblastoma (GBM) characteristically display a basally active amiloride sensitive cation current not seen in normal human astrocytes. We hypothesize this current to be mediated by a hybrid channel composed of a mixture of Epithelial Sodium Channel (ENaC)/ Degenerins (Deg) subunits. Using semi‐quantitative RT‐PCR we have shown higher expression of ASIC1, αENaC, and γENaC in primary GBM cells compared to primary human astrocytes. We have also shown the expression of these subunits in GBM biopsies and on the plasma membrane of GBM cells using immunocytochemistry. To test our hypothesis we made dominant negative (DN) cDNAs for ASIC1, αENaC, γENaC, and δENaC. D54‐MG cells transfected with the DN constructs for ASIC1, αENaC or γENaC showed 50‐ 70% reduction in protein expression and significant inhibition of whole cell patch clamp current compared with untransfected D54‐MG. Knocking down δENaC in D54‐MG cells reduced δENaC protein expression by 50%, but had no effect on the whole cell patch clamp current. Using co‐immunoprecipitation we show interactions between ASIC1, αENaC, and γENaC, consistent with these subunits interacting with each other to form an ion channel in glioma cells. These data suggest that different ENaC/Deg subunits combine to form a hybrid channel that likely underlies the amiloride‐sensitive current in human gliomas. This study was supported by NIH Grant CA101952.