Growth Of Malignant Glioma Cells Research Articles

Antitumor effect of memantine is related to the formation of the splicing isoform of GLG1, a decoy FGF‑binding protein.

Drug repositioning is a strategy for repurposing the approved or investigational drugs that are outside the scope of the original medical indication. Memantine is used as a non‑competitive N‑methyl‑D‑aspartate receptor antagonist to prevent glutamate‑mediated excitotoxicity in Alzheimer's disease, and is one of the promising agents which is utilized for the purpose of cancer therapy. However, the association between memantine and Golgi glycoprotein1 (GLG1), an intracellular fibroblast growth factor receptor, in cancers has not yet been clarified. The present study analyzed the expression and location of GLG1 in tumor cells treated with memantine. Memantine was found to suppress the growth of malignant glioma and breast cancer cells in a concentration‑dependent manner. The mRNA expression of GLG1 was upregulated in a concentration‑dependent manner, and the splicing variant profiles were altered in all cell lines examined. The results of western blot analysis revealed an increase in the full‑length and truncated forms of GLG1. Moreover, GLG1 spread in the cytosol of memantine‑treated cells, whereas it localized in the Golgi apparatus in control cells. Since GLG1 functions as a decoy FGF receptor, the modulation of GLG1 may prove to be one of the mechanisms underlying the cancer‑suppressive effects of memantine.

Enzymatic activity of glycosyltransferase GLT8D1 promotes human glioblastoma cell migration

SummaryGlioblastoma (GBM) is the most aggressive primary brain tumor characterized by infiltrative growth of malignant glioma cells into the surrounding brain parenchyma. In this study, our analysis of GBM patient cohorts revealed a significantly higher expression of Glycosyltransferase 8 domain containing 1 (GLT8D1) compared to normal brain tissue and could be associated with impaired patient survival. Increased in vitro expression of GLT8D1 significantly enhanced migration of two different sphere-forming GBM cell lines. By in silico analysis we predicted the 3D-structure as well as the active site residues of GLT8D1. The introduction of point mutations in the predicted active site reduced its glycosyltransferase activity in vitro and consequently impaired GBM tumor cell migration. Examination of GLT8D1 interaction partners by LC-MS/MS implied proteins associated with cytoskeleton and intracellular transport as potential substrates. In conclusion, we demonstrated that the enzymatic activity of glycosyltransferase GLT8D1 promotes GBM cell migration.

TRPM7 Induces Tumorigenesis and Stemness Through Notch Activation in Glioma.

We have reported that transient receptor potential melastatin-related 7 (TRPM7) regulates glioma stem cells (GSC) growth and proliferation through Notch, STAT3-ALDH1, and CD133 signaling pathways. In this study, we determined the major contributor(s) to TRPM7 mediated glioma stemness by further deciphering each individual Notch signaling. We first determined whether TRPM7 is an oncotarget in glioblastoma multiforme (GBM) using the Oncomine database. Next, we determined whether TRPM7 silencing by siRNA TRPM7 (siTRPM7) induces cell growth arrest or apoptosis to reduce glioma cell proliferation using cell cycle analysis and annexin V staining assay. We then examined the correlations between the expression of TRPM7 and Notch signaling activity as well as the expression of GSC markers CD133 and ALDH1 in GBM by downregulating TRPM7 through siTRPM7 or upregulating TRPM7 through overexpression of human TRPM7 (M7-wt). To distinguish the different function of channel and kinase domain of TRPM7, we further determined how the α-kinase-dead mutants of TRPM7 (α-kinase domain deleted/M7-DK and K1648R point mutation/M7-KR) affect Notch activities and CD133 and ALDH1 expression. Lastly, we determined the changes in TRPM7-mediated regulation of glioma cell growth/proliferation, cell cycle, and apoptosis by targeting Notch1. The Oncomine data revealed a significant increase in TRPM7 mRNA expression in anaplastic astrocytoma, diffuse astrocytoma, and GBM patients compared to that in normal brain tissues. TRPM7 silencing reduced glioma cell growth by inhibiting cell entry into S and G2/M phases and promoting cell apoptosis. TRPM7 expression in GBM cells was found to be positively correlated with Notch1 signaling activity and CD133 and ALDH1 expression; briefly, downregulation of TRPM7 by siTRPM7 decreased Notch1 signaling whereas upregulation of TRPM7 increased Notch1 signaling. Interestingly, kinase-inactive mutants (M7-DK and M7-KR) resulted in reduced activation of Notch1 signaling and decreased expression of CD133 and ALDH1 compared to that of wtTRPM7. Finally, targeting Notch1 effectively suppressed TRPM7-induced growth and proliferation of glioma cells through cell G1/S arrest and apoptotic induction. TRPM7 is responsible for sustained Notch1 signaling activation, enhanced expression of GSC markers CD133 and ALDH1, and regulation of glioma stemness, which contributes to malignant glioma cell growth and invasion.

CSIG-13. TRPM7 INDUCES TUMORIGENESIS AND STEMNESS THROUGH NOTCH ACTIVATION IN GLIOMA

Abstract Our group found that the inhibitory effect of TRPM7 on proliferation and invasion of human glioma cell is mediated by multiple mechanisms. TRPM7 regulates miR-28-5p expression, which suppresses cell proliferation and invasion in glioma cells by targeting Ras-related protein Rap1b. In particular, our group found that TRPM7 channels regulate glioma stem cell (GSC) growth/proliferation through STAT3 and Notch signaling. However, which Notch component(s) is crucial for its activity regulated by TRPM7, and its relationship with other GSC markers, such as CD133 and ALDH1, remain unclear. In the current project, we elucidate the mechanisms of TRMP7’s regulation of Notch signaling pathway that contribute to the development and progression of glioma and maintenance of self-renewal and tumorigenicity of GSC using multiple glioma cell lines (GC) with different molecular subtypes and GSCs derived from the GC lines. 1) We first analyzed TRPM7 expression using the Oncomine database (https://www.oncomine.org) and found that the TRPM7 mRNA expression is significantly increased in anaplastic astrocytoma, diffuse astrocytoma, and GBM patients compared to that in normal brain tissue controls. 2) TRPM7 is expressed in GBM, and its channel activity is correlated with Notch1 activation. Inhibition of TRPM7 downregulates Notch1 signaling, while upregulation of TRPM7 upregulates Notch1 signaling. 3) GSC markers, CD133 and ALDH1, are correlated with TRPM7 in GBM. 4) Targeting TRPM7 suppresses the growth and proliferation of glioma cells through G1/S arrests and apoptosis of glioma cells. 5) Targeting Notch1 suppresses the TRPM7-induced growth and proliferation of glioma cells, as well as the expression of GSC markers CD133 and ALDH1. In summary, TRPM7 is responsible for sustained Notch signaling activation, enhanced expression of GSC markers, and regulation of glioma stemness, which contribute to malignant glioma cell growth and invasion. Notch1 and ligand DII4 are key components that contribute GSC stemness.

Evaluation of combination gene therapy with SLC22A18 upregulation and sequence binding protein 1 downregulation for glioma U251 cells in vitro and in vivo

Background and Aim: Our previous study demonstrated that SLC22A18 downregulation through promoter methylation and Sequence binding protein 1 (SATB1) upregulation are associated with the development and progression of glioma. This study aimed to examine the effect of combined SLC22A18 and short hairpin RNA (shRNA) targeting SATB1 gene therapy on glioma growth and invasion. Materials and Methods: Here, a combined gene therapy to upregulate SLC22A18 and downregulate SATB1 in malignant glioma was evaluated both in vitro and in vivo. Glioma U251 cells overexpressing SLC22A18 and underexpressing SATB1 were generated to investigate the effects of these changes on cell survival, as measured by the methyl thiazol tetrazolium assay, and on cell invasion, as measured by cell invasion and migration assays. In addition, analysis of the cell cycle was performed using flow cytometry in vitro. The animal experiments were approved by the Ethics Committee of Shanghai Ninth People's Hospital Affiliated to Shanghai Jiao Tong University School of Medicine and Zhongnan Hospital of Wuhan University Ethics Committees (approval No. ZNHWHU0389, NTPHSHJTUSM046). Results: The upregulation of SLC22A18 and downregulation of SATB1 significantly inhibited growth and invasion in vitro and reduced in vivo tumor growth of human glioma U251 cells. Furthermore, we revealed that U251 cells were arrested at the G0/G1 phase. Similar data for apoptotic glioma cell death were obtained in tumor cells from an in vivo glioma xenograft model after transfection. At the same total dose, the therapeutic effect was markedly better in the glioma xenografts transfected with both SLC22A18 gene and SATB1 short hairpin RNA (shRNA) expression vectors compared with tumors transfected with either agent alone. The levels of SLC22A18 and SATB1 protein expression were respectively increased and decreased in the glioma cells.Conclusion: These results demonstrate that combination treatment with SLC22A18 gene and SATB1 shRNA expression vectors effectively inhibits the growth of human malignant glioma cells both in vitro and in glioma xenografts in vivo, suggesting a promising novel strategy for glioma therapy that warrants further study.

Novel Chemically Synthesized, Alpha-Mangostin-Loaded Nano-Particles, Enhanced Cell Death Through Multiple Pathways Against Malignant Glioma.

Mangostin is a hydrophobic agent with potential anticancer activity. Molecular dynamics computer simulation indicated methoxy poly(ethylene glycol)-poly(lactide) (MPEG-PLA) and α-mangostin (α-M) have good compatibility. The α-M-loaded nano-particles acting as an anticancer agents against growth of glioma cells were prepared by self-assembly methods. In this study, the effects of α-M-loaded nano-particles, α-M/MPEG-PLA with drug loading of 15% and a mean particle size of 32 nm, on the growth of glioma cells were examined both in vitro and in vivo. The occurrence of changes in the cell signaling molecules and expression levels of various proteins related to cell death and glioma xenograft models (i.e., zebra fish, subcutaneous-mouse and orthotopic-mouse) growth following the administration of α-M/MPEG-PLA were investigated. The novel α-M/MPEG-PLA inhibited the growth of malignant glioma cells, induced cells apoptosis with cleaved caspases expression and DNA fragmentation, along with down-regulation of anti-apoptotic molecules and up-regulation of apoptotic molecules. Furthermore, decreased proliferation as well as vascularization of the tumor in vivo models were significantly achieved. A dramatic induction of programmed cell death was found in malignant glioma cells after treatment with synthetic α-M/MPEG-PLA. These results suggest that synthetic α-M/MPEG-PLA could be a promising novel anticancer agent for performing chemotherapy against malignant glioma.

Hyperbaric oxygen inhibits production of CD3+ T cells in the thymus and facilitates malignant glioma cell growth.

ObjectiveHyperbaric oxygen (HBO) is an emerging complementary alternative medical approach in glioma treatment. However, its mode of action is unknown, so this was investigated in the present study.MethodsWe constructed an intracranial glioma model of congenic C57BL/6J mice. Glioma growth under HBO stimulation was assessed by bioluminescent imaging and magnetic resonance imaging. Flow cytometry assessed direct effects of HBO on reactive oxygen species (ROS) signaling of transplanted glioma cells and organs, and quantified mature T cells and subgroups in tumors, the brain, and blood.ResultsHBO promoted the growth of transplanted GL261-Luc glioma in the intracranial glioma mouse model. ROS signaling of glioma cells and brain cells was significantly downregulated under HBO stimulation, but thymus ROS levels were significantly upregulated. CD3+ T cells were significantly downregulated, while both Ti/Th cells (CD3+CD4+) and Ts/Tc cells (CD3+CD8+) were inhibited in tumors of the HBO group. The percentage of regulatory T cells in Ti/Th (CD3+CD4+) cells was elevated in the tumors and thymuses of the HBO group.ConclusionHBO induced ROS signaling in the thymus, inhibited CD3+ T cell generation, and facilitated malignant glioma cell growth in vivo in the intracranial glioma mouse model.

Combinatorial therapy with adenoviral-mediated PTEN and a PI3K inhibitor suppresses malignant glioma cell growth in vitro and in vivo by regulating the PI3K/AKT signaling pathway

Glioblastoma is a highly invasive and challenging tumor of the central nervous system. The mutation/deletion of the tumor suppressor phosphatase and tensin homolog (PTEN) gene is the main genetic change identified in glioblastomas. PTEN plays a critical role in tumorigenesis and has been shown to be an important therapeutic target. The phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 is commonly used to inhibit glioma cell growth via regulation of the PI3K/AKT signaling pathway. In this study, we examined the growth inhibitory effects of a combinatorial therapy of adenoviral-mediated PTEN (Ad-PTEN) and LY294002 on LN229 and U251 glioma cells in vitro and on tumor xenografts in vivo. In vitro, LN229 and U251 glioma cells were treated by combinatorial therapy with Ad-PTEN and LY294002. The growth ability was determined by MTT assay. The cell cycle distribution was analyzed by flow cytometry. Cell invasive ability was analyzed by transwell invasion assay and cell apoptosis analysis via FITC-Annexin V analysis. In vivo, U251 subcutaneous glioblastoma xenograft was used to assay anti-tumor effect of combinatorial therapy with Ad-PTEN and LY294002 by mean volume of tumors, immunohistochemistry and TUNEL method. The combinatorial treatment clearly suppressed cell proliferation, arrested the cell cycle, reduced cell invasion and promoted cell apoptosis compared with the Ad-PTEN or LY294002 treatment alone. The treatment worked by inhibiting the PI3K/AKT pathway. In addition, the growth of U251 glioma xenografts treated with the combination of Ad-PTEN and LY294002 was significantly inhibited compared with those treated with Ad-PTEN or LY294002 alone. Our data indicated that the combination of Ad-PTEN and LY294002 effectively suppressed the malignant growth of human glioma cells in vitro and in tumor xenografts, suggesting a promising new approach for glioma gene therapy that warrants further investigation.

Enhanced inhibition of malignant glioma cell growth in vitro. By Sonochemical Internalization of Bleomycin

Enhanced inhibition of malignant glioma cell growth in vitro. By Sonochemical Internalization of Bleomycin

Hyperbaric oxygen promotes malignant glioma cell growth and inhibits cell apoptosis.

Glioblastoma multiforme (GBM) is the most frequently diagnosed intracranial malignant tumor in adults. Clinical studies have indicated that hyperbaric oxygen may improve the prognosis and reduce complications in glioma patients; however, the specific mechanism by which this occurs remains unknown. The present study investigated the direct effects of hyperbaric oxygen stimulation on glioma by constructing an intracranial transplanted glioma model in congenic C57BL/6J mice. Bioluminescent imaging (BLI) was used to assess the growth of intracranial transplanted GL261-Luc glioma cells in vivo, while flow cytometric and immunohistochemical assays were used to detect and compare the expression of the biomarkers, Ki-67, CD34 and TUNEL, reflecting the cell cycle, apoptosis and angiogenesis. BLI demonstrated that hyperbaric oxygen promoted the growth of intracranially transplanted GL261-Luc glioma cells in vivo. Flow cytometric analysis indicated that hyperbaric oxygen promoted GL261-Luc glioma cell proliferation and also prevented cell cycle arrest. In addition, hyperbaric oxygen inhibited the apoptosis of the transplanted glioma cells. Immunohistochemical analysis also indicated that hyperbaric oxygen increased positive staining for Ki-67 and CD34, while reducing staining for TUNEL (a marker of apoptosis). The microvessel density was significantly increased in the hyperbaric oxygen treatment group compared with the control group. In conclusion, hyperbaric oxygen treatment promoted the growth of transplanted malignant glioma cells in vivo and also inhibited the apoptosis of these cells.

Suppression of TRPM7 inhibits proliferation, migration, and invasion of malignant human glioma cells.

Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor with a dismal prognosis. Despite intensive study on tumor biology, the underlying mechanisms of the unlimited proliferation and progressive local invasion are still poorly understood, and no effective treatment has been developed for GBM patients. We determine the role of TRPM7 channels in the growth, migration, and infiltration of malignant glioma cells. Using a combination of RT-PCR, Western blot, and patch-clamp techniques, we demonstrated the expression of functional TRPM7 channels of A172 cells, a human glioma cell line, as well as in human glioma tissues. Furthermore, we evaluated the role of TRPM7 in growth, migration, and infiltration of A172 cells with MTT and transwell migration and invasion assays. We showed the expression of functional TRPM7 channels in both A172 cells and human glioma tissues. Suppression of TRPM7 expression with TRPM7-siRNA dramatically reduced the proliferation, migration, and invasion of A172 cells. Pharmacological inhibition of TRPM7 channel with 2-aminoethoxydiphenyl borate (2-APB) showed a similar effect as TRPM7-siRNA. We demonstrate that human glioma cells express functional TRPM7 channel and that activation of this channel plays an important role in the proliferation, migration, and invasion of malignant glioma cells. TRPM7 channel may represent a novel and promising target for therapeutic intervention of malignant glioma.

Efficacy of ribavirin against malignant glioma cell lines.

Ribavirin (1-β-D-ribofuranosy-1,2,4-triazole-3-carboxamide) has been widely administered as an antiviral agent against RNA and DNA viruses. Ribavirin, in combination with interferon, has predominantly been applied in the treatment of the hepatitis C virus infection and its potential antitumor efficacy has recently become a point of interest. The aim of the present study was to evaluate the effect of ribavirin on the growth of malignant glioma cells, to identify novel predictive genes in malignant glioma cells (by analyzing gene expression profiles) and to assess the influence of ribavirin on the cell cycle of malignant glioma cells. The present study evaluated the antitumor efficacy of ribavirin against various malignant glioma cell lines (A-172, AM-38, T98G, U-87MG, U-138MG, U-251MG and YH-13). After culturing the cells in ribavirin-containing culture medium (final concentration, 0–1,000 μM) for 72 h, the viable proliferated cells were harvested and counted. The half maximal inhibitory concentration of ribavirin, with regard to the growth of the malignant glioma cell lines, was determined from the concentration of ribavirin required for 50% growth inhibition in comparison to the untreated control cells. Furthermore, the current study identified the genes in which the gene expression levels correlated with the ribavirin sensitivity of the malignant glioma cells lines, using a high-density oligonucleotide array. Finally, cell cycle analysis was performed on the U-87MG cell line. It was identified that ribavirin inhibited the growth of all of the malignant glioma cell lines in a dose-dependent manner, although the ribavirin sensitivity varied between each cell line. Of the extracted genes, PDGFRA demonstrated the strongest positive correlation between gene expression level and ribavirin sensitivity. Cell cycle analysis of the U-87MG cell line demonstrated that ribavirin treatment induces G0/G1 arrest and thus may be an effective agent for inhibiting malignant glioma cell growth. Therefore, the results of the current study indicate that ribavirin may have potential as a therapeutic agent in the treatment of malignant gliomas.

ROS-dependent prostate apoptosis response-4 (Par-4) up-regulation and ceramide generation are the prime signaling events associated with curcumin-induced autophagic cell death in human malignant glioma

Malignant gliomas are extremely resistant to therapies that induce apoptosis, but are less resistant to therapies that induce autophagy. Therefore, drugs targeting autophagy are promising in the management of malignant gliomas. In this study, we investigated the anti-glioma potential of curcumin in vitro, and further examined the molecular mechanisms of curcumin-induced cell death in human malignant glioma. Here, we provide evidence that curcumin is cytotoxic against human malignant glioma cell lines, and the mechanism of cell death caused by curcumin is associated with features of autophagy. Curcumin suppresses the growth of human malignant glioma cells via ROS-dependent prostate apoptosis response-4 (Par-4) induction and ceramide generation. Extracellular supplementation of antioxidants such as glutathione and N-acetylcysteine to glioma cells abrogated the Par-4 induction, ceramide generation, and in turn, prevented curcumin-induced autophagic cell death. Moreover, tumor cells transfected with Par-4 gene sensitized the curcumin-induced autophagic cell death. Overall, this study describes a novel signaling pathway by which curcumin induces ROS-dependent Par-4 activation and ceramide generation, leading to autophagic cell death in human malignant glioma cells.

Mature BDNF promotes the growth of glioma cells in vitro

High-grade glioma is incurable and is associated with a short survival time and a poor prognosis. There are two forms of brain-derived neurotrophic factor (BDNF), proBDNF and mature BDNF, which exert opposite effects. Their diverse actions are mediated through two different transmembrane receptor signalling systems: p75NTR and TrkB. The important roles of the BDNF/TrkB signalling system in tumour cell proliferation and survival have been demonstrated. However, few studies have been able to distinguish mature BDNF from proBDNF due to the limitation of specific antibodies. Using specific proBDNF antibodies, we demonstrated that the proBDNF/p75NTR pathway appears to inhibit malignant glioma cell growth and migration. In the present study using specific mature BDNF antibodies, we found that mature BDNF inhibited C6 glioma cell apoptosis and increased cell growth and migration in vitro. Our data suggest that the counterbalance between mature BDNF and proBDNF may regulate tumour growth.

Effect of flupirtine on the growth and viability of U373 malignant glioma cells.

ObjectiveFlupirtine is a non-opioid analgesic without antipyretic or antiphlogistic properties but with favorable tolerability in humans. This analgesic also exhibits neuroprotective activities. Furthermore, flupirtine antagonizes glutamate- and NMDA-induced intracellular levels of Ca2+ and counteracts the effects of focal cerebral ischemia. Although flupirtine has been used to relieve pain caused by different diseases and clinical procedures, information on the safety and efficacy of flupirtine is limited. The present study was conducted to investigate the neuroprotective effects of flupirtine on U373 malignant glioma (MG) cell lines.MethodsCell viability and cell cycle analysis was performed by MTT assay and flow cytometry, respectively.ResultsVariations in the growth of U373 MG cells in 5 mM N-methyl-D-aspartate (NMDA), 1 mM flupirtine, and combined treatment indicated the antagonistic effects of NMDA and flupirtine on MG cell lines. The variation in the percentage of gated cell population in different cell cycle phases showed significant variations after 48 h of treatment.ConclusionFlupirtine has neuroprotective effect of on U373 MG cells, which limits its use in the pain management of brain tumors. This property warrants further studies using animal models and large-scale clinical trials.

Alphaxalone inhibits growth, migration and invasion of rat C6 malignant glioma cells

Malignant gliomas are the most devastating and aggressive brain tumors affecting the central nervous system. The insidious growth and infiltration are the most prominent characteristics of malignant gliomas, which render the current therapies for malignant gliomas including surgery, radiation and chemotherapy unsuccessful. Inhibition of infiltration as well as proliferation in combination with surgery might be more effective in the treatment of malignant gliomas. In the current study, we demonstrate the alphaxalone (3-hydroxypregnane-11,20-dione) could effectively inhibit the proliferation of C6 glioma cells in a concentration dependent manner. Moreover, this compound could also suppress the migration and invasion of C6 glioma cells at a concentration without causing significant cytotoxicity. Except the in vitro anti-glioma activity, alphaxalone effectively delayed the growth of rat C6 malignant glioma xenografts in vivo. Together, these findings suggest alphaxalone might be a promising candidate for the treatment of malignant gliomas and may also provide helpful clues for anti-glioma drugs development in future.

Antisense MMP-9 RNA inhibits malignant glioma cell growth in vitro and in vivo

The matrix-degrading metalloproteinases (MMPs), particularly MMP-9, play important roles in the pathogenesis and development of malignant gliomas. In the present study, the oncogenic role of MMP-9 in malignant glioma cells was investigated via antisense RNA blockade in vitro and in vivo. TJ905 malignant glioma cells were transfected with pcDNA3.0 vector expressing antisense MMP-9 RNA (pcDNA-ASMMP9), which significantly decreased MMP-9 expression, and cell proliferation was assessed. For in vivo studies, U251 cells, a human malignant glioma cell line, were implanted subcutaneously into 4- to 6-week-old BALB/c nude mice. The mice bearing well-established U251 gliomas were treated with intratumoral pcDNA-AS-MMP9-Lipofectamine complex (AS-MMP-9-treated group), subcutaneous injection of endostatin (endostatin-treated group), or both (combined therapy group). Mice treated with pcDNA (empty vector)-Lipofectamine served as the control group. Four or eight weeks later, the volume and weight of tumor, MMP-9 expression, microvessel density and proliferative activity were assayed. We demonstrate that pcDNA-AS-MMP9 significantly decreased MMP-9 expression and inhibited glioma cell proliferation. Volume and weight of tumor, MMP-9 expression, microvessel density and proliferative activity in the antisense-MMP-9-treated and therapeutic alliance groups were significantly lower than those in the control group. The results suggest that MMP-9 not only promotes malignant glioma cell invasiveness, but also affects tumor cell proliferation. Blocking the expression of MMP-9 with antisense RNA substantially suppresses the malignant phenotype of glioma cells, and thus can be used as an effective therapeutic strategy for malignant gliomas.

Nicotine enhances proliferation, migration, and radioresistance of human malignant glioma cells through EGFR activation

It has been suggested that continued tobacco use during radiation therapy contributes to maintenance of neoplastic growth despite treatment with radiation. Nicotine is a cigarette component that is an established risk factor for many diseases, neoplastic and otherwise. The hypothesis of this work is that nicotine promotes the proliferation, migration, and radioresistance of human malignant glioma cells. The effect of nicotine on cellular proliferation, migration, signaling, and radiation sensitivity were evaluated for malignant glioma U87 and GBM12 cells by use of the AlamarBlue, scratch healing, and clonogenic survival assays. Signal transduction was assessed by immunoblotting for activated EGFR, ERK, and AKT. At concentrations comparable with those found in chronic smokers, nicotine induced malignant glioma cell migration, growth, colony formation, and radioresistance. Nicotine increased phosphorylation of EGFR(tyr992), AKT(ser473), and ERK. These molecular effects were reduced by pharmacological inhibitors of EGFR, PI3K, and MEK. It was therefore concluded that nicotine stimulates the malignant behavior of glioma cells in vitro by activation of the EGFR and downstream AKT and ERK pathways.

Regulation of the FABP7 gene by PAX6 in malignant glioma cells

Brain fatty acid-binding protein (FABP7) and PAX6 are both expressed in radial glial cells and have been implicated in neurogenesis and glial cell differentiation. FABP7 and PAX6 have also been postulated to play a role in malignant glioma cell growth and invasion. Here, we address the role of PAX6 in regulating FABP7 gene expression in malignant glioma cells. We report that PAX6 and FABP7 RNA are generally co-expressed in malignant glioma cell lines, tumors and tumor neurospheres. Using the CAT reporter gene assay, we show that FABP7 promoter activity is upregulated by PAX6. Sequential deletion analysis of the FABP7 promoter, combined with gel shift and supershift assays demonstrate the presence of a PAX6 responsive region located upstream of the FABP7 gene, at −862 to −1033bp. Inclusion of sequences between −1.2 and −1.8kb reduced CAT activity, suggesting the presence of a repressor element within this region. While PAX6 overexpression did not induce endogenous FABP7 expression in FABP7-negative cells, knock-down of PAX6 in PAX6-positive malignant glioma cells resulted in reduced FABP7 levels. These data provide the first evidence of direct transactivation of the FABP7 proximal promoter by PAX6 and suggest a synergistic mechanism for PAX6 and other co-factor(s) in regulating FABP7 expression in malignant glioma.

Abstract 5357: Evaluation of colony formation and growth of malignant glioma cells in soft agar using advanced colony counter system

Abstract [Introduction] Malignant gliomas are the most common primary tumors of the central nervous system. The prognosis of patients with malignant gliomas is poor in spite of current intensive therapy because of inevitable regrowth or recurrence. Since glioma stem-like cells (GSCs) are thought to play critical role in initiation, regrowth and recurrence, effective therapy against GSCs is sought for. Efficient anticancer therapies should suppress not only the growth of tumor mass but also colony formation initiated by single cancer stem cell. Therefore, it is essential to analyze the effect of anticancer therapies on both tumor growth and colony formation. Regarding tumor growth, multicellular tumor spheroid (MCTS) model is thought to be more plausible method than 2-D culture because MCTSs resemble intervascular regions of solid tumors, that is, MCTSs reproduce 3-D structure-specific features such as intense paracrine, cell-cell adhesion related signaling, direct intercellular transport of materials, gradient of oxygen and nutrients, and lower drug penetration into the spheroids. Since GSCs form MCTSs in the standard stem cell culture media, viability and growth of GSCs have been analyzed mainly in this culture condition. However, regarding analysis of clonogenicity, colony formation assay using GSCs has been a challenge because GSCs aggregate in the stem cell culture media and evaluation of the accurate colony number requires single cell culture system that might need large number of wells/plates, especially for combination treatment, and might not be always appropriate for high-throughput screening. Therefore, a better approach is necessary. GelCountTM colony counter was developed to analyze both number and volume of colonies. In this study, we used soft agar and GelCountTM to analyze colony formation and growth of malignant glioma cells including GSCs. [Materials and Methods] Human glioma stem-like cells GSC11 and GSC20, or human malignant glioma cell line U87-MG were cultured in agarose gel and treated with erlotinib, sorafenib, imatinib or a polynuclear platinum compound BBR3610. The colony number and the calculated total colony volume (total biomass) were analyzed using GelCountTM colony counter system (Oxford Optronix Inc., UK). [Results] Glioma cells formed 3-D colonies in agarose, and the colony number and the total biomass increased according to the Gompertz function that is known to correlate with tumor growth in vivo. Each drug suppressed increase of colony number and total biomass in comparable concentration to that in 2-D culture. [Conclusions] GelCountTM allowed accurate, rapid monitoring of colony number and size at many time points without harm to the cultures. This system can be high throughput screening method using GSCs and also bridge the gap between 2-D culture assays and in vivo tumor formation assays. 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 5357. doi:1538-7445.AM2012-5357