Glioma

Experimental evidence suggests that the transcription factor nuclear factor-Kappa B (NF-kB) plays an important role in tumor cell invasion, apoptosis suppression and growth. Malignant glioma is one of the most intractable tumors because of its invasiveness to surrounding brain tissue. Our study investigated the role ofneomycin on NF-kB activity in glioma cell cultures. We performed immunocytochemical analysis of cells with the antibody NF-kBp65 which results show that neomycin decreases significantly the activation of NF-kB when added to glioma cultures for 30 min. This finding supports an important role for neomycin in glioma invasion, apoptosis and growth. Collectively, these data suggest a rationale for clinical trials with neomycin in the treatment of gliomas. [Neurol Res 2003; 25: 271-274]

Keywords: Glioma; neomycin; nuclear factor-kappa B (NF-kB); immunocytochemistry

INTRODUCTION

A hallmark of gliomas is the ability of tumor cells to invade and infiltrate the brain parenchyma. The steps necessary for tumor cell migration and local invasion include adhesion to the local tissue, motility for migration, and ability to modify the local environment to allow for cell invasion1. Although the molecular mechanisms underlying these events remain to be elucidated, the first step in tumor cell invasion involves the adhesion of tumor cells to the host extracellular matrix. Recently an important role of the transcription factor nuclear factor kappa B (NF-kB) has been suggested for glioma cell invasion2.

NF-kB was originally identified as a transcription factor bound on the enhancer element of the K light chain gene of B lymphocytes. It has been implicated in control of transcription of several genes involved in immune and inflammatory responses and as well in cell growth, including IL-2, IL-6, IL-8, GM-CSF, G-CSF, ICAM-1, VCAM-1, iNOS, PDGF, VEGF, tissue factor, c-myc genes, etc.3. Furthermore, it has been reported that NF-kB comprises several proteins such as p50, p52, p65, c-rel and ReI B, with differential regulation in their induction4. The NF-kB is sequestered in the cytoplasm by a member of the IkB family of inhibitory proteins. IkB proteins mask the nuclear localization signal of NF-kB, thereby preventing NF-kB nuclear translocation. The activation of NF-kB occurs through site-specific phosphorylation and subsequent degradation of IkB. This allows the translocation of NF-kB into the nucleus to bind to NF-kB specific DNA-binding sites and regulate gene transcription4. Although NF-kB has been extensively investigated in hematopoietic cells, its activity and function in neoplasms have been incompletely elucidated. NF-kB plays a role in the control of cell migration and cell proliferation, because this factor is involved in the transcription of several genes encoding integrins and growth-promoting cytokines. Recently, it has been reported that NF-kB activity was implicated in glioma cell invasion2 and growth5. Several studies have suggested that neomycin is involved in regulating endothelial cell growth and proliferation6'7. It has been also documented that neomycin has a role in proliferation of glioma cells8'9. Collectively, these findings indicate that neomycin is an antiproliferative agent, which suggests that neomycin might be able to efficiently transduce its growth-suppressor signal by modulating intracellular signaling cascades. But the function of this antibiotic in cell signaling is not fully understood. These evidences led us to investigate whether neomycin has a role in NF-kB activity in glioma cell cultures. We assessed this hypothesis using immunocytochemistry assays.

MATERIALS AND METHODS

Cell culture and drug exposure

C6 glioma cells were cultured in Dulbecco's modified Eagle's medium (DMEM) (Paisley, Scotland, UK) supplemented with 7.5% fetal calf serum and antibiotics penicillin (10 units mh^sup -1^) and streptomycin (10 [mu]g mh^sup -1^), and were maintained in a humidified atmosphere of 5% CO2 at 37[degrees]C. Cells were routinely passaged using 0.025% (v/v) tripsin in HBSS (Hank's Balanced Salt Solution, Bio Whittaker Europe, Belgium) and seeded at a density of 1 x 10^sup 4^ cells/cm^sup 2^ in culture flasks medium or 1OmM neomycin sulfate (Sigma Chemicals, St. Louis, MO, USA) for 30 min which is enough to inhibit glioma cell proliferation8. For immunocytochemical staining studies C6 cells were seeded at 25.000 cells in 12 mm diameter cover glasses.

lmmunocytochemistry

Control and neomycin-treated cultures were fixed in 4% paraformaldehyde, washed two times with TBS, permeabilized and then treated with a polyclonal antiNF-kBp65 antibody (1 :100; Santa Cruz Biotechnology, Inc., CA, USA) which exclusively recognized the activated form of NF-kB10. For NF-kBp65 immunodetection cultures were treated with biotin-goat anti-rabbit IgG (Zymed, San Francisco, CA, USA). DAB (3-3-diaminobenzidine tetrahydrochloride; Dako, Glostrup, Denmark) was used as chromagen. Negative control experiments were performed in cultures that were incubated without primary antibody and subsequently processed with secondary antibody and reacted with chromagen.

Quantitation of immunocytochemical signals was carried out on a computer using a MOTICAM 1300 camera (Motic, Barcelona Spain) with software. Integrate pixel intensity was measured in 17 selected fields. A constant pixel area was used for three independent measurements that were averaged. The averaged integrate pixel intensity was statistically analyzed. Image and quantitation were blind conducted.

Statistical analysis

Data are expressed as mean + or - SE mean and paired means were compared with the Student's f-test. p

RESULTS

Effect of neomycin in NF-kB activity

The cells in Figure 1 are the C6 glioma cell line, which express high levels of growth factor receptors and produce large amounts of growth factor family of proteins11. To confirm whether NF-kB was activated in C6 cells, we performed immunocytochemical studies of the NF-kB activity using an antibody which selectively binds to the activated NF-kB. Intense immunostaining for NF-kBp65 was observed in the nuclei of tumor cells and only fine and difuse immunocytochemical signals were depicted through the cytoplasm. These findings are consistent with the fact that activated NF-kB translocates into the nucleus.

In control cultures activated NF-kB p65 was localized within the cells and more pronounced in the nuclear region (Figure 1A), thereby indicating that most of the activated NF-kBp65 was present in the nucleus. In contrast, minor staining in much fewer cells, indicating only a little activated NF-kBp65, was observed in glioma cultures treated for 30 min with 1OmM neomycin (Figure IB) In these cells, staining for activated NF-kBp65 was located outside the nucleus in the cytoplasm, close to the outer nuclear membrane. Image intensity analysis indicates that neomycin blocks NF-kB activity in almost 68% of glial cells (Figure 1C). As depicted in Figure 1, neomycin produced changes in glioma cell phenotype (compare panel A to panel B), due to a disturbance of F-actin as it has been previously reported12.

DISCUSSION

One of the most important functions of NF-kB is the activation of an anti-apoptotic gene expression program in tumor cells that may contribute to malignant progression by conferring a survival advantage through suppression of apoptotic cell death13'14. More recently, NF-kB activation has been connected to cell growth control15. Because signaling pathways that govern proliferation and survival rate are important for tumor development, NF-kB has an intrinsic oncogenic potential. Many studies reported that NF-kB activation is connected with multiple aspects of oncogenesis. The earliest support for this idea came from a work with v-rel, an oncogenic viral homolog of c-rel, which causes aggressive tumors in chickens16. In many cancers, such as Hodgkin's lymphoma, NF-kB is constitutively activated aberrantly'7. It is known that various oncoproteins activate NF-kB to oncogenesis. Transcriptional activation of NF-kB is required for Ha-ras-mediated cellular transformation18, and oncogenic fusion protein BCRABL induces nuclear translocation of NF-kB and tumorigenesis which are blocked by represser IkB[alpha]19.

Several studies have addressed the association between NF-kB and cell adhesion events on tumorigenic cells. The key role of NF-kB on tumor cell attachment and invasion was demonstrated in several human tumor cell lines including osteosarcoma, colon carcinoma, and breast carcinoma when these cells were treated with p65 antisense oligodeoxynucleotide. A significant inhibition of cell adhesion and in vitro growth was depicted after antisense treatment20. Recently, it has been reported that NF-kB participates in glioma cell attachment via an increase in the levels of mRNA for the [beta]^sup 3^ and the [alpha]v integrin subunits2. Furthermore, it has been reported that NF-kB has an important role in glioma cell invasion through the brain extracellular matrix by regulation of the matrix metalloproteinase-9 (MMP-9 ) gene21. Since MMP-9 contributes not only to tumor invasion but also to the degradation of the blood-brain barrier (BBB), and to angiogenesis, NF-kB inhibition offers therapeutic oportunities for treating gliomas. In addition, NF-kB acts as a cell survival signalling molecule through an [alpha]v[beta]^sup 3^ involvement22 Collectively, the data from these studies support an important role for NF-kB in glioma cell invasion, apoptosis suppression and growth. Recently it has been reported that constitutively activated NF-kB participates in the activation of the cell-cycle regulatory cyclins genes23~26. Since pharmacological inhibitors of cyclin dependent kinases (CDKs) are currently being evaluated for therapeutic use against cancer, neurodegenerative disorders and cardiovascular diseases26 it seems likely that further studies of CDKs inhibition by neomycin will offer new unforeseen therapeutic indications for this compound.

Consistent with previous findings showing that neomycin blocks mitogen-activated protein kinases (MAPK) pathway28, our present data provide strong evidence that other possible alternative pathways, such as NF-kB signals are also involved in the antiproliferative effects of neomycin in gliomas. Taken together these data with those from previous reports led to suggest that anti-proliferative effect of neomycin can be accomplished at least by three mechanisms: by blocking nuclear translocation of growth factors, by inhibition of MARK signaling pathways and by inactivation of NF-kB.

NF-kB-targeted therapies present opportunities and challenges for the future. It has been reported that simultaneous activation of multiple signal pathways, including NF-kB and ERK1/2 mitogen-activated protein kinases (MARK) is a prerequisite for sustained production of several pro-inflammatory cytokines asTNF-[alpha]29. TNF-[alpha] has been proposed as adjuvant inmunotherapy in malignant glioma30, but long term follow-up studies showed no significant improvement. Furthermore, TNF-[alpha] induces activation of NF-kB in malignant cells31, and this activation may contribute to the cell resistance to TNF-[alpha] by preventing apoptosis or cell cycle arrest in glioma cells. Thus refractoriness to TNF-[alpha] treatment could be prevented by inhibiting NF-kB activation31. In this particular context neomycin may play a role. Accordingly, in the clinical setting, complete inhibition of TNF-[alpha] release may require treatment with single drugs or drug combination capable of inhibiting multiple activation pathways. Since neomycin inhibited ERK1/2(28) and NF-kB in glioma cells, this antibiotic is a new promising drug for treating gliomas and other diseases in which ERK1/2 and NF-kB activation were involved.

ACKNOWLEGEMENTS

We thank Chantai Bourdier for editorial assistance, Concha Muela and Argentina Fernandez-Ayerdi for technical work. This study was partially supported by Fundacion Futuro (Madrid).

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Pedro Cuevas*[dagger], Diana Diaz-Gonzalez* and Manuel Dujovny[dagger]

* Departamento de Investigacion, Hospital Universitario Ramon y Cajal, Universidad de Alcala de Henares, Madrid, Spain

[dagger] Department of Neurosurgery, Wayne State University, Detroit, MI, USA

Correspondence and reprint requests to: Dr Pedro Cuevas, Departamento de Investigacion, Hospital Universitario Ramon y Cajal, Universidad de Alcala de Henares, 28034 Madrid, Spain. [pedro.cuevas@hrc.es] Accepted for publication November 2002.

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