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Valproic acid (VPA) has been considered as a possible treatment agent for malignant gliomas. In order to characterise the possibilities of VPA, we investigated the effects on cell migration and proliferation. Human cell lines T98G, A172, 85HG66 and 86HG39 were treated with VPA or left untreated, afterwards Boyden chamber assay was used for measuring vertical migration. In a second assay cells were stimulated to create spheroids and spheroid migration was measured. Proliferation was assessed using a cell counter. VPA decreased proliferation of 86HG39 > A172 > 85HG66 cells, whereas T98G remained uninfluenced. The influence of VPA on migration was different; whereas VPA dose-dependently stimulated migration of 86HG39 cells, migration of T98G and 85HG66 decreased, whereas A172 cells remained uninfluenced. Only 86HG39 and A172 cells created spheroids. In both cell lines Boyden-chamber-findings were confirmed by analysing the influence of VPA on spheroid migration. These non-uniform data demonstrate that the benefit of VPA in glioma treatment is not clear and needs further investigation.

Because of the outstanding importance of the glucocorticoid Dexamethasone (DEX) as supportive therapy in the management of brain tumours, the direct effect of DEX on tumour cell proliferation is of particular interest. Previous in vitro studies led to contradictory results. To characterise more precisely the influence of DEX, we investigated the glioblastoma multiforme (GM) cell lines A172, T98G and 86HG39. Cells were treated with DEX concentrations ranging from 5 x 10(-9) to 5 x 10(-5) M from 24 to 240h under different treatment conditions. Influence of DEX on glioma cell viability was assessed daily for 5 days by MTT-assay: (I) with continuous DEX incubation (acute treatment), (II) in a recultivation period without DEX after 5 days of DEX pre-incubation (pre-treatment), (III) with continuous DEX incubation after 5 days of DEX pre-incubation (combination treatment). DEX acute treatment led to strongly decreased proliferation of A172 cells, whereas T98G and 86HG39 cells remained uninfluenced. In opposite, a time-delayed inhibition of cell proliferation was observed in all three cell lines after DEX pre-treatment. Combination treatment induced a significant increase of the inhibitory effect in A172 and T98G cells. These data show a variable, partial time-dependent inhibitory effect of DEX on the proliferation of GM cells and may open new treatment strategies for malignant brain tumours.

Despite surgery and adjuvant cytotoxic therapy anaplastic astrocytoma, glioblastoma and diffuse intrinsic brain stem glioma continue to have dismal prognosis. Differentiation induction is a new approach taking into account that malignant glioma cells share many features with immature glial progenitor cells that are capable of terminal differentiation. The concept of differentiation therapy is currently evaluated for several pediatric malignancies with or without multimodal standard therapy. Valproic acid (VPA) is a branched chain fatty acid that is able to inhibit proliferation of neuroectodermal cells and to induce these cells along neuronal or glial lineage. Preclinical studies have shown that VPA inhibits growth of human and rodent glial tumor cells in vitro and induces a distinct mature glial phenotype. In addition, growth of human neuroblastoma cells is inhibited in vitro and in vivo and exhibits marked evidence of differentiation. Treatment of neuroblastoma and glioma cells with VPA was accompanied by changes of surface molecule expression that enhance immunogenicity and reduce their capability to metastasize. The antitumoral effects observed in preclinical studies were reached at concentrations that are readily achieved in patients treated with VPA for epilepsy. Epilepsy patients receiving VPA have significantly enhanced hemoglobin F levels, supporting the hypothesis that nontoxic levels of VPA can induce cellular differentiation. Broad clinical experience with VPA and its low toxicity encourage the evaluation of VPA in patients that have been submitted to postoperative combined chemo- and radiotherapy for pediatric malignant glioma.

Malignant gliomas are often treated with cisplatin (cis-diamminedichloroplatinum(II), CDDP) and radiation but results remain unsatisfactory. The development of resistance might explain the poor prognosis. The aim of this study was to investigate whether two human malignant glioma cell lines, 86HG39 and A172, develop resistance to CDDP and/or radiation after CDDP pretreatment. The cells are incubated three times with 10(-6) M CDDP for 24 h, allowed to recover from the treatment and then tested for sensitivity to CDDP and radiation (9 Gy, 60Co) using a colorimetric assay (MTT). A172 pretreated and wild-type cells showed no difference in sensitivity to CDDP, whilst 86HG39 cells became more sensitive following pretreatment. This indicates that no resistant phenotype towards CDDP developed in any of the cell lines. In contrast, the CDDP-pretreated cells, after radiation, had significantly higher growth rates compared with the wild-type cells in both cell lines (A172: 4.4 +/- 0.5 versus 2.5 +/- 0.3, respectively, 192 h after radiation; 86HG39: 6.2 +/- 0.7 versus 2.3 +/- 0.3, respectively, 216 h after radiation; P < 0.05) indicating resistance against radiation. The level of glutathione (GSH), which is known to mediate resistance against radiation, was 157.2 +/- 61.3 ng/mg protein in A172 cells and 93.2 +/- 16.9 ng/mg protein in 86HG39 cells, and there was no significant difference between levels in wild-type and pretreated cells (A172: 130.1 +/- 34; 86HG39: 81.6 +/- 10.4). These data show that CDDP pretreatment can induce resistance against radiation in vitro independently of CDDP cross-resistance mediated by a mechanism different from GSH.

Oxazaphosphorines are inactive anticancer prodrugs that are bioactivated by hepatic cytochrome P450. Besides hepatic metabolism, there is increasing interest in the possibility of intratumoral activation of oxazaphosphorines by P450. Therefore, we investigated the expression of P450 (CYP3A4, CYP3A5, CYP2C9) by RT-PCR in 10 different brain tumor samples. Because P450 may be downregulated by interleukin-1 (IL-1) and IL-6, the receptors for IL-1 and IL-6 were analyzed. None of the brain tumors was positive for CYP3A4 whereas CYP3A5 was detected in 3 out of 10 tumors (two meningeomas, one medulloblastoma grade IV). All five gliomas, an ependymoma, and a lymphoma-metastase gave no signal. CYP2C9 mRNA was present in every sample studied. All samples were positive for IL-1 and IL-6 receptors. In summary, we have demonstrated that tumors of the CNS express P450, indicating that activation of prodrugs like oxazaphosphorines may take place intratumorally. However, the most abundantly hepatically expressed CYP3A4 enzyme is absent in the brain tumor samples. The presence of the IL-1 and IL-6 receptors opens the possibility that the wellknown downregulating influence of these cytokines also takes place in brain tumors.

Background: The prognosis of children with malignant gliomas is poor. A new therapeutic strategy using interferon-gamma (IFN-g) as an immunostimulator substance will start in Germany in 1997. One of the therapeutic problems in malignant gliomas is the high invasiveness of the tumor cells. Adhesion to the extracellular matrix molecule hyaluronic acid (HA) is involved in invasion. We addressed the question of whether IFN-g (i) influences the proliferation and (ii) changes the HA-binding capacity of malignant glioma cells., Method: T98G glioblastoma cells were incubated up to 4 days with IFN-g (30 and 300 IE/ml). Proliferation was measured by a colorimetric assay (MTT) and compared with untreated controls. Expression of the HA-receptor CD44 was determined by FACS-analysis using the mouse monoclonal antibody J-173. HA-adhesion was investigated using HA-coated (3 mg/ml), bovine serum albumin blocked 24-well-plates and compared with uncoated wells., Results: FN-g (300 IE/ml) inhibited proliferation to 76.3% (p < 0.0001) after 48 hours compared with untreated controls. This effect was mediated not only by inhibition of proliferation, but also by induction of cell-death, first seen 72 h after IFN-g incubation. 24 hours later only 24% of treated cells survived. 93.1% of T98G cells expressed CD44 (FACS-analysis). A specific HA-adhesion of glioma cells was shown: 85.5% of the cells adhered to HA, 13.3% to BSA compared with controls. 300 IE/ml IFN-g decreased HA-adhesion significantly (p < 0.001) to 17%, whereas BSA-adhesion remained unchanged., Conclusion: IFN-g inhibits tumor cell proliferation and diminishes the invasive properties of glioma cells via reduction of HA binding capacity. Our results support the use of IFN-g in the therapy of malignant gliomas.