Re-evaluation of cytostatic therapies for meningiomas in vitro

Abstract

Purpose: The purpose was to re-evaluate in cell culture models the therapeutic usefulness of some discussed chemotherapies or targeted therapies for meningiomas with a special emphasis on the role of the neurofibromatosis type 2 (NF2) tumor suppressor, which had been neglected so far. In addition, the study intended to evaluate a potential benefit from a treatment with drugs which are well established in other fields of medicine and have been linked recently with tumor disease by epidemiological studies.

Methods: Meningioma cell lines corresponding to various subtypes and pairs of syngenic meningioma cell lines with or without shRNA-induced NF2 knockdown were analyzed for their dose-dependent response to the drugs in microtiter tetrazolium assays, BrdU assays and for selected cases in ELISAs measuring nucleosome liberation to specifically separate cell death from pure inhibition of cell proliferation.

Results: We confirmed a moderate efficacy of hydroxyurea (HU) in clinically relevant concentrations. Under appropriate dosing, we neither detected major responses to the alkylating compound temozolomide nor to various drugs targeting membrane receptors or enzymes (tamoxifen, erlotinib, mifepristone, losartan, metformin and verapamil). Only concentrations far beyond achievable serum levels generated significant effects with the exception of losartan, which showed no effects at all. Chemosensitivity varied markedly among meningioma cell lines. Importantly, cells with NF2 loss exhibited a significantly higher induction of cell death by HU.

Conclusions: Alternative chemotherapeutic or targeted approaches besides HU have still to be evaluated in further studies, and the role of NF2 must be taken into account.

Fig. 1

Effects of the DNA alkylating drug temozolomide on menigioma cell lines. a MTT assay shows a significant growth reduction only in IOMM-Lee cells treated with high temozolomide concentration (20 µM; p < 0.001). b Silver-stained polyacrylamid gel demonstrates methylation of the MGMT promoter only in IOMM-Lee cells, whereas in the other cell lines, the MGMT promoter is completely unmethylated. The labeled significances refer to the difference between the indicated treatment and solvent-treated controls

Fig. 2

Effects of different drugs on meningioma cell survival in MTT assays. a Hydroxyurea exhibits moderate activity in all cell lines except HBL52 cells. b Tamoxifen shows a significant dose-dependent effect in all cell lines, but only at concentrations which are not reachable in patients’ plasma. c A decrease in cell viability is seen following erlotinib treatment in all cell lines. The metabolic inhibitors metformin (d) and verapamil (e) clearly diminished the MTT conversion in a dose-dependent manner, but in all cell lines only at concentrations which cannot be reached in patient’s plasma. The labeled significances refer to the difference between the indicated treatment and solvent-treated controls

Fig. 3

Effects of different drugs on merlin-positive and merlin-negative SF4068 meningioma cells in MTT assays. a SF4068 cells were subjected to NF2 depletion by lentiviral transduction with a vector expressing an efficient NF2-targeting shRNA (shNF2) or a corresponding control vector (sh-Con). Western blot analysis confirms different merlin expression. Note that the lanes originate from a larger blot and are re-arranged for better clarity. Hydroxyurea (b), tamoxifen (c), erlotinib (d), metformin (e) and verapamil (f) all cause a significant dose-dependent decline of cell viability. The somewhat different responses of merlin-positive (sh-Con) and merlin-depleted (shNF2) cells differ, however, not statistically significant. The labeled significances refer to the difference between the indicated treatment and solvent-treated controls

Fig. 4

Nucleosome liberation assay demonstrates that cell death induction was higher (p ≤ 0.05) in HU- (a) and verapamil (b)-treated cells compared to the solvent control (set to 1). A significantly higher cell death induction in the merlin-depleted cells as compared to wt (p ≤ 0.05) is indicated by an asterisk. c In BrdU assays with NF2-depleted (sh-NF2) and control (sh-Con) SF4068 cells, inhibition of cell proliferation with a cytotoxic concentration of 50 µM verapamil was further confirmed, but the non-toxic concentration of 10 µM verapamil leads to a small increase in BrdU incorporation. The labeled significances in c refer to the difference between the indicated treatment and solvent-treated controls

Fig. 5

Combination of irradiation (5 Gy) plus hydroxyurea (a) or verapamil (b) in NF2-depleted (sh-NF2) and control (sh-Con) SF4068 meningioma cells results in a dose-dependent significant effect on cell viability (measured with MTT assay), but the difference between the two cell lines was not statistically significant. The labeled significances refer to the difference between the indicated treatment and solvent-treated controls