Inhibition of Ras farnesylation by lovastatin leads to downregulation of proliferation and migration in primary cultured human glioblastoma cells.
BACKGROUND
Malignant astrocytomas are the most common primary intracranial human tumors. All therapeutic approaches are limited due to their high proliferative capacity and their ability to diffusely invade the brain. Amplification of tyrosine kinase receptors and their signaling pathways have been implicated as contributing to the molecular pathogenesis of astrocytomas, providing possible new targets for therapeutic intervention. In particular, astrocytomas, although lacking oncogenic Ras mutations, have elevated levels of activated Ras. Lovastatin, an inhibitor of the beta-hydroxy-beta-methylglutary CoA reductase (HMG-CoA-reductase), is currently used to treat patients with hypercholesterolemia. In addition, it inhibits isoprenylation of several members of the Ras superfamily of proteins and therefore has multiple cellular effects including the reduction of proliferation.


METHODS
In this study, we investigated the impact of lovastatin on two human glioma cell lines and on 15 primary cell cultures established from biopsies of patients with glioblastoma multiforme (GBM,) Proliferation of glioma cell lines and primary tumor cells was determined by cell counting and by using the MTT assay. The cell morphology was analyzed by staining of actin filaments with phalloidin. Apoptosis was measured using the TUNEL assay. To investigate the influence of this drug on glioma cell motility, tumor cell migration was investigated using three dimensional spheroid disintegration assays. In addition, tumor cell invasion was analyzed with a confrontational assay between tumor spheroids and rat brain aggregates.


RESULTS
Inhibition of farnesyl biosynthesis using lovastatin led to a block in Ras mediated signaling, indicated by lower MAPK activity. Consequently, tumor cell proliferation was reduced up to 80%. Lovastatin appeared to decrease glioma viability by inducing apoptosis, as indicated by morphological changes and increase of TUNEL positive cells. Lovastatin acts through isoprenoid depletion, because supplementation of the media with 50-100 microM mevalonate restored all tau eta epsilon effects. Invasion of tumor cells into brain tissue was not effected while migration was reduced beta upsilon about 30-40% in cells treated with high concentrations (> or = 100 microM) of lovastatin. This was surprising because drug treatment at lower concentrations led to a disruption of the actin cytoskeleton, as indicated by Phalloidin staining.


CONCLUSIONS
Our data strongly suggest that inhibition of elevated Ras activity by lovastatin effectively targets the MAPK and probably other signaling pathways thus offering a pharmacological based approach for a potential treatment of human astrocytomas.
Bouterfa
H L
HL
Department of Neurosurgery, University of Würzburg, Germany. hakim.bouterfa@pharma.novartis.com
Sattelmeyer
V
V
Czub
S
S
Vordermark
D
D
Roosen
K
K
Tonn
J C
JC
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D001921:Brain / Q000187:drug effects / Q000473:pathology; D002455:Cell Division / Q000187:drug effects; D002465:Cell Movement / Q000187:drug effects; D015536:Down-Regulation; D005909:Glioblastoma / Q000188:drug therapy* / Q000473:pathology; D006801:Humans; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors / Q000494:pharmacology*; D008148:Lovastatin / Q000494:pharmacology*; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors; D009361:Neoplasm Invasiveness; D017368:Protein Prenylation; D051381:Rats; D014407:Tumor Cells, Cultured; D018631:ras Proteins / Q000378:metabolism*
D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors; D008148:Lovastatin; D020928:Mitogen-Activated Protein Kinases; D018631:ras Proteins