Mechanisms operative in the antitumor activity of temozolomide in glioblastoma multiforme.
OBJECTIVE
Glioblastoma multiforme (GBM) is the most frequent and incurable brain tumor in adults. Although temozolomide (TMZ) does not cure GBM, it has demonstrated anti-GBM activity and has improved survival (8-14 months) and quality of life. We studied the mechanisms by which TMZ affects 2 human GBM cell lines; U251-MG and U87-MG, aiming to unravel the drug-activated cascades to enable the development of combination therapies that will improve the efficacy of TMZ.


METHODS
The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium assay was used to assess cell viability. Modulation of gene expression by TMZ therapy was assayed by gene profiling and verified by quantitative real-time polymerase chain reaction. Protein levels influenced by the treatment were studied by Western blots and immunocytochemistry.


RESULTS
Increasing concentrations of TMZ decreased cell viability in a concentration-dependent manner. The expression of 1,886 genes was altered >2-fold after TMZ treatment. We focused on the 81 genes similarly altered by TMZ treatment in both cell lines to neutralize tissue-specific characteristics. Fourteen target genes of hypoxia-inducible factor (HIF-1), were found to be up-regulated after TMZ treatment including vascular endothelial growth factor (VEGF). HIF-1alpha expression was constant at the mRNA level; however, its post-treatment protein levels increased compared with those of untreated control cells.


CONCLUSIONS
The genetic analyses suggest that treatment with TMZ activates stress mechanisms in GBM cells that include the angiogenesis-inducing proteins HIF-1alpha and VEGF. We propose that treatment with TMZ be supplemented with either an antibody to VEGF or down-regulators of HIF-1alpha to improve clinical results of TMZ in the treatment of GBM.
Fisher
Tamar
T
Department of Pediatric Hemato-Oncology, Safra Children's Hospital and the Institute of Hematology, Tel-Hashomer, Israel. tamar.fisher@sheba.health.gov.il
Galanti
Gil
G
Lavie
Gad
G
Jacob-Hirsch
Jasmine
J
Kventsel
Iris
I
Zeligson
Sharon
S
Winkler
Robert
R
Simon
Amos J
AJ
Amariglio
Ninette
N
Rechavi
Gideon
G
Toren
Amos
A
D000970:Antineoplastic Agents / Q000494:pharmacology*; D001932:Brain Neoplasms / Q000378:metabolism / Q000473:pathology*; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D003606:Dacarbazine / Q000031:analogs & derivatives* / Q000494:pharmacology; D020869:Gene Expression Profiling; D005909:Glioblastoma / Q000378:metabolism / Q000473:pathology*; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000096:biosynthesis; D007150:Immunohistochemistry; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000077204:Temozolomide
D000970:Antineoplastic Agents; C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D003606:Dacarbazine; D000077204:Temozolomide