Mechanisms operative in the antitumor activity of temozolomide in glioblastoma multiforme

Purpose: 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. Materials and 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 (UEGF). HIF-1 alpha expression was constant at the mRNA level; however, its post-treatment protein levels increased compared with those of untreated control cells. Discussion: The genetic analyses suggest that treatment with TMZ activates stress mechanisms in GBM cells that include the angiogenesis-inducing proteins HIF-1 alpha and VEGF. We propose that treatment with TMZ be supplemented with either an antibody to VEGF or down-regulators of HIF-1 alpha to improve clinical results of TMZ in the treatment of GBM.