gms | German Medical Science

Objective: Glioblastoma multiforme (GBM) is the most malignant brain tumor characterized by its invasive growth pattern and chemoresistance. In the current study we investigated whether the diazeniumdiolate JS-K, which results in tumor cell specific release of nitric oxide (NO), sensitizes GBM cells to Temozolomide (TMZ) and inhibits their migration capacity.

Method: GBM cells were treated with JS-K (0–3 µM) for 4h. Migration capacity was assessed by cell motility assay over 96h. Regulation of matrix metalloproteases (MMP) and their inhibitors (TIMP) were analyzed by ELISA. To investigate the chemosensitizing effect cells were treated with JS-K (1–25 µM, 4h), TMZ (5–50 µM, 24h) or a combination of both. Cell viability and proliferation was analyzed by MTT and BrdU assays. Apoptosis was analyzed by immunocytochemistry for p53 and Western Blot for cleaved ©PARP1. To identify genes involved in chemoresistance of U87 cells a RNA microarray was performed. Statistical analysis was performed by student's t-test.

Results: Low dose JS-K (0–3 µM) has no effect on cell viability or proliferation. Concentrations ≥1.5 µM significantly inhibit the migration of all tested GBM cell lines, prominent after 96h. NO does not influence the expression level of MMP1/2/9 and TIMP1/2, but it temporarily inactivates MMP2/9 by nitrosylation. High doses of JS-K (15–25 µM) significantly reduce cell viability to 50% after 48h and to 35% after 72h. TMZ treatment (25 µM) causes only a slight reduction in viability. JS-K/TMZ (15 µM/25 µM) results in an earlier and stronger induction of apoptosis indicating a synergistic effect, accompanied by an earlier nuclear translocation of p53 and an increase of cPARP1 after 48h. Microarray analysis reveals a differential expression of genes associated with DANN repair and resistance.

Conclusions: Low doses of JS-K significantly reduce the migration of GBM cells in vitro via the temporary inactivation of MMPs. High doses of JS-K sensitize GBM cells to TMZ resulting in an earlier and stronger induction of apoptosis. Microarray data suggest that JS-K modulates the transcription of genes involved in DNA repair and in the induction of apoptosis, indicating that multiple mechanisms underlie the resistance of GBM cells.