Glioblastoma multiforme (GBM) continues to have a poor survival despite advances in surgical resection and the addition of temozolomide to radiation. Radiotherapy, one of the mainstays of treatment for GBM, is known to induce tumor cell killing through DNA double strand breaks (DSB). Poly (ADP-ribose) polymerase (PARP) is an enzyme involved in the repair of DSBs. Here we investigate the in vitro and in vivo radiosensitizing effects of the novel PARP inhibitor, GPI 21016, on a GBM cell line.
Materials/Methods
The U251 human GBM cell line was treated for 6 h with GPI 21016 (3 uM) pre-irradiation in all in vitro experiments. Inhibition of PARP-1 by GPI 21016 was evaluated using a PARP chemiluminescent assay. Clonogenic survival assays were performed according to standard protocol. To assess DSBs, γH2AX foci were assessed at 1, 6 and 24 h after 2 Gy. To further evaluate DSBs, neutral comet assay was performed at 0, 1, 3, 6 and 24 h after exposure to 10 Gy. Mechanism of cell death was determined by immunostaining for mitotic catastrophe and apoptosis by flow cytometry. Cell cycle changes were evaluated by staining of phospho-histone H3 and flow cytometry. For in vivo studies, U251 cells were implanted in both SC and IC models.
Results
A 6h exposure of GPI 21016 (3uM) inhibited PARP activity by 73%. Clonogenic survival resulted in a dose enhancement factor (DEF) of 1.6 at a surviving fraction of 10% (SF = 0.49 in drug alone cells) in cells treated with GPI 21016 and IR vs. IR alone. To assess DSB repair, γH2AX measured at 24 h post-IR had significantly more foci/cell in the combination group vs. the IR alone group. Neutral comet assay further suggested unrepaired DSBs with significantly greater DNA damage at 6 h post-IR in the combination group vs. IR alone, however this damage resolved by 24 h. Mitotic catastrophe (MC) staining revealed significantly greater cells in MC at 72 h post-IR in the combination group vs. the IR alone group. Drug, IR and combination groups showed little apoptosis at 24 and 72 h post-treatment. Cell cycle analysis revealed no changes with exposure to drug pre-IR. In both SC and IC in vivo models, the combination therapy had a greater than additive anti-tumor effect than either single modality.
Conclusions
GPI 21016 effectively inhibits PARP leading to decreased DSB repair and enhanced mitotic catastrophe. Thereby, GPI 21016 radiosensitizes U251 cells in vitro and results in decreased tumor growth in vivo, suggesting a potential role for this drug in the treatment of GBM.
National Cancer Institute, NIH, Bethesda, MD
Author Disclosure: A.L. Russo, None; D. Citrin, None; W.E. Burgan, None; D. Carter, None; K. Beam, None; H. Kwon, None; P.J. Tofilon, None; K. Camphausen, None.
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