Overexpression or activation of Src family kinases (SFKs) has been implicated in a number of different cancers. Because SFKs are involved in multiple cellular signaling pathways, including cell division, survival, motility, and invasion, development of SFKs inhibitors is ongoing. Here, we examine the effect of one such agent AZD0530 on response to radiation (IR) in a glioblastoma multiforme cell line.
Materials/Methods
U251 human GBM cell line was treated for 16 hours with AZD0530 (3μmol/L) pre-irradiation in all in vitro experiments. The effects of AZD0530 on the in vitro radiosensitivity of U251 was evaluated using clonogenic assay. DNA damage and repair were evaluated using γH2AX at 1, 6 and 24 hours after 2 Gy IR. Neutral comet assay was performed at 0, 3, 6, 16, 24 hours after 10 Gy IR for further evaluate DNA damages and repair. Mechanism of cell death after DNA damage was determined by immunostaining for mitotic catastrophe and flow cytometry for apoptosis. Drug induced cell cycle changes were evaluated by staining of phospho-H3 and flow cytometry.
Results
Exposure of U251 to AZD0530 for 16 hour prior to radiation resulted in an increase in radiosensitivity with dose enhancement factors at a surviving fraction of 0.1 of 1.31. AZD0530 had no effect on the number of cells in each phase of the cell cycle nor on the activation of the G2 cell cycle checkpoint after IR. As a measure of DNA double strand breaks, γH2AX foci were determined as a function of time after the AZD0530 + IR combination. The number of γH2AX foci per cell was significantly greater at 24 hours after the combined modality as compared to the individual treatments. Mitotic catastrophe, measured at 72 hours, was also significantly increased in cells receiving the AZD0530 + IR combination as compared to the single treatments.
Conclusions
These results indicate that AZD0530 can enhance tumor cell radiosensitivity in vitro and suggest that this effect involves an inhibition of DNA repair leading to an increase in mitotic catastrophe.
1Radiation Oncology Branch, National Caner Institute, Bethesda, MD
2Molecular Radiation Therapeutics Branch, National Cancer Institute, Bethesda, MD
3Science Applications International Corporation-Frederick, National Cancer Institute-Frederick, Frederick, MD
4Drug Discovery Program, H. Lee Moffitt Cancer Center, Tampa, FL
Author Disclosure: W. Kil, None; W. Burgan, None; K. Beam, None; P. Tofilon, None; K. Camphausen, None.
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