Microtubule-targeting agents can sensitize cancer cells to ionizing radiation by an interphase-based mechanism

Abstract

Background

The cytotoxic effects of microtubule-targeting agents (MTAs) are often attributed to targeted effects on mitotic cells. In clinical practice, MTAs are combined with DNA-damaging agents such as ionizing radiation (IR) with the rationale that mitotic cells are highly sensitive to DNA damage. In contrast, recent studies suggest that MTAs synergize with IR by interfering with the trafficking of DNA damage response (DDR) proteins during interphase. These studies, however, have yet to demonstrate the functional consequences of interfering with interphase microtubules in the presence of IR. To address this, we combined IR with an established MTA, mebendazole (MBZ), to treat glioma cells exclusively during interphase.

Materials and methods

To test whether MTAs can sensitize interphase cells to IR, we treated GL261 and GBM14 glioma cells with MBZ during 3–9 hours post IR (when the mitotic index was 0%). Cell viability was measured using a WST-1 assay, and radiosensitization was quantified using the dose enhancement factor (DEF). The effect of MBZ on the DDR was studied via Western blot analysis of H2AX phosphorylation. To examine the effects of MTAs on intracellular transport of DDR proteins, Nbs1 and Chk2, cytoplasmic and nuclear fractionation studies were conducted following treatment of glioma cells with MBZ.

Results

Treatment with MBZ sensitized interphase cells to the effects of IR, with a maximal DEF of 1.34 in GL261 cells and 1.69 in GBM14 cells. Treatment of interphase cells with MBZ led to more sustained γH2AX levels post IR, indicating a delay in the DDR. Exposure of glioma cells to MBZ resulted in a dose-dependent sequestration of Chk2 and Nbs1 in the cytoplasm.

Conclusion

This study demonstrates that MBZ can sensitize cancer cells to IR independently of the induction of mitotic arrest. In addition, evidence is provided supporting the hypothesis that MTA-induced radiosensitization is mediated by inhibiting DDR protein accumulation into the nucleus.

Keywords:

• microtubules
• mebendazole
• ionizing radiation
• radiosensitization
• interphase
• DNA damage response

Acknowledgments

GBM14 cells were kindly provided by Dr J Sarkaria, Mayo Clinic, Rochester, MN, USA. All data generated and analyzed during this study are included in this publication. This study was supported by grants to MS from the Swim Across America Foundation and the Project To Cure Foundation and to RR from the Zankel Foundation.

Author contributions

All authors contributed toward data analysis, drafting and critically revising the paper and agree to be accountable for all aspects of the work.

Disclosure

The authors report no conflicts of interest in this work.