Vorinostat Enhances the Radiosensitivity of a Breast Cancer Brain Metastatic Cell Line Grown In Vitro and as Intracerebral Xenografts

Article Outline

• Purpose/Objective(s)
• Materials/Methods
• Results
• Conclusions
• Copyright

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Purpose/Objective(s) 

Histone deacetylase (HDAC) inhibitors are a promising treatment option for many tumors. Multiple HDAC inhibitors have been shown to enhance tumor cell sensitivity to radiation. One such HDAC inhibitor that is currently undergoing clinical trials is vorinostat (suberoylanilide hydroxamic acid, SAHA), which has been shown to enhance the radiosensitivity of a number of tumor cell lines grown in vitro. To extend these observations to a model of brain metastases, we investigated the radiosensitivity of vorinostat on a breast cancer brain metastasis cell line grown in vitro and in vivo as subcutaneous and intracerebral xenografts.

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Materials/Methods 

MDA-MB-231-BR cells, a brain-seeking variant of a human breast cancer cell line MDA-MB-231, were used in all experiments. The acetylation status of histones H3 and H4 was determined in vorinostat-treated cells by immunoblot analysis. In vitro radiosensitivity was determined via colony forming assays in 231-BR cells. DSBs were assessed by gamma-H2AX foci and the neutral comet assay. Cell cycle changes were evaluated by flow cytometry. Tumor growth delay was assayed by measuring tumor volume of MDA-MB-231BR cells grown s.c. in the hind leg of nude mice. Survival was determined in an i.c. model of brain metastases. In both these experiments 50mg/kg of vorinostat was given 6h before IR.

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Results 

Hyper acetylation of H3 was achieved with a 16h exposure of 1uM vorinostat. Clonogenic survival assays showed that 231BR cells exposed to 1 uM of vorinostat for 16 hrs before and maintained in the media after IR had an increase in radiosensitivity with DEFs (dose enhancement factors at a surviving fraction of 0.1) of 1.57. Gamma-H2AX, as an indicator of DSBs, had significantly more foci per cell in the combination group vs. the IR alone group when measured at 24 h post-IR. Cell cycle analysis revealed no differences in cells treated with drug, IR or combination. Irradiation of 231-BR tumors in mice treated with vorinostat resulted in an increase in radiation-induced tumor growth delay. Most importantly, for xenografts grown intracerebrally there was a greater than additive effect on survival using the combination of vorinostat and IR than either treatment individually.

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Conclusions 

These results indicate that vorinostat can enhance tumor cell radiosensitivity in vitro and in vivo. In particular there was a greater than additive improvement in survival in our model of breast cancer brain metastases and therefore may be a potential treatment option for patients with breast cancer who develop brain metastases.