In vivo efficacy of a novel histone deacetylase inhibitor in combination with radiation for the treatment of gliomas

Abstract

Histone modification has emerged as a promising approach to cancer therapy. We explored the in vivo efficacy of a butyric acid derivative, pivaloyloxymethyl butyrate (AN-9), for the treatment of gliomas. Relative to control and single-modality treatments, the combination of AN-9 and radiation significantly inhibited tumor growth and prolonged time to failure in mice bearing glioma xenografts. The enhanced response to radiation was accompanied by inhibition of cellular proliferation and by increased phosphorylation of H2AX, implicating DNA double-strand breaks in the antineoplastic effects of AN-9 and radiation. The data suggest that AN-9 in combination with radiation may be an effective therapy for malignant gliomas.

Fig. 1

Combined in vivo treatment with AN-9 and radiation delays the tumor growth rate and prolongs TTF in mice bearing U251 MG xenografts. (A) Mean tumor volumes ± SEM on day 36 after treatment initiation in four cohorts receiving vehicle control, AN-9 alone, radiation alone, and AN-9 plus radiation. *Statistically significant difference between combined treatment and each of the other three conditions (two-sided t-test). (B) Tumor growth rate curves of the four experimental groups. Arrows indicate the days on which radiation was administered. The last tumor measurement was taken on day 36, the last day on which all animals were still alive. (C) TTF calculated for the first 47 days of treatment (log-rank test). Treatment failure was defined as tumor size greater than 1000 mm³ or development of severe necrosis requiring euthanasia.

Fig. 2

Analyses of tumor sections from mice bearing xenografts of U251 MG and treated with vehicle control, AN-9 alone, radiation alone, or combined AN-9 and radiation. (Left column) Hematoxylin and eosin staining. (Right column) Immunohistochemical staining for cleaved caspase-3.

Fig. 3

(A) AN-9 and radiation cooperate to augment DNA DSBs, as reflected in increased expression of phospho-γ-H2AX. Established xenografts were treated with vehicle control, AN-9 (50 mg/kg), radiation (2.5 or 5 Gy), or AN-9 and then radiation 24 h later. Phospho-γ-H2AX levels were assayed 24 h after the last treatment. (B) AN-9 functions as an HDACI in vivo and results in enhanced H3 and H4 acetylation. At designated times following each treatment, mice were euthanized; histones were purified from resected xenografts and analyzed for H3 and H4 acetylation by Western blot analysis. Total H4 was used as a loading control for histone modification. Quantitation of total H4 was done with a lower exposure film.

Fig. 4

Trimodality therapy with AN-9, radiation, and TMZ displayed the greatest antineoplastic activity. Values from MTS proliferation assays are presented relative to untreated control. *Statistically significant difference (two-tailed t-test) between the combination treatment and each other modality alone. **Statistically significant difference between the trimodality therapy and any other bimodality treatment (AN-9 + 4 Gy, AN-9 + TMZ, and 4 Gy + TMZ).