Cooperation of the HDAC inhibitor vorinostat and radiation in metastatic neuroblastoma: efficacy and underlying mechanisms

Abstract

Histone deacetylase (HDAC) inhibitors can radiosensitize cancer cells. Radiation is critical in high-risk neuroblastoma treatment, and combinations of HDAC inhibitor vorinostat and radiation are proposed for neuroblastoma trials. Therefore, we investigated radiosensitizing effects of vorinostat in neuroblastoma. Treatment of neuroblastoma cell lines decreased cell viability and resulted in additive effects with radiation. In a murine metastatic neuroblastoma in vivo model vorinostat and radiation combinations decreased tumor volumes compared to single modality. DNA repair enzyme Ku-86 was reduced in several neuroblastoma cells treated with vorinostat. Thus, vorinostat potentiates anti-neoplastic effects of radiation in neuroblastoma possibly due to down-regulation of DNA repair enzyme Ku-86.

Fig. 1

Clonogenic survival assays of neuroblastoma cell lines NB1691^luc, SY5Y, Kelly, Tet21 (MYCN overexpressed), and Tet21 (MYCN repressed) treated with vorinostat and radiation. Surviving fractions are normalized to control samples for each treatment group. Solid line: radiation alone; dashed line: 0.5 μM vorinostat pre-treatment followed by radiation. All experiments were performed in quadruplicate. Surviving fractions are shown on a log scale as mean and bars represent standard error of the mean; where errors are not visualized, the error bars are smaller than the size of the symbol.

Fig. 2

(A) In vivo model of metastatic neuroblastoma in athymic mice. Mice were injected by tail vein with 6 × 10⁶ NB1691^luc cells. Approximately 21 days after injection all mice demonstrated visible tumors by bioluminescence. Tumor volumes for each mouse were quantified by measuring the whole mouse body emission of photons in a standardized fashion. (B) Immunohistological hematoxillin–eosin (H&E) stain of a representative tumor.

Fig. 3

In vivo efficacy of combined vorinostat (vorinst) and radiation in the treatment of metastatic neuroblastoma. Seven days after tail vein injection of NB1691^luc cells, mice were treated with intraperitoneal vorinostat (150 mg/kg) every other day for a total of three doses, radiation (1 Gy) every other day for a total of 3 Gy, or combination of vorinostat (150 mg/kg) followed by radiation (1 Gy delivered 1 h after vorinostat). Each treatment group contained 10 mice. Arrows indicate treatment days. Bioluminescence imaging was performed until the first mouse died. p-Values of group comparisons are based on Wilcoxon Rank-Sum test and were calculated as vorinostat versus combination of vorinostat and radiation (p = 0.04) and radiation versus combination of vorinostat and radiation (p = 0.04). Control group is represented by dotted line, radiation (XRT) alone by solid line, vorinostat (vorinst) alone by small dashed, and combination group by large dashed line.

Fig. 4

γ-H2AX expression levels after vorinostat and radiation (XRT) treatment measured by flow cytometric analysis of NB1691^luc cells. Cells were incubated with vorinostat at (0.5 μM, 1 μM, or 2.5 μM) for 24 h and irradiated with 2 Gy 24 h later. Cells were harvested 6 h after radiation. Percent of cells positive for γ-H2AX staining is shown on the y-axis.

Fig. 5

Western blot analysis of Ku-86, XRCC4 Like Factor (XLF), and Rad51 expression in NB1691^luc cells after vorinostat and radiation treatment. Cells were treated with vorinostat (1.0 μM or 2.5 μM), 2 Gy of radiation, or combination treatment. Cells were harvested 24 h after radiation. β-Actin was used as a loading control.