Rapid Peroxide Removal Limits the Radiosensitization of Diffuse Intrinsic Pontine Glioma (DIPG) Cells by Pharmacologic Ascorbate

Abstract

Diffuse intrinsic pontine gliomas (DIPG) are an aggressive type of pediatric brain tumor with a very high mortality rate. Surgery has a limited role given the tumor's location. Palliative radiation therapy alleviates symptoms and prolongs survival, but median survival remains less than 1 year. There is no clear role for chemotherapy in DIPGs as trials adding chemotherapy to palliative radiation therapy have failed to improve survival compared to radiation alone. Thus, there is a critical need to identify tissue-specific radiosensitizers to improve clinical outcomes for patients with DIPGs. Pharmacologic (high dose) ascorbate (P-AscH-) is a promising anticancer therapy that sensitizes human tumors, including adult high-grade gliomas, to radiation by acting selectively as a generator of hydrogen peroxide (H2O2) in cancer cells. In this study we demonstrate that in contrast to adult glioma models, P-AscH- does not radiosensitize DIPG. DIPG cells were sensitive to bolus of H2O2 but have faster H2O2 removal rates than GBM models which are radiosensitized by P-AscH-. These data support the hypothesis that P-AscH- does not enhance DIPG radiosensitivity, likely due to a robust capacity to detoxify and remove hydroperoxides.

FIG. 1.

DIPG cancer cell lines do not show sensitivity to ascorbate. Pre-plating clonogenic survival assays were performed with SU-DIPG50, SU-DIPG38, SU-DIPG36, SF10693, U251, U87, MDA-MB-231 and NHA cells treated with various concentrations of ascorbate (5–20 pmol cell⁻¹). Data are normalized to the untreated condition for each cell line. Error bars represent the mean ± SD of three experiments with three replicates (n = 3+; N = 9+). ns = not significant.

FIG. 2.

Ascorbate does not sensitize DIPG cells to radiation. Pre-plating clonogenic survival assays were performed with NHA, SU-DIPG36, SF10693, SU-DIPG50 and SU-DIPG38, U251, U87, and MDA-MB-231 cells treated with a single dose of radiation (1–6 Gy), with or without ascorbate (20 pmol cell⁻¹). Data are normalized to 0 Gy for both treated and untreated samples to start at 100%. Error bars represent the mean ± SD of three experiments with three replicates (n = 3; N = 9).

FIG. 3.

DIPG cancer cell lines are more sensitive to a bolus of exogenous H₂O₂ than normal astrocytes. Pre-plating clonogenic survival assays were performed with NHA, SU-DIPG50 and SU-DIPG38 cells treated with a bolus of H₂O₂ (7–70 μM). Data are normalized to the untreated condition for each cell line. Error bars represent the mean ± SD of three experiments with three replicates (n = 3; N = 9). *P < 0.05 as analyzed by one-way ANOVA with Tukey’s Test for post hoc analysis.

FIG. 4.

Rate constant, k_cell, for removal of extracellular H₂O₂ by cells. H₂O₂ removal was monitored in NHA, SU-DIPG50, SU-DIPG38, U251 and U87 cells. Cells were treated with 20 μM H₂O₂ at different times before the addition of the stopping solution. Panel A: The removal of H₂O₂ using rate= −k_cell [H₂O₂] (number of cells L⁻¹). Panel B: A bar graph representation with statistical analysis of the mean peroxide removal rates for the cell lines tested. Error bars represent the mean ± SD (n = 5+). ns = not significant, *P < 0.05 as analyzed by One-Way ANOVA with Tukey’s Test for post hoc analysis.