Repurposing the anti-epileptic drug sodium valproate as an adjuvant treatment for diffuse intrinsic pontine glioma

<div><p>Targeting epigenetic changes in diffuse intrinsic pontine glioma (DIPG) may provide a novel treatment option for patients. This report demonstrates that sodium valproate, a histone deacetylase inhibitor (HDACi), can increase the cytotoxicity of carboplatin in an additive and synergistic manner in DIPG cells <i>in vitro</i>. Sodium valproate causes a dose-dependent decrease in DIPG cell viability in three independent <i>ex vivo</i> cell lines. Furthermore, sodium valproate caused an increase in acetylation of histone H3. Changes in cell viability were consistent with an induction of apoptosis in DIPG cells <i>in vitro</i>, determined by flow cytometric analysis of Annexin V staining and assessment of apoptotic markers by western blotting. Subsequently, immunofluorescent staining of neuronal and glial markers was used to determine toxicity in normal rat hippocampal cells. Pre-treatment of cells with sodium valproate enhanced the cytotoxic effects of carboplatin, in three DIPG cell lines tested. These results demonstrate that sodium valproate causes increased histone H3 acetylation indicative of HDAC inhibition, which is inversely correlated with a reduction in cell viability. Cell viability is reduced through an induction of apoptosis in DIPG cells. Sodium valproate potentiates carboplatin cytotoxicity and prompts further work to define the mechanism responsible for the synergy between these two drugs and determine <i>in vivo</i> efficacy. These findings support the use of sodium valproate as an adjuvant treatment for DIPG.</p></div

<i>In vitro</i> primary neuronal and glial cell toxicity analysis.

<p>Primary rat hippocampal cells were dosed with valproate for 72 hours and assessed by immunofluorescent staining of neuronal (B3 tubulin) and glial (GFAP) markers. Hippocampal cultures show intact neuronal networks at 5 mM valproate and glial cells with normal morphology indicative of no significant toxicity.</p

Valproate induced apoptosis in <i>ex vivo</i> DIPG cells <i>in vitro</i>.

<p>Cells were seeded in 12-well tissue culture plates and treated with sodium valproate for 72 hours prior to analysis by flow cytometry for Annexin V and 7-AAD. (A) Dose responses from at least 3 independent experiments were assessed and demonstrate an increase in Annexin V binding occurs from 1–10 mM valproate, with a significant induction of apoptosis occurring at doses of ≥5 mM valproate (p = <0.05). (B) Representative images of apoptosis analysis, all experiments were compensated based on single dyes alone. (C) To confirm the induction of apoptosis western blotting was carried out on all three cell lines treated with ≤5 mM valproate for 72 hours. * denotes statistical significance p = <0.05.</p