Pharmacological concentrations of ascorbate radiosensitize glioblastoma multiforme primary cells by increasing oxidative DNA damage and inhibiting G2/M arrest

Abstract

Glioblastoma multiforme (GBM) has a very poor prognosis because of its chemo- and radiation therapy resistance. Here we investigated the ability of pharmacological concentrations of ascorbate to radiosensitize primary cells isolated from six GBM patients, mouse astrocytoma cells, and mouse astrocytes. We measured cell viability by trypan blue exclusion, generation of double-stranded DNA breaks by H2AX phosphorylation using fluorescently labeled antibodies and FACS analysis, apoptosis by annexin V/propidium iodide staining, inhibition of autophagy by 3-methyladenine, and cell cycle progression by propidium iodide staining of permeabilized cells. We showed that 5 mM ascorbate in combination with 6 Gy radiation killed more GBM primary cells by generating significantly more double-stranded breaks than either treatment alone (p<0.05). Combined treatment affected viability and double-stranded break generation in normal astrocytes to a much smaller extent. Radiation, but not 5 mM ascorbate, caused G2/M arrest in GBM cells and ascorbate prevented radiation-induced G2/M arrest in combined treatment. Cell death in response to 5 mM ascorbate or combination treatment was not mediated by apoptosis or autophagy. In conclusion, pharmacological concentrations of ascorbate radiosensitize GBM primary cells to a much greater extent than astrocytes; this large therapeutic ratio may be of clinical significance in radiation-resistant cancers.