Repurposing Disulfiram for Targeting of Glioblastoma Stem Cells: An In Vitro Study

Abstract

Mesenchymal glioblastoma stem cells (GSCs), a subpopulation in glioblastoma that are responsible for therapy resistance and tumor spreading in the brain, reportedly upregulate aldehyde dehydrogenase isoform-1A3 (ALDH1A3) which can be inhibited by disulfiram (DSF), an FDA-approved drug formerly prescribed in alcohol use disorder. Reportedly, DSF in combination with Cu2+ ions exerts multiple tumoricidal, chemo- and radio-therapy-sensitizing effects in several tumor entities. The present study aimed to quantify these DSF effects in glioblastoma stem cells in vitro, regarding dependence on ALDH1A3 expression. To this end, two patient-derived GSC cultures with differing ALDH1A3 expression were pretreated (in the presence of CuSO4, 100 nM) with DSF (0 or 100 nM) and the DNA-alkylating agent temozolomide (0 or 30 µM) and then cells were irradiated with a single dose of 0–8 Gy. As read-outs, cell cycle distribution and clonogenic survival were determined by flow cytometry and limited dilution assay, respectively. As a result, DSF modulated cell cycle distribution in both GSC cultures and dramatically decreased clonogenic survival independently of ALDH1A3 expression. This effect was additive to the impairment of clonogenic survival by radiation, but not associated with radiosensitization. Of note, cotreatment with temozolomide blunted the DSF inhibition of clonogenic survival. In conclusion, DSF targets GSCs independent of ALDH1A3 expression, suggesting a therapeutic efficacy also in glioblastomas with low mesenchymal GSC populations. As temozolomide somehow antagonized the DSF effects, strategies for future combination of DSF with the adjuvant standard therapy (fractionated radiotherapy and concomitant temozolomide chemotherapy followed by temozolomide maintenance therapy) are not supported by the present study.

Keywords

• brain tumor
• primary spheroid culture
• tumor-initiating cells
• propidium iodide
• Nicoletti staining
• survival fraction