Chemical Screening Identifies EUrd as a Novel Inhibitor Against Temozolomide-Resistant Glioblastoma-Initiating Cells

Abstract

Glioblastoma (GBM), one of the most malignant human cancers, frequently recurs despite multimodal treatment with surgery and chemo/radiotherapies. GBM-initiating cells (GICs) are the likely cell-of-origin in recurrences, as they proliferate indefinitely, form tumors in vivo, and are resistant to chemo/radiotherapies. It is therefore crucial to find chemicals that specifically kill GICs. We established temozolomide (the standard medicine for GBM)-resistant GICs (GICRs) and used the cells for chemical screening. Here, we identified 1-(3-C-ethynyl-b-D-ribopentofuranosyl) uracil (EUrd) as a selective drug for targeting GICRs. EUrd induced the death in GICRs more effectively than their parental GICs, while it was less toxic to normal neural stem cells. We demonstrate that the cytotoxic effect of EUrd on GICRs partly depended on the increased expression of uridine-cytidine kinase-like 1 (UCKL1) and the decreased one of 5 0 -nucleotidase cytosolic III (NT5C3), which regulate uridine-monophosphate synthesis positively and negatively respectively. Together, these findings suggest that EUrd can be used as a new therapeutic drug for GBM with the expression of surrogate markers UCKL1 and NT5C3. STEM CELLS 2016;34:2016-2025 SIGNIFICANCE STATEMENTOne of main hurdles in cancer stem cell research is to establish a method for maintaining and enriching bona fide cancer-initiating cells (CICs) in culture. Herein we show that glioblastomainitiating cells (GICs) were enriched in the presence of temozolomide, the standard medicine for glioblastoma. Using the temozolomide-resistant GICs (GICRs), we performed chemical screening and identified 1-(3-C-ethynyl-b-D-ribopentofuranosyl) uracil (EUrd) that effectively killed GICRs. We demonstrate that the upregulated uridine-monophosphate synthesis pathway increased EUrd-cytotoxicity in GICRs. Thus, combination of anti-cancer drug-resistant CICs and chemical screening can be applied to find curable anti-cancer drugs.

Identification of antipsychotic drug fluspirilene as a potential anti-glioma stem cell drug

“…In this study, we identified fluspirilene as an agent inhibiting viability of GSCs and differentiated GBM cells, which demonstrated that our drug screening system was practical for the isolation of small-molecule compounds for the treatment of GBM. Tsukamoto et al designed a screening system for a unique compound library, containing nucleosides, sugars, peptides, kinase inhibitors, and natural products, using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay [ 23 ]. We adopted a screening system using existing drug libraries.…”

Dramatic response of BRAF V600E-mutant epithelioid glioblastoma to combination therapy with BRAF and MEK inhibitor: establishment and xenograft of a cell line to predict clinical efficacy

“…To investigate the efficacy of treatment, these mice received vehicle control (0.5% hydroxypropyl methylcellulose plus 0.2% Tween 80, and 20% dimethyl-sulfoxide (DMSO)) or combined treatment (20 mg/kg dabrafenib and 2 mg/kg trametinib solubilized in 20% DMSO) by oral gavage for 10 days. The tumors were measured daily with calipers, and tumor volume was calculated using the formula: Tumor volume (mm 3 ) = Length (mm) × Width (mm) 2 /2 [26]. Two additional mice from each treatment group were used for histopathological and Western blot analyses as described above.…”

MGMT Expression Contributes to Temozolomide Resistance in H3K27M-Mutant Diffuse Midline Gliomas

“…NGT16, SF8628, U87MG, T98G cells were grown as adherent monolayer cultures in 10% FBS DMEM. SF7761, JHH-DIPG1 cells were grown as sphere cultures in EF20 medium composed with Neurobasal Medium (Thermo), 20 ng/ml EGF (Peprotech, Rocky Hill, NJ, USA), 20 ng/ml FGF (Peprotech), 2% B27 supplement (Thermo), 0.25% N2 supplement (Thermo), 3 mM L-Glutamine (Thermo), 2 µg/ml Heparin (Sigma), and 1% Antibiotic-Antimycotic (Thermo) (30,31).…”