Targeting Folate Metabolism Is Selectively Cytotoxic to Glioma Stem Cells and Effectively Cooperates with Differentiation Therapy to Eliminate Tumor-Initiating Cells in Glioma Xenografts

Abstract

Glioblastoma (GBM) is one of the deadliest of all human cancers. Developing therapies targeting GBM cancer stem cells or glioma stem cells (GSCs), which are deemed responsible for the malignancy of GBM due to their therapy resistance and tumor-initiating capacity, is considered key to improving the dismal prognosis of GBM patients. In this study, we found that folate antagonists, such as methotrexate (MTX) and pemetrexed, are selectively cytotoxic to GSCs, but not to their differentiated counterparts, normal fibroblasts, or neural stem cells in vitro, and that the high sensitivity of GCSs to anti-folates may be due to the increased expression of RFC-1/SLC19A1, the reduced folate carrier that transports MTX into cells, in GSCs. Of note, in an in vivo serial transplantation model, MTX alone failed to exhibit anti-GSC effects but promoted the anti-GSC effects of CEP1347, an inducer of GSC differentiation. This suggests that folate metabolism, which plays an essential role specifically in GSCs, is a promising target of anti-GSC therapy, and that the combination of cytotoxic and differentiation therapies may be a novel and promising approach to effectively eliminate cancer stem cells.

HDAC Class I Inhibitor Domatinostat Preferentially Targets Glioma Stem Cells over Their Differentiated Progeny

“…The human GSCs used in this study (GS-Y01, GS-Y03, and TGS01) were maintained under previously reported monolayer stem cell culture conditions [ 8 , 39 ]. The differentiation of GSCs was induced by culturing cells in a DMEM/F-12 medium supplemented with 10% fetal bovine serum (FBS), 100 units/mL of penicillin, and 100 μg/mL of streptomycin for 2 weeks [ 8 , 39 ].…”

“…The human GSCs used in this study (GS-Y01, GS-Y03, and TGS01) were maintained under previously reported monolayer stem cell culture conditions [ 8 , 39 ]. The differentiation of GSCs was induced by culturing cells in a DMEM/F-12 medium supplemented with 10% fetal bovine serum (FBS), 100 units/mL of penicillin, and 100 μg/mL of streptomycin for 2 weeks [ 8 , 39 ]. IMR90, a human normal fetal lung fibroblast cell line, was obtained from the American Type Culture Collection (Manassas, VA, USA) and maintained in DMEM supplemented with 10% FBS.…”

“…Cell viability was evaluated using the WST-8 assay [ 7 , 8 ]. Cells (0.5–1 × 10 4 /well) plated on 96-well collagen I-coated plates (GSCs) or non-coated plates (differentiated GSCs and IMR90) were treated with domatinostat, as described in the figure legends.…”

“…Western blot analysis was conducted as previously described [ 8 , 45 ]. Cells were harvested and washed with ice-cold PBS and lysed in RIPA buffer (10 mM Tris/HCl (pH 7.4), 0.1% sodium dodecyl sulfate (SDS), 0.1% sodium deoxycholate, 1% Nonidet P-40, 150 mM NaCl, 1 mM EDTA, 1.5 mM sodium orthovanadate, 10 mM sodium fluoride, 10 mM sodium pyrophosphate, and protease inhibitor cocktail set III (FUJIFILM Wako Chemicals, Osaka, Japan)).…”

“…We previously demonstrated using GSC lines and their isogenic, differentiated counterparts that licochalcone A, a herbal medicine with known inhibitory activity against complex III of the mitochondrial electron transport chain, is selectively cytotoxic to GSCs [ 6 ], which led to the subsequent discovery that GSCs are characterized by increased oxidative phosphorylation, which they depend on heavily for survival, and can be targeted with verteporfin, a drug approved for the treatment of macular degeneration [ 7 ]. We also found recently that methotrexate, an antimetabolite which has long been used to treat a variety of human cancers, exerts preferential cytotoxicity toward GSCs over their differentiated counterparts due to their dependence on folate metabolism as represented by increased expression of the reduced folate carrier RFC-1/SLC19A1 in GSCs [ 8 ]. This is in line with the reported dependence of GSCs on purine synthesis [ 9 ] and together suggests that folate-dependent one-carbon metabolism involved in purine synthesis may be one of the vulnerabilities of GSCs.…”