Mide. MGMT directly demethylates O6-meG and is downregulated in about
Mide. MGMT directly demethylates O6-meG and is downregulated in about 45 of glioblastoma sufferers with MGMT promoter methylation in the tumor and enhanced temozolomide sensitivity [15]. A reported mechanism of temozolomide chemosensitization by disulfiram has been identified in pituitary adenoma stem-like cells [51] and in glioblastoma cell lines [44]: disulfiram covalently modifies MGMT, major to the proteasomal degradation on the DNA repair enzyme. Furthermore, disulfiram has been proposed in glioblastoma spheroid cultures to facilitate the DNA-damaging temozolomide effect by impairing DNA repair [12]. Temozolomide-mediated DNA DSBs reportedly trigger a G2 /M arrest of cell cycle [55]. In our present experiments (see Figures 4 and five), a temozolomide-mediated G2 /M arrest could not be detected in unirradiated LK7 and LK17 cells. Given the doubling occasions of exponentially growing LK7 and LK17 pGSCs in NSC medium of 1.7 and 1.0 days, respectively, (see Figure 1C) it might be assumed that all cells (LK17) or maybe a important fraction of cells (LK7) underwent two rounds of DNA RGS16 Inhibitor Molecular Weight replication (expected for temozolomidetriggered MMR-mediated DNA damage) in the course of the selected Met Inhibitor medchemexpress incubation period (48 h) with the flow cytometry experiments (see Figures 4 and five). In addition, temozolomide in the selected concentration (30 ) has been demonstrated in our preceding experiments to exert a higher tumoricidal effect in MGMT promotor-methylated pGSCs (unpublished personal observations). Therefore, the flow cytometry data on cell cycle and cell death with the present study confirms the relative temozolomide resistance of MGMT promoter-unmethylated glioblastoma. This was also evident in the statistically insignificant effects of temozolomide on clonogenic survival in both pGSC cultures (see Figures 6A and 7A). When confirming the tumoricidal action of disulfiram/Cu2+ in temozolomide-resistant glioblastoma stem-cell cultures, our present study didn’t observe a temozolomidesensitizing effect of disulfiram/Cu2+ (see Figures 6A and 7A). Quite the contrary, in each cell models, temozolomide markedly or had a tendency to attenuate the inhibitoryBiomolecules 2021, 11,16 ofeffect of disulfiram on clonogenic survival. Such a disulfiram effect-diminishing action of temozolomide was also suggested by our flow cytometry experiments on the cell cycle (see Figures 4 and 5). One may well speculate that temozolomide interferes with lethal pathways triggered by disulfiram. Independent of your underlying molecular mechanisms, the present observations don’t help future therapy techniques pursuing a concomitant disulfiramtemozolomide chemotherapy. Moreover, this observation suggests that the tumoricidal effect of disulfiram may well be sensitive to pharmaco-interactions with co-medications. The understanding of such pharmaco-interactions, having said that, is usually a prerequisite for the achievement of future clinical trials applying disulfiram for second-line therapy in glioblastoma patients with tumor progression during temozolomide maintenance therapy. The analysis from the molecular mechanism of such pharmaco-interactions (here, the temozolomide-disulfiram interaction), on the other hand, goes beyond the scope of the present study. 4.2. Disulfiram as a Radiosensitizer Likewise, our present study did not recognize any radiosensitization of each glioblastoma stem-cell cultures by disulfiram/Cu2+ . This really is in seeming contrast to prior studies that show a disulfiram/Cu2+ -mediated radiosensitization in patient-derived spheroid glioblas.