Mide. MGMT straight demethylates O6-meG and is downregulated in about
Mide. MGMT straight 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 for the proteasomal degradation in the DNA repair enzyme. Furthermore, disulfiram has been proposed in glioblastoma spheroid cultures to facilitate the DNA-damaging temozolomide impact 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 5), a temozolomide-mediated G2 /M arrest couldn’t be detected in unirradiated LK7 and LK17 cells. Given the doubling instances of exponentially expanding LK7 and LK17 pGSCs in NSC medium of 1.7 and 1.0 days, respectively, (see Figure 1C) it may be assumed that all cells (LK17) or maybe a considerable fraction of cells (LK7) underwent two rounds of DNA replication (essential for temozolomidetriggered MMR-mediated DNA harm) through the selected incubation period (48 h) from the flow cytometry experiments (see Figures four and 5). Furthermore, temozolomide at the chosen concentration (30 ) has been demonstrated in our preceding experiments to exert a higher tumoricidal effect in MGMT promotor-methylated pGSCs (unpublished own observations). Therefore, the flow cytometry information on cell cycle and cell death on the present study confirms the relative temozolomide resistance of MGMT promoter-unmethylated glioblastoma. This was also evident from the statistically insignificant effects of temozolomide on clonogenic survival in both pGSC cultures (see Figures 6A and 7A). While confirming the tumoricidal action of disulfiram/Cu2+ in temozolomide-resistant glioblastoma stem-cell cultures, our present study did not observe a temozolomidesensitizing impact of disulfiram/Cu2+ (see Figures 6A and 7A). Very the contrary, in both 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 around the cell cycle (see Figures four and five). One could speculate that temozolomide interferes with lethal pathways triggered by disulfiram. Independent on the underlying molecular mechanisms, the present observations usually do not assistance future TXA2/TP Antagonist drug therapy tactics pursuing a concomitant disulfiramtemozolomide chemotherapy. Furthermore, this observation suggests that the tumoricidal impact of disulfiram may perhaps be sensitive to pharmaco-interactions with co-medications. The understanding of such pharmaco-interactions, however, is usually a prerequisite for the achievement of future RIPK3 Activator review clinical trials applying disulfiram for second-line therapy in glioblastoma individuals with tumor progression during temozolomide maintenance therapy. The evaluation from the molecular mechanism of such pharmaco-interactions (here, the temozolomide-disulfiram interaction), having said that, goes beyond the scope from 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 is in seeming contrast to prior research that show a disulfiram/Cu2+ -mediated radiosensitization in patient-derived spheroid glioblas.