Three Thiol-Reactive Reagents drive Synergistic Lethality in Glioblastoma cells.

Glioblastoma (GBM) is the most prevalent malignant primary brain tumor. Disruption of the redox state of the cell through cysteine (Cys) reactive residues has been suggested to play a role in the progression of GBM. Here, we demonstrate that the addition of acrylamide (ACR), a thiol-reactive molecule that covalently modifies redox-related proteins and disrupts critical redox signaling pathways, further amplifies the cytotoxic effects and the synergistic lethality induced by auranofin (Auf) and buthionine sulfoximine (DL-BSO). We established a minimal triple-thiol cocktail of ACR/Auf/BSO, which enhances suppression of cell growth in various cancer cell lines. This combination of ACR, BSO, and Auf, which causes a complete cell death in U87MGMG, U87MGΔEGFR, A431, PC12 and SH-SY5Y cells induces significantly less toxicity in primary rat cortex neuronal cultures. The synergy observed is strongly associated with increased activation of ERK1/2 and p38MAPK phosphorylation, as well as the inhibition of the STAT3 signaling pathways, which are both critical for cell survival and proliferation. In contrast, primary neuronal cortical cells, which exhibit minimal toxicity with the minimal ACR/Auf/BSO combination, display no activation of these antioxidant/anti-inflammatory molecular pathways. The combination of the oxidizing reagents induces whole-cell and mitochondrial reactive oxygen species (ROS) production in patient-derived GBM cells and reduces proliferation of a human GBM organoid model. We suggest that combining multiple Cys-associated redox targets is a putative therapeutic strategy for GBM, which offers a promising approach for improving treatment outcomes in GBM and other malignancies.