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Cold Atmospheric Plasma Increases Temozolomide Sensitivity of Three-Dimensional Glioblastoma Spheroids via Oxidative Stress-Mediated DNA Damage.

ABSTRACT: Glioblastoma multiforme (GBM) is the most frequent and aggressive primary malignant brain tumor in adults. Current standard radiotherapy and adjuvant chemotherapy with the alkylating agent temozolomide (TMZ) yield poor clinical outcome. This is due to the stem-like properties of tumor cells and genetic abnormalities in GBM, which contribute to resistance to TMZ and progression. In this study, we used cold atmospheric plasma (CAP) to enhance the sensitivity to TMZ through inhibition of antioxidant signaling (linked to TMZ resistance). We demonstrate that CAP indeed enhances the cytotoxicity of TMZ by targeting the antioxidant specific glutathione (GSH)/glutathione peroxidase 4 (GPX4) signaling. We optimized the threshold concentration of TMZ on five different GBM cell lines (U251, LN18, LN229, U87-MG and T98G). We combined TMZ with CAP and tested it on both TMZ-sensitive (U251, LN18 and LN229) and TMZ-resistant (U87-MG and T98G) cell lines using two-dimensional cell cultures. Subsequently, we used a three-dimensional spheroid model for the U251 (TMZ-sensitive) and U87-MG and T98G (TMZ-resistant) cells. The sensitivity of TMZ was enhanced, i.e., higher cytotoxicity and spheroid shrinkage was obtained when TMZ and CAP were administered together. We attribute the anticancer properties to the release of intracellular reactive oxygen species, through inhibiting the GSH/GPX4 antioxidant machinery, which can lead to DNA damage. Overall, our findings suggest that the combination of CAP with TMZ is a promising combination therapy to enhance the efficacy of TMZ towards the treatment of GBM spheroids.

SUBMITTER: Shaw P

PROVIDER: S-EPMC8068248 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundGlioblastoma (GBM) is the most common primary malignant brain tumor in adults. Ubiquitously expressed volume-regulated anion channels (VRAC) are thought to play a role in cell proliferation, migration, and apoptosis. VRAC are heteromeric channel complexes assembled from proteins belonging to the leucine-rich repeat-containing 8A (LRRC8A through E), among which LRRC8A plays an indispensable role. In the present work, we used an RNAi approach to test potential significance of VRAC and LRRC8A in GBM survival and sensitivity to chemotherapeutic agents.MethodsPrimary GBM cells were derived from a human surgical tissue sample. LRRC8A expression was determined with quantitative RT-PCR and downregulated using siRNA. The effects of LRRC8A knockdown on GBM cell viability, proliferation, and sensitivity to chemotherapeutic agents were determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide and Coulter counter assays. Cell cycle progression was further explored using fluorescence-activated cell sorting analysis of propidium iodide-stained cells.ResultsTemozolomide (TMZ), carmustine, and cisplatin reduced GBM cell survival with the IC50 values of ~1,250, 320, and 30?µM, respectively. Two of three tested gene-specific siRNA constructs, siLRRC8A_3 and siLRRC8A_6, downregulated LRRC8A expression by >80% and significantly reduced GBM cell numbers. The most potent siLRRC8A_3 itself reduced viable cell numbers by ?50%, and significantly increased toxicity of the sub-IC50 concentrations of TMZ (570?µM) and carmustine (167?µM). In contrast, the effects of siLRRC8A_3 and cisplatin (32?µM) were not additive, most likely because cisplatin uptake is VRAC-dependent. The results obtained in primary GBM cells were qualitatively recapitulated in U251 human GBM cell line.ConclusionDownregulation of LRRC8A expression reduces GBM cell proliferation and increases sensitivity to the clinically used TMZ and carmustine. These findings indicate that VRAC represents a potential target for the treatment of GBM, alone or in combination with the current standard-of-care.

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Cold Atmospheric Plasma Increases Temozolomide Sensitivity of Three-Dimensional Glioblastoma Spheroids via Oxidative Stress-Mediated DNA Damage.

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