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.

In Vitro and In Vivo Enhancement of Temozolomide Effect in Human Glioblastoma by Non-Invasive Application of Cold Atmospheric Plasma

“…As anticipated, TMZ treatment alone resulted in a~50-60% reduction in cell viability relative to controls. However, pre-treatment with the CAP jet for 30 and 60 s followed by TMZ treatment enhanced the TMZ-induced cytotoxicity, indicating that the CAP jet sensitizes GBM cells to TMZ [13,22,23,30]. Interestingly, this potential sensitization effect was evident not only in TMZ-sensitive, but also TMZ-resistant (T98G) cell lines.…”

“…We next investigated the responsiveness of three GBM cell lines (two TMZ sensitive and one resistant; A172, U87-MG, and T98G, respectively) [14][15][16][17][18][19][20] to direct CAP jet treatment at different time exposures of 30, 60, and 90 s (frequency of 12.5 kHz and 10 V and He flow rate of 3-4 LPM) under TMZ (400 µM) exposure alone and a combination treatment of CAP + TMZ [13,22,23]. The four treatment conditions for each time point were: control (untreated), CAP jet treated, TMZ treated, and co-treatment with CAP + TMZ.…”

“…Hence, it is important to sensitize cells to TMZ using novel treatments to overcome the issue of resistance [14][15][16][17][18][19][20]. Therefore, using a series of in vitro and in vivo experiments in GBM models, we introduced co-treatment with CAP and TMZ through the bone as a new approach and non-invasive strategy to potentially sensitize GBM to chemotherapy [21][22][23]. Furthermore, we also demonstrated the utility of a novel, non-invasive He-based CAP jet to target GBM through the skull [9].…”

ROS Pleiotropy in Melanoma and Local Therapy with Physical Modalities

“…The concentration of the ROS can be tuned by changing the ionization variables and the target exposure time [217], while the ambient air condition was found to have a lower impact [218]. As a mechanism of action, gas plasmaderived ROS modulate the expression of redox-regulating enzymes and pathways [219][220][221] and was found to show combinatorial effects with chemotherapy [222][223][224], radiotherapy [225][226][227], and antibody [228] and topical immunotherapy [229]. We have recently also reported for the first time apoptotic effects in patient-derived melanoma tissues [230] and that ROS-derived oxidative posttranslational protein modifications (oxPTMs) generated with gas plasma technology have immunogenic properties and protect from melanoma growth in vivo [231].…”

Aberrant Expressional Profiling of Small RNA by Cold Atmospheric Plasma Treatment in Human Chronic Myeloid Leukemia Cells

“…), and reactive nitrogen species (RNS), such as nitric oxide (NO) and nitrate (NO 3 − ), both intracellularly and extracellularly after CAP treatment (Yan et al, 2017). Among these diverse species, H 2 O 2 has been proved to be the main anti-cancer reactive species causing the death of cancer cells (Bekeschus et al, 2014), which are able to induce DNA and mitochondrial damages that activate cell cycle checkpoints and initiate signaling cascades leading to cell death (Shaw et al, 2021). RNS, such as NO, also plays a non-negligible role since synergistically using H 2 O 2 /NO 2 in solution can generate an anti-cancer effect more close to that generated by plasma than just using H 2 O 2 alone (Girard et al, 2016).…”