A novel role of liquid plasma (LP) induced RONS triggers the endoplasmic reticulum stress response and is associated with GSDME-mediated pyroptosis in anaplastic thyroid cancer.

Background: Anaplastic thyroid cancer (ATC) is an aggressive cancer with a poor prognosis and resistance to multimodal treatments. Various drugs have a short response time and are likely ineffective against ATC. Therefore, new molecular therapies and novel targets are needed. Recently, plasma medicine has been used in biomedical applications on skin, wound healing, and cancer treatment of the liver, colon, and head and neck. However, direct plasma has difficulty treating, sustaining, and permeating the various affected areas. In this study, we prepared Liquid-type plasma (LP) and investigated their effect on ATC cells.

Methods: The physicochemical properties and ion composition of liquid-type plasma (LP) were analyzed using ion chromatography, after which anaplastic thyroid cancer (ATC) cell lines, primary fibroblasts, and keratinocytes were employed to evaluate its biological effects. Cell viability, apoptosis/pyroptosis, and redox status were assessed by biochemical assays, flow cytometry, and Western blotting, while transcriptomic profiling (RNA-seq) with gene ontology analysis was performed to identify regulated pathways. Finally, an orthotopic ATC xenograft mouse model was used to validate the therapeutic efficacy of LP in vivo.

Results: The pH of LP decreased to 1.92 And remained stable for 14 days, while electrical conductivity was measured at 4.76 mS/cm without significant Change. Additionally, the oxidation-reduction potential was maintained at 652.2 mV for 14 days. Cl, NO2, and NO3 Anions were identified as 62 ppb, 12.6 ppm, And 1044 ppm, respectively, And Na, K, And Ca cations were measured as 169 ppb, 27 ppb, And 25 ppb, respectively. Therefore, a significant concentration of nitrogen species is involved in the LP, which is attributed to the secondary reaction of reactive oxygen species (ROS) formed during this treatment. LP reduced ATC cell viability, which exhibited cell swelling and bubble formation morphologically. Gasdermin E (GSDME) was cleaved by LP and induced pyroptosis in ATC cells. Inhibiting caspase activity and knock down of GSDME showed reduced caspase-3 dependent cleavage of GSDME, consequently increasing cell viability and decreasing lactate dehydrogenase release. LP enhanced the production of intracellular RONS and induced phosphorylation of IRE1α that is associated with endoplasmic reticulum (ER) stress. Therefore, GSDME-mediated LP induced pyroptosis downstream of the RONS/ER stress pathway and activated caspase-3. We then confirmed tumor shrinkage through in vivo and histological analysis, with a focus on the increasing GSDME-N terminal.

Conclusion: LP induces pyroptosis via GSDME activation and RONS generation in vitro and in vivo in ATC models, suggesting a novel mechanism and indicating a potential chemotherapeutic approach for the treatment of ATC.