Διερεύνηση των αποκρίσεων των καρκινικών κυττάρων μετά από οξειδωτικό στρες που προκαλείται από CAP, με έμφαση στον μεταγραφικό παράγοντα NRF2 και στα γονίδια-στόχους του

Cold atmospheric plasma (CAP) is an innovative technology that has attracted increasing interest in the field of oncology. It is a partially ionized gas that can be applied at temperatures compatible with biological tissues and consists of electrons, ions, excited particles, and reactive oxygen and nitrogen species (RONS). Experimental evidence has demonstrated its ability to selectively induce cell death. Although the molecular mechanisms underlying its anticancer activity have not yet been fully elucidated, disruption of intracellular redox homeostasis by CAP-generated RONS is considered a key contributing factor. The transcription factor NRF2 plays a central role in the cellular response to oxidative stress by inducing the expression of antioxidant and cytoprotective genes. In cancer cells, however, NRF2 activation may serve as an adaptive mechanism that promotes cell survival and enhances resistance to oxidative and therapeutic challenges. The aim of the present Master’s thesis was to investigate the role of reactive species and NRF2 signaling in the response of breast cancer cells to oxidative stress induced by the direct application of dielectric barrier discharge cold atmospheric plasma (DBD-CAP). For this purpose, three breast cancer cell lines with distinct molecular and phenotypic characteristics were employed: Mcf-7, an estrogen receptor-positive cell line with low metastatic potential, and the more aggressive triple-negative breast cancer cell lines MDA-MB-231 and Hs578T. The involvement of RONS in the effects of DBD-CAP was confirmed by pre-treating the cells with the scavenger N-acetylcysteine (NAC). In parallel, the contribution of NRF2 was investigated using its established inhibitors, chrysin and brusatol. Cell viability and morphology, the subcellular distribution of NRF2, the transcriptional expression of NRF2 and its antioxidant target genes HO-1, NQO-1, and GSR, as well as NRF2 protein levels and the activation of the redox-sensitive ERK1/2 and Akt signaling pathways, were evaluated. The results demonstrated that direct DBD-CAP treatment significantly reduced cell viability in all three cell lines, although the degree of sensitivity differed among them. NAC pre-treatment markedly attenuated, but did not completely reverse, DBD-CAP-induced cytotoxicity and contributed to the preservation of cellular morphology. Furthermore, DBD-CAP induced pronounced nuclear accumulation of NRF2, which was attenuated in the presence of NAC. NAC also generally suppressed the induction of the antioxidant target genes HO-1, NQO-1, and GSR. Collectively, these findings confirm that CAP-generated RONS are key mediators of both DBD-CAP-induced cytotoxicity and the associated activation of the cellular antioxidant response. Pre-treatment with either chrysin or brusatol enhanced the DBD-CAP-induced reduction in cell viability in all three cell lines, with brusatol generally exerting a more pronounced effect. Both compounds attenuated the nuclear accumulation of NRF2 following plasma treatment. Nevertheless, alterations in the expression of NRF2 and its downstream target genes, as well as in the activation of the ERK1/2 and Akt signaling pathways, varied considerably depending on the cell line and the compound employed. Collectively, our results reveal that CAP-generated RONS and NRF2-dependent antioxidant responses are critical determinants of breast cancer cell sensitivity to plasma treatment, and suggest that targeting NRF2 signaling could improve the therapeutic efficacy of plasma-based anticancer approaches, particularly in aggressive breast cancer subtypes such as triple-negative breast cancer.