## 4 Discussion
Endogenously, ROS are formed in physiologically highly active and fast-growing cancer cells, mostly during oxidative ATP synthesis, but can be neutralized by cellular detoxification systems [38; 39]. NIPP generated ROS lead to higher ROS concentrations into cancer cells and overcome the capacity of the biochemical detoxification machinery. Therefore, NIPP-induced ROS can damage the cytoplasmic membrane and intracellular membrane compartments such as the nuclear membrane, the endoplasmic reticulum, and the mitochondria. The exact mechanism of NIPP-induced ROS on membranes is not yet fully understood [40]. However, the ROS-mediated damage to mitochondrial functionality after NIPP treatment has already been demonstrated [41]. Therefore, in addition to the observed decrease in ATP levels, cellular ATP biosynthesis in mitochondria would also be impeded. Restrictions in cancer cell energy metabolism and severe disruption of energy metabolism by NIPP treatment induce apoptosis and diminish proliferation [41], as shown in this study at the cellular and molecular level (Figure 9). This observation goes in line with other studies, showing that sustained cellular (redox) stress leads to adaptations in gene expression in breast cancer cells at the level of DNA methylation [42; 43] and apoptotic and proliferative signaling cascades [39]. These NIPP effects were demonstrated in both malignant and non-malignant human breast cells [35; 44; 45]. However, our study also showed that some apoptotic background is already induced during the experimental handling of the control approaches. It is unknown by what means these secondary effects are triggered (e.g., separation and seeding of the cells, temperature variations, mechanical stress). On the other hand, it should be noted that, compared with these artificial effects, NIPP treatment also induces significantly higher apoptosis rates. The biochemical activity of ROS both as oxidizing agents as well as cellular signaling molecules can explain a higher sensitivity of cancer cells to NIPP treatment. As mentioned above, NIPP application leads to a significant increase in ROS concentrations, which in turn cannot be neutralized efficiently by the cellular detoxification systems of physiologically highly active cancer cells. However, since adjacent non-malignant cells of the tissue still have a significantly higher detoxification capacity, they are affected considerably less or even not at all by NIPP treatment [5]. This hypothesis may account for a cancer-specific efficacy of NIPP in terms of a significantly higher sensitivity compared to physiologically healthy cells. On the other hand, this phenomenon explains the frequently observed good tolerability of NIPP in clinical use. So far, no significant side effects of NIPP treatment have been described [46], which is of great relevance for the use of NIPP, especially in otherwise very invasive and cytotoxic therapies of neoplasia. Recent in vitro data, including the present study, suggest promising clinical applications of NIPP in gynecology [47; 48; 49; 50]. The local effect of NIPP makes its use in combination with established procedures especially promising. The broad spectrum of mechanisms of action and thus of therapeutic targets could potentially also lead to increased therapeutic efficiency. In addition, there is the option of using NIPP-treated liquids for therapy. In this application, very comparable effects to direct NIPP treatment of cancer cells have been demonstrated in vitro [7; 51]. Furthermore, a combination of NIPP treatment and chemotherapy rinsing (hyperlemic intraperitoneal chemotherapy, HIPEC; pressurized intraperitoneal aerosol chemotherapy, PIPAC) would be feasible. The enhanced permeability of the cytoplasmic membrane may enhance drug delivery into cancer cells. This could lead to a mitigation of the dose and thus to a reduction in the undesirable side effects of the drug [52; 53; 54].