# Medical Gas Plasma Treatment in Head and Neck Cancer--Challenges and Opportunities
## 4 In Vitro and In Vivo Studies
Thereby, p53 and its downstream pathway are activated via phosphorylation through the ataxia-telangiectasia mutated (ATM) kinase, inducing cellular responses, such as apoptosis, indicating the importance of the ATM/p53 pathway for plasma-mediated head and neck cancer (HNC) cell inactivation [37]. Moreover, [PERSON] et al. and [PERSON] et al. demonstrated the contribution of p53 during plasma-induced apoptosis. They showed that plasma-mediated activation of mitogen-activated protein kinase (MAPK) proteins, like c-Jun N-terminal kinase (JNK) and p38, induces p53 phosphorylation, which resulted in apoptosis of the HNSCC cell line HSC3 [29; 33]. In addition to the different signaling pathways that are induced through the physical plasma application, the redox state of the cancer cell lines is also influenced by the plasma treatment. It has been reported that the antioxidative activity of cancer cells is generally lower than in non-malignant cells due to a compromised redox state, showing decreased levels of the two major redox state regulators glutathione (GSH) and nicotinamide adenine dinucleotide phosphate (NADPH) [30; 33]. The additional intracellular oxidative stress that is generated by plasma-derived ROS leads to further imbalances in the cellular redox state and the loss of the MMP. This results in cell damage of squamous carcinoma cells after a plasma-mediated increase of intracellular ROS levels [26; 28; 30; 32]. Furthermore, physical-plasma-induced ROS selectively enhances the iron-dependent lipid peroxidation and mitochondrial superoxide formation in HNSCC cells since they harbor more catalytic Fe(II) than non-malignant cells. Consequently, the amount of catalytic Fe(II) matters for the killing efficacy of physical plasma, suggesting the presence of ferroptoosis processes during cancer cell inactivation [27]. Besides inactivation, several studies also point to a decrease in the motility of squamous cell carcinoma cells after plasma treatment [6]. [PERSON] et al. revealed that the migration and invasion of the head and neck cancer cell lines MSKQLL1 and SCC1483 were significantly reduced after plasma treatment through the downregulated expression of the focal adhesion kinase (FAK), integrin, and paxillin, especially when utilizing N\({}_{2}\) as a feed gas [31]. The ability to form colonies was also impaired by plasma treatment in various HNSCC cell lines [19; 27; 30]. The fact that the application of different feed gases influences the effectivity of plasma, as shown by [PERSON] et al., demonstrates the possibility to optimize physical plasma further for head and neck cancer treatment [31]. In contrast to the in vitro studies, only two in vivo studies have been performed regarding the physical plasma treatment of HNSCC. The first study confirmed preliminary in vitro results by in vivo experiments using a syngeneic mouse model, as well as a xenograft mouse model. It was demonstrated that the application of a physical-plasma-conditioned liquid applied over one week reduced the head and neck tumor progression through an increase of mitochondrial E3 ubiquitin-protein ligase 1 (MUL1) expression, ultimately resulting in a decline of p-AKT levels and AKT kinase activity. Correspondingly, the tumor weight and volume were significantly reduced in the plasma-treated group compared to the control group [28]. Since AKT contributes to the regulation of cell survival and death in terms of apoptosis and cancer development, it is not surprising that it plays an essential role in plasma-induced HNC cell damage [71]. AKT degradation is mediated by MUL1, which binds to the kinase domain of phosphorylated AKT, thereby inhibiting its downstream signaling. This finally leads to the suppression of cell proliferation and migration. Since the expression of MUL1 was enhanced by plasma-derived ROS, the reactive species were also assumed to be responsible for the increased MULI/AKT binding observed. In untreated HNSCC cells, changes in AKT and MUL1 levels were not noticed [28].