## 4 Discussion  
Recent studies claimed the important role of CAP in various kinds of cancer treatments. Nowadays, CAP-induced RONS, or its derived short and long-lived free radical species on interaction with liquid (in case of PAM), might be a key factor leading to anticancer effects [34]. Our _in vivo_ results suggested a decrease in tumor volume and tumor weight after 21 days when giving PAM intradermally at the tumor site. This could be attributed due to the effect of the conversion of short-lived OH\({}^{\bullet}\) into long-lived hydrogen peroxide (H\({}_{2}\)O\({}_{2}\)) inside the PAM. H\({}_{2}\)O\({}_{2}\) is the main stable species responsible for most of the activity in PAM [35]. It interacts with some other organic peroxides, which may disrupt the permeability of the melanoma cell membrane and cause cell injury, followed by an invasion of extracellular reactive species from PAM. Studies on breast cancer cell lines demonstrated the role of H\({}_{2}\)O\({}_{2}\) and OH\({}^{\bullet}\) radical-induced apoptosis after plasma treatment [19]. These reactive species not only initiated a molecular cascade of generating cellular apoptosis but also initiated certain immunogenic treatment approaches, like the release of Damage Associated Molecular Patterns (DAMPs) or inducing Immunogenic Cell Death (ICD) and macrophage activation, which helps in the reduction of melanoma progression inside the body [36; 37; 38; 39; 40; 41; 42; 43]. The redox reactions are very important sources of energy in the biological system, and its alteration leads to cell death [44]. PAM changed the normal machinery of the tumor by alleviating protein carbonylation, which is an irreversible post-translational modification induced by oxidative stress [45]. Another very important component, nitric oxide (NO), present in PAM (NO-PAM) accumulates inside the cells and quickly converts to NO\({}_{2}\)\({}^{-}\) and NO\({}_{3}\)\({}^{-}\) in PAM [46]. NO\({}_{2}\)\({}^{-}\) to H\({}_{2}\)O\({}_{2}\) gives a synergistic effect and reacts to produce peroxitrite (ONOO\({}^{-}\)), which is toxic and leads to protein carbonylation in  
Figure 5: Antitumor effect of PAM in mice model. (**A**) Representative hematoxylin and eosin (H & E) staining of tissue sections of both groups (magnification, \(\times\) 40). Immunohistochemistryal analysis of \(p53\), _Parp_, _Casp-8_, and _Akt_ expression levels in control and PAM treated tumors tissues. Three independent slides were checked in each case. Scale bar = 100 μm. (**B**) Graphical representation of IHC score of both groups. (**C**) Western blot analysis for \(p53\), _Parp_, _Casp 8_, and _Akt_. _B_-Actin is used for normalization. cells [47]. PAM also showed an increase in lipid peroxidation, which was estimated by the increase in MDA concentration, which directly relates to the cell in response to induced oxidative stress [48]. Excessive RONS formation (specially OH\({}^{\bullet}\)) can cause macromolecule oxidation, such as membrane lipids, by abstracting a hydrogen atom from the methylene group, thus leading to the cross-linking of the fatty-acid side chain to form transient pores in the cell membrane, leading to membrane lipid peroxidation and depolarization of cell membrane potential [49]. Melanoma showed some very peculiar, effective prognostic markers, which helps melanoma cells to differentiate it from normal tissues [50]. The level of LDH and I-DOPA both decreased significantly as compared to their respective controls. LDH is responsible for the interconversion of pyruvate to lactate during glycolysis and gluconeogenesis and gives energy to the cancer cells [51]. PAM can be attributed to the reduction in LDH level because of the presence of the OH\({}^{\bullet}\) radical, which is the only important species known to inactivate the LDH effect [52], which correlates with decreased tumor burden, and hence, consequently decreases the tumor's growth and invasive potential.