**1. Introduction**

Cold atmospheric plasma (CAP) consists of a heterogeneous mixture of reactive oxygen (ROS) and nitrogen (RNS) species, as well as other ions, uncharged particles, and small amounts of radiation in ultraviolet and infrared ranges. Manifold effects of CAP have been described in the past, mainly referring to strong antibacterial, antiviral, and antifungal action [1–3]. Significant efforts have been made to evaluate and use its positive impact on wound healing [4–6], dental health [7,8], and regenerative medicine [9]. Furthermore, beneficial effects of plasma treatment have been reported both in vitro and in vivo for numerous cancer types, including malignant melanoma [10–12], colon [13,14], and brain tumors [15–17]. Given the constantly high incidence and mortality of such malignancies, and their enormous burden on the patient as well as the health care system, CAP technology displays a promising approach to the development of novel therapeutic treatments.

**Citation:** Zimmermann, T.; Gebhardt, L.A.; Kreiss, L.; Schneider, C.; Arndt, S.; Karrer, S.; Friedrich, O.; Fischer, M.J.M.; Bosserhoff, A.-K. Acidified Nitrite Contributes to the Antitumor Effect of Cold Atmospheric Plasma on Melanoma Cells. *Int. J. Mol. Sci.* **2021**, *22*, 3757. https://doi.org/10.3390/ijms22073757

Academic Editor: Akikazu Sakudo

Received: 11 March 2021 Accepted: 2 April 2021 Published: 4 April 2021

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The potential impact of an oncological application is highlighted by recent studies showing that chemo-resistance might be challenged directly via apoptosis [18,19] and indirectly by restoration of chemo-sensitivity [20]. Furthermore, it was reported that CAP shows strong selectivity against cancer cells, while healthy cells remain largely unaffected [21–23]. Such preferential killing of tumor cells, however, remains controversial as treatment conditions and cell culture media largely affect the potency of CAP [24]. Despite extensive research and ongoing advances in plasma medicine, the exact molecular mechanisms of CAP treatment are still unknown. In terms of malignant melanoma, multiple studies have contributed to a better understanding of CAP effects and underlying molecular mechanisms. For example, it was shown that melanoma cells enter apoptosis in response to DNA damage and mitochondrial dysfunction caused by CAP-induced ROS and RNS [25,26]. Our group previously reported dose-dependent effects of CAP ranging from senescence to apoptosis [11]. On a molecular level, the establishment of cellular senescence was tightly linked to an immediate elevation of cytoplasmic Ca2+ levels, mainly originating from intracellular stores [27]. In comparison to direct treatment of melanoma cells, indirect treatment by application of CAP-treated physiological buffers showed similar but attenuated results, indicating that many of the activating agents are able to dilute in aqueous solutions and remain fairly stable. This was supported by the finding that such CAP-treated buffers do not lose their biological effect if their application is delayed for one hour. Another study revealed strong extracellular acidification during CAP treatment to be essential for its effect on melanoma cells [28]. However, since the exact molecular and cellular mechanisms are still unknown, further research is required to enable the development of plasma-based tumor therapy and valid plasma devices for such approaches. The aim of this study was to characterize reactive species involved in the CAP effect on melanoma cells and to further understand the CAP-induced mechanisms as a basis for the generation of personalized plasma therapy.

#### **2. Results**

#### *2.1. CAP Induces Production of Nitrate and Nitrite in Aqueous Solutions*

Recently, we described the effects of surface micro discharge (SMD) generated CAP on tumor cells, linking these to the induction of reactive species. For an unbiased characterization of CAP-induced production of ROS and RNS, we used Raman spectroscopy. Even though this approach was not able to detect reactive species with a short lifetime, significantly elevated levels of nitrate (1048 cm−<sup>1</sup> ) and nitrite (817 cm−<sup>1</sup> and 1336 cm−<sup>1</sup> ) were found after 2 min CAP treatment (Figure 1A). The validity of these findings was confirmed using standard solutions of potassium nitrate and sodium nitrite (Figures S1 and S2). An established colorimetric assay based on the transnitration of salicylic acid [29] was then used to validate and quantify the observed nitrate production. We determined a dosedependent increase in nitrate levels reaching approximately 1 mM nitrate after 2 min CAP treatment (Figure 1B). Nitrite levels were assessed using a colorimetric assay based on the Griess diazotization reaction. A similar dose-dependency was observed, resulting in nitrite levels of 2 mM after 2 min CAP (Figure 1C). In previous studies, we could show that CAP-treated solutions retained a substantial fraction of their cellular effects even 1 h after incubation [27]. We, therefore, assessed the stability of nitrate and nitrite and found high stability of the CAP-induced substances (Figure 1B,C).

**Figure 1.** CAP treatment causes the production of nitrate and nitrite. (**A**) Raman spectroscopy of untreated extracellular solution (Ctrl) versus a solution treated with 2 min CAP. Traces are mean only, and box plots are mean with 95% confidence interval (Student's *t*-test, *n* = 300). (**B**) Photometric quantification of nitrate and (**C**) nitrite after different doses of CAP. Stability of these molecules was assessed 1 h after treatment (F(4,10) = 29.31 and F(4,10) = 29.63, both *p* < 0.0001). Bars are shown as mean ± SEM (ANOVA followed by Tukey's HSD post-hoc test vs. no treatment, *n* = 3, \*: *p* <0.05, ns: not significant (*p* > 0.05)). **Figure 1.** CAP treatment causes the production of nitrate and nitrite. (**A**) Raman spectroscopy of untreated extracellular solution (Ctrl) versus a solution treated with 2 min CAP. Traces are mean only, and box plots are mean with 95% confidence interval (Student's *t*-test, *n* = 300). (**B**) Photometric quantification of nitrate and (**C**) nitrite after different doses of CAP. Stability of these molecules was assessed 1 h after treatment (F(4,10) = 29.31 and F(4,10) = 29.63, both *p* < 0.0001). Bars are shown as mean ± SEM (ANOVA followed by Tukey's HSD post-hoc test vs. no treatment, *n* = 3, \*: *p* <0.05, ns: not significant (*p* > 0.05)).

*2.2. Nitrite and Acidification Have Synergistic Effects on Ca2+ Influx and Cytotoxicity* 

Next, we assessed the effects of nitrate and nitrite on the cytoplasmic Ca2+ levels using the fluorescent calcium indicator fura-2 AM. Cells of the melanoma cell line Mel Im (derived from metastasis) were treated with 1 mM nitrate or 2 mM nitrite in a physiological
