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Melanoma is the deadliest form of cutaneous neoplasia. With a five-year survival rate of only 5–19%, metastatic melanoma presents severe challenges in clinical therapies. In addition, palliation is often problematic due to large numbers of fast growing metastasis. This calls for new therapeutic avenues targeting highly aggressive melanoma in palliative patients. One recently suggested innovative approach for eradication of topical tumor lesions is the application of cold physical plasma. This partially ionized gas emits a cocktail of reactive oxygen and nitrogen species (ROS/RNS). ROS/RNS have been shown to be a double-edged sword in fueling cancer growth at low doses but abrogating it at higher doses. The ROS/RNS output of plasma devices is tunable, and many studies have successfully decreased cancer cell growth in vitro and tumor burden in vivo. In general, increasing numbers of clinical trials suggest combination therapies to outperform monotherapies with regard to prognosis in patients. This review describes current challenges in melanoma treatment and highlights the concept of plasma therapy in experimental studies performed in melanoma research. Future perspectives are given that combine the usage of physical plasma with e.g. chemotherapy, immunotherapy, and ionizing radiation in melanoma medical oncology.

Cutaneous melanoma is a highly aggressive malignancy and has rapidly increased over the past several decades [1]. Although the clinical biology and pathogenesis of melanoma are well understood, the prognosis remains poor with limited therapeutic options in the metastatic stage of the disease [2]. Reactive oxygen species (ROS) may be drivers of carcinogenesis and may cause oxidative damage to several cellular components, so it is generally considered to be deleterious [3]. However, ROS, as well as the products generated during redox reactions can act as second messengers and participates of signaling pathways, activating redox-sensitive transcription factors and gene expressions leading to cell proliferation, metastasis, and therapy resistance [4]. Although antioxidants can prevent the onset of melanoma [5], studies have shown that a systemic pro oxidant status is necessary to prevent distant metastases [6]. Consequently, some therapies aim at the generation of ROS as a mechanism of death [7]. Despite advances in chemotherapy, immunotherapy, and radiotherapy, the success in drug treatment of disseminated disease remains limited. The large number of side effects and resistance to treatment are the main causes of unsuccessful therapy. Thus, it is necessary to search for new strategies that can increase patient survival and quality of life. Cold plasma is a partially ionized gas proved its effectiveness for different applications in health care and medicine. It has been shown to exert various biologic effects to living tissue and cells ranging from cytoprotective to cytotoxic potency depending on the applied technique [8]. The gas generates reactive oxygen and nitrogen species [9] that are being deposited in cell culture media [10]. Such oxidant-enriched media selectively kills cancer cells in vitro by targeting the mitochondrial network [11], but the exact mechanism is still unclear. We aim at therapeutic strategies combining chemotherapy/radiotherapy with cold plasma as a strategy against melanoma. In addition, we studied the mitochondrial action mechanism of plasma, elucidating its possible target.

Due to its functional duality in toxicity as well as signaling, research on therapeutic manipulation of reactive oxygen species (ROS) in cancer has been futile. Targeting melanoma cell death with excess amounts ROS, however, is difficult as these cells are highly resistant towards oxidative stress, creating a need for alternative and combinational approaches. Tumor metabolism mainly relies on aerobic glycolysis. We investigated a potential synergism of mitochondrial complex IV inhibition and exogenous-derived oxidants with both single treatment modalities being essentially non-toxic in melanoma cells. In this study, we investigated murine (metastatic B16F10 and non-metastatic B16F0) as well as human (SK-Mel 28) melanoma cells, and compared them with murine (primary fibroblasts) and human (HaCaT keratinocytes) non-cancer cells. Mitochondrial complex IV inhibition was realized using sodium azide (NaN3) and potassium cyanide (KCN) and pharmacological inhibitor ADDA5. Exogenous oxidants were generated using cell culture medium that has been treated with the cold physical plasma effluent of an atmospheric pressure argon plasma jet (kINPen). This plasma-treated medium was subsequently applied to different cells. Metabolic activity was assessed by fluorescence quantification of resazurin-to-resorufin transformation. ATP levels were monitored by a luminescence assay. Total cell counts, intracellular ROS levels (DCF), mitochondria membrane potential (TMRE), cell membrane integrity (sytox green) and cellular morphology were quantitatively assessed using live cell and high throughput confocal imaging (Operatta; Perkin Elmer). For tumor cells, also 3D spheroid models were investigated for viability by sytox green staining. ADDA5, NaN3 and KCN treatment alone failed to induce a significant alteration in metabolic activity in cancer cells. We then exposed the cells pretreated with NaN3 (100-500 µM) or KCN (250-500 µM) to plasma-treated medium. There was a significant inhibition of metabolic activity and rapid induction of cell death 6 h following treatment in melanoma cells compared to cell receiving plasma-treated medium alone. Increase in cellular superoxide levels and decrease in mitochondrial membrane potential and ATP levels was observed preceding cell death. However, no caspase 3 cleavage was observed upon immunoblotting. siRNA mediated knockdown of COX4a led to increased sensitivity of tumor cells towards plasma-treated medium. Furthermore, we validated our findings using a pharmacological inhibitor of complex IV ADDA5 in a tumor spheroid model and demonstrated that complex IV inhibition supplemented with plasma-treated medium, triggered significant cell death via caspase independent mechanism in melanoma cells.