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Caffeine has been studied as a potentiating agent in chemotherapy against some types of cancer, but there are few reports on its effects on melanoma. This study aimed to investigate caffeine's ability to enhance the effects of dacarbazine in vitro.Murine melanoma B16F10 cells were treated 24 h with 1-40 µM caffeine. We evaluated cytotoxicity, DNA damage, apoptosis, and oxidative lesion induced by dacarbazine associated with caffeine. The metabolization of these drugs, as well as immunocytochemical labeling, were also evaluated.The pre-treatment with caffeine showed to be more effective. Caffeine potentiated dacarbazine-induced cytotoxic effects by increasing dacarbazine biotransformation, apoptosis, DNA damage, and malondialdehyde levels; also, caffeine reduced Ki67 and ERK1/2 nuclear labeling and increased p53 labeling in B16F10 cells. In our experiment, caffeine promoted modifications associated with dacarbazine metabolism by viable cells potentiating this antineoplastic drug. These promising results should be further evaluated in experimental models in vivo.

Despite continuous advances in therapy, malignant melanoma is still among the deadliest types of cancer. At the same time, owing to its high plasticity and immunogenicity, melanoma is regarded as a model tumor entity when testing new treatment approaches. Cold physical plasma is a novel anticancer tool that utilizes a plethora of reactive oxygen species (ROS) being deposited on the target cells and tissues. To test whether plasma treatment would enhance the toxicity of an established antitumor therapy, ionizing radiation, we combined both physical treatment modalities targeting B16F10 murine melanoma cell in vitro. Repeated rather than single radiotherapy, in combination with gas plasma-introduced ROS, induced apoptosis and cell cycle arrest in an additive fashion. In tendency, gas plasma treatment sensitized the cells to subsequent radiotherapy rather than the other way around. This was concomitant with increased levels of TNF&alpha, IL6, and GM-CSF in supernatants. Murine JAWS dendritic cells cultured in these supernatants showed an increased expression of cell surface activation markers, such as MHCII and CD83. For PD-L1 and PD-L2, increased expression was observed. Our results are the first to suggest an additive therapeutic effect of gas plasma and radiotherapy, and translational tumor models are needed to develop this concept further.

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.

TGF-β1 and oxidative stress are involved in cancer progression, but in melanoma, their role is still controversial. Our aim was to correlate plasma TGF-β1 levels and systemic oxidative stress biomarkers in patients with melanoma, with or without disease metastasis, to understand their participation in melanoma progression. Thirty patients were recruited for melanoma surveillance, together with 30 healthy volunteers. Patients were divided into two groups: Non-metastasis, comprising patients with tumor removal and no metastatic episode for 3 years; and Metastasis, comprising patients with a metastatic episode. The plasmatic cytokines TGF-β1, IL-1 β, and TNF-α were analyzed by ELISA. For oxidative stress, the following assays were performed: malondialdehyde (MDA), advanced oxidation protein products (AOPP) levels, total radical-trapping antioxidant parameter (TRAP) and thiol in plasma, and lipid peroxidation, SOD and catalase activity and GSH in erythrocytes. Patients with a metastatic episode had less circulating TGF-β1 and increased TRAP, thiol, AOPP and lipid peroxidation levels. MDA was increased in both melanoma groups, while catalase, GSH, and IL-1β was decreased in Non-metastasis patients. Significant negative correlations were observed between TGF-β1 levels and systemic MDA, and TGF-β1 levels and systemic AOPP, while a positive correlation was observed between TGF-β1 levels and erythrocyte GSH. Lower levels of TGF-β1 were related to increased oxidative stress in Metastasis patients, reinforcing new evidence that in melanoma TGF-β1 acts as a tumor suppressor, inhibiting tumor relapse. These findings provide new knowledge concerning this cancer pathophysiology, extending the possibilities of investigating new therapies based on this evidence.

Malignant melanoma is the most dangerous form of skin cancer. Despite new therapies for melanoma treatment, effective therapy is mainly limited by excessive metastasis. Currently, the factors determining metastasis development are not elucidated, but oxidative stress was suggested to be involved. To this end, we analyzed oxidative stress parameters during the metastatic development using the syngeneic B16F10 melanoma model. An increase in blood plasma lipid peroxidation occurred at the earliest stage of the disease, with a progressive decrease in oxidative damage and an increase in antioxidant defense. Vice versa, increased lipid peroxidation and 3-nitrotyrosine, and decreased antioxidant parameters were observed in the metastatic nodules throughout the disease. This was concomitant with a progressive increase in vascular endothelial growth factor and proliferating cell nuclear antigen. We conclude that the oxidative stress in the bloodstream decreases during the metastatic process and that nitrosative stress increases during the proliferation and growth of metastatic nodules in the tumor microenvironment. These results will help to better understand the role of oxidative stress during melanoma metastasis.

Malignant melanoma is an aggressive cancer that develops drug resistance leading to poor prognosis. Efficient delivery of chemotherapeutic drugs to the tumor tissue remains a major challenge in treatment regimens. Using murine (B16) and human (SK-MEL-28) melanoma cells, we investigated traditional cytotoxic agents in combination with cold physical plasma-derived oxidants. We report synergistic cytotoxicity of doxorubicin and epirubicin, and additive toxicity of oxaliplatin with plasma exposure in coefficient of drug interaction analysis. The combination treatment led to an increased DNA damage response (increased phosphorylation of ATM, γ-H2AX foci, and micronuclei formation). There was also an enhanced secretion of immunogenic cell death markers ATP and CXCL10 in cell culture supernatants following combination treatment. The observed synergistic effects in tumor cells was due to enhanced intracellular doxorubicin accumulation via upregulation of the organic cationic transporter SLC22A16 by plasma treatment. The doxorubicin uptake was reversed by pretreating cells with antioxidants or calcium influx inhibitor BTP2. Endoribonuclease-prepared siRNAs (esiRNA)-mediated knockdown of SLC22A16 inhibited the additive cytotoxic effect in tumor cells. SK-MEL 28 and THP-1 monocytes co-culture led to greater THP-1 cell migration and SK-MEL-28 cytotoxicity when compared with controls. Taken together, we propose pro-oxidant treatment modalities to sensitize chemoresistant melanoma cells towards subsequent chemotherapy, which may serve as therapeutic strategy in combination treatment in oncology.

Despite striking advances in the treatment of metastasized melanoma, the disease is often still fatal. Attention is therefore paid towards combinational regimens. Oxidants endogenously produced in mitochondria are currently targeted in pre-clinical and clinical studies. Cytotoxic synergism of mitochondrial cytochrome c oxidase (CcO) inhibition in conjunction with addition of exogenous oxidants in 2D and 3D melanoma cell culture models were examined. Murine (B16) and human SK-MEL-28 melanoma cells exposed to low-dose CcO inhibitors (potassium cyanide or sodium azide) or exogenous oxidants alone were non-toxic. However, we identified a potent cytotoxic synergism upon CcO inhibition and plasma-derived oxidants that led to rapid onset of caspase-independent melanoma cell death. This was mediated by mitochondrial dysfunction induced by superoxide elevation and ATP depletion. This observation was validated by siRNA-mediated knockdown of COX4I1 in SK-MEL-28 cells with cytotoxicity in the presence of exogenous oxidants. Similar effects were obtained with ADDA 5, a recently identified specific inhibitor of CcO activity showing low toxicity in vivo. Human keratinocytes were not affected by this combinational treatment, suggesting selective effects on melanoma cells. Hence, targeting mitochondrial CcO activity in conjunction with exogenous pro oxidant therapies may constitute a new and effective melanoma treatment modality.

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.