Mitochondrial Complex IV Inhibition and Exogenous Oxidants Potently Synergize in Melanoma Cell Death Induction

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 ...
Source: Clinical Plasma Medicine - Category: Research Source Type: research