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Cell stimulation versus cell death induced by sequential treatments with pulsed electric fields and cold atmospheric pressure plasma
Pulsed electric fields (PEFs) and cold atmospheric pressure plasma (CAP) are currently both investigated for medical applications. The exposure of cells to PEFs can induce the formation of pores in cell membranes and consequently facilitate the uptake of molecules. In contrast, CAP mainly acts through reactive species that are generated in the liquid environment. The objective of this study was to determine, if PEFs combined with plasma-treated cell culture medium can mutually reinforce effects on viability of mammalian cells. Experiments were conducted with rat liver epithelial WB-F344 cells and their tumorigenic counterpart WB-ras for a direct comparison of non-tumorigenic and tumorigenic cells from the same origin. Viability after treatments strongly depended on cell type and applied field strength. Notably, tumorigenic WB-ras cells responded more sensitive to the respective treatments than non-tumorigenic WB-F344 cells. More cells were killed when plasma-treated medium was applied first in combination with treatments with 100-μs PEFs. For the reversed treatment order, i.e. application of PEFs first, the combination with 100-ns PEFs resulted in a stimulating effect for non-tumorigenic but not for tumorigenic cells. The results suggest that other mechanisms, besides simple pore formation, contributed to the mutually reinforcing effects of the two methods.
Impact of the hypoxic phenotype on the uptake and efflux of nanoparticles by human breast cancer cells
Breast cancer cells adapt to the hypoxic tumoral environment by undergoing changes in metabolism, cell signalling, endo-lysosomal receptor uptake and recycling. The resulting hypoxic cell phenotype has the potential to undermine the therapeutic efficacy of nanomedicines designed for endocytic uptake and specific intracellular trafficking. The aim of this study was to examine the impact of hypoxia and simulated reperfusion on the in vitro uptake and release of nanomedicines by human breast cancer cells. Cells were exposed to a hypoxic preconditioning treatment in 1% oxygen for 6 and 24 hours to induce temporal changes in the hypoxic circuit (e.g. HIF-1α expression). The preconditioned cells were then dosed with nanoparticles for 45 or 180 minutes emulating nanomedicine access following tumor reperfusion. Hypoxic preconditioning significantly increased nanoparticle retention by up to 10% when compared to normoxic cultures, with the greatest relative difference between normoxic and hypoxic cultures occurring with a 45 minute dosing interval. Exocytosis studies indicated that the preconditioned cells had a significantly increased nanoparticle efflux (up to 9%) when compared to normoxic cells. Overall, we were able to show that hypoxic preconditioning regulates both the endocytosis and exocytosis of nanomedicines in human breast cancer cells.
Cytochrome C oxidase Inhibition and Cold Plasma-derived Oxidants Synergize in Melanoma Cell Death Induction
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.