Peroxiredoxins as biomarkers of oxidative stress in metastatic melanoma cells

Abstract

Melanoma is one of the most common forms of skin cancer, and is particularly prevalent in New Zealand and Australia. If detected early, surgery may be curative; however, once metastatic there are very few effective treatments and the five-year survival rate declines drastically from 90% to 20%. Increased oxidant production has been associated with oncogenic transformation, and as a result, antioxidant systems in cancer cells are often upregulated in order to overcome this oxidative insult and promote cell survival. Disruption of these cellular antioxidant defences in melanoma cells could provide a therapeutic target. To validate the viability of this approach, it is first important to determine the extent of oxidative stress within melanoma cells. In this thesis, I have investigated the ability of peroxiredoxins to act as biomarkers of oxidative stress in metastatic melanoma cells. Peroxiredoxins (Prx) are a family of antioxidant enzymes that clear hydrogen peroxide from cells. Oxidized Prx dimers form during the catalytic cycle, and using non-reducing polyacrylamide gel electrophoresis, oxidized Prx dimers can be separated from reduced Prx monomers, allowing the percentage of oxidized Prx to be quantified. A pilot study using the NCI60 panel of cancer cell lines showed that Prxs were mostly reduced in cancer cells, with the exception of melanoma cells, which had variable levels of Prx oxidation. Using reducing and non-reducing polyacrylamide gel electrophoresis, I measured protein expression and oxidation of Prx1, Prx2 and Prx3 in eight metastatic melanoma cell lines from the New Zealand melanoma cell panel. These cells have been grown at 5% oxygen since isolation, and expression data is available on each cell line. Protein expression levels varied across metastatic melanoma cell lines for each Prx. Prx1, found in the nucleus and cytosol, was mostly oxidized in all cell lines, including normal melanocytes, ranging from 60-97% oxidized. Prx2, which is also found in the nucleus and cytosol, showed varying levels of oxidation, ranging from 40-97%. Mitochondrial Prx3 oxidation similarly varied from 35-85%. Prx3 oxidation levels were higher in metastatic melanoma cells compared to normal melanocytes, while Prx1 and Prx2 in metastatic melanoma cells had similar oxidation levels to normal melanocytes, with the exception of two metastatic melanoma cell lines that had increased Prx2 oxidation. Analysis of RNA-Seq expression data demonstrated that Prx oxidation was correlated with superoxide dismutase, an enzyme that converts superoxide to hydrogen peroxide; as well as tyrosinase, a key enzyme in melanin synthesis. Levels of extracellular hydrogen peroxide produced by melanoma cells were below detectable levels, suggesting that hydrogen peroxide is being efficiently cleared by cellular antioxidant systems. In summary, metastatic melanoma cells appear to be under high nuclear and cytosolic oxidative stress, and moderate to high mitochondrial oxidative stress, with variation between cell lines. Therefore, Prx may be used as a biomarker of oxidative stress in metastatic melanoma cells, and to determine which melanomas will be sensitive to treatments that target antioxidant defences.