A 2018/2019 Summer Studentship research project
New Zealand has one of the highest incidences of melanoma in the world, and an ability to decrease invasiveness and metastasis would have significant health benefits. A likely role for peroxidasin in melanoma cell invasion has only recently been identified and how it acts is completely unexplored. Results from this project will help us understand the function of peroxidasin in melanoma cells and thus help provide information that is needed to assess whether targeting peroxidasin activity in melanoma has therapeutic potential.
Student: Daisy O’Connor
Supervisors: Dr Martina Paumann-Page, Professor Christine Winterbourn
Sponsor: Cancer Society of New Zealand Canterbury/West Coast Division
Project brief
Introduction
Invasion and metastasis are fundamental hallmarks of cancer and the main causes of cancer deaths. It has recently been discovered that a peroxidase enzyme called peroxidasin is expressed at much higher levels in metastatic melanoma cells derived from human tumours that are highly invasive than in less invasive ones. Other studies also support a role for peroxidasin in promoting tumour cell invasion in melanoma and several other types of cancer. However, no-one know how peroxidasin affects melanoma cell function in order to make them invasive.
The summer student project is part of a larger study investigating how peroxidasin acts in melanoma cells. We know that the enzymatic activity of peroxidasin produces reactive oxygen species including hypobromous acid, a chemical similar to household bleach. We hypothesise that it causes oxidative changes to cell or extracellular matrix constituents that activate cell mechanisms involved in invasion. Our overall objective is to identify these changes and their importance in metastasis. An important part of this is to use biomarkers to establish the extent to which peroxidasin activity causes general oxidative modifications in melanoma cells.
Aim
To measure oxidative stress biomarkers in cultured melanoma cell lines and determine whether these markers are elevated in cell lines that show high peroxidasin expression.
Method
Metastatic melanoma cell lines derived from human tumours will be grown in the Centre for Free Radical Research cell culture laboratory. Cells will be harvested and analysed for general oxidative stress markers, using established assays that involve ELISA (enzyme-linked immune-sorbent analysis), SDS-polyacrylamide gel electrophoresis, Western blotting and mass spectrometric methods. Results will be related to peroxidasin expression and the effect of peroxidasin inhibition on marker levels will be measured.
Student researcher’s component of the study
The student will grow, maintain, harvest and fractionate melanoma cells, carry out SDS-PAGE and ELISA assays on the samples and analyse results. He/she will observe basic mass spectrometric techniques and assist with sample preparation and data analysis.
