The development of nanotechnology-based detection systems for the diagnosis of breast cancer

Abstract

Breast cancer is one of the major causes of death in South Africa. About 1 in 29 South African women are at risk of developing this type of cancer in their lifetime. The global incidence of breast cancer also increases annually with over 1 million new cases diagnosed every year. Molecular diagnostic techniques such as qRT-PCR, Fluorescent In Situ Hybridization (FISH), Immunohistochemistry (IHC) and ELISA are used to diagnose breast cancer. Some of these diagnostic techniques make use organic fluorophores as fluorescent reporter molecules. The principle of all these diagnostic techniques is reliant on the detection of molecular biomarkers that are associated with the disease. In most cases these molecular biomarkers are DNA, RNA or proteins that are up-regulated in response to or as a result of the disease. The first aim of this study was therefore to identify membrane proteins that are up-regulated in cancers that can potentially be used as biomarkers for the detection of breast cancer. The second aim of this study was to investigate the application of quantum dots in the development of a molecular diagnostic test that can detect a breast cancer biomarker. The most commonly used method to identify molecular biomarkers for diseases have traditionally been gene expression analysis using technologies such as DNA microarray. These technologies have certain limitations and have therefore not been very successful in identifying useful disease biomarkers. Biomarker II discovery by proteomics can overcome some of these limitations and is potentially a more suitable method to identify molecular biomarkers for breast cancer. In this study proteomics in combination with Stable Isotope Labelling with Amino Acids in Cell Culture SILAC was used to do a comparative analysis of the expression levels of membrane proteins present in a human breast cancer cell line (MCF-7) derived from a breast cancer patient and a human breast cell line (MCF- 12A) derived from a healthy individual. This led to the identification of the transmembrane protein, GFRA1 as potential new biomarker for breast cancer. This study showed that this protein is over expressed in MCF-7 cells as compared to MCF-12A cells and that it is also highly expressed in the myoepthelial cells of the milk ducts of breast cancer patients. This study also demonstrates the use of molecular beacon technology to develop a DNA probe for the detection of cDNA encoding the CK19 gene, which is a known biomarker for breast cancer. In the development of this probe, quantum dots were used as the fluorescence reporter. This molecular beacon probe was able to demonstrate the over expression of CK19 in MCF-7 cells. This study shows that this technology can potentially be used as a diagnostic test for breast cancer and since quantum dots are used in the development of these molecular beacon probes, this diagnostic test can potentially facilitate the development of multiplex detection systems for the diagnosis of breast cancer. Molecular beacon technology can potentially also be used to detect novel biomarkers such as GFRA1.