Date

2015

Author

Aşık, Elif

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The understanding of how magnetic nanoparticles (MNPs) interact with living system is one of the prerequisite pieces of information needed to be obtained before any further development for desired biomedical applications. In this study, Cobalt Ferrite magnetic nanoparticles (CoFe-MNPs) in their naked and silica-coated forms were characterized. In vitro cell culture for their likely cytotoxicity and genotoxicity potential were examined. The apoptosis, lipid peroxidation, ROS formation and oxidative stress related gene expression levels of some drug metabolizing enzymes in human cancer (MDA-MB- 231, MCF-7) and non-cancer (MCF-10A) breast cell lines were analyzed in response to MNPs treatment. Our results revealed that, uptake of the highest amounts of CoFe-MNPs was observed in metastatic cells and the uptake of the silica coated CoFe-MNPs were higher than the naked ones in all cells. Naked CoFe-MNPs represented higher levels of cytotoxicity, genotoxicity and ROS generation compared to silica coated CoFe-MNPs. Silica coated CoFe-MNPs were functionalized with COOH groups for further modifications. 2-amino-2-deoxy-glucose (2DG), which is a potential targeting molecule for cancer treatment, was conjugated on silica coated CoFe-MNPs surface through - vi COOH groups. Internalization and accumulation of both -COOH modified (COOHMNPs) and 2DG conjugated (2DG-MNPs) were studied in MDA-MB-231 and MCF-7 cancer and MCF-10A non-cancer breast cells by transmission electron microscopy (TEM), Prussian blue staining and Inductively Coupled Plasma Optical Emission Spectroscopy (ICP-OES). According to our results, it was apparent that 2DG-MNPs were internalized more efficiently than COOH-MNPs under same conditions, in all cell types studied. Moreover, the highest amount of uptake was observed in MDA-MB-231 cells, which is followed by MCF-7 and normal MCF-10A for both MNPs. The apoptotic effects of 2DG-MNPs was further evaluated, and it was found that apoptosis was not induced at low concentration of 2DG-MNPs, regardless of the cell types, whereas dramatic cell death was observed at higher concentrations. In addition, the gene expression levels of some drug metabolizing enzymes, two Phase I (CYP1A1, CYP1B1) and two Phase II (GSTM3, GSTZ1) were also seen to increase at high concentration of 2DG-MNPs, whereas at low concentration no induction was observed. Eukaryotic elongation factor-2 kinase (eEF2K) is gaining potential as a prognostic marker and therapeutic target to treat breast cancer. Hence, eEF2K siRNA was decorated on silica coated CoFe-MNPs through -COOH groups and the expression and role of this gene investigated in the BRCA1 mutated breast cancer cells (MDA-MB-436, HCC- 1937). According to our results, the silencing of eEF2K using siRNA decorated MNPs inhibited colony formation; proliferation, migration and invasion of BRCA1 mutated cells. Furthermore, the down modulation of eEF2K was investigated in an orthotropic model of BRCA1 mutated breast cancer in nude mice in vivo by through systematically administration of eEF2K siRNA decorated MNPs. In vivo silencing of eEF2K lead to inhibition of molecules and pathways that are involved in migration/invasion (Src/FAK/paxillin), angiogenesis (VEGF), proliferation (c-Myc), cell cycle (CyclinD1), survival/drug resistance (PI3K/Akt) translational regulation (4EBP1). Taken together, our data suggest, for the first time, which eEF2K is associated with tumorigenesis and progression of BRCA1 mutated breast cancer and may be a novel potential therapeutic target in this cancer

Subject Keywords

Breast, Metastasis., Nanoparticles., Genetic toxicology., Cancer invasiveness., Cell-mediated cytotoxicity.

URI

http://etd.lib.metu.edu.tr/upload/12619553/index.pdf
https://hdl.handle.net/11511/25273

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Graduate School of Natural and Applied Sciences, Thesis