Apple Phytochemicals In The Prevention Of Breast Cancer: Effects On Cell Proliferation And Apoptosis
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Among women in the United States, breast cancer is the most frequently occurring cancer and is the second-leading cause of cancer deaths. Approximately 30% of cancer risk is estimated to be attributed to diet. Apple consumption has been correlated with reduced breast cancer risk as well as more favorable prognostic factors. The first objective was to develop a more efficient methylene blue assay for cell counting which could be applied to a broader range of cell densities and various cell culture plates. A linear relationship (r2>0.99) was established between cell number and absorbance at 570 nm wavelength when the modified methylene blue assay was applied to three cell lines plated in a broad range of cell densities in 4 different types of culture plates. This modified methylene blue assay is a powerful tool for any application requiring a versatile, efficient, and accurate method of cell counting, especially in the research of natural products, bioactive compounds, phytochemicals, functional foods and nutraceuticals. The second objective was to determine whether whole apple extracts and purified compounds from apples interfered with IGF-Imediated signal transduction pathways in MCF-7 human breast cancer cells. Quercetin-3-Glucoside (Q-3-G) proved to be surprisingly effective, inhibiting IGF-Iinduced MCF-7 cell proliferation more potently than FBS-stimulated proliferation, as well as more potently than quercetin inhibited cell growth. Q-3-G blocked cell cycle progression and induced apoptosis via down-regulated Akt phosphorylation, Cyclin D1 and CDK-4 expression, and increased Caspase-9 and PARP cleavage. The results help to explain the mechanisms by which apple consumption may reduce breast cancer risk. Additionally, such studies of purified compounds contribute to the understanding of cancer cell behavior and may inform future endeavors in chemotherapeutic drug design. The third objective was to determine the effects of apple phytochemical extracts on cell proliferation and apoptosis in mammary tissues in DMBA-treated Sprague-Dawley rats in vivo. The expression of PCNA, BrdU-labeled cells, Cyclin D1 and Bcl-2 decreased, and Bax expression and apoptosis increased in the apple treated groups. These results demonstrate the potent capacity of fresh apples to suppress promotion and progression of DMBA-initiated mammary carcinogenesis in rats.
dissertation or thesis