Quantitative Proteomic Analysis of 2-Methoxyestradiol Induced Apoptosis in MCF-7 Human Breast Cancer Cells

Ellisor, Michael Barrett

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Year:: 2013
Contributor:: Ellisor, Michael Barrett (creator); Bazemore-Walker, Carthene (Director); Salomon, Arthur (Reader); Sello, Jason (Reader); Brown University. Chemistry (sponsor)
Genre:: theses
Subject:: Label-free Quantification; 2-Methoxyestradiol; Proteomics
Extent:: xii, 284 p.
DOI: https://doi.org/10.7301/Z0PZ574H

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Abstract:: Reliable quantitation of protein abundance is fundamental in accurately determining differential protein expression associated with biological phenomena such as cell growth and differentiation, disease progression, and drug treatment. Furthermore, the ability to quantitatively monitor global and site-specific changes in phosphorylation and other post-translational modifications are required in order to discern the dynamic molecular events and signaling cascades involved in governing these systems in response to particular stimuli. Mass spectrometry-based approaches have proven particularly effective for large-scale proteomic and phosphoproteomic analyses. However, many of the conventional quantitative strategies are typically not suitable for investigations pertaining to hormone sensitive cell lines such as estrogen-dependent MCF-7 human breast cancer cells. It is therefore critical to develop flexible and accurate quantitative proteomic and phosphoproteomic strategies that may be easily adapted to a wide range of scientific applications We have successfully developed a practical and statistically validated two-dimensional LC-MS/MS platform (RP-RP-nanoESI-MS/MS) for proteomic analysis using label-free quantification. An additional advantage of this methodology is the use of an intermediate-pH during the first dimension of separation which prevents possible β-elimination of phosphopeptides in support of phosphoproteomic investigations. However, with the increase in experimental variation associated with TiO2 phosphopeptide enrichment, we have established a robust and flexible phosphoproteomic strategy utilizing a simple and cost-effective quantification method based on reductive isotopic diethylation. 2-Methoxyestradiol has shown considerable promise as an anticancer therapeutic agent with a broad spectrum of activity against multiple tumor cell lines. Evidence has shown that various cellular targets and pathways have been implicated in 2-ME induced apoptosis. However, conflicting reports suggest that the cytotoxic effects of 2-ME may operate through multiple mechanisms of action with varying degrees of contribution depending upon cell type. The MCF-7 human breast cancer cell line is commonly used as a cellular model for breast cancer investigations and has shown to be particularly sensitive to 2-ME treatment (IC₅₀ – 0.45 μM). Utilizing our global proteomic and phosphoproteomic strategies, we aimed to determine potential therapeutic targets and signaling pathways associated with 2-ME-induced apoptosis that may provide evidence for a unifying mechanism of action. Although our data supports many of the previously described mechanisms associated with 2-ME-induced apoptosis, we provided additional evidence that these individual mechanisms may be related to the effects generated by the disruption of the Mevalonate pathway which coincides with an increase in ER-related stress.
Notes:: Thesis (Ph.D. -- Brown University (2013)

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Ellisor, Michael Barrett, "Quantitative Proteomic Analysis of 2-Methoxyestradiol Induced Apoptosis in MCF-7 Human Breast Cancer Cells" (2013). Chemistry Theses and Dissertations. Brown Digital Repository. Brown University Library. https://doi.org/10.7301/Z0PZ574H

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Chemistry Theses and Dissertations

Theses and Dissertations for the Chemistry department.
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