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Quantitative Proteomic Analysis for Assessing the Mechanisms of Action of Anti-Cancer Drugs and Arsenite

  • Author(s): Zhang, Fan
  • Advisor(s): Wang, Yinsheng
  • et al.
Abstract

Mass spectrometry is a powerful tool used in proteomic analysis. By combining with different isotope labeling methods, protein quantification at the entire proteome scale can be achieved. In this dissertation, I employed an LC-MS/MS coupled with stable isotope labeling by amino acid in cell culture (SILAC) strategy, to explore the molecular mechanisms of action of anti-cancer drugs and an environmental toxicant, arsenic.

In Chapters 2 and 3, I performed a quantitative assessment of global proteome changes upon treatment with two anti-cancer drugs, both are nucleoside analogues, i.e. 5-aza-2'-deoxytidine (5-Aza-CdR) and 6-thioguanine (SG). We found, for the first time, that 5-Aza-CdR exerts its cytotoxic effects in leukemia and melanoma cells through epigenetic reactivation of DPP4 gene and the resultant inhibition of cholesterol biosynthesis. In addition, proteome-wide analysis suggested that SG may exert its cytotoxic effect by inducing mitochondrial dysfunction and reactive oxygen species formation in acute lymphoblastic leukemia cells.

In Chapters 4 and 5, we utilized the same MS-based quantitative proteomic method, to study the mechanisms underlying trivalent arsenic-induced carcinogenic effect. Our results confirmed the previous findings and provided a more complete picture about the biological pathways that are altered upon arsenite treatment in human skin fibroblast cells. To further understand how arsenic affects RING-finger proteins that are known to be important in DNA damage repair, we performed in vitro and in vivo arsenite binding experiments. Our studies revealed, for the first time, that arsenite may exert its carcinogenic effect by targeting cysteine residues in the RING finger domains of the RNF20-RNF40 histone E3 ubiquitin ligase, thereby altering histone epigenetic mark and compromising DNA DSB repair.

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