Cytotoxicty, Cellular Localization And Fluorescent Microscopy Of Malignant Cells Treated With Ruthenium (II) Complexes Of The Tetraazatetrapyridopentacene Ligand
Dayoub, Adam S.
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The ruthenium (II) polypyridyl complexes [(phen)2Ru(tatpp)Ru(phen)2]4+, (P) and [(phen)2Ru(tatpp)]2+, (MP) are promising anticancer activity candidates due to their observed cytotoxic effect against multiple cancer cell lines. These complexes contain a redox-active (tatpp) bridge and a cytotoxic selectivity against cancer lines that are of interest in cellular biological systems. They also exhibit the ability to regress tumor growth in mouse models and have been shown to cleave DNA with an inverse dependence with (O2). Significantly, these complexes may show a higher cytotoxic action in a hypoxic environment similar to that of tumors in vivo. This thesis is a direct test of the following hypothesis. We postulate that the ruthenium(II) polypyridyl complexes P and MP exhibit enhanced cytotoxicity in a hypoxic environment. We believe that against multiple cancer cell lines, P and MP will show enhanced cytotoxicity in a reduced (O2) environment. We also postulate that dsDNA, mitochondrial potential damage or a combination of both are responsible for the cytotoxic values exhibited by P and MP. The hypothesis of this thesis is that one or multiple compartments are being damaged as well as different methods of cell membrane entry attributing to the separate mechanistic ability of these complexes in situ. Chapters 2 and 3 of this thesis develop the hypothesis by an analysis of prior literature research and our biological screening approach to test the cytotoxic and intracellular mechanistic ability of these complexes. In chapter 2, the details of the enhanced cytotoxicity in a hypoxic environment are discussed for complexes P and MP against cell lines H358, HCC-2998, HOP-62 and Hs766T. The study also contrasts and compares these findings in a normoxic environment as well as against complexes that show low cytotoxicity in the same environments. Chapter 3 presents the method of cellular entry, compartment localization and possible location of cytotoxic action within the cancer cells utilizing ICP-MS and fluorescent microscopy to determine if their location corresponds to their cytotoxic effect. In 3rd chapter we will show stark contrasts between complexes P which is localized between several compartments vs. MP which was found to be highly in the cytoskeleton of cell lines H358 and HCC-2998. It was also found that these complexes exhibit active cellular membrane transport and separate endocytosis transport channels in Hs766T cells. Whereas P and MP, were both found to utilize the clathrin mediated transport channels, MP exhibited the use of lipid raft dependent endocytosis vs. P utilizing GTP couple protein transport. This study also shows the mitochondrial potential damage by complexes P and MP, utilizing fluorescent microscopy live and fixed cell imaging. Propidium iodide and JC-1 mitochondrial potential stains were used to discern the location of action between the nuclear dsDNA and mitochondrial membrane potential within H358 cells.