Graduation Semester and Year

2016

Language

English

Document Type

Dissertation

Degree Name

Doctor of Philosophy in Chemistry

Department

Chemistry and Biochemistry

First Advisor

Frederick M MacDonnell

Abstract

The ruthenium (II) polypyridyl complexes [(phen)2Ru(tatpp)Ru(phen)2]4+, RPC4 and [(phen)2Ru(tatpp)]2+, RPC3 are promising anticancer candidates due to their observed cytotoxic effect against multiple cancer cell lines. These complexes contain a reactive oxygen species (ROS) creating redox-active tetraazatetrapyridopentacene (tatpp) bridge inducing cytotoxic activity against multiple cancer lines and types. Also, they have shown a selectivity towards malignant and normal cell tissue types that are of interest in cellular biological systems and treatment care. They exhibit the ability to regress tumor growth in mouse models, cleave DNA in gel assays and exhibit efficacies in a hypoxic environment similar to that of tumors in vivo. This thesis is a direct test of the following hypothesis: Ruthenium(II) polypyridyl complexes RPC3 and RPC4 both having similar structures and gel based DNA cleaving ability act similarly in cells. The putative targets are the nuclear DNA and/or the mitochondria. Also, we will explore doublets of RPC3 and RPC4 with a number of standard care chemo drugs to determine if the RPCs can potentiate a positive response in certain chemo drug resistant cell lines and potentially act in a synergistic fashion. This thesis develops both hypotheses by an analysis of prior literature research and our biochemical screening approach to test the cytotoxic and intracellular mechanistic ability of these complexes. In this thesis work, the details of mechanism of action are discussed for complexes RPC3 and RPC4 against multiple cancer cell types. Briefly, this examination included methods of cellular entry, RPC cellular compartment localization, their effects on mitochondrial fitness, and the intracellular ROS production levels after inoculation with RPC3 and RPC4 and quantification of their DNA cleaving ability in vitro. We first looked at how these complexes differ in their use of active cellular membrane transport and active endocytosis channels. RPC3 was found to use a great deal of clatherin active transport whereas RPC4 used a modest amount in comparison. RPC3 also utilized appreciable lipid raft endocytosis and also gains more entry in ppb into a whole cell vs. RPC4 which was not found to use any other major transport channel in comparison and passively diffuses through the cell membrane. We also found stark contrasts between intracellular compartmentalization. Our studies elucidate that RPC4, is localized heavily in the nucleus of a cell vs. RPC3, which was found to be highly localized in the cytoskeleton. This study also exhibits RPCs effects to mitochondrial fitness, ROS production and DNA cleavage ability utilizing confocal fluorescent microscopy of live and fixed cells. We show that RPC3 and RPC4 effect mitochondrial membrane potential as well as impair intracellular ATP production against a dosing gradient. We also present evidence of a timed intracellular ROS H2O2 production which markedly increases after inoculation with RPCs. Not only was the increase of ROS appreciable but a direct and indirect ROS signal was also found with these RPCs. In the nuclear region of the cells, where RPC4 is particularly localized, we exhibit an increase of ROS at the 2 h mark vs RPC3, highly localized in the cytoskeletal cellular compartment, showing an appreciable ROS increase at 22 h. Last, we demonstrate the DNA cleaving ability using H2AX to identify double stranded breaks (DSBs) production by RPC3 and RPC4. Very similar to our ROS study, a direct correlation with RPC4 showed appreciable DSBs in 2 h vs. RPC3 exhibiting an indirect yet equal DSB foci formation at 22 h. We also tested these RPCs in multi-cell tumor spheroids (MCTS) as 3D cell culture more closely resembles in vivo micro-tumor environments. We successfully grew non-small cell lung carcinoma (NSCLC) cells into 3D tumor spheroids and tested RPC3 and RPC4 where we identified a shift in inhibitory concentration (IC50) to the right of 2D cell culture inhibition curves. We also noticed in our models these RPCs exhibit differences in morphological effects upon the MCTS. While at IC50 doses or above them, RPC4 did not affect the MCTS spheroid in shape or morphology whereas RPC3 at small doses was able to disassemble the 3D spheroid. Lastly, we also examined successful synergy studies with RPC3 and RPC4 against a variety of standard care chemotherapy drugs including: cisplatin, etoposide, docetaxel, pemetrexed and gemcitabine. We show in this study that successful doublet combinations with RPCs, cisplatin, etoposide and docetaxel all shifted inhibition curves to the left in a variety of cell lines and types. Not only were they effective but the Loewe synergy index was used to measure these effects as a quantitative synergy proof. We also demonstrate that these RPCs are successful at potentiating cytotoxic efficacies in chemo resistant NSCLC in 2D and 3D cell cultures. Notably, cell lines which were docetaxel resistant, showed a 2-fold or higher synergy when combined with either RPC3 or RPC4 and potentiation effects were also found with cisplatin and etoposide resistant cell lines. In this work we provide evidence that suggests while RPC3 and RPC4 are exhibiting similar cytotoxic IC50 curves in multiple cell lines and relatively have similar chemical structures, they are behaving in multiple different cellular mechanistic responses in 2D and 3D intracellular environments. We also show that when combined with multiple different standard care chemotherapeutic agents, they are effective as synergistic drug combinations and effective at potentiating responses against chemotherapeutic resistant cancer cell types.

Keywords

Ruthenium, DNA cleavage, Double stranded breaks, Synergy, Potentiation, 3D cell culture

Disciplines

Chemistry | Physical Sciences and Mathematics

Comments

Degree granted by The University of Texas at Arlington

Included in

Chemistry Commons

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