Title

Use of Pyrococcus Furiosus Rubredoxin as a Potential Targeted Cancer Therapeutic

Date of Award

6-2014

Degree Type

Thesis

Degree Name

Master of Science (MS)

Department

Basic Sciences

First Advisor

Francis E Jenney, Jr, PhD

Second Advisor

Kimberly Baker, PhD

Third Advisor

Abigail Hielscher, PhD

Fourth Advisor

Brian M Matayoshi, PhD

Abstract

Cancer is a disease that affects approximately one million people around the world each year. Though there are both traditional and targeted therapies currently available, there is still a need for therapeutics that will specifically target cancer cells and not cause toxicity by killing normal cells in the body. Pyrococcus furiosus rubredoxin could potentially be the agent for such therapies as it is a small (5.9 kDa), water-soluble protein that is a hyperthermostable, non-heme iron protein with an iron cofactor bound by cysteine residues. The focus of this project is to use mutated forms of this protein and replace the iron cofactor with a cytotoxic metal, for example ruthenium, giving it the potential to be a successful cancer delivery system. A mutant of P. furiosus rubredoxin that contains the integrin-binding tag, RGD, and another mutant that contains an E-tag that can be easily visualized are being used. RGD is a sequence that will bind to tumor cells overexpressing integrins. Both of these tags were previously incorporated after the D20 residue of the protein. These wild type and mutant proteins were purified using anion-exchange DEAE and sizeexclusion G-75 Sephadex chromatography. The proteins were then analyzed via absorption spectroscopy, SDS-PAGE, and protein assays. Mutants were then denatured and refolded in the presence of ruthenium. The metal content of the proteins was analyzed via inductively coupled plasma mass spectrometry (ICP-MS). Analyses via ICP-MS also show that both the wild type and mutant rubredoxins were successfully able to unfold and refold with ruthenium as the new cofactor with few contaminants. PANC-1 cancer cells were stimulated with phorbol ester in order to overexpress integrins on the membrane. The cells were incubated with the ruthenium-containing wild type rubredoxin, the D20-RGD mutant, and the D20 Etag mutant in varying concentrations and then assayed for cell viability using Trypan blue and for apoptosis using gel electrophoresis. The mutant RGD rubredoxin had a higher affinity for the phorbol ester stimulated cells than the wild type rubredoxin. The mutant RGD rubredoxin always showed preliminary results that indicated some cell death of PANC-1 cells.

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