Title

Eliciting the Apoptotic Mechanism of Ruthenium-Substituted, NGR-Tagged Rubredoxin on CD13-Expressing Cancer Cells

Date of Award

6-2018

Degree Type

Thesis

Degree Name

Master of Science in Biomedical Sciences

First Advisor

Francis E Jenney Jr, PhD

Second Advisor

Kimberly Baker, PhD

Third Advisor

Abigail Hielscher, PhD

Fourth Advisor

Richard White, PhD, FAHA

Abstract

Cancer research continues to reveal the complex nature of the disease and influences how diagnostics and treatments are developed. More traditional treatments, first-line chemotherapies and surgery, are effective but are not without drawbacks; therefore, targeted therapeutics that minimize off-target effects and maximize treatment efficacy have garnered much attention. This project focuses on using a mutated protein, rubredoxin from Pyrococcus furiosus, containing the tumor homing peptide tag Asp-Gly-Arg (NGR) as a targeted therapeutic drug delivery system. Escherichia coli cells are transfected with a plasmid and cultured to express a mutant rubredoxin with an internal (020) CNGRCG tag. Following purification, the native iron atom is substituted for a ruthenium atom, a known cytotoxic metal, to create the drug treatment: NGR-tagged, ruthenium-substituted rubredoxin (RuRdNGR). The human fibrosarcoma cell line, HT-1080, expresses the membrane protein aminopeptidase N (APN/CD13) -the target for the NGR tumor homing peptide tag - is treated with various concentrations of RuRdNGR to identify an effective treatment concentration as well as the specificity of RuRdNGR treatment via flow cytometric analysis. Further, determination of which apoptotic pathway is induced is examined via western blot analysis of caspases 3, 8, and 9. Preliminary data suggest that a RuRdNGR dose of 8 μM provides sufficient apoptotic effects on CD13-expressing cancer cell lines.

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