Localization of Ruthenium Substituted Protein in HT-1080 Human Fibrosarcoma Cells

Date of Award

8-2023

Degree Type

Thesis

Degree Name

Master of Science in Biomedical Sciences

First Advisor

Francis E Jenney, Jr

Second Advisor

Kimberly Baker

Third Advisor

Richard White

Fourth Advisor

Brian DeHaven

Abstract

The prevalence of cancer is slowly rising, projecting to increase considerably in the future. Today, there are many treatments used to combat cancer. One of these includes metal-based chemotherapies, such as cisplatin. While these treatments have been effective in the past, they cause cytotoxic side effects while also lacking specificity. Therefore, these side effects have led to the investigation of other metal-based chemotherapy treatments, and one metal under review is ruthenium (Ru). Ruthenium has a similar electron structure to iron. In addition, ruthenium produces lower cytotoxic side effects when utilized in other chemotherapy treatments. Therefore, an iron-containing protein found in Pyrococcus furiosus called rubredoxin (Rb) could be a suitable carrier of Ru when exchanged with the iron present in Rb—resulting in a metalloprotein-based treatment that will possibly lead to reduced cytotoxicity. However, lack of specificity remains an issue. This treatment's specificity is essential for reducing cytotoxic side effects. Thus, an NGR (Asp-Gly-Arg) tumor-homing peptide can be attached to the ruthenium-substituted rubredoxin (RSR). This tumor-homing peptide is known to interact with the aminopeptidase-N/CD13 (APN/CD13) receptor that is highly expressed in some types of cancer. Previous studies have shown a strong interaction between the RSR-NGR complex and human fibrosarcoma cells (HT-1080). This study has led to further investigation of the complex. Specifically, localization of this complex after NGR/APN binding.

Incorporating a small peptide (Strep II) into rubredoxin-NGR makes localization of the protein possible. Purification of the Ru-Strep II and Ru-NGR-Strep II was performed, which resulted in a purified protein determined through SDS-PAGE and UV visible spectroscopy. Additionally, verification of APN/CD13 receptor expression needed to be determined on human breast cancer (MCF-7) (control) and human fibrosarcoma (HT-1080) treated cells. Confirmation of APN/CD13 expression concluded that the MCF-7 cells showed no expression, while the HT-1080 cells did show expression of the receptor. Through fractionation and the immunofluorescence of HT-1080, cells treated with Ru-NGR-Strep II showed extracellular localization of the protein, indicating that the tumor-homing peptide shows specificity to the HT-1080 cells. However, further investigation of the ruthenium-substituted form of rubredoxin is needed to confirm that extracellular localization is still occurring.

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