Determining the Potential Cytotoxic Effects of NGR-tagged Ruthenium Substituted Rubredoxin on HT-1080 Cells

Date of Award

6-2025

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

Xinyu Wang, PhD

Fourth Advisor

Bonnie Buxton, PhD

Abstract

Rubredoxin is a small, iron-containing electron transfer protein commonly found in anaerobic bacteria. In this study, rubredoxin was engineered to incorporate a ruthenium (Ru) metal center in place of its native iron (Fe) and an Asn-Gly-Arg (NGR) targeting peptide. The NGR sequence selectively binds to CD13, a cell surface aminopeptidase that is overexpressed in certain malignant tumors, including HT-1080 fibrosarcoma cell line. The resulting construct, referred to as NGR-tagged ruthenium substituted rubredoxin (NGR-RuRd), was designed to evaluate the potential of metal-substituted proteins as selective anticancer agents.


The cytotoxic effects of NGR-RuRd were assessed in vitro using the PrestoBlue™ cell viability assay in both CD13-positive HT-1080 cells and CD13-negative MCF-7 breast cancer cells. Cells were treated with increasing concentrations of NGR-RuRd (50 µM to 500 µM), and viability was compared to multiple controls, including untreated cells, Triton X-100 as a positive control for cell death, wild-type rubredoxin (WtRd), and iron-containing NGR-tagged rubredoxin (NGR-Rd).

In HT-1080 cells, NGR-RuRd caused a statistically significant, dose-dependent decrease in cell viability. At the highest concentration tested (500 µM), viability was reduced to approximately 66 percent compared to the untreated control group (p < 0.0001). Significant cytotoxic effects were also observed at lower concentrations, including 300 µM and 50 µM. In contrast, MCF-7 cells did not show a significant reduction in viability at any tested concentration, with viability remaining above 90 percent across all treatment groups. WtRd and NGR-Rd had no significant cytotoxic effects in either cell line suggesting that the presence of both the ruthenium center and the NGR targeting sequence may be important for selective activity.

Statistical analysis using two-way ANOVA and Dunnett’s multiple comparisons confirmed the significance of the results. These findings support the conclusion that NGR-RuRd selectively targets and induces cytotoxicity in CD13-expressing cancer cells while sparing non-targeted cell types. This suggests that engineered rubredoxin variants have potential as a platform for the development of targeted cancer therapies.

Future work will investigate the underlying mechanisms of cell death, including the roles of apoptosis and necrosis of NGR-RuRd. Overall, this research contributes to the growing interest in metal-based and protein-based therapeutic strategies in oncology.

Rubredoxin is a small, iron-containing electron transfer protein commonly found in anaerobic bacteria. In this study, rubredoxin was engineered to incorporate a ruthenium (Ru) metal center in place of its native iron (Fe) and an Asn-Gly-Arg (NGR) targeting peptide. The NGR sequence selectively binds to CD13, a cell surface aminopeptidase that is overexpressed in certain malignant tumors, including HT-1080 fibrosarcoma cell line. The resulting construct, referred to as NGR-tagged ruthenium substituted rubredoxin (NGR-RuRd), was designed to evaluate the potential of metal-substituted proteins as selective anticancer agents.

The cytotoxic effects of NGR-RuRd were assessed in vitro using the PrestoBlue™ cell viability assay in both CD13-positive HT-1080 cells and CD13-negative MCF-7 breast cancer cells. Cells were treated with increasing concentrations of NGR-RuRd (50 µM to 500 µM), and viability was compared to multiple controls, including untreated cells, Triton X-100 as a positive control for cell death, wild-type rubredoxin (WtRd), and iron-containing NGR-tagged rubredoxin (NGR-Rd).

In HT-1080 cells, NGR-RuRd caused a statistically significant, dose-dependent decrease in cell viability. At the highest concentration tested (500 µM), viability was reduced to approximately 66 percent compared to the untreated control group (p < 0.0001). Significant cytotoxic effects were also observed at lower concentrations, including 300 µM and 50 µM. In contrast, MCF-7 cells did not show a significant reduction in viability at any tested concentration, with viability remaining above 90 percent across all treatment groups. WtRd and NGR-Rd had no significant cytotoxic effects in either cell line suggesting that the presence of both the ruthenium center and the NGR targeting sequence may be important for selective activity.

Statistical analysis using two-way ANOVA and Dunnett’s multiple comparisons confirmed the significance of the results. These findings support the conclusion that NGR-RuRd selectively targets and induces cytotoxicity in CD13-expressing cancer cells while sparing non-targeted cell types. This suggests that engineered rubredoxin variants have potential as a platform for the development of targeted cancer therapies.

Future work will investigate the underlying mechanisms of cell death, including the roles of apoptosis and necrosis of NGR-RuRd. Overall, this research contributes to the growing interest in metal-based and protein-based therapeutic strategies in oncology.

This document is currently not available here.

Share

COinS