Ruthenium-Substituted, NGR-tagged Rubredoxin: A potential targeted drug delivery for cancer.

Introduction: Current cancer treatments include chemotherapy, radiation, monoclonal antibodies, and surgery. The challenges that these therapies pose to cancer patients are compromised immunity, frequent infections, degradation of healthy cells and nephrotoxicity. Most of these side effects are due to the lack of specificity of these therapies. Pyrococcus furiosus is a hyperthermophile containing thermostable proteins that show strong potential for being used in industrial processes for drug development. Previous studies have shown successful interactions between an amino peptidase (CD13) that is over-expressed in certain cancer cell lines such as HT1080 CD13) and the homing peptide NGR. It is hypothesized that a protein having an NGR tag and a cytotoxic metal will be effective in treating particular types of cancer.

Objectives: The objective for this project is to add a peptide tag (NGR) to rubredoxin that is known to interact with a receptor (APN/CD13) commonly found on cancer cell lines (HT1080) and replace the iron cofactor found in rubredoxin with a cytotoxic metal (ruthenium). Cancer cells will be treated with the modified rubredoxin and outcomes will be observed.

Methods: Anion exchange and size exclusion will be used to purify WT and mutated rubredoxin. Metal reconstitution will be conducted to replace the native iron cofactor metal with ruthenium. Western blots will show the expression of CD13 on HT-1080 fibrosarcoma cell lines. HT-1080 cell lines will be treated with wild type and NGR-rubredoxin, both with the native iron and ruthenium substituted forms, as well as the common cancer drug, cisplatin as a control, at serial concentrations. Apoptosis, necrosis cytotoxicity assays will be performed to observe the mechanism of action.

Results: UV/Visible spectrophotometry has confirmed purification of rubredoxin. Western blot has shown CD13 is present on HT-1080 fibrosarcoma cell line.