Event Title
Cytotoxic mechanisms in HT-1080 human fibrosarcoma cells treated with ruthenium-substituted protein
Location
Suwanee, GA
Start Date
11-5-2023 1:00 PM
End Date
11-5-2023 4:00 PM
Description
Introduction: Treatment of cancerous tissue in patients typically involves non-specific therapies that target cancerous and healthy tissues. Among these treatments are chemotherapy, radiation therapy, and surgical resection. Currently, metal-based chemotherapy treatments such as cisplatin are commonly used treat some cancers. However, it is a non-specific chemotherapy treatment that causes side effects such as nausea, vomiting, and hair loss. These side effects have led to the study of more specific metal-based chemotherapy treatments. One potential treatment involves a protein from Pyrococcus furiosus (Pf) called rubredoxin. Rubredoxin is a non-heme iron-containing protein with an iron atom coordinated by four cysteine sulfur ligands. Pf rubredoxin is known to be stable in high-temperature environments. In addition, it can also mainly maintain its structure with the removal of iron from the protein. Due to this stability, metals with a structure similar to iron can replace the iron core.
One example is the ruthenium ion. Ruthenium-substituted rubredoxin, when attached to a homing peptide such as NGR (Asparagine-Glycine-Arginine), allows for a possible specific metalloprotein-based chemotherapy treatment. The homing peptide (NGR) binds favorably to the Aminopeptidase-N (APN/CD13) receptor on the surface of cancer cells such as HT-1080 human fibrosarcoma cells.
Objective: This research project investigates the interaction between the ruthenium-substituted rubredoxin with the NGR tag and HT-1080 cells. Interaction mechanisms will be evaluated to determine if this metalloprotein enters the HT-1080 cells.
Methods: Fractionation is one form of testing to separate the cells into membrane/cytosol, membrane-bound organelles, and nuclear fractions. Western blotting will be used to determine the location of the metalloprotein within these fractions.
Furthermore, immunohistochemistry will also be used to determine the location of the metalloprotein. Another test will involve Inductively Coupled Plasma Mass Spectroscopy (ICPMS). This testing will help determine if the metal ruthenium is being separated from the rubredoxin protein if it enters the cell. Once it is determined what happens to the metalloprotein after entering the cell, we will test the HT-1080 cells to determine if cell death occurs by apoptosis or necrosis after exposure.
Results: Previous results indicate there is interactions between the wild-type rubredoxin protein with the NGR homing peptide and the HT-1080 APN/CD13 receptor. Through fractionation/immunohistochemistry testing, we hypothesize that the lysosomes will take up the metalloprotein due to the ability of the lysosomes to pick up and degrade foreign substances that enter the cell. In addition, it is hypothesized that ICPMS will indicate separation of the ruthenium ion from the rubredoxin protein in the cell. Furthermore, apoptosis is thought to occur rather then necrosis.
Conclusion: These findings will help determine if ruthenium-substituted rubredoxin is an excellent candidate for treating cancerous tissue while avoiding the surrounding healthy tissue.
Embargo Period
5-31-2023
Cytotoxic mechanisms in HT-1080 human fibrosarcoma cells treated with ruthenium-substituted protein
Suwanee, GA
Introduction: Treatment of cancerous tissue in patients typically involves non-specific therapies that target cancerous and healthy tissues. Among these treatments are chemotherapy, radiation therapy, and surgical resection. Currently, metal-based chemotherapy treatments such as cisplatin are commonly used treat some cancers. However, it is a non-specific chemotherapy treatment that causes side effects such as nausea, vomiting, and hair loss. These side effects have led to the study of more specific metal-based chemotherapy treatments. One potential treatment involves a protein from Pyrococcus furiosus (Pf) called rubredoxin. Rubredoxin is a non-heme iron-containing protein with an iron atom coordinated by four cysteine sulfur ligands. Pf rubredoxin is known to be stable in high-temperature environments. In addition, it can also mainly maintain its structure with the removal of iron from the protein. Due to this stability, metals with a structure similar to iron can replace the iron core.
One example is the ruthenium ion. Ruthenium-substituted rubredoxin, when attached to a homing peptide such as NGR (Asparagine-Glycine-Arginine), allows for a possible specific metalloprotein-based chemotherapy treatment. The homing peptide (NGR) binds favorably to the Aminopeptidase-N (APN/CD13) receptor on the surface of cancer cells such as HT-1080 human fibrosarcoma cells.
Objective: This research project investigates the interaction between the ruthenium-substituted rubredoxin with the NGR tag and HT-1080 cells. Interaction mechanisms will be evaluated to determine if this metalloprotein enters the HT-1080 cells.
Methods: Fractionation is one form of testing to separate the cells into membrane/cytosol, membrane-bound organelles, and nuclear fractions. Western blotting will be used to determine the location of the metalloprotein within these fractions.
Furthermore, immunohistochemistry will also be used to determine the location of the metalloprotein. Another test will involve Inductively Coupled Plasma Mass Spectroscopy (ICPMS). This testing will help determine if the metal ruthenium is being separated from the rubredoxin protein if it enters the cell. Once it is determined what happens to the metalloprotein after entering the cell, we will test the HT-1080 cells to determine if cell death occurs by apoptosis or necrosis after exposure.
Results: Previous results indicate there is interactions between the wild-type rubredoxin protein with the NGR homing peptide and the HT-1080 APN/CD13 receptor. Through fractionation/immunohistochemistry testing, we hypothesize that the lysosomes will take up the metalloprotein due to the ability of the lysosomes to pick up and degrade foreign substances that enter the cell. In addition, it is hypothesized that ICPMS will indicate separation of the ruthenium ion from the rubredoxin protein in the cell. Furthermore, apoptosis is thought to occur rather then necrosis.
Conclusion: These findings will help determine if ruthenium-substituted rubredoxin is an excellent candidate for treating cancerous tissue while avoiding the surrounding healthy tissue.