Localization and cytotoxicity of ruthenium substituted protein in human fibrosarcoma cells
Location
Suwanee, GA
Start Date
7-5-2024 1:00 PM
End Date
7-5-2024 4:00 PM
Description
INTRODUCTION: Many current cancer treatments such as chemotherapy lack a targeting mechanism - leading to side effects such as nausea, vomiting, and hair loss. In efforts to increase targeting associated with these treatments, the exploration of metal-based therapies is currently an area of interest. One potential vector associated with this method involves the hyperthermophilic archaeon Pyrococcus furiosus that contains thermostable proteins such as rubredoxin (Rd). This non-heme iron protein has the ability to unfold and refold allowing for substitution of its core iron (Fe) center, while maintaining its protein structure. A tumor homing peptide known as NGR (Asn-Gly-Arg) will bind an amino peptidase receptor (CD13/APN) overexpressed on Human Fibrosarcoma (HT-1080) cells. By substituting a cytotoxic metal such as ruthenium into an NGR-tagged version of Rd, a more targeted cancer therapeutic mechanism may be established.
OBJECTIVES: The focus of this research is to determine localization of NGR tagged rubredoxin (Mu-Ru) in HT-1080 cells. To determine the fate of the Mu-Ru, APN expression levels in HT-1080 and MCF-7 cells need confirmation. Followed by verification of NGR binding to the APN receptor on both cell lines. Furthermore, Mu-Ru needs to be introduced to the cells to determine localization and cytotoxicity of the metalloprotein.
METHODS: To compare APN receptor expression in HT-1080 cells and MCF-7 western blotting was performed. Next, verification of NGR binding was done via immunofluorescence analysis. The localization of the Mu-Ru was performed two ways. Both utilized a Strep-II tagged Mu-Ru for determining localization by fractionation of HT-1080 followed by western blotting on each fraction. In addition, immunofluorescence was performed to ascertain visual localization.
RESULTS: It was determined that the HT-1080 cells expressed the APN receptors at various cellular confluences, while the MCF-7 cells showed no detectable expression of the receptor. Furthermore, WT-Ru binding compared to Mu-Ru verified as the concentration increased so did Mu-Ru binding to the APN receptor on HT-1080 cell, while there was little binding of the WT-Ru to the cells. A comparison of binding to the MCF-7 cells also determined little binding at the same concentrations. Preliminary studies on the localization of the protein using fractionation did not show the presence of Mu-Ru in any cellular compartments by western blot analysis, while the immunofluorescent analysis showed possible intracellular localization.
DISCUSSION: The NGR-tagged rubredoxin metalloprotein can lead to a more specific and less cytotoxic chemotherapy treatment. To support this claim the research done in this study indicates the APN receptor is highly expressed on HT-1080 cells. Additionally, immunofluorescent studies show that NGR bound rubredoxin binds to APN leading to greater specificity of the Mu-Ru. However, the localization of the Mu-Ru has not been completely determined and further studies are in progress. Further studies will address dosage, localization, and cytotoxic effects.
Embargo Period
5-23-2024
Localization and cytotoxicity of ruthenium substituted protein in human fibrosarcoma cells
Suwanee, GA
INTRODUCTION: Many current cancer treatments such as chemotherapy lack a targeting mechanism - leading to side effects such as nausea, vomiting, and hair loss. In efforts to increase targeting associated with these treatments, the exploration of metal-based therapies is currently an area of interest. One potential vector associated with this method involves the hyperthermophilic archaeon Pyrococcus furiosus that contains thermostable proteins such as rubredoxin (Rd). This non-heme iron protein has the ability to unfold and refold allowing for substitution of its core iron (Fe) center, while maintaining its protein structure. A tumor homing peptide known as NGR (Asn-Gly-Arg) will bind an amino peptidase receptor (CD13/APN) overexpressed on Human Fibrosarcoma (HT-1080) cells. By substituting a cytotoxic metal such as ruthenium into an NGR-tagged version of Rd, a more targeted cancer therapeutic mechanism may be established.
OBJECTIVES: The focus of this research is to determine localization of NGR tagged rubredoxin (Mu-Ru) in HT-1080 cells. To determine the fate of the Mu-Ru, APN expression levels in HT-1080 and MCF-7 cells need confirmation. Followed by verification of NGR binding to the APN receptor on both cell lines. Furthermore, Mu-Ru needs to be introduced to the cells to determine localization and cytotoxicity of the metalloprotein.
METHODS: To compare APN receptor expression in HT-1080 cells and MCF-7 western blotting was performed. Next, verification of NGR binding was done via immunofluorescence analysis. The localization of the Mu-Ru was performed two ways. Both utilized a Strep-II tagged Mu-Ru for determining localization by fractionation of HT-1080 followed by western blotting on each fraction. In addition, immunofluorescence was performed to ascertain visual localization.
RESULTS: It was determined that the HT-1080 cells expressed the APN receptors at various cellular confluences, while the MCF-7 cells showed no detectable expression of the receptor. Furthermore, WT-Ru binding compared to Mu-Ru verified as the concentration increased so did Mu-Ru binding to the APN receptor on HT-1080 cell, while there was little binding of the WT-Ru to the cells. A comparison of binding to the MCF-7 cells also determined little binding at the same concentrations. Preliminary studies on the localization of the protein using fractionation did not show the presence of Mu-Ru in any cellular compartments by western blot analysis, while the immunofluorescent analysis showed possible intracellular localization.
DISCUSSION: The NGR-tagged rubredoxin metalloprotein can lead to a more specific and less cytotoxic chemotherapy treatment. To support this claim the research done in this study indicates the APN receptor is highly expressed on HT-1080 cells. Additionally, immunofluorescent studies show that NGR bound rubredoxin binds to APN leading to greater specificity of the Mu-Ru. However, the localization of the Mu-Ru has not been completely determined and further studies are in progress. Further studies will address dosage, localization, and cytotoxic effects.