Pt(II) based anticancer drugs—cisplatin, carboplatin, and oxaliplatin—are widely used in the treatment of a variety of cancers. Unfortunately, the clinical efficacy of these drugs is currently hindered by the development of undesirable side effects and resistance during treatment. The molecular mechanisms underlying these effects are still unclear. For decades, research has focused on DNA as the main cellular target of Pt(II) compounds. However, there is increasing interest in proteins as alternative targets of Pt(II) and contributors to cytotoxic and resistance mechanisms of cisplatin. In this work, I utilize Pt(II) compounds that have been functionalized to participate in the azide-alkyne cycloaddition ‘click’ reaction to study protein targets of platinum reagents.
First, I describe the use of an azide-modified Pt(II) compound to fluorescently label and isolate Pt(II)-bound bovine serum albumin in vitro. Additionally, we discover that Pt(II) compounds form monofunctional adducts on BSA that can crosslink to DNA oligonucleotides. I then use the click-functionalized Pt(II) compound, azidoplatin, to enrich for Pt(II)-bound proteins in Saccharomyces cerevisiae using a biotin-streptavidin pull-down. I identified 152 proteins that are significantly enriched in AzPt-treated samples by LC-MS/MS analysis. A subset of these proteins are involved in proteostasis and ER stress, which I confirm is induced in both AzPt- and cisplatin-treated yeast. Of interest was the identification of the ER protein folding chaperone protein disulfide isomerase (PDI), which I observe is inhibited by Pt(II) binding in vitro. Finally, I investigate PDI activity in human cancer cell lines HeLa and MDA-MB-468 following treatment with Pt(II) compounds. Extracts from platinum-treated MDA-MB-468 cells show significant PDI inhibition at low concentrations of Pt(II), and these cells appear to have constitutive activation of the unfolded protein response. PDI activity in extracts from platinum-treated HeLa cells is inhibited only at high concentrations of Pt(II), and HeLa cells do not show significant XBP1 mRNA splicing during Pt(II) treatment. Additionally, MDA-MB-468 cells are nearly three times as sensitive to Pt(II) compounds than HeLa cells. From these data, I hypothesize that basal ER stress increases sensitivity to PDI inhibition by Pt(II) binding and that this interaction enhances Pt(II)-induced cell death. / 10000-01-01
| Identifer | oai:union.ndltd.org:uoregon.edu/oai:scholarsbank.uoregon.edu:1794/22652 |
| Date | 06 September 2017 |
| Creators | Cunningham, Rachael |
| Contributors | Hawley, Diane |
| Publisher | University of Oregon |
| Source Sets | University of Oregon |
| Language | en_US |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Rights | All Rights Reserved. |
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