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Interaction between anticancer drug Cisplatin and copper chaperone Atox1 in human melanoma cells
Umeå University, Faculty of Science and Technology, Department of Chemistry. (Pernilla Wittung-Stafshede)
Umeå University, Faculty of Medicine, Department of Surgical and Perioperative Sciences, Surgery. (Peter Naredi)
Umeå University, Faculty of Science and Technology, Department of Chemistry.
Kirurgi, Sahlgrenska sjukhuset, Göteborgs universitet.
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2014 (English)In: Protein peptide letters, ISSN 0929-8665, Vol. 21, no 1, 63-68 p.Article in journal (Refereed) Published
Abstract [en]

Cisplatin (CisPt) is one of the most common anticancer drugs used against many severe forms of cancers. However, treatment with this drug causes many side effects and often, it results in the development of cell resistance. A majority of side effects as well as cell resistance are thought to develop due to CisPt interactions with proteins prior to reaching the nucleus and the DNA target. The copper (Cu) transport proteins Ctr1 and ATP7A/B have been implicated in cellular resistance of CisPt, possibly exporting the drug out of the cell. Recent in vitro work demonstrated that CisPt also interacts with the cytoplasmic Cu-chaperone Atox1, binding in or near the Cu-binding site, without expulsion of bound Cu. Whereas Ctr1 and ATP7B interactions with CisPt have been shown in vivo or ex vivo, there is no such information for Atox1-CisPt interactions. To address this, we developed a method to probe if CisPt interacts with Atox1 in human melanoma cells. Atox1-specific antibodies were linked to magnetic beads and used to immune-precipitate Atox1 from melanoma cells that had been pre-exposed to CisPt. Analysis of extracted Atox1 with inductively coupled plasma mass spectrometry demonstrated the presence of Pt in the protein fraction. Thus, CisPt-exposed human melanoma cells contain Atox1 molecules that bind some derivative of CisPt. This study gives the first indication for the intracellular presence of Atox1-CisPt complexes ex vivo.

Place, publisher, year, edition, pages
2014. Vol. 21, no 1, 63-68 p.
Keyword [en]
Copper-chaperone, Cisplatin, Atox1, anticancer, resistance
National Category
Biochemistry and Molecular Biology
Research subject
biological chemistry
URN: urn:nbn:se:umu:diva-80714DOI: 10.2174/09298665113209990036ISI: 000329022400011OAI: diva2:651190
Available from: 2013-09-24 Created: 2013-09-24 Last updated: 2014-03-04Bibliographically approved
In thesis
1. Copper-transporting proteins and their interactions with platinum-based anticancer substances
Open this publication in new window or tab >>Copper-transporting proteins and their interactions with platinum-based anticancer substances
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

  Cisplatin (CisPt) is an important drug that is used against various cancers, including testicular, ovarian, lung, head, and neck cancer. However, its effects are limited by cellular resistance. The resistance is believed to be multifactorial, and may be mediated to varying degree by multiple systems in cells, one of the proposed systems being the copper (Cu) transporting system. The Cu-importer Ctr1 has proven importance for cellular sensitivity to CisPt by regulating its influx, while the Golgi-localized Cu-ATP:ases ATP7A/B can putatively mediate CisPt efflux and/or drug sequestration. Atox1 is a small Cu-chaperone that normally transfers Cu between Ctr1 and ATP7A/B, prior to delivery of Cu to the proteins in the secretory pathway. Since Ctr1 and ATP7A/B are reportedly involved in CisPt-resistance, CisPt interaction with Atox1 was the focus of the project this thesis is based upon.

  Using a variety of techniques, Atox1 was found to bind CisPt, also simultaneously with Cu. The Atox1-CisPt complexes were further probed using selected mutants in studies demonstrating that only the two cysteines (Cys12 and Cys15) in the Cu-binding site of Atox1 are essential for CisPt interactions. A proposed Atox1 di-metal complex containing both Cu and CisPt was found to be monomeric, and no loss of Cu was observed. In vitro experiments demonstrated that CisPt could also bind to metal-binding domain 4 of ATP7B (WD4), and that the drug could be transferred from Atox1 to the domain. These findings indicated that Atox1 may transfer CisPt to ATP7A/B in vivo, utilizing the same transport pathway as Cu. However, the CisPt-bound Atox1 complexes were not stable over time; upon incubation, protein unfolding and aggregation were observed. Thus, in vivo, Atox1 might alternatively be a dead-end sink for CisPt.

  The effects of the ligands around the Pt-center of Pt-based anticancer drugs and drug derivatives on Atox1 binding and unfolding were also investigated. The ligands’ chemistry and geometry were shown to dictate the extent and rate of the Pt-based substances interactions with Atox1. Finally, the occurrence of Atox1-CisPt interactions in a biological environment was demonstrated by developing and applying an antibody-based method allowing analysis of metals associated with Atox1 extracted from CisPt-treated cells.

  The findings presented in this thesis show that CisPt binds to Atox1 and WD4, also simultaneously with Cu, in vitro. The results support the hypothesis that Cu-transporting proteins can mediate cellular resistance to CisPt in vivo, and provide a deeper chemical understanding of the interactions between the proteins and the drug.

Place, publisher, year, edition, pages
Umeå: Umeå Universitet, 2013. 96 p.
Cisplatin, Atox1, copper transport, anticancer drug, resistance, platinum, spectroscopy.
National Category
Other Chemistry Topics
Research subject
biological chemistry
urn:nbn:se:umu:diva-80717 (URN)978-91-7459-705-9 (ISBN)
Public defence
2013-10-18, KBC-huset, KB3B1, Umeå universitet, Umeå, 09:00 (English)
Available from: 2013-09-27 Created: 2013-09-24 Last updated: 2013-09-27Bibliographically approved

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Palm-Espling, MariaLundin, ChristinaBjörn, ErikNaredi, PeterWittung-Stafshede, Pernilla
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