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Reaction of platinum anticancer drugs and drug derivatives with a copper transporting protein, Atox1
Umeå University, Faculty of Science and Technology, Department of Chemistry.
Umeå University, Faculty of Science and Technology, Department of Chemistry.
2012 (English)In: Biochemical Pharmacology, ISSN 0006-2952, E-ISSN 1873-2968, Vol. 83, no 7, 874-881 p.Article in journal (Refereed) Published
Abstract [en]

Platinum (Pt) containing anticancer drugs have been used in cancer treatment for several decades as they trigger cell death upon DNA binding. Pt-containing anticancer drugs and drug derivates with a variety of ligands around the Pt center (with Cisplatin being most well known) exist today in clinics and in clinical trials. However, a major drawback with these drugs is limited efficacy due to side reactions resulting in cell resistance. The cellular copper (Cu) transport pathway is proposed to be responsible for part of these side reactions through interactions with the Pt-containing drugs and possibly cellular export of Pt. The cytoplasmic Cu chaperone, Atox1, was recently found to bind Cisplatin in vitro and, when over-expressed in Escherichia coli, in vivo. Here we investigate how the chemical properties of six Pt-substances differentially affect binding, unfolding, and aggregation of Atox1 in vitro using near- and far-UV circular dichroism (CD) spectroscopy and SDS-PAGE. The results show that both ligand type and orientation dictate the interactions with Atox1. Only substances with two good leaving groups in cis-configuration result in near-UV CD changes that report on Cu–Pt interactions. The different substances promote Atox1 unfolding in a pattern that can be explained by ligand chemistry and geometry. Our work emphasize that ligands around the Pt-center have decisive roles in tuning protein interactions (prior to DNA binding) and therefore they also dictate the level of drug side effects and cellular resistance.

Place, publisher, year, edition, pages
Elsevier, 2012. Vol. 83, no 7, 874-881 p.
Keyword [en]
Atox1, Cisplatin, Anticancer, Protein unfolding, Spectroscopy
National Category
Chemical Sciences
URN: urn:nbn:se:umu:diva-52617DOI: 10.1016/j.bcp.2012.01.018OAI: diva2:506180
Available from: 2012-02-28 Created: 2012-02-28 Last updated: 2013-09-26Bibliographically 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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