Please use this identifier to cite or link to this item: http://dl.umsu.ac.ir/handle/10616/39343
Title: Exploring thioredoxin reductase as an anticancer drug target
Authors: Prast-Nielsen, Stefanie
Year: 18-Sep-2009
9-Oct-2009
Publisher: Institutionen för medicinsk biokemi och biofysik (MBB) / Department of Medical Biochemistry and Biophysics
Description: Mammalian thioredoxin reductase (TrxR; EC 1.8.1.9) is a homodimeric NADPH-dependent selenium-containing flavoenzyme with disulfide oxidoreductase activity. The mammalian thioredoxin (Trx) system exerts an impressive spectrum of functions either directly through reactions catalyzed by TrxR or by its prime substrate Trx. It is involved in redox regulation, antioxidant defense, cell growth, replication and regulation of transcription factors. TrxR was found to be overexpressed in a variety of tumor cells and may be involved in several, if not all, steps of carcinogenesis. Therefore, TrxR has been proposed as a potential target for anticancer therapy. In fact, inhibition of TrxR has been shown by a number of chemotherapeutic drugs as part of their mechanism of action. The rather unique biochemistry of TrxR indeed supports efficient targeting by electrophiles. TrxR has an N-terminal active site with two cysteines (Cys) and a Cterminal active site with a Cys and a selenocysteine (Sec). Both active sites are necessary for native TrxR function. Sec is a rare, naturally occurring amino acid encoded by a UGA codon. As this codon normally signals a termination of translation, a recoding signal, the Sec insertion sequence is present in the 3´ untranslated region of the mRNA. A specific translation machinery then guides the insertion of the Sec into the polypeptide chain. Sec is a highly reactive Cys analog with a selenium atom in place of the sulfur, resulting in a significantly lower pKa value. Therefore, Sec is mainly deprotonated at physiological pH and thus highly reactive towards alkylation by electrophilic agents. This results in abrogation of redox activity of the enzyme´s C-terminal active site that is essential for reduction of Trx and most other substrates. However, specific targeting of the Sec does not render a completely inactive protein. With an intact N-terminal active site, inhibited TrxR may still induce a rapid cell death in cancer cells presumably by redox cycling with an endogenous substrate inducing oxidative stress as shown in this thesis. We named this cytotoxic form of TrxR SecTRAPs (selenium compromised thioredoxin reductase-derived apoptotic proteins). Introducing SecTRAPs directly into A549 (lung carcinoma) or HeLa (cervical adenocarcinoma) cells using the protein delivery reagent BioPORTER® we observed DNA condensation, phosphatidylserine exposure prior to loss of membrane integrity and production of reactive oxygen species. Cell death was caspase-dependent but independent of novel protein synthesis. Cisplatin was shown to inhibit TrxR and form SecTRAPs in vitro. We investigated the elemental differences in TrxR inhibition of simple Pt, Pd and Au salts, showing Pd and Au to be superior in this aspect. All salts seemingly target the C-terminal active site of TrxR in a specific manner, potentially forming SecTRAPs. Inhibition of cellular TrxR was shown to correlate with cytotoxicity of nitroaromatic and quinone compounds involving caspase activation. However, the extent of cell death was in some cases, but not all, dependent on TrxR1 levels. Using siRNA constructs to specifically knock down TrxR1 by 90% in A549 cells resulted in increased susceptibility towards DNCB and menadione, and decreased sensitivity to cisplatin. This may indicate that different TrxR inhibition mechanisms can lead to different treatment outcomes and that cisplatin may indeed form toxic SecTRAPs within cells. This occurs possibly at a greater extent in tumors where high expression of TrxR is prevalent. The potential of TrxR as an anticancer drug target is discussed in this thesis with special focus on drugs likely to form SecTRAPs and possibly to induce selective tumor killing.
URI: https://openarchive.ki.se/xmlui/handle/10616/39343
Standard no: 978-91-7409-576-0
20091009pras
Appears in Collections:Dept of Medical Biochemistry and Biophysics

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