Mammalian thioredoxin reductase as a drug target in anticancer therapy through direct apoptosis induction by selenium compromised forms of the protein

The mammalian thioredoxin system, consisting of thioredoxin(s), thioredoxin reductase(s) and NADPH, participates in the regulation of many important systems in a mammalian cell. The mammalian thioredoxin reductases (TrxR) are selenoproteins containing a selenocysteine (Sec) residue within a carboxyl terminal -Gly -Cys -Sec -Gly -COOH motif. Sec is encoded by a UGA codon, normally functioning as a stop of translation, which necessitates an expansion of the genetic code. The Sec residue is thus inserted by an intricate translation machinery dependent on a Sec insertion sequence (SECIS) element located in the 3' untranslated region of the mRNA.

A dysfunctional SECIS element would likely generate a form of TrxR with a truncated carboxyl terminal motif consisting of Gly-Cys-COOH lacking its normal enzymatic activity. Alkylating the selenocysteine with electrophilic compounds, a reaction that is highly favoured at physiological pH due to a high reactivity of Sec, can also generate an inactive enzyme. The aim of this thesis project was to examine how different forms of TrxR affect cell viability, especially in cancer cells.

The BioPORTER technique, which makes it possible to introduce fully active proteins into cells, was used to deliver different preparations of pure TrxR1 protein derivatives into A549 (lung carcinoma) cells. Interestingly we found that selenium compromised TrxR1, either in the truncated form or produced by alkylating the Sec residue by electrophilic compounds, provoked a very rapid cell death in A549 cells. This effect could not be detected after introduction of fully active TrxR1 enzyme nor upon introduction of glutathione reductase. The selenium compromised TrxR1 forms initiating cell death were suggested to be collectively termed SecTRAPs (selenium compromised thioredoxin reductase-derived apoptotic proteins).

The cell death provoked by SecTRAPs was demonstrated to trigger exposure of phosphatidyl-serine, a phenomenon characteristic of apoptotic cell death. It was also found that the apoptosis provoked by SecTRAPs required both caspase-2 and caspase-3/7 activation but not induction of protein synthesis. Either human or rat SecTRAPs could induce apoptosis in human A549 (lung adenocarcinoma) or HeLa (cervix carcinoma) cancer cells. HeLa cells overexpressing Bcl-2, which were resistant to staurosporine, were still susceptible to apoptosis provoked by SecTRAPs. Notably, human embryonic kidney-derived cells (HEK293) were resistant to SecTRAPs, illustrating that not all human cell types are susceptible to cell death provoked by SecTRAPs.

We subsequently evaluated the importance of a redox-active disulfide/dithiol motif involving Cys59 and Cys64 within a CVNVGC sequence present in TrxR for the apoptotic effects of SecTRAPs. This was performed by generation of a mutant where the two redox active cysteins were replaced by Ser residues. The findings indicated that the two Cys residues of the CVNVGC motif, found in both TrxR1 and SecTRAPs, are required for the apoptotic features of the latter. This may suggest that SecTRAPs redox cycle with an endogenous substrate leading to oxidative stress and apoptosis or, alternatively, that local effects at the mitochondrial membrane may directly signal apoptosis through release of cytochrome c in a process involving caspase-2. The critical Cys residues could also be susceptible to posttranslational modifications.

In addition we found by in vitro analysis that mammalian TrxR may easily be targeted by all representative compounds of the major classes of clinically used anticancer alkylating agents and most platinum compounds whereas this was not the case with glutathione reductase. In conclusion, our results suggest that TrxR may be a prime target for anticancer alkylating agents, producing SecTRAPs that can directly induce apoptosis in cancer cells.

List of scientific papers

I. Anestal K, Arner ES (2003). Rapid induction of cell death by selenium-compromised thioredoxin reductase 1 but not by the fully active enzyme containing selenocysteine. J Biol Chem. 278(18): 15966-72. Epub 2003 Feb 6
https://pubmed.ncbi.nlm.nih.gov/12574159

II. Witte AB, Anestal K, Jerremalm E, Ehrsson H, Arner ES (2005). Inhibition of thioredoxin reductase but not of glutathione reductase by the major classes of alkylating and platinum-containing anticancer compounds. Free Radic Biol Med. 39(5): 696-703.
https://pubmed.ncbi.nlm.nih.gov/16085187

III. Anestal K, Arner ESJ (2006). SecTRAPs are potent inducers of a caspase-dependent apoptosis in cancer cells that can not be prevented by overexpression of Bcl2. [Submitted]

IV. Anestal K, Arner ESJ (2006). The apoptotic effects of SecTRAPs require an intact CVNVGC motif in the aminoterminal domain. [Manuscript]