High levels of thioredoxin reductase 1 modulate drug-specific cytotoxic efficacy

doi:10.1016/j.freeradbiomed.2009.09.016

Free Radical Biology and Medicine

Volume 47, Issue 11, 1 December 2009, Pages 1661-1671

https://doi.org/10.1016/j.freeradbiomed.2009.09.016 Get rights and content

Abstract

The selenoprotein thioredoxin reductase 1 (TrxR1) is currently recognized as a plausible anticancer drug target. Here we analyzed the effects of TrxR1 targeting in the human A549 lung carcinoma cell line, having a very high basal TrxR1 expression. We determined the total cellular TrxR activity to be 271.4 ± 39.5 nmol min⁻¹ per milligram of total protein, which by far exceeded the total thioredoxin activity (39.2 ± 3.5 nmol min⁻¹ per milligram of total protein). Knocking down TrxR1 by approx 90% using siRNA gave only a slight effect on cell growth, irrespective of concurrent glutathione depletion (≥98% decrease), and no increase in cell death or distorted cell cycle phase distributions. This apparent lack of phenotype could probably be explained by Trx functions being maintained by the remaining TrxR1 activity. TrxR1 knockdown nonetheless yielded drug-specific modulation of cytotoxic efficacy in response to various chemotherapeutic agents. No changes in response upon exposure to auranofin or juglone were seen after TrxR1 knockdown, whereas sensitivity to 1-chloro-2,4-dinitrobenzene or menadione became markedly increased. In contrast, a virtually complete resistance to cisplatin using concentrations up to 20 μM appeared upon TrxR1 knockdown. The results suggest that high overexpression of TrxR has an impact not necessarily linked to Trx function that nonetheless modulates drug-specific cytotoxic responses.

Section snippets

Chemicals and reagents

Recombinant rat TrxR1 was produced as described [50] and human wt Trx [51] was kindly provided by Arne Holmgren (Karolinska Institutet, Stockholm, Sweden). Yeast glutathione reductase (GR) (Cat. No. G3664), reduced glutathione (GSH), and oxidized glutathione (GSSG) were obtained from Sigma–Aldrich Chemicals (Steinheim, Germany). Chemicals used in the drug sensitivity assays were as follows: auranofin from Alexis Biochemicals (San Diego, CA, USA), cDDP (Platinol) from Bristol–Myers Squibb

Confirming knockdown of TrxR1

The aim of this study was to assess the cellular phenotype upon siRNA-mediated knockdown of the selenium-dependent TrxR1 in the highly TrxR1-overexpressing A549 lung carcinoma cells. First an Alexa 488-labeled AllStar negative siRNA (Qiagen) was used as a control for transfection efficiency, and under the utilized transfection conditions the efficiency was at least 95% (data not shown). Specific siRNA-mediated knockdown of TrxR1 was first confirmed using ⁷⁵Se labeling of all cellular

Discussion

In this study we found that knocking down the endogenous overexpression of TrxR in a cancer cell line using siRNA leads to diverse, complex, and unexpected cellular phenotypes. The basal nonstressed growth of A549 cells was unaffected by an efficient (approx 90%) TrxR1 knockdown, even in combination with GSH depletion, illustrating that these cells have a tremendous reserve capacity in vital redox systems. We also found that the very high TrxR1 activity in A549 cells exceeded by severalfold the

Acknowledgments

Determination of selenium content in the fetal calf serum was kindly performed by Marie Vahter and collaborators, Karolinska Institutet. Oxaliplatin used in the drug sensitivity assays was a kind gift from Maria Shoshan, and human thioredoxin was kindly provided by Arne Holmgren, both at Karolinska Institutet. The kind help of Qing Cheng and Olle Rengby in the production of TrxR1 is also acknowledged. This study was supported by funding from the Swedish Research Council (Medicine), the Swedish

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Original ContributionHigh levels of thioredoxin reductase 1 modulate drug-specific cytotoxic efficacy

Abstract

Section snippets

Chemicals and reagents

Confirming knockdown of TrxR1

Discussion

Acknowledgments

Free Radic. Biol. Med.

Free Radic. Biol. Med.

Dev. Biol.

J. Biol. Chem.

Biochem. Pharmacol.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

Arch. Biochem. Biophys.

Semin. Cancer Biol.

Semin. Cancer Biol.

J. Biol. Chem

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

Toxicol. In Vitro

J. Biol. Chem.

J. Biol. Chem.

Free Radic. Biol. Med.

J. Biol. Chem.

Exp. Cell Res.

Free Radic. Biol. Med.

Free Radic. Biol. Med.

J. Biol. Chem.

J. Biol. Chem.

Free Radic. Biol. Med.

Methods Enzymol.

Methods Enzymol.

Anal. Biochem.

Free Radic. Biol. Med.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Physiological functions of thioredoxin and thioredoxin reductase

Eur. J. Biochem.

Thiol-based mechanisms of the thioredoxin and glutaredoxin systems: implications for diseases in the cardiovascular system

Am. J. Physiol. Heart Circ. Physiol.

Original Contribution
High levels of thioredoxin reductase 1 modulate drug-specific cytotoxic efficacy