Inhibition of thioredoxin reductase by alantolactone prompts oxidative stress-mediated apoptosis of HeLa cells Junmin Zhang, Ya Li, Dongzhu Duan, Juan Yao, Kun Gao*, Jianguo Fang* State Key Laboratory of Applied Organic Chemistry and College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou 730000, China A R T I C L E I N F O Article history: Received 29 October 2015 Accepted 8 December 2015 Available online 11 December 2015 Chemical compounds studied in this article: Alantolactone (PubChem CID: 72724) Racemosalactone C (PubChem CID: 71665531) Keywords: Thioredoxin reductase Alantolactone Oxidative stress Redox regulation Apoptosis A B S T R A C T The mammalian thioredoxin reductase (TrxR) isoenzymes, TrxR1 in cytosol or nucleus, TrxR2 in mitochondria, and TrxR3 in testis, are essential seleno-flavoenzymes with a conserved penultimate selenocysteine (Sec) residue at the C-terminus, and have attracted increasing interests as potential targets for development of cancer chemotherapeutic agents. The sesquiterpene lactone alantolactone (ATL), an active component from the traditional folk medicine Inula helenium, has been documented possessing multiple pharmacological functions, especially the anticancer activity. However, the underlying mechanism has not been well defined. We reported that ATL inhibits both the recombinant TrxR and the enzyme in the cellular environment. The alpha-methylene-gamma-lactone moiety in ATL and the Sec residue in TrxR are critical for targeting TrxR by ATL. By employing our newly developed pull down assay, we demonstrated the remarkable elevation of the oxidized thioredoxin in HeLa cells after ATL treatment. In addition, ATL elicits accumulation of reactive oxygen species, and eventually induces apoptosis of HeLa cells. Importantly, overexpression of the functional TrxR attenuates the cytotoxicity of ATL, while knockdown of the enzyme sensitizes the cells to ATL treatment. Targeting TrxR thus discloses a novel molecular mechanism underlying the cellular action of ATL, and sheds light in considering the usage of ATL as a potential cancer chemotherapeutic agent. ã 2015 Elsevier Inc. All rights reserved. 1. Introduction Plant-derived natural products have provided and continue to provide a rich and unique source of therapeutic agents [1,2]. Sesquiterpene lactones are a large and diverse family of natural products widely present in a variety of herbal plants, and display multiple pharmacological functions [3,4]. Alantolactone (ATL, Fig. 1), a sesquiterpene lactone containing an a-methylene- g-lactone moiety, is the principal active component from Inula helenium, a traditional medicinal herb that has been extensively used to treat diverse ailments for centuries [5]. The presence of ATL in I. helenium, especially in the root, is highly abundant (>2% of the dry root) [6,7]. ATL has been documented having antibacterial activity [8], targeting Nrf2 to activate detoxifying enzymes [9], and possessing anti-inflammatory property by suppressing inducible nitric oxide synthase and cyclooxygenase-2 [10] or inhibiting chemokine production [11]. In recent years, increasing interests have been attracted by the potential anticancer activity of ATL in different types of tumor cells, and the putative mechanisms have been reported, including arrest of cell cycles [12], suppression of STAT3 activation [13], downregulation of testes-specific protease 50 [14], induction of reactive oxygen species (ROS) [15], inhibition of Bcr/Abl [16], and modulation of activin/SMAD3 signaling [17], and others [7]. Despite the potent anticancer efficacy of ATL, its molecular mechanism is still elusive, and the primary cellular target and mode of action of this small molecule remain unclear. The mammalian thioredoxin reductase (TrxR) isoforms, includ- ing the cytosolic/nuclear enzyme TrxR1, the mitochondrial enzyme TrxR2, and the testis-specific isoform TrxR3, are essential selenocysteine (Sec)-containing flavoenzymes with an indispens- able Sec residue at the penultimate C-terminal position. TrxR, thioredoxin (Trx) and NADPH comprise the thioredoxin system, which is a highly conserved and ubiquitous system and plays pivotal roles in maintaining intracellular redox homeostasis [18–20]. TrxRs catalyze the transfer of electrons from NADPH to the active site of Trxs generating the reduced Trxs, which interacts with a panel of downstream target proteins ranging from metabolic enzymes to kinases to transcription factors, to regulate a wide array of cellular redox events involved in cell proliferation, differentiation and death [21,22]. Expanding evidence has * Corresponding authors. Fax: +86 931 8915557. E-mail addresses: npchem@lzu.edu.cn (K. Gao), fangjg@lzu.edu.cn (J. Fang). http://dx.doi.org/10.1016/j.bcp.2015.12.004 0006-2952/ ã 2015 Elsevier Inc. All rights reserved. Biochemical Pharmacology 102 (2016) 34–44 Contents lists available at ScienceDirect Biochemical Pharmacology journa l home page : www.e lsevier.com/loca te/biochempharm