Original Contribution Upregulation of phase II enzymes through phytochemical activation of Nrf2 protects cardiomyocytes against oxidant stress Danielle J. Reuland a , Shadi Khademi a , Christopher J. Castle a , David C. Irwin b , Joe M. McCord c , Benjamin F. Miller a,1 , Karyn L. Hamilton a,*,1 a Department of Health and Exercise Science, Colorado State University, Fort Collins, CO 80523, USA b Cardiovascular Pulmonary Research Group, Division of Cardiology, School of Medicine, University of Colorado at Denver Health Science Center, Aurora, CO 80045, USA c Pulmonary Sciences and Critical Care Medicine, University of Colorado, Denver Anschutz Medical Campus, Aurora, CO 80045, USA article info Article history: Received 9 November 2012 Accepted 11 November 2012 Available online 30 November 2012 Keywords: Nrf2 Phytochemicals Oxidative stress Antioxidants Free radicals abstract Increased production of reactive oxygen species has been implicated in the pathogenesis of cardiovas- cular disease (CVD), and enhanced endogenous antioxidants have been proposed as a mechanism for regulating redox balance. Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a transcriptional regulator of phase II antioxidant enzymes, and activation of Nrf2 has been suggested to be an important step in attenuating oxidative stress associated with CVD. A well-defined combination of five widely studied medicinal plants derived from botanical sources (Bacopa monniera, Silybum marianum (milk thistle), Withania somnifera (Ashwagandha), Camellia sinensis (green tea), and Curcuma longa (turmeric)) has been shown to activate Nrf2 and induce phase II enzymes through the antioxidant response element. The purpose of these experiments was to determine if treatment of cardiomyocytes with this phytochemical composition, marketed as Protandim, activates Nrf2, induces phase II detoxification enzymes, and protects cardiomyocytes from oxidant-induced apoptosis in a Nrf2-dependent manner. In cultured HL-1 cardiomyocytes, phytochemical treatment was associated with nuclear accumulation of Nrf2, significant induction of phase II enzymes, and concomitant protection against hydrogen peroxide- induced apoptosis. The protection against oxidant stress was abolished when Nrf2 was silenced by shRNA, suggesting that our phytochemical treatment worked through the Nrf2 pathway. Interestingly, phytochemical treatment was found to be a more robust activator of Nrf2 than oxidant treatment, supporting the use of the phytochemicals as a potential treatment to increase antioxidant defenses and protect heart cells against an oxidative challenge. & 2012 Elsevier Inc. All rights reserved. Oxidative stress has been implicated in the development or exacerbation of over 100 human diseases [1], including cardio- vascular disease (CVD), the leading cause of death and disability within the Western world [2]. Cells contain enzymatic and non- enzymatic antioxidants to prevent damage caused by reactive oxygen species (ROS). Antioxidants may act by directly scaven- ging ROS, by recycling or reducing other direct antioxidants, or by indirectly upregulating endogenous antioxidant defenses. Direct exogenous antioxidants including vitamin C, b-carotene, and vitamin E, have been the focus of extensive research but are still only presumed effective in the treatment of CVD [3]. Recent clinical trials [4,5] fail to show therapeutic benefit of exogenous antioxidant supplementation in CVD and suggest the need for a new approach to regulating cellular redox status. As a result of the apparent ineffectiveness of antioxidant vitamins in attenuating oxidative stress, recent research has focused on novel ways to induce endogenous antioxidant responses [6,7]. The upregulation of endogenous antioxidant defenses provides the potential for more profound cellular pro- tection than antioxidant vitamin supplementation because of the enhanced ability of enzymatic antioxidants to scavenge ROS compared to traditional antioxidant vitamins. Some phytochem- icals can increase endogenous antioxidant enzyme activity through the activation of the transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2) [6,8]. Nrf2 is a member of the basic leucine zipper transcription factor family [8,9] and controls both basal and inducible expression of more than 200 genes [10]. Because of the profound number of genes it transcriptionally regulates, Nrf2 has been termed the ‘‘master regulator’’ of antioxidant defenses [11]. Under normal conditions, Nrf2 is sequestered in the Contents lists available at SciVerse ScienceDirect journal homepage: www.elsevier.com/locate/freeradbiomed Free Radical Biology and Medicine 0891-5849/$ - see front matter & 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.freeradbiomed.2012.11.016 Abbreviations: ARE, antioxidant response element; CVD, cardiovascular disease; GR, glutathione reductase; HO-1, heme oxygenase-1; Nrf2, nuclear factor (erythroid-derived 2)-like 2; ROS, reactive oxygen species; tBH, tert-butylhydroperoxide * Corresponding author. Fax: þ1 970 491 0445. E-mail address: karyn.hamilton@colostate.edu (K.L. Hamilton). 1 These authors contributed equally to this work. Free Radical Biology and Medicine 56 (2013) 102–111