S-1-propenylmercaptocysteine protects murine hepatocytes against oxidative stress via persulfidation of Keap1 and activation of Nrf2

Free Radical Biology and Medicine 143 (2019) 164–175

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Original article

S-1-propenylmercaptocysteine protects murine hepatocytes against oxidative stress via persulfidation of Keap1 and activation of Nrf2

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Restituto Tocmo∗, Kirk Parkin Department of Food Science, University of Wisconsin-Madison, Babcock Hall, 1605 Linden Drive, Madison, WI, 53706, USA

ARTICLE INFO

ABSTRACT

Keywords: S-1-Propenylmercaptocysteine Keap1-Nrf2 Hydrogen sulfide Persulfidation tert-Butyl hydroperoxide

The onion-derived metabolite, S-1-propenylmercaptocysteine (CySSPe), protects against oxidative stress and exhibits anti-inflammatory effects by modulating cellular redox homeostasis. We sought to establish whether CySSPe activates nuclear factor erythroid 2–related factor 2 (Nrf2) and whether activation of Nrf2 by CySSPe involves modification of the Kelch-like ECH-associated protein-1 (Keap1) to manifest these effects. We found that CySSPe stabilized Nrf2 protein and facilitated nuclear translocation to induce expression of antioxidant enzymes, including NQO1, HO-1, and GCL. Moreover, CySSPe attenuated tert-butyl hydroperoxide-induced cytotoxicity and dose-dependently inhibited reactive oxygen species production. Silencing experiments using Nrf2-siRNA confirmed that CySSPe conferred protection against oxidative stress by activating Nrf2. CySSPe enhanced cellular pool of reduced glutathione (GSH) and improved GSH:GSSG ratio. Pretreatment of cells with Lbuthionine-S,R-sulfoximine (BSO) confirmed that CySSPe increases de novo synthesis of GSH by upregulating expression of the GSH-synthesizing enzyme GCL. Treatment of cells with CySSPe elevated hydrogen sulfide (H2S) production. Inhibition of H2S-synthesizing enzymes, cystathionine-gamma-lyase (CSE) and cystathionine-betasynthase (CBS), by pretreating cells with propargylglycine (PAG) and oxyaminoacetic acid (AOAA) revealed that H2S production was partially dependent on a CSE/CBS-catalyzed β-elimination reaction with CySSPe that likely produced 1-propenyl persulfide (RSSH). Depleting cells of their GSH pool by exposure to BSO and diethylmaleate attenuated H2S production, suggesting a GSH-dependent formation of H2S, likely via the reduction of RSSH by GSH. Finally, treatment of cells with CySSPe persulfidated Keap1, which may be the mechanism involved for the stabilization of Nrf2 by CySSPe. Taken together, our results showed that attenuation of oxidative stress by CySSPe is associated with its ability to produce H2S or RSSH, which persulfidates Keap1 and activates Nrf2 signaling. This study provides insights on the potential of CySSPe as an onion-derived dietary agent that modulates redox homeostasis and combats oxidative stress.

1. Introduction Oxidative stress arises when cells and tissues cannot adequately detoxify excessive levels of reactive oxygen species (ROS) upon exposure to environmental stressors (e.g., X-rays, ozone, cigarette smoking, air pollutants, and industrial chemicals) and unhealthy lifestyles [1,2]. ROS impair cellular capacity to maintain redox homeostasis (balance between electrophiles/nucleophiles) resulting in disruption of normal cellular signaling mechanisms and immune function [3]. Oxidative stress can cause damage to macromolecules (e.g., proteins, DNA and lipids) and is implicated in the pathophysiology of major chronic and degenerative disorders such as cancer, cardiovascular diseases, and neurodegenerative disorders [1]. Therefore, identifying dietary agents or diet-based therapeutic interventions that have

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the capacity to defend against oxidative and/or redox stresses by maintaining cellular redox homeostasis may help reduce risk of diseases where oxidative stress is a pathological factor. Cellular homeostasis is maintained in cells through the regulation of the antioxidant response element (ARE), which mediates transcriptional activation of some 250 genes including those encoding for proteins involved in antioxidant defense, drug transport, and detoxification [4,5]. Up-regulation of the ARE involves a redox-signaling pathway, often referred to as a phase 2 response, that activates the nuclear factorerythroid 2 p45-related factor 2 (Nrf2), a bZIP (basic-leucine zipper) protein transcription factor. Under quiescent conditions, protein level regulation of Nrf2 occurs by repression in association with the Kelchlike ECH-associated protein-1 (Keap1), which signals Nrf2 ubiquitination by the Cullin3–Rbx1 (ring-box protein 1) E3 (Cul3-Rbx1) ubiquitin

Corresponding author. E-mail address: [email protected] (R. Tocmo).

https://doi.org/10.1016/j.freeradbiomed.2019.07.022 Received 30 May 2019; Received in revised form 22 July 2019; Accepted 22 July 2019 Available online 23 July 2019 0891-5849/ Published by Elsevier Inc.

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ligase and subsequent proteasomal degradation [5]. Although Nrf2 regulation can also occur at the transcriptional level [6] and in a Keap1independent manner [7], numerous studies have focused on the posttranslational modification of Keap1 cysteine (Cys) residues to derepress Nrf2 leading to its stabilization and activation [8]. The reactive Cys residues of Keap1 (those with low pKa values) act as sensors for endogenously and exogenously encountered electrophiles (also known as Nrf2 inducers or activators). For example, the well-studied Nrf2 activator, sulforaphane (SF), reacts via Michael addition with Cys151 (C151) in the BTB (Broad complex, Tramtrack, and Bric-a-Brac) domain of Keap1. Electrophilic addition to C151 causes steric hindrance that alters Keap1-Cul3 interaction, impairing Nrf2 ubiquitination, and resulting in Nrf2 stabilization [9]. Nrf2 stabilization leads to its translocation into the nucleus, where it binds to the ARE and upregulates antioxidant and cytoprotective gene transcription. Besides electrophilic attack by Michael acceptors, the sulfhydryl (-SH) group of Keap1 Cys could undergo persulfidation (also referred to as S-sulfhydration), a post-translational mechanism of Cys modification that adds an –SH group to a protein-Cys residue resulting in protein persulfide (RSSH) formation [10]. Evidence suggests the importance of persulfidation as a redox signaling mechanism involved in regulating various cellular functions [11,12]. About 10–25% of major proteins in the liver, including glyceraldehyde-3-phosphate, β-tubulin, and actin are persulfidated [13]. One of the most studied molecules that activates cell signal via protein persulfidation is the endogenous gasotransmitter, hydrogen sulfide (H2S). H2S has been shown to persulfidate C38 of NFκB p65 and mediate the anti-apoptotic effect of NF-κB [14]. In another study, H2S conferred protective effects against cellular aging process via persulfidation of C151 of Keap1, which enhanced Nrf2 nuclear translocation and ARE gene transcription [15]. In addition, synthetic H2S donors, such as sodium hydrosulfide (NaHS) and sodium sulfide (Na2S) are able to activate Nrf2 via persulfidation of the C151 of Keap1 [15,16]. Together, these studies provide compelling evidence that activation of Nrf2 via Keap1 persulfidation may commonly involve H2S donors. Thiosulfinates (TS) are alliinase-evolved organosulfur compounds (OSC) upon disruption fresh Allium (e.g., garlic and onions) tissues [17]. While these compounds are stable at the acidic pH of the stomach [17–19], TS have a half-life of < 1 min in blood [17] presumably as a result of rapid metabolism. Because TS are generally unstable, and are rapidly transformed in situ and metabolized in vivo, multiple metabolic products are believed to contribute biological activities [18,20]. In extracellular fluids and in the luminal space of the gastrointestinal tract where Cys/cystine (Cys/CySSCy) are the predominant thiols [21], TS can rapidly react with Cys, via chemical conjugation, to form S-alk(en) ylmercaptocysteine (CySSR) [22]. For example, reaction of allicin, the major TS in crushed garlic tissues, with Cys produces S-allylmercaptocysteine (CySSA), which so far has received considerable research attention as a garlic-derived bioactive agent. CySSA has exhibited antioxidant [23], anti-apoptotic [24], anti-proliferative [25], and antiinflammatory [26] activities. Beyond the studies conducted on CySSA, there is limited information on whether other CySSR species, such as those derived from onions, have similar bioactivities. In crushed onion tissues, 1-propenylbearing TS and related analogues are more dominant than any other alk (en)yl-containing OSC species (45% of total TS) [27]. However, despite their abundance, obtaining high amounts of 1-propenyl OSC derivatives is challenging. In onion, the alliinase reaction product sulfenic acid (1propenylSOH) is largely diverted to propanethial S-oxide by lachrymatory factor synthase [28] instead of forming TS. S-1-propenyl TS species are also difficult to synthesize and prepare in pure form [27] and are not commercially available. Recently, our group developed a gram-scale tissue homogenate-based method [29] to prepare CySSPe, enabling comparative studies of bioactivities relative to CySSA. We found that CySSPe exhibited superior anti-inflammatory and antioxidant properties compared to CySSA and was more potent in

modulating cellular thiol redox status in lipopolysaccharide (LPS)-activated macrophages [30] and in inducing quinone reductase (QR, a representative Nrf2-mediated enzyme) activity in hepatocytes [26]. These studies suggested that CySSPe could be a major OSC derivative responsible for health-beneficial effects of onion. In the present study, we show that CySSPe exhibits cytoprotective effects in murine hepatoma cells. Our findings indicate that CySSPe upregulates cellular H2S production and cause persulfidation of Keap1, which appears to be a mechanism involved in CySSPe-induced Nrf2 stabilization and upregulation of ARE-coded antioxidant enzymes. Moreover, we show that CySSPe protects cells against tert-butyl hydroperoxide-induced oxidative stress in an Nrf2-dependent manner. 2. Methods 2.1. Chemicals Tert-butyl hydroperoxide (tBHP), dimethyl sulfoxide (DMSO), Lbuthionine-S,R-sulfoximine (BSO), dithiothreitol (DTT), reduced and oxidized glutathione (GSH, GSSG), GSH reductase (GR), 2-vinylpyridine, triethanolamine, bovine serum albumin (BSA), N,N-dimethyl-p phenylenediaminedihydrochloride (N,N-DPD) dye, 5,5′-dithiobis-(2nitrobenzoic acid (DTNB), and 2′, 7′-dichlorofluorescein diacetate (DCFH-DA) were purchased from Millipore Sigma (St. Louis, MO, USA). NADPH was purchased from Santa Cruz Biotechnology (SCBT). Enhanced chemiluminescent reagent Clarity Western ECL substrate was purchased from Bio-Rad Laboratories (Hercules, CA, USA). All other chemicals and solvents used were reagent/analytical grade purchased from Millipore Sigma (Milwaukee, WI), Santa Cruz Biotechnology, or Fisher Scientific (Chicago, IL) unless otherwise noted. 2.2. Preparation and isolation of CySSPe Highly pure (> 90%) CySSPe was prepared following a method previously developed in our lab [30]. Identity confirmation was based on obtained NMR and LC-MS data that were matched with previously reported data [22,26]. 2.3. Cell culture and MTT assay Hepa1c1c7 (Hepa) cells (ATCC® CRL-2026™) were obtained from ATCC (Manassas, VA). Cell cultures were maintained in 75-cm2 tissue culture flasks in a Dulbecco's Modified Eagle Media (DMEM) supplemented with 10% fetal bovine serum (FBS), streptomycin (100 μg/ml), penicillin (100 U/ml), and incubated at 37 °C with 5% CO2 humidified atmosphere. Cells (80–90% confluence) were subcultured every 3 days, passaging at a 1:5 split ratio and cell passages 4 to 20 were used for all experiments. Cell viability was measured using a standard MTT assay [30]. 2.4. Small interfering RNA (siRNA) transfection Hepa cells (4 × 105/well, 6-well plate, 60% confluence) were transfected with mouse Nrf2-siRNA (sc-37049, Santa Cruz Biotechnology) or a non-targeting control-siRNA (sc-37007) using a Lipofectamine® RNAiMAX reagent (Life Technologies) and Opti-MEM® (1058-021; Gibco) according to the manufacturer's protocol. Final concentration of siRNA was 75 pmol in 9 μl Lipofectamine® RNAiMAX reagent. After 24 h, the transfected cells were treated with CySSPe (as indicated in the figures) followed by cell lysate preparation and immunoblotting (section 2.6). 2.5. Extraction of cytosolic and nuclear proteins Hepa cell cytoplasmic and nuclear protein extracts were obtained using a nuclear and cytoplasmic extraction kit (GBiosciences, Inc., St. 165

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Louis, MO), following the manufacturer's protocol, before being used for immunoblot analysis.

control) were homogenized in lysis buffer (150 mM NaCl, 0.5% v/v Tween 20, 50 mM Tris, pH 7.5, and 1 mM EDTA) containing freshly added protease inhibitors and immunoprecipitated with anti-Keap1 antibody (10503-2-AP, Proteintech) following the manufacturer's instructions. After washing of the beads with the same buffer, they were incubated with Alexa Fluor 680 conjugated C2 maleimide (Life Technologies; Cat. No.: A-20344) (2 mM final concentration) and kept for 2 h at 4 °C in the dark with occasional gentle mixing. The beads were pelleted (5000 rpm for 5 min) and washed (4x) with the same buffer, and the suspended beads were then treated with or without DTT (1 mM final concentration) for 1 h at 4 °C. Beads were pelleted, washed (4x), suspended in 2x Laemmli buffer, and boiled prior to gel electrophoresis. Gels were transferred to Immobilon-P membranes (Millipore) using a Transblot® Turbo™ Transfer System (Bio-Rad), which were scanned in a Li-COR Odyssey system. The intensity of red fluorescence of Keap1 was quantified using AzureSpot Analysis Software (Azure Biosystems, CA). The same membranes were used for western blotting with anti-Keap1 antibody.

2.6. Western blot analysis The antibodies for Nrf2 (16396-1-AP) and Keap1 (10503-2-AP) were purchased from ProteinTech (Rosemont, IL, USA). NQO1 (sc32793), GCLc (sc-390811), β-actin (sc-47778), and Lamin B1 (#12586S) antibodies were purchased from Santa Cruz Biotechnology or Cell Signaling Technology (CST). Anti–HO–1 (NBP19750705) and Lamin B1 (#12586S) antibody was purchased from Novus Biologicals. Anti-rabbit IgG, horseradish peroxidase (HRP)-conjugated secondary antibody or mouse IgGκ mouse binding protein-HRP were purchased from CST and SCBT. Proteins in cell lysates were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and subjected to immunoblot analysis. Protein concentration was measured with a BCA protein assay kit (R&D Systems) according to the manufacturer's instructions. Blot images were captured using a ChemiDoc™ Touch Imaging System (Bio-Rad, Hercules, CA) and the intensities were quantified by densitometric analysis using Image Analysis (Bio-Rad, Hercules, CA).

2.10. Statistical analysis Statistical analyses were performed using SigmaPlot 13 software (Systat Software, Inc., San Jose, CA). Data from at least three independent experiments were subjected to one-way ANOVA followed by a post hoc analysis with the Tukey's test to determine significant differences among treatments. Differences at P ≤ 0.05 were considered statistically significant.

2.7. GSH/GSSG and cellular ROS assays GSH and GSSG levels in Hepa cells were determined following a method previously described [31], with modifications. Cells were collected in cold extraction buffer (1x RIPA buffer with 5 mM EDTA, pH 7.4), and the cell lysates obtained after centrifugation were diluted with ice-cold extraction buffer containing sulfosalicylic acid (0.6% final concentration) and centrifuged (5000×g for 5 min, at 4 °C). Total GSH in 20 μl cell lysate was determined after incubating with 2.4 mM DTNB, 300 μM NADPH and 10 U/ml GR in extraction buffer for 25 min, with TNB measured by absorbance at 405 nm. To measure GSSG, the same procedure was applied for lysates derivatized and neutralized with 2vinylpyridine (1 h at 20–22 °C) and triethanolamine (diluted 1:4 in distilled H2O), respectively. GSH and GSSG were quantified from calibration curves using GSH and GSSG standards and normalized for total protein content using the BCA protein assay. Total ROS was measured using DCFH-DA, which oxidizes to fluorescent dichlorofluorescein (DCF) in the presence of ROS, as described previously [32]. Briefly, cells pre-treated with CySSPe for 6 h were washed with PBS prior to addition of 10 μM DCFH-DA in serum- and phenol red-free DMEM. After 40 min, cells were washed twice with PBS and treated with 2.5 mM tBHP. Fluorescence was monitored every 5 min for 30 min using a Varioskan Flash (Thermo Scientific, Vantaa, Finland) microplate reader with excitation and emission settings of 485 nm and 535 nm, respectively. The resulting data were normalized using the control values and results were expressed as % of control.

3. Results 3.1. CySSPe induced Nrf2 nuclear translocation by increasing protein stability To investigate whether CySSPe induces stabilization and/or activation of Nrf2, we treated Hepa cells with non-toxic levels (see section 3.3) of CySSPe (0–40 μM) and measured Nrf2 and Keap1 proteins by immunoblotting. CySSPe treatment resulted in a dose-dependent increase (Fig. 1a) in total Nrf2 levels (in whole cell lysates). Time-course experiments showed a ∼70% increase in Nrf2 expression as early as 3 h of CySSPe (25 μM) incubation, which gradually decreased to basal levels at 6–12 h (Fig. 1b). For both dose range and time-course experiments, CySSPe did not affect Keap1 protein levels (Fig. 1a and b). These results suggest that CySSPe causes dissociation of Nrf2 from Keap1, enabling Nrf2 protein to evade Keap1-facilitated cytosolic proteasomal degradation, resulting in Nrf2 stabilization. To determine whether the observed CySSPe-induced Nrf2 stabilization results in cyto-nuclear translocation of Nrf2, nuclear protein extracts were probed for Nrf2 levels. Time-course experiments using 25 μM CySSPe revealed significant increases in nuclear Nrf2 accumulation as early as 30 min, reaching 4- to 5-fold greater levels than controls between 2 and 4 h of incubation (Fig. 1c and d). Nuclear accumulation of Nrf2 was also demonstrated in cells treated with tertbutyl hydroquinone (tBHQ), a known Nrf2 inducer and positive control. As a result of cyto-nuclear translocation, the nuclear:cytosolic (nuc:cyt) ratio of Nrf2 increased significantly compared to the controls reaching a maximum after 2h. (Fig. 1e). To confirm Nrf2 stabilization by CySSPe in the absence of an increased rate of transcriptional activity, we treated cells with the protein synthesis inhibitor, cycloheximide (CHX). Exposure of cells to CHX (5 μg/ml) for up to 3 h blocked the basal expression of Nrf2 in whole cell lysates by 36% as early as 15 min, reaching the highest inhibition of 86% after 180 min of incubation (Fig. 1f). Pretreatment with 25 μM CySSPe resulted in significantly higher levels of Nrf2 at 15–60 min of CHX exposure. These results show that in the absence of Nrf2 protein synthesis, Keap1-sequestered Nrf2 became dissociated and was stabilized likely due to a CySSPe-mediated post-translational mechanism.

2.8. Measurement of H2S-releasing activity H2S generation from CySSPe was determined in cystine/methionine-depleted culture media collected at timed intervals (0–3 h). The concentration of H2S (determined as Σ H2S, HS−, S2−) was measured using a spectrophotometer as described previously [33]. Briefly, medium was mixed with an equal amount of 1% w/v zinc acetate solution/3% NaOH mixture (1:1 ratio). N,N-DPD dye (20 mM, 25 μl) in 7.2 mM HCl and FeCl3 (25 μl) in 1.2 mM HCl were combined to form methylene blue, which was measured by absorbance at 670 nm after 10 min of incubation. The H2S concentration of each sample was calculated using a standard curve of NaHS (0–15.6 μM; R2 = 0.998). 2.9. Measurement of Keap1 persulfidation Persulfidation of Keap1 was determined as previously described [14,34]. Briefly, cells treated without or with CySSPe or NaHS (positive 166

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Fig. 1. CySSPe-induced Nrf2 activation by increasing protein stability and nuclear translocation. Hepa cells were treated with (a) CySSPe (0–40 μM) for 3 h or (b) 25 μM CySSPe over a 24-h period. Cells were lysed and immunoblotted with anti-Nrf2 and anti-Keap1 antibodies; (c-e) Cells were treated with 25 μM CySSPe or 25 μM t-BHQ (known Nrf2 activator) over a 4-h period. Cytosolic and nuclear proteins were collected at each time point and immunoblotted with anti-Nrf2 antibody; (f) Stabilization of Nrf2 in Hepa cells after treatment with either CHX alone or CHX + CySSPe. Cells were pretreated with 25 μM CySSPe for 6 h followed by exposure to 5 μg/ml CHX over a 180-min period. Cell lysates were collected at each time point and immunoblotted with anti-Nrf2 antibody. Relative protein densities were normalized to β-actin or Lamin B1. The blots shown are representatives of at least three independent experiments. Data are expressed as mean values ± SD (one-way ANOVA with Tukey's post-test) from triplicate experiments. *P < 0.05, **P < 0.01 vs control.

3.2. CySSPe upregulates Nrf2-mediated antioxidant enzymes

exposure to 25 μM CySSPe progressively increased levels of all three proteins as early as 3 h of incubation peaking at 3–6 h (HO-1) or 12–24 h (NQO1 and GCLc) (Fig. 2a,c). Nrf2-siRNA was used to knock down the Nrf2 gene in Hepa cells to confirm the requirement for Nrf2 in the effect of CySSPe on ARE protein expression. Hepa cells were transiently transfected with control-siRNA or Nrf2-siRNA and subsequently treated with 25 μM CySSPe at incubation times corresponding to maximal expression levels (Figs. 1b

Induction of Nrf2-regulated antioxidant enzymes is a cytoprotective response conferred by many synthetic and natural compounds [35,36]. To test if CySSPe induces upregulation of ARE enzymes, Hepa cells were exposed to the 0–40 μM CySSPe levels that caused Nrf2 nuclear translocation (Fig. 1). CySSPe dose-dependently increased protein levels of Nrf2-dependent enzymes GCLc, NQO1 and HO-1 (Fig. 2a and b). The 167

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Fig. 2. CySSPe upregulates Nrf2-mediated antioxidant enzymes. (a) Hepa cells were treated with 0–40 μM CySSPe for 6 h (HO-1), 12 h (NQO1 and GCLc) [left panel], or with 25 μM CySSPe over a 24-h period [right panel]. (b,c) Cell lysates were collected and immunoblotted with anti-NQO1, anti–HO–1, and anti-GCLc antibodies; relative protein densities normalized to β-actin. Data are expressed as mean values ± SD (one-way ANOVA with Tukey's post-test) from triplicate experiments. *P < 0.05, **P < 0.01 vs control.

and 2a). As expected, CySSPe increased Nrf2 protein expression in both untreated (control) and control-siRNA-transfected cells (Fig. 3a). Transfection with Nrf2-siRNA inhibited basal Nrf2 protein expression by 63% relative to untreated control. As a result of Nrf2-siRNA-mediated blockade of transcriptional activity for Nrf2, CySSPe-induced Nrf2 protein stabilization was not observed (Fig. 3b). While CySSPe upregulated both NQO1 and HO-1 (Fig. 3c and d), this effect was attenuated in samples transfected with Nrf2-siRNA. Collectively, these results confirmed that the upregulation of antioxidant enzymes in CySSPetreated Hepa cells is dependent on the ability of CySSPe to activate Nrf2.

was unaffected by CySSPe (0–40 μM) exposure as well as the antioxidant N-acetyl cysteine (NAC, 2.5 mM) (Fig. 4a). Treatment with tBHP for 3 h decreased cell viability by 50% (Fig. 4b), but pre-treatment with 25 μM CySSPe for 12 h rescued about 48% of the cell viability lost from the toxic effect of tBHP. CySSPe (0–40 μM) or 2.5 mM NAC alone did not affect DCF fluorescence compared to the non-tBHP-stressed controls (data not shown), indicating that CySSPe and NAC alone do not induce ROS production in Hepa cells. As early as 5 min, exposure of cells to 2.5 mM tBHP induced a 4-fold increase in total ROS levels relative to controls, reaching 6-fold after 30 min (Fig. 4c). Pretreatment of cells with CySSPe (5–20 μM) suppressed tBHP-induced ROS production in a dose-dependent manner from 7 to 39% after 30 min, with the highest dose similar in effectiveness as 2.5 mM, NAC, a commonly used antioxidant in cell studies. To determine whether the attenuation of tBHP-induced ROS production by CySSPe is associated with the activation of Nrf2, Nrf2 knockdown cells were exposed to tBHP. As expected, cells exposed to 2.5 mM tBHP or control-siRNA caused significant increases in ROS

3.3. CySSPe protects Hepa cells against tBHP-induced oxidative stress in an Nrf2-dependent manner To assess the broader cytoprotective action of CySSPe in Hepa cells, we monitored cellular responses to 2.5 mM tBHP, a widely used toxic agent to study cellular responses to oxidative stress [37]. Cell viability 168

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Fig. 3. Nrf2-dependent upregulation of ARE-coded enzymes by CySSPe. (a) Representative immunoblots for Nrf2, NQO1 and HO-1 in untreated (control), control-siRNA-treated, or Nrf2-siRNAtreated Hepa cells. Cells of 60% confluency were either untreated or transfected with either controlor Nrf2-siRNA for at least 24 h; analysis of protein expression following treatment with 25 μM CySSPe was conducted for 3 h (Nrf2), 12 h (NQO1), or 6 h (HO-1). Cell lysates were collected and immunoblotted with anti-Nrf2, anti-NQO1, and anti–HO–1 antibodies. Relative protein densities of (b) Nrf2, (c) NQO1, and (d) HO-1 were normalized to β-actin. Data are expressed as mean values ± SD (one-way ANOVA with Tukey's posttest) from triplicate experiments. **P < 0.01 vs control.

levels (Fig. 4d). The ROS level in cells transfected with Nrf2-siRNA was higher than the untreated controls or those transfected with controlsiRNA. Cells pre-treated with 25 μM CySSPe had a significantly lower ROS level compared to the controls or cells transfected with scrambled or Nrf2-siRNA. This ROS-inhibitory effect of CySSPe was attenuated in cells transfected with Nrf2-siRNA prior to CySSPe treatment (Nrf2siRNA + CySSPe). Aside from the Nrf2-upregulated phase 2 enzymes, GSH – the most abundant, low molecular weight antioxidant in cells – plays an important role in cellular defense against ROS, free radicals and electrophilic metabolites [38,39]. The observed induction of GCL (Fig. 2) would predict that de novo synthesis of GSH should be enhanced and may represent another feature of how CySSPe confers cytoprotection. Quiescent Hepa cells expressed basal levels of total glutathione (TotGSH = GSH + GSSG)) of 41 nmol/mg protein, which was further increased by ∼50% upon treatment with 50 μM CySSPe (Fig. 4e). Exposure of cells to 2.5 mM tBHP for 4 h decreased TotGSH levels by ∼80% relative to the control. About 45% of these losses in TotGSH were rescued by pre-incubation with 50 μM CySSPe prior to tBHP exposure (tBHP + CySSPe). Cellular GSSG levels were not affected among all treatments and ranged 1.5–2.0 nmol/mg protein. As a result of GSH depletion by tBHP, GSH:GSSG ratio significantly decreased (25 for control vs 4.4 in tBHP-treated cells) (Fig. 4f); CySSPe treatment of controls and prior to tBHP exposure elevated GSH:GSSG ratio by a magnitude of 6.4 and 7.3 nmol/mg protein, respectively. Patterns of changes in TotGSH and GSH:GSSG for the three treatments groups and controls were quantitatively similar (Fig. 4e and f).

At 200 μM CySSPe, significant amounts of H2S were evolved as early as 30 min, and peaked at 2 h over the 3-h observation period (Fig. 5b). Pre-treatment of cells with a selective CSE inhibitor, propargylglycine (PAG) [40] prior to incubation with CySSPe modestly decreased H2S levels compared to samples treated with CySSPe alone, while a greater inhibitory effect on H2S evolution was registered for with aminooxyacteic acid (AOAA), an inhibitor of both CSE and CBS (Fig. 5c). H2S levels in cells sequentially pre-treated with BSO and DEM (to deplete cellular GSH) decreased by 74% compared to cells treated with CySSPe alone (Fig. 5d). These results indicate that the H2S-synthesizing enzymes CSE/CBS and cellular GSH are both involved in cellular metabolism of CySSPe to generate H2S. 3.5. CySSPe persulfidates Keap1 in Hepa cells We investigated whether Keap1 is persulfidated in cells treated with CySSPe. After incubation of cells with CySSPe or NaHS, immunoprecipitated Keap1 proteins were subjected to maleimide assay using an Alexa Fluor conjugated C2 maleimide (red maleimide), which labels both persulfidated and intrinsic Cys residues (Fig. 6a and b). Red maleimide-labeled Keap1 protein was subsequently treated with DTT, which selectively cleaves disulfide bonds in persulfidated Cys, resulting in a decrease in fluorescence signal relative to persulfidated Keap1, but not nonpersulfidated protein [14]. The degree of persulfidation is proportional to the extent to which the original fluorescent signal (-DTT) is extinguished following DTT treatment for each of the three samples, normalized to protein. Keap1 from untreated (control) cells had no detectable persulfidation (Fig. 6b). In contrast, treatment with 50 μM CySSPe or 50 μM NaHS (as positive control) persulfidated Keap1 to similar extents.

3.4. CySSPe release H2S in CSE/CBS- and GSH-dependent manner We determined whether CySSPe is metabolized in cells to release H2S and whether H2S-evolving enzymes, cystathionine-γ-lyase (CSE) and cystathionine-β-synthase (CBS), are involved in H2S production. H2S was detectable in submicromolar range in culture media collected from control (untreated) cells, while those treated with CySSPe (0–200 μM) for 2 h showed dose-dependent production of H2S (Fig. 5a).

4. Discussion This study probed for a mechanistic basis of the cytoprotective effect of CySSPe, a 1-propenyl-bearing CySSR species formed between dominant onion TS and CySH. Our initial structure-activity studies 169

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Fig. 4. CySSPe protects Hepa cells against tBHPinduced oxidative stress in a dose- and Nrf2-dependent manner. (a) Treatment with CySSPe (0–40 μM) or NAC (2.5 mM, as positive control) for 24 h did not decrease cell viability of Hepa cells; (b) CySSPe attenuates t-BHP-induced cytotoxicity. Cells were pretreated without or with 25 μM CySSPe for 12 h, followed by exposure to 2.5 mM tBHP for 3 h. Cell viability was determined by standard MTT assay; (c) CySSPe attenuated t-BHPinduced ROS production in a dose-dependent manner. (d) CySSPe inhibits tBHP-induced ROS production in an Nrf2-dependent manner. Cells were transfected without or with scrambled-siRNA or Nrf2-siRNA for at least 24 h followed by preincubation without or with 25 μM CySSPe for 6 h, and treatment with 2.5 mM tBHP. ROS production was determined 30 min after tBHP treatment. (e,f) CySSPe attenuates tBHP-induced depletion of the total [GSH + GSSG] pool and decrease in GSH:GSSG ratio. Hepa cells were pre-treated without or with 40 μM CySSPe followed by treatment with 2.5 mM tBHP for 3 h. Data are expressed as mean values ± SD (one-way ANOVA with Tukey's post-test) from at least three independent experiments. *P < 0.05, **P < 0.01.

revealed CySSPe to be similarly potent as the major garlic analogue (CySSA) in inducing phase II enzymes in Hepa cells and attenuating inflammation in LPS-activated RAW 264.7 cells using respective biomarkers of enhanced NQO1 levels and attenuated NO levels [22,26]. More recently, we found that CySSPe and CySSA inhibition of the LPSactivated canonical NF-κB signaling pathway in RAW 264.7 cells was associated with a reduction in ROS levels, and enhanced GCL and GSH levels [30]. These findings implicate activation of Nrf2 by CySSR species that confers a cytoprotective response to inflammatory stress. CySSPe was more potent than CySSA in that study, but the (bio)chemical mechanism by which CySSR caused induction of ARE-coded genes was not investigated. Many garlic-derived OSC have received considerable research attention in the context of elevating cellular defenses to

oxidative, environmental and inflammatory stresses in cells and in vivo. For example, diallyl trisulfide (DATS), diallyl disulfide (DADS), allicin and CySSA have all been shown to activate Nrf2 [41–44]. The knowledge that these analogues with related but distinct structural units share similar biological effects suggests some commonality in mechanism of action. However, the putative Nrf2 activation step(s) remain to be established and our study was designed to address this gap in knowledge, specifically for CySSR species. Although onions are recognized as being abundant in the Nrf2 activator quercetin [45–48], there is a dearth of information relative to garlic on whether onion OSC also confers this function. Thus, to follow up on evidence showing CySSR species activating Nrf2 in LPS-activated macrophage cells [30], we chose to evaluate the mechanistic features of CySSPe-mediated activation of Nrf2 in 170

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Fig. 5. CySSPe upregulates H2S production in CSE/CBSand GSH-dependent manners. (a) Dose-dependent increase in H2S in Hepa cells treated with CySSPe (0–40 μM) for 2 h; (b) H2S production over a 3-h period in cells treated with 200 μM CySSPe; (c) H2S production in cells treated with 5 mM PAG or 1 mM AOAA for 4 h, prior to incubation with 200 μM CySSPe for 2 h; (d) H2S levels in cells sequentially pretreated with 200 μM BSO for 18 h and 10 mM DEM for 40 min prior to incubation with 200 μM CySSPe. Data are expressed as mean values ± SD (one-way ANOVA with Tukey's post-test) from at least three independent experiments. *P < 0.05, **P < 0.01.

Hepa cells with and without oxidative stress imposed. In this study, we routinely used 25–50 μM of CySSPe to elicit the observed responses of Hepa cells. This concentration range appears to be physiologically attainable. If all the organosulfur units originating as ACSO (∼80 mol% as 1-propenyl groups) were transformed to CySSPe, then consumption of 250 g onion would yield about 4 mmol CySSPe [49,50]. Total absorption (CySSA is ∼100% bioavailable; 51) and partitioning into 20 L extracellular fluid (for a 75 kg human) would yield 200 μM CySSPe. Cellular uptake of the CySSR pool (through the cysteine transporter xCT; 26) would cause further dilution to 100 μM intracellularly. xCT is also upregulated by the Nrf2-ARE axis [52],

providing selective enrichment of CySSR in cells responsive to Nrf2activators. Treatment of Hepa cells with CySSPe resulted in increased Nrf2 levels in whole cell lysates without changes in Keap1 protein levels (Fig. 1a,b), and a rapid accumulation (as early as 30 min) of Nrf2 in the nucleus (Fig. 1c,d,e). Exposure of cells to the protein synthesis inhibitor CHX led to progressive depletion of Nrf2, but pretreatment with CySSPe maintained significantly elevated Nrf2 levels (Fig. 1f). A similar pattern was observed for the collective effects of 1,2-dithiole-3-thione (D3T) and CHX on murine keratinocytes [53]. These results indicate that the effect of CySSPe is principally post-translational stabilization of Nrf2 by

Fig. 6. CySSPe persulfidates Keap1 in Hepa cells. (a) Schematic diagram for detection of Keap1 persulfidation with red maleimide (details of the experiment are described in section 2.9); (b) Persulfidation of Keap1 in cells treated with 50 μM CySSPe or with 50 μM NaHS (as positive control) for 2 h. 171

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dissociation from the Keap1-Nrf2 complex without transcriptional regulation of Nrf2. This pattern of CySSPe effect is consistent with behavior of other potent Nrf2 activators, such as tBHQ and diethylmaleate (DEM) that modify Keap1 Cys residues without affecting Nrf2 mRNA transcription [54,55]. Nrf2-mediated induction of ARE-coded antioxidant enzymes, and those involved in synthesis and recycling of GSH (e.g., GCL, GR), play a crucial role in summoning cellular defenses that counter oxidative stress caused by xenobiotics and ROS [56,57]. Exposure to CySSPe enhanced HO-1, NQO1, and GCLc protein expression in Hepa cells in a time- and dose-dependent manner (Fig. 2). Maximum induction of these enzymes occurred between 6 and 12 h with subsequent declines observed at 24 h, and upregulation of HO-1 was particularly robust (over 13-fold). HO-1 is inducible by a great diversity of stimuli and a plurality of transcription factors including Nrf2, and the activator protein-1 and nuclear factor kappa-B protein families [58,59]. A common feature of non-carcinogenic, diet-derived Nrf2 inducers, such as SF and quercetin is their ability to transiently activate Nrf2 compared to carcinogens (e.g. arsenic), which can sustain prolonged (up to 60 h) Nrf2 activation [60]. The pattern of Hepa cell response to CySSPe is consistent with that of other “soft” electrophilic agents (SF and quercetin) that target redox sensing protein thiols [7,61]. Nrf2-knockdown cells using siRNA reduced Nrf2 protein to ∼40% of basal levels and ablated Nrf2-inducing capacity by CySSPe (Fig. 3), consistent with Nrf2 being an autoregulatory transcription factor [53]. In contrast, NQO1 and HO-1 protein levels were similar in control and control Nrf2-knockdown cells, with the CySSPe-inducing effect retained 40% and 52% for these respective enzymes in knockdown cells compared to controls. Both HO-1 [58,59] and NQO1 [62] are regulated by transcription factors in addition to Nrf2. Collectively, these results show that CySSPe is an effective inducer of antioxidant enzymes, largely conferred by its ability to activate Nrf2 signaling. Based on levels required for doubling NQO1 in Hepa cells (CD value), the potency of CySSPe (25 μM; 29) is 1% that of sulforaphane, 10% that of quercetin and curcumin, and similar to resveratrol. However, when one factors in human “bioavailability” [51,63], CySSPe (∼100% bioavailable) may be more effective than quercetin (by 3-fold), curcumin (by 10-fold) and resveratrol (by > 100-fold). Nrf2 is expressed throughout mammalian tissues, especially in organs involved in detoxification, such as the liver [64,65]. Thus, Nrf2 serves as a major pathway that regulates redox homeostasis in hepatic cells, making it one of the most studied molecular targets for discovering diet-derived agents with hepaprotective effects. The induction of antioxidant defenses by CySSPe in Hepa cells was effective and meaningful as evidenced by the lack of cell toxicity at doses up to 40 μM, a partial rescuing of losses in cell viability, and the reduction of cellular ROS evoked by the oxidative stressor tBHP (Fig. 4a–c). In studies involving hepatocytes, tBHP is favored over its analogue lipid hydroperoxide because it is not metabolized by catalase, but is readily metabolized by cytochrome P450 to free radical intermediates (e.g. peroxyl and alkoxyl radicals) [66,67]. These free radicals can cross membranes, react with macromolecules and damage cells ([66,68]). Reduction of cellular ROS levels by 20 μM CySSPe was as effective as 100-fold greater levels of NAC, which is often used as an “effective” antioxidant [69,70]. Similar to other low molecular weight thiols, NAC reacts slowly with physiological ROS, but functions uniquely as a source of CySH for cellular GSH synthesis and a reductant of protein disulfide bonds [70]. CySSR can be imported by cells via the cysteine/glutamate antiporter (xCT, another ARE-coded protein) and reduced by glutaredoxin or thioredoxin reductase into CySH and RSH [22,26]. However, since 80% of the CySH equivalents from 20 μM CySSPe is exported into the extracellular space within 3 h in cells [26], the remaining intracellular CySH equivalents cannot begin to account for the equivalent antioxidant effect of 2.5 mM NAC. Thus, the antioxidant ability of CySSPe must be largely accounted for as Nrf2-mediated induction of AREregulated enzymes (Fig. 4d). NQO1 functions by reducing quinones,

replenishing antioxidant capacity of ubiquinone and tocopherol, and scavenging superoxide, albeit in a less efficient manner than superoxide dismutase (SOD) [71]. HO-1 is a cellular stress protein that regulates inflammatory signaling through pleiotropic antioxidant effects of heme degradation products, including carbon monoxide, biliverdin, bilirubin (by action of biliverdin reductase) and ferritin [72]. The in vivo importance of the antioxidant functions of NQO1 and HO-1 are demonstrated by increased susceptibilities to quinone toxicities and oxidative stress in NQO1- and HO-1-knockout animals [73,74]. Consistent with the GCLc-inducing effect of CySSPe (Fig. 2) was the corresponding 50% increase in total cellular GSH + GSSG and 20% increase in GSH:GSSG relative to control cells (Fig. 4e). Furthermore, CySSPe pre-treatment could restore more favorable redox status upon the imposition of tBHPinduced oxidative stress (Fig. 4 e,f). As the rate-limiting enzyme in the de novo synthesis of GSH, GCL serves an important role in cytoprotection by upregulating and maintaining cellular GSH pool [75]. Thus, a benefit of this response to CySSPe is the enhanced capacity to maintain and/or restore cellular redox homeostasis [76]. The increase in GSH levels also furnish co-substrate and reducing power for antioxidant enzymes such as peroxiredoxins, thioredoxin reductase, glutaredoxin, glutathione reductase, and glutathione peroxidase, all of which are regulated by the ARE [77]. Rate constants of enzyme reactions using GSH to detoxify ROS are up to 8-9 orders of magnitude greater than scavenging by low molecular weight thiols like NAC [70]. We recently showed that CySSPe upregulates GCL and GSH in LPS-activated RAW macrophages, and pre-treatment of cells with BSO indicated the antiinflammatory effect of CySSPe is linked to GSH-dependent processes [30]. The most widely accepted mechanism of Nrf2 activation involves the interaction of electrophilic agents with Keap1 sensor thiols. This “cysteine code” involves four critical and highly reactive murine Keap1 Cys residues including, C257, C273, C288 andC297 located in the intervening region (IVR) domain and C151, located in the BTB domain [78–80]. A common feature among these Cys residues is their proximity to basic amino acid residues and H-bond donors, which lowers their pKa and increases their nucleophilicity [7,9,76]. Several diet-derived Nrf2 inducers are Michael acceptors or compounds that possess an electron-withdrawing group enabling participation in reversible alkylating reactions with Keap1 redox sensing thiols [81]. For example, natural products such as SF, xanthohumol, isoliquiritigenin, and 10shogaol readily modify Keap1 C151 via Michael addition to activate Nrf2 [78,82]. Relative to the extensively studied Nrf2 activators SF and D3T, the mechanism of Nrf2 activation by Allium OSC, in general, is poorly understood. Natural OSC are a structurally diverse class of chemicals, which implies that various OSC chemotypes may differ in the mechanism by which they activate Nrf2. In cells, it is unlikely that CySSPe could kinetically compete in SH/SS exchange reactions to Salkylate Keap1 thiols because of multiple enzymic reactions (reduction by glutaredoxin and thioredoxin reductase) that yield 1-propenyl mercaptan (PeSH) and CySH [26], or scission by CSE and/or CBS that yield electrophilic metabolites. Therefore, we explored alternative mechanisms to clarify Nrf2 activation by CySSPe. Sulfur-containing natural products are known to generate H2S when metabolized in cells or tissues. For example, allyl sulfides from Alliums (e.g. DATS, DADS) and isothiocyanates from Brassicas have been shown to release H2S via non-enzymatic and enzymatic (e.g. via CSE and CBS) action [83,84]. Allyl sulfides produce H2S in a GSH-dependent manner, producing hydropersulfide, a key intermediate in the formation of H2S [84]. We found that CySSPe significantly increased H2S levels in a timeand dose-dependent manner in Hepa cells (Fig. 5a and b). This led us to hypothesize an H2S-dependent mechanism of Nrf2 activation by CySSPe, likely via persulfidation, which involves the addition of sulfur atom to Cys residues of proteins to yield protein persulfides (RSSH/RSS) [12,85]. Also, at physiological pH, RSSH (sulfane sulfur (S0)) may persulfidate the –SH groups of protein Cys residues to form protein persulfides (PSSH) [86]. Mammalian CSE/CBS metabolize CySSCy to 172

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activation of Nrf2 have inferred an important role of matrix-activated protein kinases (MAPK) for DATS, DADS and diallyl sulfide [95,96], and an autophagy response involving p21 for DAS in a skin cancer model [97] and p62 for CySSA in a colon cancer model [98]. There is also some consensus that Allium OSC may modulate redox sensitive proteins [89] and/or cellular GSH:GSSG status [99]. We showed CySSPe to have both of these effects, and others have shown allyl sulfides [100,101] and allicin [94] to have these effects. Our findings suggest that activation of Nrf2 may be a potential therapeutic strategy conferred by onion-rich diet. Specifically, exogenous administration of CySSPe evolves persulfidating agents (i.e H2S or RSSH) via intrinsic cellular metabolism, which may be of potential therapeutic benefit for multiple adverse conditions related to sustained oxidative and inflammatory stress. Finally, we showed for the first time, that a major onion-derived OSC could activate the Nrf2 pathway.

Fig. 7. Proposed mechanism of Nrf2 activation by CySSPe. a) Schematic representation of H2S production involving CSE, CBS and GSH; (b) Proposed mechanism for the activation of Nrf2 via persulfidation of Keap1 protein in CySSPe-treated cells. CySSPe undergoes β-elimination catalyzed by CSE and CBS and form 1-propenyl-SSH, which are further reduced to GSSH or H2S. The sulfane sulfur in RSSH (1-propenyl-SSH or GSSH) then persulfidates the –SH groups of Keap1 Cys to produce Keap1-SSH, which alters the Keap1-Nrf2 interaction leading to dissociation of Nrf2.

Acknowledgement

generate RSSH (e.g. CySSH, GSSH) and/or H2S [85]. CySSH was shown to protect SH-SY5Y cells against methylglyoxal-induced toxicity via activation of Nrf2 [16]. CSE is known to catalyze β-elimination reactions with cysteinyl S-conjugates, including CySSA, to produce ASSH, ammonia and pyruvate [87]. We examined if CSE/CBS in Hepa cells could catalyze β-elimination reaction with CySSPe to generate the reactive RSSH species. Results indicate that both CSE and CBS were involved in metabolizing CySSPe to generate RSSH, which upon reaction with GSH could generate H2S (or GSSH). Since H2S production was not completely abated by the inhibitors, it is possible that CSE/CBS were not completely inhibited, or alternative routes to H2S production from CySSPe exist in Hepa cells, including the action of 3-mercaptopyruvate sulfurtransferase (3-MST), a H2S-producing enzyme which is present in large amounts in liver and hepatocytes [88]. In addition, pretreatment of cells with BSO and DEM reduced H2S evolution by 87% compared to controls upon exposure to CySSPe, indicating that GSH is virtually essential for H2S generation from CySSPe. The H2S and/or RSSH evolved by β-lyase metabolism of CySSPe indicate that Nrf2 activation could involve Keap1 persulfidation. Treatment of hepa cells with CySSPe or NAHS resulted in a majority of the fluorescently labeled Keap1 groups being persulfides (Fig. 6a and b) compared to Keap1 control cells being almost exclusively labeled to thiol groups. This persulfidation of Keap1 at 2 h of CySSPE or NAHS incubation (Fig. 6a and b) corresponds with a meaningful extent of Nrf2 activation (Fig. 1) and H2S evolution (Fig. 5) following CySSPe exposure for 2 h. In conclusion, our study provided evidence that CySSPe can upregulate Nrf2 in quiescent Hepa cells and as an adaptive response to tBHP-induced oxidative stress. Mechanistically, this effect can partly be attributed to the direct formation PeSSH as a persulfidating agent from CySSPe, or further transformation by GSH to yield GSSH as the persulfidating agent (Fig. 7a and b). In either case, PeSH is expected to be formed, and this has been observed in macrophages exposed to a series of CySSR species, including CySSPe [26]. CySSA and S-propyl mercapto-L-cysteine (CySSP) have been shown to be substrates for β-lyase present in rat liver cytosol [87,89], and it stands to reason CySSPe would be also. The evolution of H2S during the process of persulfidation may simply be a marker for this event, rather than H2S having a direct role, a concept advanced previously [85]. Despite the abundance of research on garlic allyl-OSC activating Nrf2, the mechanism by which this occurs is poorly understood [90,91]. Recently, DATS was suggested to modify Keap1 Cys288 in human gastric cells through an Cys-S-allyl conjugate [41]. However, evidence of this modification was gleaned from a simple binary reaction mixture of Keap1 with DATS for 30 min prior to gel electrophoresis and digestion prior to mass spectroscopy analysis. Prior studies have also reported direct reactions with proteins in simple solutions for CySSA and DATS with tubulin [92,93] and for CySSA and GSSA with papain [94]. Other studies on allyl-OSC

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