Applying an Electrophilicity-Based Strategy to Develop a Novel Nrf2

Article Cite This: J. Agric. Food Chem. 2018, 66, 7983−7994

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Applying an Electrophilicity-Based Strategy to Develop a Novel Nrf2 Activator Inspired from Dietary [6]-Shogaol Yu-Ting Du, Ya-Long Zheng, Yuan Ji, Fang Dai,* Yong-Jing Hu, and Bo Zhou* State Key Laboratory of Applied Organic Chemistry, Lanzhou University, 222 Tianshui Street South, Lanzhou, Gansu 730000, China

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S Supporting Information *

ABSTRACT: Activation of nuclear factor erythroid-2-related factor 2 (Nrf2) is a crucial cellular defense mechanisms against oxidative stress and also an effective means to decrease the risk of oxidative stress-related diseases including cancer. Thus, identifying novel Nrf2 activators is highly anticipated. Inspired from [6]-shogaol (6S), an active component of ginger, herein we developed a novel potent Nrf2 activator, (1E,4E)-1-(4-hydroxy-3-methoxyphenyl)-7-methylocta-1,4,6-trien-3-one (SA) by an electrophilicity-based strategy. Compared with the parent 6S, SA bearing a short but entirely conjugated unsaturated ketone chain manifested the improved electrophilicity and cytoprotection (cell viability for the 10 μM 6S- and SA-treated group being 48.9 ± 5.3% and 76.1 ± 3.2%, respectively) against tert-butylhydroperoxide (t-BHP)-induced cell death (cell viability for the tBHP-stimulated group being 42.4 ± 0.4%) of HepG2. Mechanistic study uncovers that SA works as a potent Nrf2 activator by inducing Keap1 modification, inhibiting Nrf2 ubiquitylation and phosphorylating ERK in a Michael acceptor-dependent fashion. Taking 6S as an example, this works illustrates the feasibility and importance of applying an electrophilicity-based strategy to develop Nrf2 activators with dietary molecules as an inspiration due to their low toxicity and extraordinarily diverse chemical scaffolds. KEYWORDS: [6]-shogaol, Nrf2, electrophilicity, Michael acceptor, oxidative stress

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INTRODUCTION Nuclear factor erythroid-2 related factor 2 (Nrf2), a redox responsive transcription factor, plays a crucial role in coordinating the expression of antioxidant genes in mammalian cell.1 Under physiological conditions, Nrf2 is maintained at low levels due to its ubiquitination and proteasomal degradation mediated by its cytosolic inhibitor Kelch-like ECH-associated protein 1 (Keap1). In response to oxidative and electrophilic stress resulting in the modification of the cysteine residues of Keap1, Nrf2 liberates from Keap1 to translocate into the nucleus. Once in the nucleus, it combines with the antioxidant response element (ARE) sequence to regulate the transcription of a battery of genes encoding cytoprotective proteins (also known as phase II detoxifying enzymes) including hemeoxygenase-1 (HO-1), NAD(P)H, quinone oxidoreductase 1 (NQO1), γ-glutamylcysteine synthetase (γ-GCS), and γglutamyl cysteine ligase (γ-GCL).2−4 Accordingly, Nrf2 activators can be considered as indirect antioxidants and offer significant superiority over traditional direct antioxidants since they can initiate sustained antioxidant activity via transcriptional induction of the above phase II detoxifying enzymes even through transient treatment.5 Because oxidative stress induced by excessive generation of reactive oxygen species (ROS) is a central component of various chronic diseases,6,7 activation of Nrf2-Keap1/ARE signaling has been confirmed to be effective for preventing oxidative stress-related diseases including cancer.8 However, the signaling pathway is a double-edged sword, because constitutive Nrf2 activation promotes cancer cell survival and resistance to anticancer drugs.9 Therefore, the last few decades have witnessed much interest in developing both Nrf2 activators and inhibitors.9−11 © 2018 American Chemical Society

A plethora of natural products and their analogues have been identified as Nrf2 activators,10,11 and some of them, such as bardoxolone methyl, have undergone human clinical trials.12 The majority of these known Nrf2 activators are chemically different classes of electrophiles including Michael acceptors (such as curcumin and chalcone derivatives), isothiocyanates and sulfoxythiocarbamates, polyphenol-based pro-electrophiles, and so on,10,11,13 due to the fact that up to 27 cysteine residues (especially Cys151) in human Keap1, a cytosolic inhibitor of Nrf2, are susceptible to covalent modification by electrophiles.14,15 Concerning the importance of electrophilic natural products in drug discovery, Sieber and co-workers have commented: “In nature, the roles of nucleophile and electrophile appear to be well split up due to the striking lack of intrinsic highly electrophilic groups in proteins and nucleic acids”.16 Thus, designing natural product-guided anticancer agents including Nrf2 activators by chemical finetuning of their electrophilicity might be a promising strategy. As part of our effort to develop natural product-guided anticancer agents by an electrophilicity-based strategy,17−20 we proposed to broaden this strategy to designing Nrf2 activators. As already noted, biological effects of electrophiles, being therapeutic to toxic, are driven by two factors, the rate and selectivity of interactions with various nucleophiles which are defined by their hardness (“hard” and “soft”).11 For example, Michael acceptors are soft electrophiles that could react Received: Revised: Accepted: Published: 7983

May 8, 2018 July 9, 2018 July 10, 2018 July 10, 2018 DOI: 10.1021/acs.jafc.8b02442 J. Agric. Food Chem. 2018, 66, 7983−7994

Article

Journal of Agricultural and Food Chemistry

glutamate cysteine ligase catalytic subunit (GCLC) and glutamate cysteine ligase modulatory subunit (GCLM) were obtained from Abcam (Shanghai, China). All the phosphorylated and nonphosphorylated kinase antibodies used in this study and U0126 were from Cell Signaling Technology (Danvers, MA). HRP-labeled secondary antibody was obtained from TransGen Biotech Co., Ltd. (Beijing, China). The other compounds used in the paper were of the highest quality available. Synthesis. Chemical synthesis of 6S and its analogue 6-DHS was described in our previous paper,32 and that of SA and SA-1 was conducted according to the route outlined in Scheme 233 whose

predominantly with soft nucleophiles such as the cysteine residues of Keap1. In this work, we selected [6]-shogaol (6S, an active constituent of ginger, Scheme 1) as the lead based on Scheme 1. Molecular Structures of 6S and Its Designed Analogues

Scheme 2. Synthesis of SA and SA-1a

the following reasons: (1) this Michael acceptor-type molecule has been identified as a potent Nrf2 activator.21,22 (2) Depending on its Michael acceptor unit, it demonstrates increased activity than another molecule derived as well from ginger ([6]-gingerol without the Michael acceptor unit) in activating Nrf2,21 antioxidant,23 anti-inflammatory,23−25 and antitumorigenic26 assays as well as in inhibiting proliferation,25,27,28 impairing tubulin polymerization,29 inducing apoptosis,28 and restraining invasion of cancer cells.30,31 Considering that the presence of a short side chain length in 6S favors its increased activity for the Nrf2 activation and the modification of conjugated unsaturated ketone further enhances this activity,21 we designed its conjugated unsaturated ketone analogues, [6]-dehydroshogaol (6-DHS) and (1E,4E)-1-(4-hydroxy-3-methoxyphenyl)-7-methylocta-1,4,6trien-3-one (SA) (Scheme 1). Compared with 6S and 6-DHS, the novel molecule SA is characterized by the presence of a short but entirely conjugated unsaturated ketone chain. These molecules were used to probe the possibility in developing natural product-inspired Nrf2 activators by an electrophilicitybased strategy. Additionally, multiple mechanisms including Keap1 modification, inhibition of Nrf2 ubiquitylation and cellular kinase pathways are likely involved in the Nrf2 activation. Another question considered here is thereby whether the above mechanisms determining Nrf2 activation are Michael acceptor unit-dependent. Thus, 1-(4-hydroxy-3methoxyphenyl)-7-methyloctan-3-one (SA-1, Scheme 1), a completely reduced analogue of SA (Scheme 1) was also used as the control to clarify this point.

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a

Reagents and conditions: (a) acetone, 75% aqueous NaOH; (b). nBuLi, THF, −78 °C; LDA, THF, 3-methyl-2-butenal, −78 °C; 10% HCl, r.t.; (c) H2, Pd/C. details are described as below. 1H and 13C NMR spectra were recorded on a Bruker AVANCE III 400 MHz NMR spectrometer (Bruker Daltonic Inc., Bremen, Germany). High-resolution mass spectra (HRMS) were measured with Orbitrap Elite (Thermo Scientific). The melting points were determined on an X-4B melting point apparatus and are uncorrected. The purity analysis of SA and SA-1 was carried out by high-performance liquid chromatography using a Waters liquid chromatograph system coupled with a 2998 photodiode array detector and a reverse phase C-18 column (Sun Fire, 4.6 mm × 150 mm). Concerning 1H NMR, 13C NMR, and HRMS spectra as well as HPLC chromatograms of SA and SA-1, see the Supporting Information. Synthesis of (E)-4-(4′-Hydroxy-3′-methoxyphenyl)-3-buten-2one (Step a in Scheme 2). Aqueous sodium hydroxide (20%, 25 mL) was added dropwise to a solution of vanillin (5.0 g, 32.89 mmol) and acetone (30 mL), and then this mixture was stirred at room temperature for 12 h. The reaction mixture was diluted with ice water and neutralized with HCl. Then the precipitate was filtered, washed with cold water, and dried under vacuum. The products were refined by flash column chromatography (petroleum ether/ethyl acetate, v/v = 5/1) to obtain a yellow solid (E)-4-(4′-hydroxy-3′-methoxyphenyl)-3-buten-2-one. Synthesis of SA (Step b in Scheme 2). To a solution of (E)-4-(4′hydroxy-3′-methoxyphenyl)-3-buten-2-one (768 mg, 4 mmol) in dry THF (20 mL) was added dropwise n-BuLi (5.5 mL, 4.8 mmol) under argon at −78 °C. Then the mixture was stirred for 30 min followed by the dropwise addition of lithium diisopropylamide (LDA) (2.5 mL, 4.8 mmol). After stirring for 3 h, a solution of 3-methyl-2-butenal (0.5 mL, 5.2 mmol) in THF (5 mL) was added to the reaction system at −78 °C. The reaction mixture was stirred for another 3 h at the same temperature and allowed to warm up to room temperature, continuing to stir for 2 h. The mixture was quenched with 10% HCl to adjust its pH to a value between 3 and 5 and then extracted with ethyl acetate (3 × 20 mL). The combined organic layers were washed with brine (50 mL), dried over sodium sulfate, and concentrated. The yellow oil was refined by silica gel column (petroleum ether/ethyl acetate, v/v from 10/1 to 5/1) to give SA (230 mg). (1E,4E)-1-(4-Hydroxy-3-methoxyphenyl)-7-methylocta-1,4,6trien-3-one (SA). Yellow solid, yield 22.3%; Mp 96−97 °C; purity 99.36% (H2O/MeOH = 35/65, Rt = 7.807 min). 1H NMR (400 MHz, CD3OD), δ 7.70 (dd, J = 11.6, 15.2 Hz, 1 H), 7.65 (d, J = 15.6

MATERIALS AND METHODS

Chemicals. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was from Amresco, Inc. (Solon, OH). Roswell Park Memorial Institute (RPMI)-1640, tert-butylhydroperoxide (tBHP), 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA), yeast glutathione reductase (GR), 2-vinylpyridine (97%), L-glutathione reduced (GSH), L-glutathione oxidized (GSSG), trigonelline (TRG), zinc protoporphyrin IX (ZnPP), L-buthionine-(S,R)-sulfoximine (BSO), SP600125, and cycloheximide (CHX) were purchased from Sigma-Aldrich (St. Louis, MO). MG132 was acquired from Selleckchem (Houston, TX). Bicinchoninic acid (BCA) protein assay kit, radio immunoprecipitation assay (RIPA) lysis buffer, and phenylmethanesulfonyl fluoride (PMSF) were from Beyotime Institute of Biotechnology (Jiangsu, China). Nuclear and Cytoplasmic Protein Extraction Kit were obtained from Viagene Biotech Inc. (Beijing, China). The antibodies against β-actin, ubiquitin, Keap1, Nrf2, Lamin A, and Protein A/G plus-agarose were from Santa Cruz Biotechnology (CA). Rabbit polyclonal antibody to HO-1 was purchased from Enzo Life Sciences (NY). Rabbit antibodies against 7984

DOI: 10.1021/acs.jafc.8b02442 J. Agric. Food Chem. 2018, 66, 7983−7994

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Figure 1. Electrophilicity assessment for SA (A), 6-DHS (B), and 6S (C) (50 μM) by monitoring their UV−visible absorption changes (interval = 5 min for SA and 6-DHS within 100 min, interval = 10 min for 6S within 200 min) in the presence of GSH (1 mM) in TBS buffer (100 mM, pH 7.4) containing 2 mM EDTA at 30 °C. was analyzed a FACSCanto flow cytometer at λex 488 nm and λem 530 nm. Imaging of ROS Types in Living Cells by Fluorescence Microscopy. HepG2 cells (5 × 105 cells/well) seeded in six-well plates for 24 h were preincubated with CAT (0.5 mg/mL) or SOD (0.5 mg/mL) for 1 h and treated with 900 μM t-BHP for 3 h followed by replacement with fresh culture medium containing 10 μM DCFHDA and incubation for another 30 min at 37 °C in the dark. The fluorescence images were generated on a microscope Leica DM 4000B (Leica Microsystems CMS GmbH, Wetzlar, Germany) with a ×20 objective lens. Measurement of Glutathione (GSH) and Glutathione Disulfide (GSSG). HepG2 cells (5 × 105 cells/well) cultured in six-well plates were incubated with SA for 24 h followed by replacement with fresh culture medium containing 900 μM t-BHP and incubation for another 3 h. Then the GSH and GSSG levels were determined by the cyclic DTNB-GR assay.34 Concerning the experimental details, please see our previous papers.19,35 Assay for Immunoprecipitation and Western Blotting. HepG2 cells (2 × 106 cells/dish) were incubated with 6S and its analogues, washed with PBS twice, and lysed with ice-cold Western and IP lysis buffer for 5 min at 4 °C. The nuclear proteins were prepared according to the protocol of Nuclear and Cytoplasmic Protein Extraction Kit. For immunoprecipitation, the whole-cell lysates (0.5 mg) were incubated with 1 μg of primary antibody against Nrf2 overnight at 4 °C under rotation followed by addition of 40 μL of Protein A/G plus-agarose and incubation for another 4 h at 4 °C. The mixture was washed with Western and IP lysis buffer five times. After centrifugation, the deposit was dissolved in 20 μL of 1× SDSPAGE Loading buffer. For immunoprecipitation and Western blotting, the whole sample and the total protein extracts (40 μg) were separate by 10% SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (Bio-Rad Laboratories, Hercules, CA). The membrane was blocked for 2 h with 5% (w/v) nonfat dry milk in TBS-Tween 20 (TBST; 0.05%) and incubated with the primary antibodies (1:1000 in TBST) at 4 °C overnight. After washing three times with TBST, the membrane was incubated with the appropriate HRP-conjugated secondary antibodies (1:2000) for 2 h at room temperature. The immunoreactive bands were developed with enhanced chemiluminescence kit (Amersham Co., Bucks, U.K.) and detected using the Image Quant 400 capture imaging system (GE Healthcare, Little Chalfont, U.K.). The band intensities were analyzed with ImageJ software (National Institutes of Health, Bethesda, MD). Determination of Nrf2 Half-Life. In order to calculate the halflife of Nrf2, the cells were treated with CHX (5 μg/mL) for 15, 30, and 60 min in the absence and presence of pretreatment with 10 μM SA for 12 h. The total Nrf2 levels in Western blotting images were densitometrically quantified using ImageJ software. Statistical Analysis. Data are expressed as the mean ± standard deviation (SD). Differences between groups were analyzed with ANOVA, followed by the LSD test. P-values 6S, and modification of conjugated unsaturated ketones indeed improves the electrophilicity of the parent 6S. Protective Effects of 6S and Its Analogues against tBHP-Induced Death of HepG2 Cells. With the electrophilicity data in hand, we used the t-BHP-induced death of HepG2 cells as an oxidative stress model to investigate the cytoprotective effects of 6S and its analogues and to further probe whether the electrophilic modification helps to increase

Synthesis and electrophilicity comparison of 6S and Its Analogues. Compounds 6S and 6-DHS were prepared as previously reported.32 The novel molecule SA was constructed from vanillin by a two-step aldol condensation, followed by catalytic hydrogenation to afford its reduced analogue SA-1 (Scheme 2). Electrophilicity of molecules can be easily estimated by their reactivity with nucleophilic GSH. To clarify whether the modification of conjugated unsaturated ketones could improve the electrophilicity of the parent 6S, we compared the electrophilicity of 6S, 6-DHS, and SA upon monitoring their UV−visible absorption changes induced by GSH. As shown in Figure 1A, addition of GSH induced decrease of the absorption bands of SA centered at 376 nm along with the development of 7986


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Figure 4. Identification of ROS types by fluorescence changes of DCFH-DA (10 μM) in HepG2 cells pretreated with CAT or SOD for 1 h and treated with 900 μM t-BHP for 3 h. Representative images are shown from three independent experiments. The fluorescence intensity was quantified by ImageJ. (***) p < 0.001 versus the control; (#) p < 0.05 and (##) p < 0.01 versus the t-BHP-stimulated group.

Figure 5. Cytoprotective effect of SA depends on Nrf2 activation and its target genes expression in HepG2 cells. (A) Effect of SA on Nrf2 nuclear translocation. HepG2 cells were treated with 10 μΜ SA for the indicated time points. (B) Effect of TRG on the cytoprotection exerted by SA. HepG2 cells were pretreatment with TRG for 1 h and then incubated with SA for 24 h, followed by their removal and replacement with 900 μM tBHP for another 6 h. (C) Time-dependent effect of SA on expression of HO-1, GCLC, and GCLM. HepG2 cells were treated with 10 μM SA for the indicated time points. (D) Dose-dependent effect of SA on expression of HO-1, GCLC, and GCLM. HepG2 cells were treated with SA at 24 h for the indicated concentrations. Effect of BSO (E) or ZnPP (F) on the cytoprotection exerted by SA. Treatment of HepG2 cells was performed by the same means as described in Figure 5B with a replacement of TRG with BSO or Znpp. Values in parts B, E, and F are the mean ± SD, n = 3. (*) p < 0.05 and (**) p < 0.01 versus the SA and t-BHP-treated group. Other experiments, n = 3. (*) p < 0.05, (**) p < 0.01 and (***) p < 0.001 versus the control.

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Figure 6. A comparison of SA with t-BHQ in the cytoprotection (A), Nrf2 activation (B), and its downstream gene expression (C). All data were expressed as the mean ± SD, n = 3. (**) p < 0.01 of t-BHP-treated group versus the vehicle group; (##) p < 0.01 versus the t-BHP-stimulated group. Other experiments, n = 3. (**) p < 0.01 and (***) p < 0.001 versus the control.

Figure 7. SA activates Nrf2 via ERK-MAPK signaling pathway. (A) Immunoblot analysis of Akt, p-Akt, ERK, p-ERK, JNK, p-JNK, p38, and p-p38 in HepG2 cells treated with 10 μM SA for the indicated time points. (B) Effect of ERK (U0126) and JNK (SP600125) inhibitors on expression of GCLC and GCLM induced by SA. U0126 or SP600125 inhibitor was used to pretreat HepG2 cells for 1 h before the cells were exposed to 10 μM SA for another 24 h. (C) Effect of U0126 or SP600125 inhibitor on the cytoprotection exerted by SA. Treatment of HepG2 cells was performed by the same means as described in Figure 5B with a replacement of TRG with U0126 or SP600125. Values in part C is the mean ± SD, n = 3. (**) p < 0.01 versus the SA and t-BHP-treated group. Other experiments, n = 3. (**) p < 0.01 and (***) p < 0.001 versus the control; (###) p < 0.001 versus the SA-stimulated group.

slight but not statistically significant cytoprotection under the same experimental conditions (cell viability for the 10 μM 6Streated group being 48.9 ± 5.3%, Figure 2A). The unobvious cytoprotection of 6S compared with SA and 6-DHS is in tune with its low electrophilicity and indicates clearly the importance of the electrophilic modification. Considering that SA was the most potent cytoprotective agents among the tested compounds, we subsequently focused our attention on SA and performed mechanistic studies on its cytoprotective action via Nrf2 activation. Effects of SA on Cellular Redox Status. To explore the connection between oxidative stress and cell death, two

the cytoprotective activity via Nrf2 activation. To rule out the possibility of direct interaction of the test compounds with tBHP, HepG2 cells were pretreated with different but nontoxic concentrations (0.5−10 μM) of 6S and its analogues for 24 h, followed by exposure to fresh medium containing 900 μM tBHP for another 6 h. Under the current experimental conditions, t-BHP led to about 60% cell death (cell viability being 42.4 ± 0.4%, Figure 2A−C). However, pretreatment with either SA or 6-DHS protected the cells from the oxidative death in a dose-dependent manner (cell viability for the 10 μM 6-DHS- and SA-treated group being 58.5 ± 5.1% and 76.1 ± 3.2%, respectively, Figure 2B,C). In comparison, 6S showed a 7988


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Figure 8. SA increases stability of Nrf2 by inhibiting its ubiquitination. Time- (A) and dose-dependent (B) effects of SA on the expression of the total Nrf2. Change of total Nrf2 levels with time (C) in the absence or presence SA and its semiquantitative histogram analysis (D) using ImageJ software. HepG2 cells were pretreated with or without 10 μM SA for 12 h, followed by treatment with 5 μg/mL CHX for the indicated time points. (E) Effect of SA on Nrf2 ubiquitylation. HepG2 cells were incubated with MG132 for 12 h in the absence or presence of SA. n = 3. (*) p < 0.05 and (***) p < 0.001 versus the control.

SA Activates Nrf2 and up-Regulates Expression of Phase II Detoxifying Enzymes. To clarify the role of Nrf2 activation and subsequent induction of phase II detoxifying enzymes in the cytoprotection of SA, we first examined whether it stimulates Nrf2 accumulation in the nucleus, a pivotal step for activating Nrf2,3 by using immunoblotting. Upon treatment of HepG2 cells with SA, both nuclear and cytosolic levels of Nrf2 were up-regulated as early as 1−3 h, to the maximum until 12 h, and then decreased gradually over time (Figure 5A). Subsequently, we used trigonelline (TRG), an Nrf2 inhibitor derived from coffee,36,37 to observe whether it could abrogate the cytoprotection of SA. As expected, TRG succeeded in achieving the abrogation (Figure 5B), providing direct evidence that SA indeed works as a potent Nrf2 activator to afford the cytoprotection against oxidative stress. This is also supported by the fact that multiple phase II detoxifying enzymes including GCLC, GCLM, and HO-1 were upregulated by SA in a time- and dose-dependent fashion (Figure 5C,D), and its cytoprotective effect was weakened by pretreatment with either ZnPP (a selective inhibitor of HO138) or BSO (a synthesis inhibitor of GCL39) (Figure 5E,F).

important indices of redox status, ROS levels and the ratios of GSH to its oxidized form (GSSG) were assayed. Incubation of HepG2 cells with 900 μM t-BHP for 3 h increased obviously the ROS levels along with significant decrease in the GSH/ GSSG ratios in a double fashion of down-regulation of GSH levels and up-regulation of GSSG levels (Figure 3). The produced ROS were also identified to be mainly hydrogen peroxide and superoxide radical anion as suggested by the fact that the green DCF fluorescence induced by t-BHP was obviously weakened by their specific scavenging enzymes CAT and SOD (Figure 4). However, SA pretreatment revised dosedependently the above changes. Specifically, 10 μM SA reversed the increased ROS levels to the basal level and enhanced remarkably the GSH/GSSG ratios by up-regulating GSH levels and down-regulating GSSG levels (Figure 3). Noticeably, the electrophilic SA induced GSH increase rather than depletion, highlighting that under the current experimental conditions, this molecule might not react with intracellular GSH, instead of activating Nrf2 to afford the cytoprotection against oxidative stress. 7989


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Figure 9. Comparison of SA with SA-1 in the abilities to modify covalently Keap1 (A), up-regulate the expression of p-ERK (B), activate nuclear translocation of Nrf2 (B), induce phase II detoxifying enzymes (C) and exert the final cytoprotection (D). Different treatment duration was scheduled for modification of Keap1 (12 h), expression of p-ERK (15 min), nuclear translocation of Nrf2 (12 h), induction of phase II detoxifying enzymes (24 h), and cytoprotection (24 h). Values in part D is the mean ± SD, n = 3. (**) p < 0.01 of t-BHP control versus the negative control. Other experiments, n = 3. (**) p < 0.01 and (***) p < 0.001 versus the control.

electrophiles, such as treatment of HepG2 cells with t-BHQ and sulforaphane, 41 as well as treatment of murine keratinocytes with 3H-1,2-dithiole-3-thione.42 SA Increases Stability of Nrf2 by Inhibiting Its Ubiquitination. Keap1 works as an adaptor for a Cul3based E3-ubiquitin ligase to regulate ubiquitination of Nrf2.1,4 Since ubiquitination of Nrf2 is dependent on distinct cysteine residues in Keap1, numerous electrophilic Nrf2 activators such as t-BHQ and sulforaphane have been reported to induce Nrf2 accumulation by inhibiting its ubiquitination.15 To explore whether SA could stabilize Nrf2 levels, we affirmed using immunobloting that the total Nrf2 levels were increased by SA at 1 h, to the maximum until 12 h followed by its subsequent decrease (Figure 8A). Furthermore, a dose-dependent increase in the total Nrf2 levels by SA was also observed at the timepoint of 12 h (Figure 8B). The increase in the total Nrf2 levels by SA hints at its ability to decrease the turnover rate of Nrf2. To clarify this point, we analyzed the half-life of Nrf2 in the presence of CHX, a protein synthesis inhibitor. As expected, CHX treatment alone decreased the total Nrf2 levels to about 50% within about 15 min, whereas pretreatment with SA led to an extension of the half-life of Nrf2 from 15 to 60 min (Figure 8C,D). The turnover rate of Nrf2 is very fast due to its rapid degradation via the ubiquitin-proteosome system,43 we thus investigated the ability of SA to inhibit ubiquitination of Nrf2 using immunoprecipitation. When HepG2 cells were treated with MG132 (a proteasome inhibitor) for 12 h, Nrf2 ubiquitination was significantly potentiated but abolished by SA (Figure 8E). Taken together, the above results demonstrate clearly that SA can effectively improve stability of Nrf2 by inhibiting its ubiquitination, resulting in its activation. SA Activates Nrf2 via Covalent Modification of Keap1. Nrf2 is usually sequestered in the cytoplasm by its inhibitor protein, Keap1. Therefore, covalent binding to one or more of the cysteine residues in Keap1 stands out as the foremost mechanism for Nrf2 activation by electrophiles.14,15,44,45 The fact that Keap1-dependent ubiquitination of Nrf2 was inhibited by SA (see before) also hints at its ability to modify covalently Keap1. To further clarify this point, we

To emphasize the importance of SA as a novel Nrf2 activator, tert-butylhydroquinone (t-BHQ), a canonical Nrf2 activator, was used as a positive control. A comparison of 10 μM SA with 20 μM t-BHQ among the results of cytoprotection, Nrf2 activation and its downstream gene expression clearly indicates that SA is a more effective cytoprotector via Nrf2 activation and its downstream gene expression than t-BHQ (Figure 6). SA Activates Nrf2 via ERK-MAPK Signaling Pathway. Having established that SA exhibits the cytoprotection via Nrf2 activation, we asked how this molecule activates Nrf2. It is known that Nrf2 accumulation in the nucleus can be facilitated by multiple cellular kinase pathways: mitogen-activated protein kinase (MAPK, including extracellular signal-regulated protein kinase (ERK), c-Jun N-terminal kinase (JNK) and p38), protein kinase C (PKC), and phosphatidylinositol 3-kinase (PI3K/Akt) pathways.10 For example, upon exposure to tBHQ, PKC-mediated phosphorylation of Nrf2 at serine 40 promotes its dissociation from Keap1.40 Since the kinasemediated Nrf2 activation depends significantly on types of activators and cells, we performed immunoblotting to identify the role of MAPK and PI3K/Akt pathways on Nrf2 activation by SA. As shown in Figure 7A, SA up-regulated the levels of pERK and p-JNK to the maximum values at 15 and 30 min, respectively, but showed no obvious effect on p-Akt and p-p38. To verify which signaling cascade controls Nrf2 activation, we used pharmacological inhibitors of ERK (U0126) and JNK (SP600125) to examine their effects on the expression of phase II detoxifying enzymes and the cytoprotection induced by SA. Interestingly, the expression of phase II detoxifying enzymes and the cytoprotection induced by SA were obviously suppressed by U0126 but not by SP600125 (Figure 7B,C). The above results support that stimulating phosphorylation of ERK by SA contributes to its Nrf2 activation and final cytoprotection, also suggesting that phosphorylation of Nrf2 is one of the mechanisms by which SA activates Nrf2. The enhancement in phosphorylation of ERK by SA may involve its ability to activate the upstream Raf-1 kinase41 and depends on its Michael acceptor units (see below). Additionally, the ERKdependent Nrf2 activation was also observed in other 7990


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Figure 10. Comparison of SA with 6S and 6-DHS in the abilities to induce phase II detoxifying enzymes (A), activate nuclear translocation of Nrf2 (B), modify covalently Keap1 (C), and up-regulate the expression of p-ERK (D). Different treatment duration was scheduled for modification of Keap1 (12 h), expression of p-ERK (15 min), nuclear translocation of Nrf2 (12 h), and induction of phase II detoxifying enzymes (24 h). n = 3. (*) p < 0.05 versus the control.

activation.15 In the case of [10]-shogaol, Cys151, 257, and 368 were suggested to be the most readily modified sites,47 whereas modification of Cys23, 38, 395, and 406 was also found for 6S.22 Structure−Activity Relationship Analysis for the Nrf2-Dependent Cytoprotection of 6S and Its Analogues: Dependency of Michael Acceptor Units and Importance of the Electrophilic Modification. Since the electrophilicity of SA is determined by its Michael acceptor units, its reduced analogue, SA-1, was also applied to clarify

examined the levels of high-molecular-weight Keap1 (HMW Keap1) from normal 70 to 150 kDa, because the protein band potentiation indicates Keap1 modification.15,46 As illustrated in Figure 9A, a more obvious expression of HMW Keap1 was clearly detected in SA-pretreated cells than in unpretreated cells (Figure 9A), supporting that it is capable of modifying covalently Keap1 to activate Nrf2 by virtue of its electrophilicity. There are 27 cysteine residues reactive to electrophiles in human Keap1.15 Among these cysteine residues, Cys151, 273, and 288 are crucial for stress sensing and Nrf2 7991


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Journal of Agricultural and Food Chemistry Scheme 3. Proposed Mechanisms for the Nrf2-Dependent Cytoprotection of SA

This molecule exhibited more effective cytoprotection against t-BHP-induced cell death of HepG2 than the parent 6S and tBHQ (a canonical Nrf2 activator) via Nrf2 activation and subsequent induction of phase II detoxifying enzymes including GCLC, GCLM, and HO-1 (Scheme 3). Mechanistic study uncovers that all of the above processes including Nrf2 activation (by modifying Keap1, inhibiting Nrf2 ubiquitylation and phosphorylating ERK), subsequent induction of phase II detoxifying enzymes and ultimate cytoprotection are Michael acceptor-dependent (Scheme 3). This work emphasizes the importance of electrophiles and gives us useful information on designing dietary natural product-guided Nrf2 activators by an electrophilicity-based strategy.

whether the Nrf2-dependent cytoprotection of SA is Michael acceptor unit-dependent. It can be clearly seen that unlike SA, SA-1 is almost powerless to modify covalently Keap1 (Figure 9A) and up-regulate the expression of p-ERK (Figure 9B), thereby having little capacity to activate nuclear translocation of Nrf2 (Figure 9B), induce phase II detoxifying enzymes (Figure 9C) and exert the final cytoprotection (Figure 9D). The Michael acceptor unit-dependent Nrf2 activation and final cytoprotection illustrate the importance of electrophiles in developing Nrf2 activators. To further rationalize the mechanisms determining the Nrf2dependent cytoprotection of 6S and its analogues, we last compared their activities in inducing phase II detoxifying enzyme, activating Nrf2, phosphorylating ERK, and modifying Keap1 by using immunoblotting. In line with the cytoprotective ability of 6S and its analogues, their activity in inducing phase II detoxifying enzymes including GCLC, GCLM, and HO-1 follows the order of SA > 6-DHS > 6S (Figure 10A). Furthermore, in activating Nrf2, SA and 6-DHS show nearly equal ability, but they are superior to 6S (Figure 10B), underlining again the importance of the electrophilic modification. Noticeably, higher Keap1-modifying (Figure 10C) but lower ERK-phosphorylating activities (Figure 10D) of SA than those of 6-DHS are responsible for its stronger activities in inducing phase II detoxifying enzymes and final cytoprotection. These results highlight that despite covalent modification of Keap1 and phosphorylation of ERK contributing to Nrf2-dependent cytoprotection of SA, these two effects are not equivalent. The former plays a more important role in the Nrf2-dependent cytoprotection than the latter. Additionally, almost equal electrophilicity was observed for SA and 6-DHS (Figure 1A,B), but SA exhibited higher activities in modifying Keap1 and inducing phase II detoxifying enzyme (Figure 10C) than 6-DHS, probably due to SA harboring more electrophilic sites to facilitate nucleophilic attack of the cysteine residues of Keap1. In summary, we developed a novel 6S-inspired Nrf2 activator, SA, bearing a short but entirely conjugated unsaturated ketone chain, by an electrophilicity-based strategy.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.8b02442. 1

H NMR,

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13

C NMR, and HRMS spectra as well as

HPLC chromatograms of SA and SA-1 (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Fax: +86-931-8912500. Phone: +86-931-8915557. *E-mail: [email protected]. ORCID

Bo Zhou: 0000-0002-8713-5906 Funding

This work was supported by the National Natural Science Foundation of China (Grant Nos. 21372109 and 31100607) and the 111 Project. Notes

The authors declare no competing financial interest. 7992


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Applying an Electrophilicity-Based Strategy to Develop a Novel Nrf2

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