Nrf2 induction potency of plant-derived compounds demonstrated by an ARE luciferase reporter and conventional assay of NAD(P) H-quinone acceptor oxidoreductase 1 activity

doi:10.21203/rs.3.rs-4204747/v1

Objective

Various plants have been reported to contain compounds that promote the nuclear accumulation of Nrf2 to induce a set of xenobiotic detoxifying enzymes such as NAD(P)H-quinone acceptor oxidoreductase 1 (NQO1) via antioxidant response element (ARE). While conventional methods for evaluating the Nrf2 induction potency of compounds include NQO1 activity, recently, an ARE luciferase reporter was developed to directly assess the Nrf2 induction potency of compounds of interest. In this study, the ability of these two assays to evaluate and determine Nrf2 induction potency of plant-derived compounds was compared.

Results

Although the compounds overall showed a high degree of consistency between the assays, several compounds did not. The results suggest that although the NQO1 assay can be used as an evaluation method to estimate the Nrf2 induction potency of a compound, an ARE luciferase reporter may offer greater precision. In summary, the inconsistency in Nrf2 induction potency evaluated by the reporter and NQO1 assays for some of the plant-derived compounds evaluated herein, including resveratrol, may be due to a variety of factors that regulate NQO1 expression and activity other than Nrf2, with each compound having a different degree of effect on these factors.

Nrf2

NQO1

ARE luciferase reporter

NQO1 assay

comparative method

plant-derived compounds

electrophilic compounds

isothiocyanates

hepatocyte

screening

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that binds to the promoter’s antioxidant response element (ARE) to induce phase II detoxifying enzymes, such as NAD(P)H-quinone acceptor oxidoreductase 1 (NQO1) and Glutathione S-transferase (GST), which protect cells from oxidative damage [1]. Nrf2 is modified by the cytoplasmic protein Kelch-like ECH-associated protein 1 (Keap1) and localized in the cytoplasm under normal cellular conditions [2]. However, under conditions of oxidative stress or electrophilic insult, Keap1 undergoes conformational changes, leading Nrf2 to stabilization, nuclear accumulation, and binding to ARE, which activates the transcription of genes involved in antioxidant response and cellular detoxification [3, 4].

Various plants contain compounds that promote the nuclear accumulation of Nrf2 [5]. The conventional method for evaluating the Nrf2 induction potency of compounds is the assay of NAD(P)H-quinone acceptor oxidoreductase 1 activity (NQO1 assay), which indirectly evaluates Nrf2 induction potency by measuring NQO1 activity as an alternative indicator of Nrf2 activity [6–10]. Recently, an ARE luciferase reporter (reporter assay) was developed [11, 12], enabling the direct assessment of the Nrf2 induction potency [13–16]. The reporter assay is more advantageous than the NQO1 assay because it requires less time for evaluation and can be performed while cells remain viable [17]. As a result, the reporter assay is becoming a popular alternative to the NQO1 assay for evaluating Nrf2 induction potency [18]. To the best of our knowledge, no studies have investigated the consistency of the Nrf2 induction potency of plant-derived compounds evaluated by reporter and NQO1 assays. Therefore, in this study, we compared the Nrf2 induction potencies evaluated by the reporter and NQO1 assays for various plant-derived compounds and examined the consistency between them.

The Nrf2 or NQO1 induction potency of a compound can be expressed using the above two assays; for example, the enzymatic activity of NQO1 [19], the concentrations required for the double activities of Nrf2 or NQO1 of untreated cells (CD values) [8–10, 20], and the relative absorbance or luminescence to untreated cells (fold induction, relative ratio) [21–23]. This study used 12 plant-derived compounds to compare the CD values evaluated by the reporter assay with those of the NQO1 assay reported in previous reports [8–10]. Furthermore, the Nrf2 induction potency of an additional 21 plant-derived compounds that have been reported to induce Nrf2 or NQO1 was also evaluated using a reporter assay. Ultimately, 33 compounds were sorted in order of their Nrf2 induction potency.

Materials

The 33 compounds used in the reporter assay are commercially available standards (Additional File 1). tert-Butylhydroquinone (tBHQ) was purchased from FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan.

Cell culture

The Nrf2 antioxidant pathway ARE reporter-HepG2 cell line (BPS Bioscience, San Diego, CA) was used in passages 6 to 12 after cell activation. Cell activation and the media used were in accordance with the manufacturer’s instructions [24]. Briefly, cells were activated in Thaw Medium (10% FBS, 1% non-essential amino acids, 1 mM sodium pyruvate, 1% PSA-containing MEM) and grown in Growth Medium (600 µg/ml Geneticin-containing Thaw Medium). The cells were cultured at 37℃ with 5% CO₂.

ARE luciferase reporter

The cells were seeded at a density of 4 × 10⁴ cells/100 µl/well in 96-well plates with clear bottoms (PerkinElmer, Waltham, MA) and cultured for 24 h in Thaw Medium before adding the compounds. All compounds were prepared as 50 mM stock solutions in dimethyl sulfoxide (FUJIFILM Wako Pure Chemical Corporation) (DMSO). The solution was then diluted in the Growth Medium at various concentrations while maintaining a DMSO concentration of 0.5% (w/v). As a positive control, 100 µM tBHQ was used. After an 18-h incubation in Growth Medium containing compounds, 100 µl/well of Steady-Glo® Luciferase Assay System (Promega, Madison, WI) was added in serial dilution (0, 0.1, 0.3, 1, 3, 10, 30, 100 µM) under light-shielded conditions. After reacting for 5 min at room temperature, the cumulative luminescence values in each well for 5 s were measured in duplicate using a Centro XS3 LB960 luminometer (Berthold Technologies, Bad Wildbad Baden, Württemberg). The concentrations required to double the activity of Nrf2 (CD values, µM) were calculated for the 33 compounds as previously reported [8–10]. The top 20 CD values were evaluated three times in finer serial dilutions, and the average was used as the CD value of the compound.

Nrf2 induction potencies of the plant-derived compounds

An approximate consistency was observed when comparing the CD values obtained from the previously reported NQO1 assay [8-10] with those obtained from the reporter assay for 12 of the compounds evaluated in this study (Table 1, Figure 1). In contrast, some compounds, such as resveratrol, showed relatively large differences in CD values between the reporter and NQO1 assays.

Table 1. Nrf2 induction potency evaluated by reporter and NQO1 assays of 12 plant-derived compounds

		CD values (µM)
	Compounds	Reporter assay	NQO1 assay
(1)	Sulforaphane	0.22	0.2 [10], 0.22 [8]
(2)	Withaferin A	0.37	0.19 [8]
(3)	Erucin	0.77	0.65 [8]
(4)	Resveratrol	3.79	23.8 [9], >150 [8]
(5)	Genistein	7.78	16.2 [9]
(6)	Piceatannol	10.53	>21.1 [9]
(7)	Curcumin	14.82	2.7 [9], 7.3 [10]
(8)	Quercetin	20.78	2.6 [9], 3-20 [10]
(9)	Benzyl-isothiocyanate	29	>30 [8]
(10)	Kaempferol	52.25	7 [10]
(11)	Epigallocatechin-3-gallate	>100	>50 [9]
(12)	Myricetin	>100	58-NA [10]

The values are the concentrations required for double activity (CD values). Numbers in parentheses are linked with Figure 1.

The CD values obtained from the reporter and NQO1 assays were plotted as a double logarithmic graph. The CD values in the reporter assay were evaluated in HepG2 cells, and those in the NQO1 assay were evaluated in Hepa1c1c7 cells (●: Ushida et al. [8], ■: Gerhäuser et al. [9], 〇: Young, et al. [10]). Values with greater than sign in Table 1 were plotted as the maximum values, and the values shown as widths were plotted as the median.

ARE luciferase reporter

The CD values of 33 plant-derived compounds (including the 12 compounds listed in Table 1) were determined by a reporter assay and sorted in order of the strongest Nrf2 induction potency (Table 2). Sulforaphane was the strongest Nrf2 inducer, followed by withaferin A, andrographolide, erucin, and tanshinone IIA, while epigallocathechin-3-gallate, rosmarinic acid, caffeic acid, mangiferin, myricetin, ginsenoside Rd, salvianolic acid B, ferulic acid, and naringin did not show Nrf2 induction potency.

Table 2. Nrf2 induction potency of 33 plant-derived compounds in HepG2 cells, expressed as CD values

Rank	Compounds	Chemical classes	CD (µM)
1	Sulforaphane	Isothiocyanates	0.22 ± 0.03
2	Withaferin A	Triterpenoids	0.37 ± 0.06
3	Andrographolide	Diterpenoids	0.53 ± 0.23
4	Erucin	Isothiocyanates	0.77 ± 0.24
5	Tanshinone IIA	Diterpenoids	1.13 ± 0.37
6	Pterostilbene	Stilbenes	2.50 ± 0.37
7	Luteolin	Flavones	3.09 ± 1.13
8	Apigenin	Flavones	3.11 ± 0.42
9	Xanthohumol	Chalcones	3.23 ± 0.20
10	Resveratrol	Stilbenes	3.79 ± 0.36
11	Galangin	Flavonols	5.28 ± 1.21
12	Emodin	Anthraquinones	5.56 ± 1.22
13	3H-1,2-dithiole-3-thione	Dithiolethiones	5.75 ± 2.69
14	Hesperetin	Flavanones	7.72 ± 1.36
15	Genistein	Isoflavones	7.78 ± 1.06
16	Piceatannol	Stilbenes	10.5 ± 1.7
17	Curcumin	Curucuminoids	14.8 ± 2.6
18	Baicalein	Flavones	15.2 ± 2.0
19	Quercetin	Flavonols	20.8 ± 6.6
20	Isorhamnetin	Flavonols	25.5
21	Benzyl-isothiocyanate	Isothiocyanates	29.0
22	Caffeic acid phenethyl ester	Phenylpropanoids	31.1 ± 3.4
23	Kaempferol	Flavonols	52.3
24	Morin.	Flavonols	60.5
25	Epigallocatechin-3-gallate	Flavanols	>100
	Rosmarinic acid	Phenylpropanoid	>100
	Caffeic acid	Phenylpropanoids	>100
	Mangiferin	Glucosylxanthones	>100
	Myricetin	Flavonols	>100
	Ginsenoside Rd	Steroid Glycosides	>100
	Salvianolic acid B	Stilbenoids	>100
	Ferulic acid	Phenylpropanoids	>100
	Naringin	Flavanone Glycosides	>100

For all 33 compounds, the CD values were calculated from the luminescence values at 0, 0.1, 0.3, 1, 3, 10, 30, and 100 µM. For compounds in the top 20, the luminescence values were measured three times in finer serial dilutions, respectively, and the mean ± SD values are shown.

To confirm the consistency of the Nrf2 induction potency of the plant-derived compounds evaluated using the two assays (ARE luciferase reporter and the conventional NQO1 assay), the CD values obtained for the 12 plant-derived compounds using these assays were compared. Most compounds showed an overall consistency, although several compounds did not. Therefore, although the NQO1 assay can be used as an evaluation method to estimate the Nrf2 induction potency of a compound, a reporter assay may offer greater precision.

A relatively high consistency was observed for isothiocyanates, such as sulforaphane, erucin, and benzyl isothiocyanate. In contrast, no consistency was observed for several of the compounds evaluated, including resveratrol and kaempferol. The reporter assay directly assesses the potency of a compound in promoting the nuclear accumulation of Nrf2, whereas the NQO1 assay indirectly assesses the Nrf2 induction potency by measuring the enzymatic activity of NQO1, which is induced by Nrf2 in response to oxidative stress or exposure to electrophilic compounds. NQO1 is also known to be induced by other transcription factors, such as the aryl hydrocarbon receptor (AhR) [25]. Among the 12 compounds, resveratrol and genistein have previously been reported to induce AhR at > 10 µM, while curcumin, quercetin, and myricetin have also been reported to slightly induce AhR at > 100 µM [26, 27]. Since the concentrations at which Nrf2 induction was observed for curcumin, quercetin, and myricetin (CD values shown in Table 1) were below the concentration at which AhR is induced (> 100 µM), it can be assumed that the NQO1 or Nrf2 induction potency evaluated in this study was not affected by AhR. The results of the NQO1 assay for resveratrol and genistein were assumed to be affected by both Nrf2 and AhR, and the CD values of the NQO1 assay were expected to be lower than those of the reporter assays. Contrary to this expectation, the CD values of the NQO1 assay were higher than those of the reporter assays. In particular, resveratrol showed the largest difference in CD values between the reporter and NQO1 assays. Previous reports showed that the NQO1 induction potency of resveratrol was CD > 50 µM (induction potency expressed as fold change converted to CD value) [21] and CD > 100 µM (induction potency expressed as nmol/min/mg protein converted to CD value) [19], suggesting that the NQO1 induction potency of resveratrol is not particularly high. Although the precise reason for this is unknown, it may be due to the influence of the 20S proteasome, which contributes to NQO1 degradation. NQO1 is degraded by the 20S proteasome at low concentrations of flavin adenine dinucleotide (FAD) [28], and the 20S proteasome can be activated by low molecular weight compounds [29]. Although the effects of resveratrol on the FAD concentration or the 20S proteasome remain unclear, our results suggest that one reason for the low NQO1 induction potency of resveratrol is the fact that resveratrol induces NQO1 via Nrf2 and AhR, while simultaneously decreasing FAD concentrations. This may in turn promote the degradation of NQO1 by the 20S proteasome. However, further studies are needed to confirm this hypothesis. In conclusion, the inconsistency in Nrf2 induction potency evaluated by the reporter and NQO1 assays for some plant-derived compounds, including resveratrol, may be due to multiple factors regulating NQO1 expression and activity other than Nrf2, and each compound has a different degree of effect on these factors. Further research is needed because the factors that regulate NQO1 expression and activity, and the effects of each plant-derived compound on each of these factors are not yet known.

Many of the compounds in the upper ranks were electrophilic, including isothiocyanates (sulforaphane and erucin), triterpenoids (withaferin A), diterpenoids (andrographolide and tanshinone IIA), and curcuminoids (curcumin). Isothiocyanates and α, β-unsaturated carbonyl groups can promote the nuclear accumulation of Nrf2 by Michael addition reactions to the thiol group of Keap1, forming covalent bonds with the cysteine residue of Keap1 [20, 30–32]. Many of the lower-ranked compounds were glycosides, such as naringin, ginsenoside Rd, and mangiferin. The NQO1 induction potency of flavonoids is known to be weakened when in glycoside form [33], which supports the low Nrf2 induction potency of various glycosides observed in the present study. Among the flavonoids, luteolin and apigenin tended to show a relatively high Nrf2 induction potency, whereas kaempferol, morin, myricetin, and epigallocathechin-3-gallate showed a relatively low potency. Thus, it is possible that the 3-C hydroxyl modification of the flavone backbone, a structure common to flavonols and flavanols, may be an obstacle to their interaction with Keap1, which may reduce their Nrf2 induction potency. Several induction and activation mechanisms of Nrf2 by flavonoids, other than its interaction with Keap1, have been reported, such as Nrf2 phosphorylation by kinases, including ERK1/2, Akt, and p38MAP kinase [34]. Therefore, each chemical structure of the compounds may lead to differences in the primarily activated pathway, depending on various factors (i.e., electron affinity, hydrophobicity, and molecular size). However, further studies are needed to elucidate the details of this hypothesis.

Limitations

Since only a limited number of studies were compared in this report, the consistency of Nrf2 induction potencies of plant-derived compounds evaluated by the reporter and NQO1 assays has not been fully investigated. In addition, the NQO1 induction potency has been reported to vary between cell types, which this study did not consider [11]. Despite these limitations, to the best of our knowledge, this study is the first to examine the consistency of Nrf2 induction potency evaluated by reporter and NQO1 assays for multiple compounds. Future comparisons of CD values by each assay across cell types may be used to demonstrate consistency between the assays, as well as reveal those factors that influence Nrf2 induction potency.

Despite an approximate degree of consistency in the Nrf2 induction potencies as evaluated by the reporter assay and the NQO1 assay for the 12 plant-derived compounds, some compounds showed inconsistent results. This suggests that although the NQO1 assay is useful for investigating the Nrf2 induction potency of a compound, the reporter assay can achieve a more accurate evaluation. In addition, using a reporter assay for 33 plant-derived compounds, the chemical structure of the compounds was shown to be potentially related to the strength of their Nrf2 induction potency. This suggests that evaluating the Nrf2 induction potency of various compounds using reporter assays may be useful for elucidating the structures that affect the Nrf2 induction potency.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Availability of data and materials

Raw data, including imaging files, and reagents described in this study will be made available upon request to the corresponding author.

Competing interests

Not applicable.

Funding

We declare that no financial support and funding were received during the preparation of this manuscript.

Authors' contributions

ET, DK, and YU designed the experiments. ET performed the experiments, analyzed the data, and wrote the manuscript. DK, YU, and KI contributed to the development of the manuscript. All authors read and approved the final manuscript.

Acknowledgments

Not applicable.

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No competing interests reported.

AdditionalFile1.docx
Additional file1. List of the 33 plant-derived compounds
AdditionalFile2.pptx
Additional file 2. Chemical structures of the 33 plant-derived compounds

Nrf2 induction potency of plant-derived compounds demonstrated by an ARE luciferase reporter and conventional assay of NAD(P) H-quinone acceptor oxidoreductase 1 activity

Status:

Version 1

Abstract

Objective

Results

Figures

Introduction

Materials and Methods

Materials

Cell culture

ARE luciferase reporter

Results

Discussion

Limitations

Conclusion

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1