Marine Natural Product Honaucin A Attenuates Inflammation by Activating the Nrf2-ARE Pathway

Article abstract of DOI:10.1021/acs.jnatprod.7b00734

The cyanobacterial marine natural product honaucin A inhibits mammalian innate inflammation in vitro and in vivo. To decipher its mechanism of action, RNA sequencing was used to evaluate differences in gene expression of cultured macrophages following hon

Full text of DOI:10.1021/acs.jnatprod.7b00734

Article
pubs.acs.org/jnp
Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX
Marine Natural Product Honaucin A Attenuates Inflammation by
Activating the Nrf2-ARE Pathway
Samantha J. Mascuch,† Paul D. Boudreau,† Tristan M. Carland,‡ N. Tessa Pierce,† Joshua Olson,§
Mary E. Hensler,§ Hyukjae Choi,⊥ Joseph Campanale,† Amro Hamdoun,† Victor Nizet,§,∥
William H. Gerwick,†,∥ Teresa Gaasterland,*,† and Lena Gerwick*,†
§
†Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography, Department of Pediatrics, School of
∥
Medicine, and Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, California
92093, United States
‡Illumina Inc., San Diego, California 92122, United States
⊥College of Pharmacy, Yeungnam University, Gyeongsan 38541, Republic of Korea
S
* Supporting Information
ABSTRACT: The cyanobacterial marine natural product
honaucin A inhibits mammalian innate inflammation in vitro
and in vivo. To decipher its mechanism of action, RNA
sequencing was used to evaluate differences in gene expression
of cultured macrophages following honaucin A treatment. This
analysis led to the hypothesis that honaucin A exerts its anti-
inflammatory activity through activation of the cytoprotective
nuclear erythroid 2-related factor 2 (Nrf2)-antioxidant
response element/electrophile response element (ARE/
EpRE) signaling pathway. Activation of this pathway by
honaucin A in cultured human MCF7 cells was confirmed
using an Nrf2 luciferase reporter assay. In vitro alkylation
experiments with the natural product and N-acetyl-L-cysteine
suggest that honaucin A activates this pathway through covalent interaction with the sulfhydryl residues of the cytosolic repressor
protein Keap1. Honaucin A presents a potential therapeutic lead for diseases with an inflammatory component modulated by
Nrf2-ARE.
resolution/high-throughput microscopy, digital computing, and
proteomics have helped expand the tool kits used for this
task.5−12
atural products produced by cyanobacteria have ther-
Napeutic potential in the treatment of many human
diseases.1,2 The broadly bioactive nature of these compounds
may be attributed, in part, to their impressive structural
diversity. In addition, these specialized metabolites are thought
to serve important ecological functions, including cell−cell
communication and chemical defense.3 It is therefore likely that
bioactive cyanobacterial natural products have been subject to
evolutionary selection for increased binding specificity to
conserved biological targets across phyla.4
Advancement of a bioactive compound with therapeutic
potential is greatly facilitated by knowledge of its cellular
target(s) and mechanism(s) of action (MOA). Natural
products are most often identified as entities of interest by
means of phenotypic cell, tissue, or whole organism screens. As
such, at the stage of discovery, they lack clearly defined MOAs.
Identification of the MOA(s) of a natural product can be
difficult and time-consuming and, for some compounds, has not
yet been possible.5 Fortunately, methods for MOA determi-
nation are constantly evolving. In addition to classical
approaches, such as biochemical affinity assays, methods arising
from technological advances in DNA/RNA sequencing, high-
Honaucin A is one bioactive small molecule for which there
was a lack of mechanistic understanding. Originally isolated
from the marine filamentous cyanobacterium Leptolyngbya
crossbyana found overgrowing a Hawaiian coral reef, it was
previously reported to inhibit both mammalian innate
inflammation and bacterial quorum sensing in vitro.13 Given
the promising bioactivity of honaucin A in mammalian cells and
the ongoing need to develop novel and effective therapeutics to
treat chronic inflammation and/or diseases with an inflamma-
tory component, we further evaluated the molecule’s anti-
inflammatory properties in vivo as well as the molecular basis of
its activity. Following transcriptomic analysis, we tested the
hypothesis that honaucin A activates the cytoprotective nuclear
erythroid 2-related factor 2 (Nrf2)-antioxidant response
element/electrophile response element (ARE/EpRE) pathway.
Special Issue: Special Issue in Honor of Susan Horwitz
Received: August 26, 2017
© XXXX American Chemical Society and
American Society of Pharmacognosy
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Figure 1. Honaucin A dose response in the mouse ear edema model. Low doses of honaucin A (A) caused a significant reduction in PMA-induced
mouse ear edema, whereas the highest dose tested increased swelling significantly (B). Ears treated with honaucin A and PMA were compared to the
paired PMA-treated ear, and the honaucin A-free, PMA positive control was compared to the paired vehicle-treated ear. All mean values between
treated and untreated ears were significantly different when compared using the Wilcoxon’s signed ranks test (p ≤ 0.05).
Figure 2. Differential gene expression in murine macrophages exposed to honaucin A. Significant differences in transcript abundance (red points)
were observed in cells treated with a low or high concentration of honaucin A at two time points (A). Relative to LPS, RAW 264.7 cells treated with
honaucin A and LPS displayed a general decrease in the abundance of transcripts involved in the inflammatory response and an increase in the
abundance of transcripts related to phase II enzymes (B). Scale depicts the log2 fold change in abundance. See also Table S1.
The Nrf2-ARE/EpRE pathway serves as an environmental
sensor for the cell, allowing it to monitor for the presence of
oxidants, electrophiles, and xenobiotic compounds that may
react with and thereby damage cellular components.14 In the
presence of these insults, pathway activation results in
translocation of transcription factor Nrf2 into the nucleus,
triggering a detoxifying antioxidant response aimed at
neutralizing the threat. Some weak electrophilic compounds,
by virtue of their chemical structures, trigger this pathway in the
absence of appreciable cellular damage and thus induce a net
cytoprotective effect.
hypersensitivity mouse ear edema model.15 The ability of the
natural product to attenuate innate inflammation and
subsequent ear edema in the presence of the irritant phorbol
12-myristate 13-acetate (PMA) was dependent upon the
amount of natural product applied and displayed a biphasic
profile (Figure 1).
Exposure to 1 μg of PMA per ear (1.62 nmol/ear) in the
absence of the natural product caused a significant increase in
swelling with the mean increase equal to 32.5 7.5% of the
average punch weight of the vehicle-treated ears (p = 0.02).
Treatment with the greatest amount of honaucin A (3 μmol/
ear) also caused significant swelling (p = 0.02; mean increase in
swelling = 51.4 14.4%). Lower concentrations of honaucin A
caused a significant reduction in PMA-induced ear edema;
application of 1 μmol/ear resulted in a 27.6 11.0% reduction
RESULTS AND DISCUSSION
■
Honaucin A Modulates in Vivo Inflammation. The in
vivo activity of honaucin A was assayed in a generalized
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Figure 3. Nrf2-ARE signaling pathway. Under normoxic conditions, cytoplasmic Keap1 is complexed to the transcription factor Nrf2. The protein
complex is ubiquitinated and degraded by the proteasome (A). If an oxidant or electrophile such as honaucin A is present, it may interact with
cysteine residues on Keap1 leading to Nrf2 stabilization and translocation into the nucleus where it can bind to the antioxidant response element/
electrophile response element (ARE/EpRE) initiating the transcription of cytoprotective genes (B).
(p = 0.03); application of 0.3 μmol/ear resulted in a 10.0
4.1% reduction (p = 0.03), and application of 0.1 μmol/ear
resulted in a 17.1 3.5% reduction (p = 0.02).
increased expression, only two genes, sulfiredoxin 1 (Srxn1)
and heme oxygenase 1 (Hmox1), were shared (Table S1).
Srxn1, an enzyme involved in antioxidant metabolism, which
may be expressed under conditions of oxidative stress to
regenerate peroxiredoxins, was upregulated in the 4 μM
treatment at both early and late time points as well as in the
12 μM treatment at the early time point. Although Srxn1 also
displayed increased expression in the 12 μM late time point
treatment, the regulation did not meet the threshold of
significance as determined by the R statistical package DESeq2.
Hmox1, an inducible regulator of inflammatory processes
whose activation results in production of the antioxidant
bilirubin, was upregulated in RAW 264.7 cells exposed to the
higher concentration of honaucin A at both time points.
Of the downregulated genes, only two were shared among
treatments. These two genes, suppressor of cytokine signaling 3
(Socs3), a regulator of STAT3 activation for cytokines
belonging to the IL-6 family, and RasGEF domain family,
member 1B (Rasgef1), a signaling network regulator that is
induced via TLR activation by LPS, were downregulated at 30
min for both high and low honaucin A concentrations (Table
S1).18,19 The other downregulated genes were all observed
from the 12 μM treatment.
Overall, honaucin A treatment resulted in a trend of
decreased abundance of transcripts of proinflammatory genes,
including cytokines and chemokines (Figure 2B, Table S1).
The finding mirrored previous in vitro findings in which
honaucin A treatment of RAW 264.7 cells resulted in a
dampened response to LPS. This included downregulation of
nitric oxide production as well as decreased transcription of
tumor necrosis factor alpha (Tnfα), interleukin-1-beta (Il1β),
interleukin-6 (Il6), and inducible nitric oxide synthase
(Nos2).13
The biphasic dose response for honaucin A was not wholly
unexpected. The finding mimicked previous in vitro observa-
tions of cytotoxicity at higher compound concentrations when
honaucin A was tested for its ability to diminish LPS-induced
nitric oxide production in murine RAW 264.7 cells.13 Biphasic
dose response curves have been reported for other chemical
compounds with anti-inflammatory properties, including the
natural products chloroquine and curcumin.16,17 Despite the
augmented inflammation at its highest dose, the significant
inhibition of inflammation induced by lower doses of honaucin
A justified further exploration of its mechanism of action.
Treatment of Murine Macrophages with Honaucin A
Results in Differential Regulation of Genes Involved in
Inflammation and Redox Balance in a Manner
Suggestive of Nrf2-ARE Activation. Transcriptomic meth-
ods have proven to be valuable tools for generating hypotheses
regarding the mechanisms of small molecules.8,11 RNA
sequencing of the transcriptome of murine macrophages
exposed to honaucin A allowed us to pinpoint differences in
gene expression that resulted from treatment. Exposure of the
murine macrophage cell line RAW 264.7 to honaucin A in the
presence of proinflammatory lipopolysaccharide (LPS) induced
the differential regulation of several genes as compared that of
the LPS-only control (Figure 2).
The concentration of honaucin A was a greater determinant
of differential gene expression than was incubation time.
Exposure of cells to 4 μM honaucin A, the half-maximal
inhibitory concentration (IC50) for nitric oxide production in
LPS-stimulated RAW 264.7 cells,13 resulted in the differential
expression of a small number of genes at both the earlier and
later time points. In contrast, exposure of cells to 12 μM
honaucin A elicited a greater degree of differential gene
expression.
Time did influence the character of differential gene
expression. At 30 min, the majority of differentially expressed
genes in the 12 μM treatment were downregulated. At 180 min,
the trend was reversed, and the majority of differentially
expressed genes in the 12 μM treatment were upregulated.
Few statistically significant, differentially expressed genes
were shared between treatments. Among shared genes with
An increase in the abundance of transcripts involved in redox
balance was somewhat at odds with the anti-inflammatory
profile observed from the diminished transcription of immune
genes. It implied that honaucin A acts as an oxidant, inducing
oxidative stress in macrophages (Figure 2B, Table S1).
Transcriptional activation of many of the genes whose
transcripts were observed in greater abundances, such as
Hmox1, Srxn1, Txrdn1, Prdx1, Sqstm1, Gclm, Slc7a11, and
Akr1b8, is known to result from Nrf2 binding to the antioxidant
response element (ARE/EpRE).20−22 One plausible explan-
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Figure 4. Activation of the Nrf2-ARE pathway in MCF7 cells in the presence of honaucin A. Honaucin A displayed a robust and dose-dependent
activation of Nrf2-ARE relative to untreated cells (A). Less activation at higher concentrations of the natural product coincides with a cytotoxic effect
at these doses (B).
ation that reconciles the apparent incongruity between
honaucin A’s oxidant and anti-inflammatory effects is that the
natural product activates the Nrf2-ARE/EpRE pathway.
Nrf2 is a transcription factor that, under basal conditions, is
sequestered in the cell cytoplasm by its repressor protein Kelch-
like ECH-associated protein 1 (Keap1).23 Keap1 additionally
binds Cul3, serving as an adapter protein between Nrf2 and the
E3 ubiquitin ligase.24 The complex is ubiquitinated and
degraded by the 26S proteasome during the normal turnover
of these proteins in the cell (Figure 3A).
Keap1 contains either 25 (murine) or 27 (human) cysteine
residues that act as environmental sensors.25 If oxidants or
electrophiles are present in the cytoplasm, they are able to
interact with the sulfhydryl groups of these cysteines. The
number and identities of the residues to which they bind
depend on the architecture of the interacting molecule and its
strength as a Michael acceptor. Moreover, a molecule’s pattern
of cysteine residue engagement may be referred to as its
“cysteine code”.26 Dynamics may differ depending on the
character of this interaction and other factors (see refs 27 and
28), but interaction with these cysteine residues ultimately
promotes dissociation of Nrf2 from Keap1. Nrf2 then enters
the nucleus where, in concert with small Maf proteins, it forms
a complex with the ARE/EpRE promoter and initiates the
transcription of cytoprotective genes (Figure 3B).
Ultimately, transcription of Nrf2-activated genes results in
the increased presence of detoxifying and antioxidant phase II
enzymes. For this reason, they have been termed “indirect
antioxidants”.20,29 Classical antioxidants administered through
diet or otherwise may not reach the site at which they are
needed and, in the absence of regenerating enzymes, are
consumed when they react with an electrophile restricting their
usefulness to a single chemical reaction. These factors limit the
therapeutic utility of classical antioxidants. Indirect antioxidants,
conversely, prompt the production of antioxidants such as
glutathione and bilirubin in situ. They also prompt the
production of enzymes able to regenerate these antioxidants
so that they may react multiple times to neutralize chemical
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Figure 5. Reaction of honaucin A with the cysteine-bearing small molecule N-acetylcysteine (NAC) through 1,4-conjugate addition to produce two
products. The later eluting peak was centered at 4.3 min (A) and had an [M + H]+ m/z 368.0572 (C13H19ClNO7S) for the SNAC addition product,
whereas the earlier eluting peak was centered at 3.8 min (B) and showed an [M + H]+ m/z 332.0801 (C13H18NO7S) for the SNAC addition plus loss
of HCl to produce the proposed cyclopropyl ring compound. This indicates that honaucin A may stabilize Nrf2 by engaging sulfhydryl residues of
the repressor protein Keap1. See also Figures S1 and S2.
threats. This renders indirect antioxidants much more potent
than classical antioxidants. Thus, although the pathway is
triggered by oxidant species and electrophiles, it ultimately has
a protective outcome; this is consistent with the reduction of
inflammation observed upon honaucin A exposure.
Because of the generalized response that results from Nrf2-
ARE/EpRE activation, activating molecules exert pleiotropic
effects. Neuroprotective, chemoprotective, and anti-inflamma-
tory effects have been reported for these compounds.30 There is
also evidence of increased proteasomal activity as well as
antibacterial and antifungal properties.31,32 One example of a
synthetic Nrf2-ARE activator, dimethyl fumarate, is prescribed
to treat relapsing multiple sclerosis under the trade name
Tecfidera. Besides honaucin A, other marine natural product
activators of Nrf2-ARE include halomadurones C and D from
an Actinomadura sp. cultured from an ascidian, zonarol from the
brown alga Dictyopteris undulate, and strongylophorine-8 from
the sponge Petrosia (Strongylophora) corticata, all of which were
investigated for their potential neuroprotective properties.33−35
Confirmation of ARE/EpRE Engagement by Nrf2 in
the Presence of Honaucin A. For evaluating our prediction
of Nrf2-ARE/EpRE involvement in the MOA of honaucin A,
the ability of the natural product to stabilize the transcription
factor Nrf2 was assessed using an in vitro luciferase reporter
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assay. When honaucin A was added to the human breast cancer
cell line MCF7 into which an Nrf2-responsive luciferase
construct had been stably integrated, an increase in
luminescence in the presence of firefly luciferase substrate
was observed relative to that of untreated cells (Figure 4A).
The level of Nrf2 activation was compared to the known Nrf2
activators tert-butylhydroquinone (tBHQ) and dimethyl
fumarate (DMF).
thione (GSH) (Figure S1). Addition products were inter-
rogated using high-resolution mass spectrometry.
Honaucin A reaction with NAC formed the predicted adduct
and its derivative (Figure 5). This likely occurred through 1,4-
conjugate addition in which the electron-withdrawing carbonyl
oxygen and chlorine atom of honaucin A contributed to make
the 4-position carbon of the Michael acceptor highly electro-
positive and thus very reactive toward the nucleophilic thiolate
of NAC (Figure S2). Interestingly, there was no evidence that
honaucin A reacted with glutathione (data not shown).
Nrf2-ARE/EpRE pathway activators often share the common
structural feature of an electrophilic Michael acceptor motif.
This motif enables electrophilic Nrf2-ARE activators to react
covalently with cysteine residues on Keap1 through S-
alkylation. The sulfhydryl group of cysteine is ionizable and
can be deprotonated to yield a reactive thiolate group; in
addition, the local protein environment around cysteine
sulfhydryl residues can stabilize nucleophilic thiolates.40
Cysteine-mediated reactivity and post-translational modifica-
tion of Keap1 make it an ideal environmental sensor able to
detect reactive electrophile species (RES) (and also oxidants
such as reactive oxygen species and reactive nitrogen species) in
the reducing cellular environment. Potential threats may then
be neutralized to avoid damage to the cell.
Honaucin A is composed of one Michael acceptor system, an
unsaturated ester motif with an electron-withdrawing group
(Figure S2). We hypothesize that honaucin A’s structural
architecture and electronic configuration facilitate interaction
with sulfhydryl groups on Keap1, thereby liberating Nrf2 to
enter the nucleus and initiate transcription of cytoprotective
genes. Mass spectrometric (MS) analysis of the reaction
products that resulted from the interaction of honaucin A and
NAC confirmed the ability of the marine natural product to
interact with cysteine through 1,4-conjugate addition in the
manner predicted. This indicates that honaucin A might also
alkylate the cysteine thiols of Keap1 to activate Nrf2.
Preparation of a fluorescent honaucin A analogue and
observation of its cellular localization in murine macrophages
demonstrated that the compound was extranuclear, supporting
its activation of Nrf2 through Keap1 engagement (Supple-
mentary Experimental Procedures, Figures S3−S5). The
concept that honaucin A operates through this mechanism is
also supported by the results of a previous structure−activity
relationship study in which structural alterations to the
molecule’s Michael acceptor system diminished its ability to
attenuate LPS-induced nitric oxide production in murine
macrophages.13
Honaucin A, at all but the highest concentration (15 μM),
caused Nrf2 stabilization and subsequent binding to the ARE/
EpRE comparable to that caused by tBHQ and DMF. The
contribution of some cytotoxicity at higher compound
concentrations confounded the interpretation of honaucin A-
induced Nrf2 activity levels in the reporter assay at these doses
(Figure 4B). tBHQ and DMF were exempt from major
cytotoxicity and displayed a linear increase in activation with
the greatest activation occurring at the highest concentrations.
ARE/EpRE promoter binding confirmed that honaucin A
activates the Nrf2-ARE pathway. More robust pathway
activation at lower concentrations was in contrast to higher
concentrations of honaucin A at which cytotoxicity prevailed.
These findings mimicked prior results from in vitro assays of
nitric oxide inhibition13 and the in vivo ear edema assay results
reported here. Bell-shaped dose−response curves are not
uncommon for Nrf2 activators.35,36 It appears that an Nrf2
stabilizing effect predominates at low honaucin A concen-
trations, whereas at higher concentrations, other factors
contribute to a shift toward cytotoxicity.
Although the precise cause of honaucin A cytotoxicity at
higher concentrations is unknown, it has generally been
recognized that electrophiles can exert either cytoprotective
or cytotoxic effects. When cytoprotective effects predominate
and cells detoxify alkylating agents through Nrf2 stabilization,
this has been termed “electrophile counterattack”.37 In contrast,
some electrophiles decrease the overall reductive capacity of the
cell and thereby induce apoptosis.37 This can occur if the
electrophile reacts with the sulfhydryl groups of glutathione,
compromising the cell’s response to oxidants and alkylating
agents. Other mechanisms through which this type of
electrophile response can occur include macromolecule
damage, reactive oxygen species production, dysregulation of
signaling pathways, and mitochondrial impairment.37,38
Gloire et al. have proposed a hierarchical model of oxidative
stress, wherein the cellular response to a stressor is dependent
upon the intensity of the stressor.39 Low stress initiates
signaling through Nrf2, intermediate stress engages NFκB, AP-
1, and MAP kinase pathways, and high levels of oxidative stress
overwhelm the electrophile counterattack response and
culminate in cell death. This high stress type of response is
typified by strong electrophiles or by persistent electrophile
bombardment. Electrophile counterattack is generally elicited
by weak electrophiles such as tBHQ and DMF and, as shown
here, honaucin A.
The lack of in vitro reaction between glutathione and
honaucin A in our hands suggests that honaucin A does not
exert toxic effects through depletion of the cell’s reductive
capacity. Rather, its initiation of Nrf2-ARE signaling as well as
its induction of phase II enzymes and glutathione synthesis
signify that at low doses it is a weak electrophile that elicits an
electrophile counterattack from cells. Cytotoxicity at higher
concentrations may arise from an independent mechanism.
Broader Implications of Nrf2-ARE Activation for
Honaucin A Bioactivity. The Nrf2-ARE signaling pathway
is highly conserved among eukaryotes.41 Bacteria also possess
an oxidative stress system that relies on sulfur chemistry.42,43 It
has been shown that, in addition to maintaining cellular redox
balance, reactive thiols in bacteria are used in signaling to
initiate expression of genes not directly involved in redox
homeostasis. Vibrio cholerae employs one such thiol switch to
Covalent Interaction of Honaucin A and N-Acetylcys-
teine and a Proposed Structural Basis for Honaucin A
Nrf2 Activation. One mechanism of Nrf2 stabilization by
small molecules involves their covalent interaction with the
cysteine residues of the Kelch-like ECH-associated protein 1
(Keap1) repressor.23 We hypothesized that honaucin A might
also interact with Keap1 in this way. For this hypothesis to be
tested, honaucin A was incubated with the cysteine-bearing
small molecules N-acetylcysteine (NAC) and reduced gluta-
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activate expression of virulence-related genes.44 Perhaps of even
greater interest, redox sensing and the quorum sensing
accessory gene regulator (agr) signaling system are linked in
Staphylococcus aureus.45 Oxidation of Cys-199 on the quorum
sensing response regulator AgrA induces transcription of genes
associated with oxidative stress resistance. Also related to
quorum sensing, halogenated furanone natural products from
the red alga Delisea pulchra were shown to alkylate the cysteine
residues on LuxS and thereby inactivate AI-2 quorum sensing.46
We have previously observed quorum sensing inhibition in
Vibrio harveyi by honaucin A.13 It may be that this activity as
well is regulated through modification of cysteine thiols,
thereby providing a structural and chemical basis for
interkingdom signaling by honaucin A.47
In conclusion, honaucin A has been shown to inhibit
inflammation both in vitro and in vivo. Experimental evidence
indicates that, at low doses, honaucin A targets the NRF2-ARE/
EpRE pathway and activates cytoprotective genes, generating
an anti-inflammatory response. Further investigation of
honaucin A as an anti-inflammatory agent would be of great
interest.
preparation using standard Illumina reagents and protocols, Illumina
Hiseq2000 sequencing was completed to generate 100 base pair single-
end reads. RNA quality assessment, library preparation, and
sequencing were completed at the Next Generation Sequencing
Core at The Scripps Research Institute in La Jolla, California. Data
were deposited to the National Center for Biotechnology Information
Gene Expression Omnibus database (GEO: GSE93558).
Read Processing and Differential Expression Analysis.
Quality of the sequenced reads was inspected using FastQC
(http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Se-
quencing adapters were removed from reads using the fastx toolkit
clipper tool (http://hannonlab.cshl.edu/fastx_toolkit/index.html).
Quality trimming was done using Prinseq (http://prinseq.
sourceforge.net/). All reads with a phred quality score less than 20
and all reads shorter than 55 base pairs after polyA trimming were
excluded from subsequent analysis. The ultrafast universal RNA-seq
aligner STAR was used to map reads to the Ensembl Mus musculus
GRCm38.75 reference genome.48 The Python package HTSeq
(http://wwwhuber.embl.de/HTSeq/doc/overview.html), specifically
htseq-count, was used to quantify the number of reads that mapped
to each feature in the genome.49 Once counts were obtained for the
different experimental treatments, the R statistical package DESeq2
(https://bioconductor.org/packages/release/bioc/html/DESeq2.
html) was used to perform differential expression analysis.50 This was
accomplished by performing pairwise comparisons of each treatment
to its appropriate LPS control.
NRF2 Luciferase Reporter MCF7 Stable Cell Line Assay.
NRF2 luciferase reporter MCF7 stable cells (Signosis) were grown in
Dulbecco’s modified Eagle medium (Gibco) supplemented with 10%
fetal bovine serum, 10,000 units/mL of penicillin, 100 μg/mL of
streptomycin, and 75 μg/mL of G418 (Life Technologies). The day
before the assay, cells were seeded into white 96-well flat-bottomed
plates at a density of 5 × 104 cells/220 μL/well and allowed to adhere
and grow overnight at 37 °C with 5% CO2. Honaucin A and controls
were solubilized in DMSO and added to the plate the following day in
a volume of 2 μL. Concentrations tested ranged from 1.98 to 15.0 μM.
The known Nrf2-ARE/EpRE activators tBHQ and DMF served as
positive controls and DMSO as the negative control. After compound
addition, the plates were again incubated overnight at 37 °C with 5%
CO2. After incubation, the media were removed, and the cells were
washed in phosphate buffered saline. Twenty-five microliters of passive
lysis buffer (Promega) was added to cells, which were then incubated
at room temperature for 15 min. Firefly luciferase substrate (Signosis)
was added via injector, and the relative luminescence of each well was
read using a GloMax Microplate Luminometer (Promega). Three
biological replicates of honaucin A and tBHQ and two biological
replicates of DMF, each consisting of three technical replicates, were
assayed.
Relative luminescence units (RLU) for the technical replicates were
averaged and compared to the average RLU of the untreated cells to
arrive at the percentage of Nrf2-ARE activation over background via
the formula (treatment − cell background/cell background) × 100.
Biological replicates were then averaged, and the values for DMSO
vehicle-treated cells were subtracted from the overall percentage for
each treatment. Standard error for each treatment was calculated.
MTT Assay for Cell Viability. 3-(4,5-Dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT, Sigma, St. Louis, MI) was used
to measure the viability of the treated MCF7 cells. NRF2 luciferase
reporter MCF7 stable cells (Signosis) were grown, seeded, and treated
in an identical manner as for the luminescent assay with the exception
that clear plates were used. The day after cell treatment, medium was
aspirated, and the cells were washed with PBS. Then, 60 μL of 1 mg/
mL MTT in serum-free DMEM was added to each well, and the plates
were incubated for 25 min at 37 °C. After the incubation, the medium
with MTT was aspirated, and the plates were dried. Then, 100 μL of
DMSO was added to each well, and the absorbance was measured at
630 and 570 nm. Background absorbance was subtracted, and viability
for each treatment was calculated as a percentage of cell survival
compared to that of an untreated control. Data are expressed as mean
standard error.
EXPERIMENTAL SECTION
■
General Experimental Procedures. Synthetic honaucin A was
used in all assays and measurements. For bioassays, lipopolysacchar-
ides from Escherichia coli (O26:B6) as well as phorbol 12-myristate 13-
acetate and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide were purchased from Sigma-Aldrich (St. Louis, MO). NRF2
luciferase reporter MCF7 stable cells were purchased from Signosis
(Santa Clara, CA). RNA sequencing was carried out using an Illumina
Hiseq2000 (San Diego, CA) following RNA quality assessment via
Bioanalyzer 2100 (Agilent, Santa Clara, CA). For in vitro alkylation
experiments, reaction products were separated via liquid chromatog-
raphy using a Phenomenex Kinetex 5 μm EVO C-18 column. High-
resolution mass analysis was accomplished with an Agilent 6230 ESI-
TOF-MS operating in positive mode.
Dose Response Ear Edema Assay. Eight-week old female CD1
mice were divided into treatment groups of six animals. Animals were
anesthetized with isoflurane, and the underside of the right ear was
treated with 10 μL of either water/ethanol/polysorbate 20 vehicle or
honaucin A at 3.0, 1.0, 0.3, or 0.1 μmol/ear. The underside of the left
ear was treated with vehicle only. Ten minutes after application of the
compound and/or vehicle, 1 μg of PMA in 10 μL was applied to the
underside of both ears except in the case of a PMA-only control. For
this control, no test compounds were used, and the irritant was applied
to the right ear and vehicle was applied to the left ear. At 6 h, the
animals were euthanized, and 6 mm biopsy punches were collected
from both ears and weighed. The percent inhibition of edema was
calculated as (vehicle − treatment/vehicle) × 100. Statistical
significance was evaluated with GraphPad Prism 6 software using
the one-tailed Wilcoxon signed rank test. The significance level was set
at 0.05, and p-values were rounded to two decimals. The experiment
was carried out under a protocol approved by the Animal Subjects
Committee at the University of California, San Diego.
Treatment of RAW 264.7 Cells with Honaucin A and RNA
Isolation. RAW 264.7 murine macrophages were grown in Dulbecco’s
modified Eagle medium (Gibco) supplemented with 10% fetal bovine
serum, penicillin, and streptomycin. Cells were seeded in 6-well plates
and grown to near confluency. EtOH or honaucin A (4 or 12 μM) in
EtOH were added to the appropriate wells, and plates were incubated
for 1 h at 37 °C with 5% CO2. Lipopolysaccharide (0.5 μg/mL) was
then added to all treatments. Three replicates comprised each
treatment. The treated cells were incubated for 30 or 180 min after
which total RNA was isolated and purified with TRIzol (Life
Technologies) according to the manufacturer’s protocol.
RNA Sequencing. RNA quality was determined using an Agilent
Technologies Bioanalyzer 2100. Following rRNA depletion and library
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In Vitro Alkylation of Honaucin A. N-Acetyl-L-cysteine (NAC,
Spectrum Chemical) was dissolved in milli-Q water, and synthetic
honaucin A was solubilized in DMSO. Honaucin A was incubated with
NAC in either 2- or 50-fold excess with stirring under argon for 2 h at
room temperature (after Wang et al., 2013).22 At the conclusion of the
reaction, vial contents were passed over a Bond Elut-C18 SPE column
(Agilent) that had been washed with 3 column volumes of methanol
and then equilibrated with 3 column volumes of water. The reaction
products were subsequently eluted with increasing percentages of
methanol. Elutions were dried using rotary evaporation and then
reconstituted at a concentration of 1 mg/mL in 50:50 methanol/water.
This preparation was analyzed for the presence of the hypothesized
addition products via high-resolution MS at the Molecular Mass
Spectrometry Facility at the University of California, San Diego.
Briefly, reaction products were separated via liquid chromatography
using a Phenomenex Kinetex 5 μm EVO C-18 column prior to being
introduced to an Agilent 6230 ESI-TOF-MS running in positive mode
for high-resolution mass measurement.
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ASSOCIATED CONTENT
* Supporting Information
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Accession Codes
All data used in the study may be accessed through the
National Center for Biotechnology Information Gene Ex-
pression Omnibus database (GEO: GSE93558).
AUTHOR INFORMATION
Corresponding Authors
*For bioinformatics, e-mail: tgaasterland@ucsd.edu.
*For other information, e-mail: lgerwick@ucsd.edu.
■
ORCID
(25) Dinkova-Kostova, A. T.; Holtzclaw, W. D.; Cole, R. N.; Itoh, K.;
Wakabayashi, N.; Katoh, Y.; Yamamoto, M.; Talalay, P. Proc. Natl.
Acad. Sci. U. S. A. 2002, 99, 11908−11913.
Hyukjae Choi: 0000-0002-7707-4767
William H. Gerwick: 0000-0003-1403-4458
Lena Gerwick: 0000-0001-6108-9000
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Kaneko, H.; Nakayama, Y.; Eguchi, M.; Wada, Y.; Kumagai, Y.;
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Notes
The authors declare no competing financial interest.
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RNA sequencing and subsequent data analyses were supported
by NIEHS/NIH R21ES024105 and NEI/NIH R01EY022306
to T.G. NIH CA100851 and NIH ES024105 to L.G. Additional
research support was provided to S.J.M. through NIH Marine
Biotechnology Training Fellowship NIH GM067550, P.E.O.
Scholar Award, Claude E. Zobell Fellowship, Robert Scripps
Fellowship, SIO Cheng An Lun Fellowship, SIO Haymet First
Year Fellowship, and Wyer Family Fellowship.
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