Chemistry and biology of deoxynyboquinone, a potent inducer of cancer cell death

Article abstract of DOI:10.1021/ja100610m

Deoxynyboquinone (DNQ) is a potent antineoplastic agent with an unknown mechanism of action. Here we describe a facile synthetic route to this anthraquinone, and we use this material to determine the mechanism by which DNQ induces death in cancer cells. DNQ was synthesized in seven linear steps through a route employing three palladium-mediated coupling reactions. Experiments performed on cancer cells grown in hypoxia and normoxia strongly suggest that DNQ undergoes bioreduction to its semiquinone, which then is re-oxidized by molecular oxygen, forming superoxide that induces cell death. Furthermore, global transcript profiling of cells treated with DNQ shows elevation of transcripts related to oxidative stress, a result confirmed at the protein level by Western blotting. In contrast to most other antineoplastic agents that generate reactive oxygen species (ROS), DNQ potently induces death of cancer cells in culture, with IC₅₀ values between 16 and 210 nM. In addition, unlike the experimental therapeutic elesclomol, DNQ is still able to induce cancer cell death under hypoxic conditions. This mechanistic understanding of DNQ will allow for a more comprehensive evaluation of the potential of direct ROS generation as an anticancer strategy, and DNQ itself has potential as a novel anticancer agent.

Full text of DOI:10.1021/ja100610m

Published on Web 03/26/2010
Chemistry and Biology of Deoxynyboquinone, a Potent
Inducer of Cancer Cell Death
Joseph S. Bair, Rahul Palchaudhuri, and Paul J. Hergenrother*
Department of Chemistry, Roger Adams Laboratory, UniVersity of Illinois at
Urbana-Champaign, Urbana, Illinois 61801
Received January 22, 2010; E-mail: hergenro@illinois.edu
Abstract: Deoxynyboquinone (DNQ) is a potent antineoplastic agent with an unknown mechanism of action.
Here we describe a facile synthetic route to this anthraquinone, and we use this material to determine the
mechanism by which DNQ induces death in cancer cells. DNQ was synthesized in seven linear steps
through a route employing three palladium-mediated coupling reactions. Experiments performed on cancer
cells grown in hypoxia and normoxia strongly suggest that DNQ undergoes bioreduction to its semiquinone,
which then is re-oxidized by molecular oxygen, forming superoxide that induces cell death. Furthermore,
global transcript profiling of cells treated with DNQ shows elevation of transcripts related to oxidative stress,
a result confirmed at the protein level by Western blotting. In contrast to most other antineoplastic agents
that generate reactive oxygen species (ROS), DNQ potently induces death of cancer cells in culture, with
IC50 values between 16 and 210 nM. In addition, unlike the experimental therapeutic elesclomol, DNQ is
still able to induce cancer cell death under hypoxic conditions. This mechanistic understanding of DNQ will
allow for a more comprehensive evaluation of the potential of direct ROS generation as an anticancer
strategy, and DNQ itself has potential as a novel anticancer agent.
Reactive oxygen species (ROS) are generated through mech-
anisms incidental to cellular respiration, or from environmental
insults such as UV irradiation.1 The predominant initial reactive
species is the superoxide radical anion (O2•-), produced by the
one-electron reduction of molecular oxygen, generally by
mitochondrial complex I or III of the electron transport chain.
As part of the endogenous antioxidant system, superoxide
dismutase converts superoxide radicals into less reactive
hydrogen peroxide, which is further degraded to water and
oxygen by catalase or reduced by glutathione as mediated by
glutathione peroxidase.
notion that cancer cells already have elevated ROS levels and
therefore have a reduced antioxidant capacity relative to normal
cells.2,7 Thus, an increase in ROS sufficient to kill cancer cells
would still be within the antioxidant capacity of healthy cells.
There is reasonable data to support this hypothesis, and indeed
a handful of compounds whose mode of action is strongly
related to ROS induction are in advanced clinical trials
(elesclomol, fenretinide, motexafin gadolinium, menadione,
ꢀ-lapachone) or are FDA approved (As2O3) for the treatment
of cancer (Figure 1A).8-10 Notably, fenretinide,11,12 motexafin
gadolinium,13 menadione,14-19 ꢀ-lapachone,20,21 and As2O322-25
Cancer cells are known to have elevated levels of ROS.2 This
increase is due in large part to increased metabolism and, hence,
increased superoxide formation in the mitochondria. The role
of hydrogen peroxide in activating pro-growth pathways3-6 is
consistent with evidence that suggests increased ROS levels
correlate with aggressive tumor growth.2-4 It has been postu-
lated that disrupting the equilibrium concentration of ROS in
cancer cells may be an effective anticancer strategy.2,5-8 The
cancer-selective toxicity of enhanced ROS levels rests on the
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A R T I C L E S
Bair et al.
Figure 1. (A) Anticancer experimental therapeutics that have ROS
generation as part of their mechanism of cell death induction. (B) Quinones
can be bioreduced to semiquinones via a one-electron process. In the
presence of oxygen the quinone can be regenerated, forming superoxide.
Alternatively, a two-electron bioreduction of quinones to leads to the
hydroquinone anion.36
Figure 2. (A) Some anticancer quinones. (B) The structures of DNQ and
SCH 538415.
are relatively modest inducers of death in cancer cells, with IC50
values in the low micromolar range versus cancer cell lines in
culture. Importantly, some of the most drug-refractory cancers
are known to have the highest levels of ROS, including
metastatic melanoma and pancreatic cancer.8,26-28
A mechanism for intracellular ROS generation is the bio-
reduction and subsequent oxidation of a quinone, typically
occurring through a one-electron mechanism (Figure 1B). The
semiquinone can then be oxidized by molecular oxygen, forming
superoxide and the parent quinone. A complicating factor in
determining the value of quinone redox cycling in cancer therapy
is that quinone-containing anticancer agents (Figure 2A) typi-
cally have other more important modes of action such as DNA
alkylation (e.g., mitomycin C, EO9),29-32 Hsp90 inhibition (e.g.,
geldanamycin),7,33 and topoisomerase inhibition (e.g., strep-
tonigrin, doxorubicin, mitoxantrone).34,35 These mixed modes
of action make it difficult to assess the effectiveness of a single
anticancer strategy.
Anthraquinone 1 (Figure 2B) was originally synthesized
during studies of the antibiotic nybomycin;37 we have named
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Chemistry and Biology of Deoxynyboquinone
A R T I C L E S
Scheme 1. Retrosynthetic Analysis of SCH 538415
this compound deoxynyboquinone (DNQ). The only published
biological study on DNQ showed that it potently induces
apoptosis in cancer cell lines through cytochrome c release.38
Although cells treated with DNQ contained ROS, it was not
known if ROS was a cause or effect of the antineoplastic activity
of DNQ.38 A nearly identical compound, SCH 538415, is a
natural product that has recently been isolated and characterized
(compound 2, Figure 2B).39,40 Interestingly, assays reveal DNQ
to be ∼10-fold more potent in inducing death of cancer cells
than SCH 538415,38,39 although they differ in structure by only
a single methyl group.
Intrigued by the potency of DNQ and the potential of ROS-
generating compounds as anticancer agents, we sought to
develop a concise and modular synthesis of DNQ and to use
the resulting material in mode-of-action studies. Herein is
detailed a convenient synthetic route to DNQ, followed by the
evaluation of this compound in a battery of biological experi-
ments. These experiments reveal DNQ to be an extremely potent
antineoplastic agent that induces death of cancer cells primarily
through a ROS-based mechanism, likely due to a uniquely stable
semiquinone species that enables facile ROS generation. As the
only known quinone that potently induces cancer cell death
predominately through a ROS-based mechanism, DNQ will be
an excellent tool by which to further probe the value of direct
ROS generation as an anticancer strategy and itself has potential
as a therapeutic agent.
devise a new route to the diazaanthraquinones that draws heavily
on palladium-mediated cross-couplings, shown retrosynthetically
for SCH 538415 in Scheme 1. Tricycle 3 was envisioned
through an intramolecular aryl amidation of intermediate 4
preceded by a double intermolecular Suzuki coupling between
vinyl iodide 5 and aryl bis(pinacolboronate) 6. Bisboronate 6
would be formed by a double Miyaura borylation of tetrahalide
7, which was predicted to arise from a double directed ortho-
metalation/silylation of 2,6-dichloroanisole to make 8 followed
by iododesilylation by an electrophilic iodine reagent.
Isomerically pure ꢀ-iodoamide coupling partner 5 was easily
accessed on a multigram scale starting from ethyl 2-butynoate
(Scheme 2). Direct amidation with methylamine in methanol
produced alkyne 9 in 87% yield. Treatment of 9 with sodium
iodide in acetic acid produced 5 in 96% yield, with only the
Z-isomer being detected by NMR.44
The specific disposition of halides in compound 7 was
envisioned to arise through directed ortho-lithiation of 2,6-
dichloroanisole. As diiodination under such conditions is
difficult, intermediate disilane 8 was targeted. Chloride is known
to be a weak directing group for ortho-lithiation but has been
successfully utilized in a number of settings.45,46 However,
lithium-chloride exchange appeared to be the dominant reaction
in a variety of n- and sec-butyllithium-mediated reactions,
presumably due to the strong directing effects of the methoxy
group. Fortunately, deprotonation with lithium diisopropyl amide
(LDA) was both efficient and selective for the 3- and 5-positions
(Scheme 2). In addition, the two-step sequence could be carried
out in one pot. Trimethylsilyl chloride was an effective in situ
quench reagent, with highest conversions when additions of
reagent were sequential, beginning with LDA.47 Iododesilylation
of 8 by the action of iodine monochloride was rapid and
quantitative, producing 7.
Results
Synthesis. The synthesis of SCH 538415 was first developed
and then applied to the nonsymmetric DNQ. Although synthetic
routes analogous to those used to make structurally related
compounds were considered,41-43 we ultimately elected to
Miyaura borylation conditions then provided cross-coupling
partner 6 in good yield but contaminated with variable amounts
of bis(pinacolborane).48 The two-step yield after the subsequent
cross-coupling was higher when 6 was used without purification;
thus, after workup, 6 was typically taken directly into the cross-
(37) Rinehart, K. L.; Renfroe, H. B. J. Am. Chem. Soc. 1961, 83, 3729–
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Scheme 2. Total Synthesis of SCH 538415
coupling. The Suzuki coupling between 5 and 6 is a challenging
process representing two individual cross-coupling events and
linking a relatively unactivated aryl boronate with base-sensitive
vinyl iodides. This reaction provided product 4 in a 55% yield.
A brief survey of known conditions for aryl chloride coupling
with amides revealed a Pd/X-Phos precatalyst recently described
by Buchwald,49 and this reagent was used to convert 4 to
diazaanthracene 3 in 96% yield. Oxidation of 3 by brief heating
in concentrated nitric acid produced SCH 538415 as a bright
red-orange solid in 40% yield. By this route the first total
synthesis of the natural product was completed in six steps and
9.7% overall yield from 2,6-dichloroanisole. Spectral data matched
those of the natural product (see Supporting Information).
To apply this route to the synthesis of DNQ, primary amide
11 was synthesized but found to be an unreactive partner in the
Suzuki coupling with 6 (Scheme 3). The N-p-methoxybenzyl
amide 13 was then synthesized in 82% yield from 2-butynoic
acid by hydroiodination44,50 and treatment of the corresponding
acid chloride with p-methoxybenzyl amine (Scheme 3). This
route was employed because the reaction of ethyl 2-butynoate
with p-methoxybenzyl amine resulted primarily in 1,4-addition
to the alkyne.
acidic hydrolysis produced 17 in 76% yield over two steps.
Oxidation of 17 catalyzed by salcomine under O2 produced
DNQ in 77% yield. The synthesis of DNQ consisted of seven
steps in the longest linear sequence, 12% overall yield. This
compares to 11 steps and <0.84% yield for the previous
synthesis of DNQ.37,51
Activity versus Cancer Cells in Culture. The abilities of DNQ
and SCH 538415 to induce death of cancer cell lines in culture
were determined using the sulforhodamine B assay.52 For these
experiments, four cancer cell lines were used: SK-MEL-5
(human melanoma), MCF-7 (human breast cancer), HL-60
(human leukemia), and HL-60/ADR (doxorubicin-resistant HL-
60). In addition to DNQ and SCH 538415, doxorubicin (DOX),
elesclomol, fenretinide, ꢀ-lapachone, and arsenic trioxide were
evaluated to obtain a side-by-side comparison with compounds
that have ROS generation as part of their mechanism of
anticancer activity. As shown in Table 1, DNQ potently induces
death in these cancer cell lines, with nanomolar toxicity similar
to that of the most potent compounds, DOX and elesclomol.
DNQ is on average an order of magnitude more potent than
SCH 538415 against all cell lines. Importantly, while the HL-
60/ADR cells are extremely resistant to DOX (∼80-fold), they
are only minimally resistant (∼3-fold) to DNQ.
A mixed cross-coupling between bisboronate 6 and iodo-
amides 5 and 13 was found to be the simplest method to form
the nonsymmetric diamide 14. Separation of 14 from the
accompanying symmetric products was easily effected by
chromatography. Aryl amidation under the previously employed
conditions efficiently formed tricycle 15 along with variable
amounts of unprotected amide 16. Isolation at this step was
unnecessary, as subjection of the crude amidation products to
Hypoxia and Antioxidants. A 2003 study comparing the
biochemical and antineoplastic properties of multiple quinones
suggested that DNQ has an unusually stable semiquinone and
that ROS are generated in cells treated with DNQ.38 Thus, DNQ
could generate ROS through redox cycling in the presence of
O2 (as shown in Figure 1B), ROS could be generated through
another mechanism, or ROS could be a product of the cell death
(49) Biscoe, M. R.; Fors, B. P.; Buchwald, S. L. J. Am. Chem. Soc. 2008,
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5013.
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Chemistry and Biology of Deoxynyboquinone
A R T I C L E S
Scheme 3. Synthesis of DNQ
Table 1. Toxicity of Compounds to Cancer Cell Lines in Culturea
Table 2. IC50 Values for Compounds versus HeLa Cells under
Normoxic (20% O2) and Hypoxic (1% O2) Conditions in the
HL-60b
HL-60/ADR
Presence and in the Absence of NACa
SK-MEL-5
MCF-7
DNQ
0.024 ( 0.002 0.016 ( 0.006 0.21 ( 0.13 0.67 ( 0.26
0.87 ( 0.16 1.30 ( 0.04 2.5 ( 0.8
0.33 ( 0.04 0.064 ( 0.004 5.1 ( 2.1
normoxia
+ 5 mM NAC
hypoxia
+ 5 mM NAC
normoxia
hypoxia
SCH 538415 0.76 ( 0.25
DOX
elesclomol
fenretinide
ꢀ-lapachone
As2O3
0.16 ( 0.02
DNQ
0.20 ( 0.02 0.75 ( 0.06 1.80 ( 0.44 5.1 ( 1.3
0.11 ( 0.04 0.024 ( 0.010 0.009 ( 0.004
fc ) 3.8
fc ) 8.4
5.2 ( 2.4
fc ) 3.6
fc ) 26
7.9 ( 3.9
fc ) 5.4
>10
12.5 ( 0.7
1.5 ( 0.1
5.9 ( 1.6
3.2 ( 1.9
0.72 ( 0.26
3.7 ( 1.6
SCH 538415 1.40 ( 0.25 1.4 ( 0.2
fc ) 1.0
1.0 ( 0.4
2.8 ( 1.0
DOX
0.10 ( 0.02 0.12 ( 0.02 0.05 ( 0.01 0.07 ( 0.03
a Cell lines were incubated with compound for 72 h, biomass was
quantified using the SRB assay, and IC50 values were calculated from
logistical dose-response curves. All IC50 values are in µM. Error is
standard deviation of the mean, n g 3. b Cell viability assessed by the
MTS assay.
fc ) 1.2
1.2 ( 0.2
fc ) 1.1
fc ) -2.2
0.52 ( 0.17 0.64 ( 0.27
fc ) -2.1 fc ) -1.6
fc ) -1.4
etoposide
mitomycin C
menadione
As2O3
1.3 ( 0.7
0.10 ( 0.03 0.14 ( 0.04 0.06 ( 0.02 0.12 ( 0.04
fc ) 1.40
fc ) -1.5
fc ) 1.3
22.0 ( 5.4
fc ) 4.0
6.0 ( 1.8
fc ) 4.5
6.1 ( 3.2
fc ) 5.4
5.9 ( 1.2
19.0 ( 2.4 7.0 ( 0.7
fc ) 3.5
fc ) 1.3
2.3 ( 1.1
fc ) 1.8
3.8 ( 1.0
fc ) 3.2
process. To explore the connection between ROS and cell death,
cytotoxicity evaluations were made in the presence of antioxi-
dants and under different partial pressures of O2. Antioxidants
can quench ROS, and thus antioxidant treatment may decrease
the cytotoxicity of an anticancer compound for which ROS is
critical to cell death. In a similar fashion, if a compound
generates cytotoxic ROS through redox cycling with O2 (Figure
1B), cells grown in hypoxia should be less sensitive to the effect
of the compound. Thus, the effects of DNQ and other cytotoxins
were evaluated against HeLa cells in normoxia (∼20% O2), in
the presence of the antioxidant N-acetylcysteine (NAC, 5 mM),
under hypoxia (1% O2), or combined with NAC under hypoxia.
Tirapazamine, a compound whose cytotoxicity is more pro-
nounced under hypoxia,30,53-55 was utilized as a control.
The most striking results were found for DNQ and elesclomol
(Table 2 and Figure 3). NAC provided significant protection
1.30 ( 0.25 3.7 ( 1.2
fc ) 2.9
ꢀ-lapachone
fenretinide
elesclomol
tirapazamine
1.2 ( 0.2
1.6 ( 0.5
fc ) 1.3
4.4 ( 1.2
4.0 ( 0.5
fc ) -1.1
4.40 ( 0.95 3.90 ( 0.87
fc ) 1.0
fc ) -1.1
X
0.012 ( 0.004 0.26 ( 0.21 Xb
fc ) 20
38 ( 6
42 ( 9
fc ) 1.1
1.9 ( 0.3
fc ) -20
2.10 ( 0.06
fc ) -18
a Fold change (fc) is with respect to normoxia. Cells were treated
with compounds for 48 h, biomass was quantified using the SRB assay,
and IC50 values were calculated from logistical dose-response curves.
All IC50 values are in µM. Error is standard deviation of the mean, n g
3. b X indicates that cells were >50% viable at every concentration.
from both DNQ- and elesclomol-induced toxicity (4- and 20-
fold, respectively).56 Incubation under hypoxia also strongly
protected the cells from these two compounds, but whereas
complete cell death was still observed at the highest concentra-
tion of DNQ (10 µM), elesclomol-treated hypoxic cells were
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Transcript Profiling. To further investigate the mechanism
by which DNQ induces death in cancer cells, cells treated with
DNQ were analyzed by global transcript profiling, and the
pattern of transcriptional response was compared to that of
compounds with known modes of action. For this experiment,
U-937 cells were treated with DNQ or vehicle control at 350
nM for 6 h, at which point the mRNA was harvested and whole
genome transcript profiling was performed using the Illumina
HumanHT-12 array. This DNQ concentration and time point
were chosen such that the data could be readily compared with
the transcript profile data from cells treated with other com-
pounds, as outlined in the Connectivity Map.63 The Connectivity
Map consists of transcript profile data for >1300 compounds,
many of which have known cellular targets. Previous studies
have shown that, in many cases, compounds with similar modes
of action will induce similar patterns of transcriptional response
in cells.63,64 Comparing the transcript profile of DNQ with those
compounds in the Connectivity Map database63 showed that
none of the >1300 compounds strongly correlate with DNQ (see
Supporting Information Figure 1). Importantly, the Connectivity
Map database contains hundreds of cytotoxins and multiple
quinones including doxorubicin, daunorubicin, and mitoxantrone.
The top 20 up-regulated and repressed genes in response to
DNQ treatment are listed in Table 3. The largest individual
transcript elevated upon treatment of U-937 lymphoma cells
with DNQ was from the HMOX1 gene, encoding the antioxidant
enzyme heme oxygenase 1 (HO-1) (8.8-fold change, Table 3).
HO-1, a 32 kDa heat-shock protein, regulates cellular heme and
iron concentrations.65,66 Elevated levels of this protein prevent
cell death by converting heme to biliverdin, a potent antioxi-
dant.67 This conversion also results in the production of carbon
monoxide (a potential neurotransmitter) and free iron, which
serves as an oxidative stress signal.65 Biliverdin produced by
heme oxygenase is rapidly degraded into bilirubin, another
potent small-molecule antioxidant.67 Other transcripts elevated
in DNQ-treated cells include those for various ferritins, which
are under the transcriptional control of NRF2, a transcription
factor that may be activated under oxidative stress.68 Ferritins
are responsible for the storage of free iron in a non-toxic and
soluble form.68 Other oxidative stress-related transcripts that are
also elevated include oxidative stress-induced growth inhibitor
1 (OKL38) and sulfiredoxin 1 homologue (SRXN1).69,70 The
NRF2-oxidative stress pathway was also the pathway most
strongly implicated by Ingenuity Pathway Analysis software (see
Supporting Information Figure 2). In summary, global tran-
Figure 3. Effect of DNQ and elesclomol on HeLa cells in hypoxia and
normoxia, and in the presence of NAC. Cells were treated with compound
for 48 h, and death was determined via the SRB assay. Error is standard
deviation of the mean, n g 3.
>50% alive even at the highest concentrations (Figure 3). When
co-treated with NAC under hypoxic conditions, elesclomol was
essentially nontoxic, whereas DNQ, although 25-fold less toxic
than under normoxic conditions, still caused ∼60% cell death
at 10 µM (Figure 3). These results suggest that DNQ induces
cell death through a ROS-based mechanism that relies on the
production of superoxide radical anion from O2. SCH 538415
responded less strongly than DNQ in these assays, with no
protection being observed by co-treatment with NAC (Table
2).
The behavior of the other compounds was generally consistent
with literature reports (Table 2): tirapazamine became significantly
more toxic under 1% O2, whereas the toxicity of mitomycin C
(known to require more severe hypoxia to increase in potency)57
remained unchanged. As expected, both menadione and arsenic
trioxide were less toxic in the presence of NAC.22,23,58,59 The
remaining compounds are known to display variable and cell line-
dependent effects under these conditions.12,14-17,60-62
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Table 3. Top 20 Elevated and Repressed Transcripts from Treatment of U-937 Cells with DNQ versus 0.2% DMSO for 6 ha
symbol
protein
function
p value
fold change
HMOX1
ADM
heme oxygenase 1
adrenomedullin
interleukin 8
antioxidant
hypotensive and vasodilatator agent
inflammation
0
8.8
4.0
3.7
3.5
3.4
2.3
2.3
2.3
2.2
2.2
2.2
2.2
2.1
2.1
2.1
2.1
2.1
0.0000002
0
0
0.0000017
0.0000002
0
0.0000012
0.0000187
0.0000018
0
IL8b
IL8b
DDIT4
FTH1
interleukin 8
inflammation
DNA damage inducible transcript 4
ferritin, heavy polypeptide-like 1
cyclin-dependent kinase inhibitor 1A
ferritin, heavy polypeptide-like 12
ferritin, heavy polypeptide-like 8
facilitative glucose transporter
tumor necrosis factor
oxidative stress induced growth inhibitor 1
ferritin, heavy polypeptide-like 11
ferritin, heavy polypeptide-like 2
G0/G1 switch regulatory protein 19-2
sulfiredoxin 1 homologue
cell growth inhibition
intracellular iron storage
p53-dependent G1 phase arrest
intracellular iron storage
intracellular iron storage
glucose transport
death ligand
oxidative stress
intracellular iron storage
intracellular iron storage
cytokine
CDKN1A
FTHL12
FTHL8
SLC2A3
TNF
OKL38
FTHL11
FTHL2
CCL3L3
SRXN1
MAFB
0.0000005
0.0000399
0.0002126
0
oxidative stress
represses ETS1-mediated transcription
0
V-maf musculoaponeurotic fibrosarcoma oncogene
homologue B
0.0000005
CCL3
G0/G1 switch regulatory protein 19-1
glucose transporter type 14
HIV-suppressive factor
glucose transport
p53-mediated caspase activation/apoptosis
0.0000003
0.0000044
0.0000098
0.0000004
0.0000005
0.0000001
0.0000094
0.0000163
0.0002341
0.0000159
0.0000457
0.0000303
0.0000568
0.0003167
0.0016747
0.0001738
0.0005653
0.0045669
0.001486
2.1
2.0
2.0
SLC2A14
NDRG1
VCX
VCX-C
VCX3A
GJB2
N-myc downstream-regulated 1
variably charged protein X-B1
variably charged protein X-C
variably charged protein X-A
gap junction protein ꢀ 2
-2.0
-1.9
-1.9
-1.8
-1.7
-1.7
-1.7
-1.7
-1.6
-1.6
-1.6
-1.6
-1.5
-1.5
-1.5
-1.5
-1.5
-1.5
-1.5
-1.5
cell-to-cell channel for ion/small molecules transfer
monocyte maturation
resistance to TGF-ꢀ1 growth inhibition
MS4A7
RBBP9
ZFYVE26
DHRS9
RBM17
FARP1
CCDC26
CD47
MEF2D
CCDC136
CXCL10
CENPE
MS4A7
KIF20A
TXNIP
C5orf29
CD20 antigen
retinoblastoma-binding protein 9
zinc finger, FYVE domain containing 26
3-R-hydroxysteroid dehydrogenase
RNA binding motif protein 17
chondrocyte-derived ezrin-like protein
coiled-coil domain containing 2
CD47 molecule
myocyte enhancer factor 2D
coiled-coil domain containing 136
interferon-inducible cytokine IP-10
centromere protein E
retinoic acid biosynthesis
sickle cell anemia
membrane transport/signal transduction
transcription factor
immune cell migration
G2-phase motor protein
monocyte maturation
CD20 antigen
0.0002341
0.0005729
0.0252104
0.0003523
mitotic kinesin-like protein 2
thioredoxin-binding protein 2
GRB2-binding adaptor protein
oxidative stress mediator
transmembrane protein
a U-937 cells were treated with DNQ (350 nM) or 0.2% DMSO for 6 h, at which time the mRNA was isolated and the global transcriptional profile
was determined. b These are different probes for the same transcript.
scriptional profiling provides strong evidence that DNQ induces
the oxidative stress response, consistent with a ROS-based
mechanism of cell death.
To confirm that the treatment of cells with DNQ elevates
heme oxygenase 1 at the protein level, a Western blot for HO-1
was performed from cells treated with 0.5 or 1.0 µM DNQ for
6 h. As shown in Figure 4A, DNQ treatment elevated HO-1
protein levels in U-937 cells, an effect that could be substantially
prevented by co-treatment with the antioxidant NAC. The
induction of antioxidant genes encoding HO-1 and the ferritins
is likely in response to cellular ROS generated upon treatment
of cells with DNQ.
It has been noted that oxidative stress can activate the heat
shock response (HSR) expression pathway and upregulate
HSP-70.56,59,71 Thus, HSP-70 protein levels in cells treated with
DNQ were analyzed by Western blot. Both DNQ and elesclomol
caused an increase in HSP-70 levels in U-937 cells (Figure 4B),
consistent with oxidative stress-mediated upregulation of HSP-
Figure 4. DNQ treatment enhances the levels of proteins involved in the
oxidative stress response. U-937 cells were treated with DNQ and control
compounds, and the lysates were then probed by Western blot. (A) Western
blot for HO-1. (B) Western blot for HSP-70.
70.
Common biological properties of anthracycline-type com-
the S or G2/M phases, DNA binding, and topoisomerase
inhibition.34,72-74 Thus, various in Vitro and cell-based assays
were performed to determine the importance of these mecha-
nisms in cell death mediated by DNQ.
pounds similar in structure to DNQ include cell cycle arrest in
(71) Burdon, R. H.; Gill, V. M.; Rice-Evans, C. Free Rad. Res. Commun.
1987, 3, 129–139.
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A R T I C L E S
Bair et al.
Figure 6. (A) Ethidium bromide displacement assay reveals large differ-
ences between DNQ and doxorubicin. Ethidium bromide was preincubated
with herring sperm DNA prior to compound addition. Fluorescence was
measured 30 min after compound addition. Error bars represent the standard
deviation of the mean, n ) 3. (B) DNQ does not inhibit topoisomerase II
in Vitro. Topoisomerase II was added to a solution of catenated DNA, ATP,
and compound in assay buffer, and the reactions were allowed to proceed
for 30 min at 37 °C. Products were analyzed by agarose gel electrophoresis.
DNA Interaction. The capacity of DNQ to interact with DNA
was assessed using an ethidium bromide displacement assay;75,76
DOX and 9-aminoacridine (9-AA) were also evaluated for
comparison. Double-stranded DNA was preincubated with
ethidium bromide in buffer before compound addition (final
DMSO concentration, 5%). After 30 min the competition
reached equilibrium and the fluorescence was measured. As
shown in Figure 6A, DOX has a very strong effect in this assay,
while DNQ has a much weaker effect. Considering the nearly
equipotent cellular toxicity of DOX and DNQ, these data are
consistent with the notion that DNA binding is not of primary
importance in the antineoplastic action of DNQ.
Topoisomerase II Inhibition. The ability of DNQ to inhibit
topoisomerase II in Vitro was evaluated using a decatenation
assay77,78 and compared to the known topoisomerase II inhibi-
tors DOX and etoposide. For this assay, catenated DNA was
incubated at 37 °C for 30 min in a buffer containing purified
topoisomerase II, ATP, and various concentrations of compound
in DMSO. As shown in Figure 6B, DOX completely inhibited
the topoisomerase II reaction at 10 µM, while DNQ showed
Figure 5. (A) DNQ treatment leads to arrest of cells in the G1-phase of
the cell cycle, while (B) doxorubicin treatment leads to S-phase arrest. HL-
60 cells were treated with the indicated concentrations of compound (or
DMSO control) for 6 h, at which point DNA levels were analyzed by
propidium iodide staining. Error bars represent standard deviation of the
mean, n ) 3.
Cell Cycle Arrest. Modes of cell death may be broadly
categorized by their effects on the progression of actively
dividing cells through the cell cycle. For example, DNA
damaging agents and microtubule disrupters or stabilizers
frequently arrest cells in the mitosis (M) phase, whereas
compounds that inhibit DNA synthesis or replication arrest in
the synthesis (S) phase. Gene expression analysis of DNQ
revealed the elevation of several transcripts related to p53-
dependent pathways and G0/G1 cell cycle regulation (CDKN1A,
CCL3L3, CCL3, NDRG1, OKL38). The down-regulation of G2/
M-associated motor kinesin transcripts was also observed
(CENPE and KIF20A, Table 3). The effect of DNQ on cell
cycle progression was measured by treating HL-60 cells with
DNQ for 6 h (Figure 5A). Consistent with the transcript profiling
results, DNQ caused significant cell cycle arrest in the G1-phase.
In contrast, DOX-treated cells exhibited weak G2/M-phase arrest
at low concentrations and a pronounced S-phase arrest at higher
concentrations (Figure 5B).
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Chemistry and Biology of Deoxynyboquinone
A R T I C L E S
virtually no inhibition at this concentration; the aqueous
solubility of DNQ precluded its evaluations at higher concentra-
tions. These data, together with the transcriptional profiling, cell
cycle arrest, and DNA interaction assay results, support the
notion that DNQ-mediated death does not involve mechanisms
common to anthracycline-type compounds.
hydrogen peroxide,87,88 tert-butyl peroxide,87 elesclomol,56
motexafin gadolinium89,90) generally activate one or more of
these pathways. The transcript profiling data presented suggest
that DNQ strongly activates the NRF2 pathway, resulting in
upregulation of transcription from genes such as HMOX1 and
many ferritins which are downstream of antioxidant response
elements. Western blot analysis demonstrates that DNQ also
activates the HSR pathway, resulting in upregulation of HSP70.
Finally, experimental evidence indicates that common targets
of planar polycyclic compounds (such as the anthracyclines,
acridines, and ethidium bromide) are of minimal importance in
DNQ-mediated toxicity, as DNQ-treated cells show a lack of
S- or G2/M-phase arrest, no topoisomerase II inhibition, and
minimal DNA intercalation.
At least three important findings are derived from the
experiments described herein: (1) A facile synthetic route to
DNQ has been developed that can be used to produce large
quantities of the compound for animal testing and is flexible
enough to allow for derivative synthesis. (2) The data strongly
indicate that ROS is a direct cause, rather than a downstream
effect, of DNQ-induced cell death. (3) DNQ is considerably
more potent than other compounds that generate ROS through
a bioreduction process. Direct ROS formation is an anticancer
strategy that has generated much interest and success. However,
most compounds that act through a ROS-generating mechanism
(with the exception of elesclomol) are only moderately potent,
with IC50 values in the low micromolar range. As shown by
the data in Table 1 and Figure 3, elesclomol and DNQ are both
quite potent to multiple cancer cell lines under normoxic
conditions. However, under conditions of hypoxia (1% O2),
DNQ is still able to kill cancer cells in culture, whereas
elesclomol is almost completely ineffective. These data suggest
that even under low oxygen conditions, the DNQ semiquinone
is able to convert molecular oxygen to superoxide. This trait
has potential translational value, as hypoxic environments are
found in the interior of many solid tumors.53
In conclusion, a modern and modular synthetic route to DNQ
has allowed for the full investigation of the activity of this
compound versus cancer cells in culture. Notably, we find that
DNQ has a potency that rivals doxorubicin in cell culture, with
the added benefit of being effective against a doxorubicin-
resistant cell line. Transcript profiling and other data strongly
indicate that DNQ induces death through ROS generation and
oxidative stress, and it is considerably more potent than other
compounds that act through this mechanism. Importantly, DNQ
provides a complement to elesclomol, as both compounds induce
ROS and are potent antineoplastic agents, but through very
different mechanisms. Thus, with these two compounds there
is now an opportunity to probe the relative importance of one-
electron bioreduction versus copper chelation for the formation
of cytotoxic ROS. Given the documented elevation in ROS
levels in many tumor types, especially those cancers with few
treatment options such as melanoma and pancreas carcinoma,
the continued exploration of the therapeutic utility of ROS
generators is critical. DNQ is an important addition to the
repertoire of compounds used to study ROS generation as a
Discussion
The facile synthesis of DNQ described herein has allowed
for the comprehensive biological evaluation of this interesting
antineoplastic agent. As shown by cytotoxicity assays (Tables
1 and 2, Figure 3), DNQ induces death of cancer cells in culture
with potencies on par with the front-line anticancer drug
doxorubicin and the experimental therapeutic elesclomol. Among
compounds evaluated that are believed to induce death pre-
dominantly through a ROS-based mechanism of action, DNQ
and elesclomol are by far the most potent and respond the most
strongly to NAC and hypoxia. These latter results suggest that
DNQ and elesclomol most directly cause death by ROS
production and not by other mechanisms. However, despite these
commonalities, the mechanisms by which these compounds
produce superoxide appear to be very different. Elesclomol, a
clinically promising anticancer agent,79 is believed to produce
ROS through the chelation of copper and facilitation of copper
redox, resulting in superoxide formation.80,81 In contrast, DNQ
appears to induce death in cancer cells through rapid redox
cycling of the quinone, a process that directly generates
superoxide. ESR measurements of live cells minutes after
treatment with DNQ indicate the presence of a semiquinone.38
Semiquinones are, in general, not detected until all the oxygen
in the cell has been consumed through redox cycling;36 thus,
the semiquinone of DNQ must be remarkably stable. To the
best of our knowledge, DNQ is the most potent antineoplastic
agent that operates predominantly through this direct ROS
generation, bioreduction mechanism.
Additional evidence for the role of ROS in DNQ-mediated
cell death was uncovered by global transcript profiling. As
shown by the transcript profiles of a number of agents, oxidative
stress results in the upregulation of genes related to three
pathways: the oxidative stress response (NRF2),82 the heat shock
response,71 and metallothioneins.83,84 Small molecules that
induce oxidative stress (arsenic trioxide,59,85,86 menadione,87
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the gene transcript profiling and preliminary data processing. We
thank the W. M. Keck Cell Flow Cytometry facility at the
University of Illinois at Urbana-Champaign.
strategy for treatment of cancer and has potential as a novel
anticancer agent.
Acknowledgment. We thank Dr. Karson S. Putt (UIUC) for
initial work on DNQ. This research was supported by the University
of Illinois. We acknowledge Dr. Justin Lamb (Broad Institute) for
advice regarding experimental design of transcript profiling experi-
ments for comparison to the Connectivity Map Database. We thank
the W. M. Keck Center for Comparative and Functional Genomics
at the University of Illinois at Urbana-Champaign for performing
Supporting Information Available: Supporting figures, spec-
tral data for all new compounds, full experimental protocols,
and complete ref 63. This material is available free of charge
via the Internet at http://pubs.acs.org.
JA100610M
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