DNA Repair 12 (2013) 10001006

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DNA Repair
journal homepage: www.elsevier.com/locate/dnarepair

Brief communication

The toxicity of nitrofuran compounds on melanoma and

neuroblastoma cells is enhanced by Olaparib and ameliorated by
melanin pigment
Ewan M. McNeil, Ann-Marie Ritchie, David W. Melton
MRC Institute of Genetics and Molecular Medicine, University of Edinburgh, MRC Human Genetics Unit, Western General Hospital, Crewe Road, Edinburgh
EH4 2XU, UK

a r t i c l e

i n f o

Article history:
Received 25 June 2013
Received in revised form 29 August 2013
Accepted 31 August 2013
Available online 23 September 2013
Keywords:
ERCC1-XPF
Melanin
Melanoma
Neuroblastoma
Nitrofuran
PARP inhibitor

a b s t r a c t
Nitrofurans are commonly used for the treatment of trypanosomal diseases including Chagas disease.
More recently, following the fortuitous discovery that nifurtimox was clinically active against neuroblastoma, nitrofuran compounds are being investigated for activity against cancer. Herein, we show that
nitrofuran compounds are similarly potent to human malignant melanoma and neuroblastoma cells.
Furthermore, a recently discovered nitrofuran compound, NFN1, was 50- to 175-fold more potent than
nifurtimox against human melanoma and neuroblastoma cell lines. As nitrofuran compounds are known
to act as pro-drugs, producing DNA-damaging reactive intermediates upon activation, we investigated
the DNA repair pathways involved. We show that, contrary to research in Escherichia coli, the Nucleotide
Excision Repair pathway is not required to repair nitrofuran-induced DNA damage in mammalian cells.
Instead, we show that inhibiting repair of single-strand DNA breaks with the poly(ADP-ribose) polymerase (PARP) inhibitor, Olaparib, enhances nitrofuran toxicity in melanoma and neuroblastoma cells.
We propose that this is due to mammalian cells utilising Type 2 nitroreductases for nitrofuran activation
producing Reactive Oxygen Species which cause DNA damage that is repaired by the Single Strand Break
Repair and/or Base Excision Repair pathways, whereas in bacteria and trypanosomes, Type 1 nitroreductases are also utilised resulting in different DNA lesions. In addition we show that, consistent with
Reactive Oxygen Species being formed upon nitrofuran activation and the ability of melanin to absorb
Reactive Oxygen Species, production of melanin in melanoma cells offers some protection from NFN1and hydrogen peroxide-induced toxicity. Our data suggest that combinations of Olaparib and nitrofuran compounds may be advantageous for the treatment of melanoma and neuroblastoma, but that the
protection offered to melanoma cells by their melanin pigment must be taken into account.
2013 Elsevier B.V. All rights reserved.

1. Introduction
Nifurtimox is a nitrofuran compound widely used for the treatment of parasitic infections including Chagas disease, caused by
the Trypanosoma cruzi parasite. Chagas disease is an endemic parasitic disease in areas of Latin America where 7.7 million people are
infected with T. cruzi, with a further 109 million people living at
risk [1]. In 2006, it was discovered fortuitously that nifurtimox was
also clinically active against neuroblastoma [2], suggesting a possible important new role for this compound. It has since undergone
Phase 1 clinical trials for relapsed, or refractory neuroblastoma and
was well-tolerated by paediatric patients, giving tumour responses
as a single or combined agent [3]. Nifurtimox is currently in Phase 2

Corresponding author. Tel.: +44 1314678449; fax: +44 1314678450.

E-mail address: David.Melton@ed.ac.uk (D.W. Melton).
1568-7864/$ see front matter 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.dnarep.2013.08.017

clinical trials for refractory, or relapsed neuroblastoma and medulloblastoma.

Neuroblastoma is a cancer arising in neuroblasts that have a
developmental origin in the neural crest [46]. As melanocytes also
arise from the neural crest [7], it was hypothesised that melanoma
may also be susceptible to nitrofuran therapy [8]. This notion
was supported by evidence that nifurtimox leads to melanocytotoxicity in zebrash [8]. Furthermore, a more potent nitrofuran
compound (NFN1) was identied with increased melanocytotoxicity in zebrash [8]. NFN1 has since been shown to have greater
potency and a greater therapeutic index than nifurtimox against T.
cruzi [9].
In vivo, nitrofurans act as pro-drugs requiring bioactivation by
nitroreductases to form reactive intermediates. Type 1 nitroreductases, found predominantly in prokaryotes [10,11], are oxygen
insensitive and reduce the nitrofuran nitro-group to promote
DNA damage [1114]. Type 2 nitroreductases are oxygen sensitive
enzymes [1113] and act by reducing the nitro-group to generate

E.M. McNeil et al. / DNA Repair 12 (2013) 10001006

1001

Fig. 1. Toxicity of nitrofuran compounds on immortalised ERCC1-procient and isogenic ERCC1-decient mouse melanocytes. Toxicity of (A) cisplatin, (B) hydrogen peroxide,
(C) NFN1 and (D) nifurtimox on non-pigmented ERCC1-procient and ERCC1-decient mouse melanocyte cell lines [24]. Growth assays were performed as detailed in Section
2. Cell growth after 6 days was quantied using a Sulphorhodamine B assay [26] and expressed as percentage of the untreated control. Data from two independent experiments
were analysed using SigmaPlot 12.0 (Systat Software Inc.) and IC50 values are shown in Table 1A.

an unstable nitro-radical which, in the presence of oxygen, undergoes futile cycling to generate superoxide (O2 ) and regenerate the
parent nitrofuran [12,13]. In eukaryotes Type 1 nitroreductases are
rarely found, so the predominant mechanism of nitrofuran activation is through Type 2 nitroreductases [11]. It has also recently been
discovered that the aldehyde dehydrogenase (ALDH) 2 enzyme is
capable of bioactivating nitrofurans in zebrash and that nitrofurans are a substrate for human ALDH2 [8].
Nitrofurans cause increased reactive oxygen species (ROS) in
both trypanosomes and eukaryotic cells [12,15,16]. Similarly, the
more potent NFN1 also results in ROS formation in melanoma cells
[8]. In Escherichia coli, where both Type 1 and Type 2 bioactivation
can occur, it is proposed that the Nucleotide Excision Repair (NER)
pathway is required for repair of nitrofuran-induced DNA damage
[17], however this is controversial [18].
Melanocytes produce melanin to protect the skin from UV
radiation. Two main forms of melanin exist: red/yellow pheomelanin is usually converted to the brown/black eumelanin, but in
red-haired individuals with a mutation in the MC1R receptor, conversion does not occur and such individuals have a 3- to 8-fold
increased risk of developing melanoma [19]. Melanin protection
occurs through two distinct mechanisms, rstly by directly absorbing UV to act as a lter, and secondly by scavenging ROS formed
in cells upon UV radiation [20,21]. While eumelanin absorbs ROS,
pheomelanin may actually act as a photosensitizer by producing ROS in response to UV irradiation [22]. As ROS are produced
during nitrofuran activation, we hypothesised that eumelanin

may protect melanoma cells against nitrofuran-induced DNA

damage.
In this study we investigate the sensitivity of melanoma and
neuroblastoma to nitrofurans. NFN1 and nifurtimox were both
active against melanoma and neuroblastoma cell lines, but NFN1
was considerably more potent. NER was not required to protect against nitrofuran toxicity, suggesting instead a role for Base
Excision Repair (BER) and/or Single Strand Break Repair (SSBR).
In support, the poly(ADP-ribose) polymerase (PARP) inhibitor,
Olaparib [23], enhanced the toxicity of nitrofuran compounds, suggesting a novel combinatorial therapy. Finally, melanin did indeed
offer some protection to melanoma cells against NFN1 toxicity.
2. Materials and methods
2.1. Cell culture
ERCC1-procient and ERCC1-decient mouse melanocyte cell
lines were cultured in supplemented RPMI medium as described
[24]. The A375 human melanoma cells (The European Collection
of Cell Cultures) and SH-5Y5Y human neuroblastoma cells [25]
were cultured in supplemented DMEM medium. Toxicity assays
were performed in 96-well plates by seeding cells directly into
compound at 1000 or 1500 (ERCC1-procient and ERCC1-decient
mouse melanocytes) and 500 or 1500 (A375 and SH-5Y5Y) cells per
well. Plates were incubated at 37 C and 5% CO2 for 5 or 6 days then
cell growth was quantied using a Sulphorhodamine B assay [26].

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E.M. McNeil et al. / DNA Repair 12 (2013) 10001006

Table 1
(A) IC50 values and fold difference in sensitivity between ERCC1-procient and ERCC1-decient mouse melanocytes (data shown in Fig. 1). (B) IC50 values for NFN1 and
nifurtimox on the human A375 melanoma and SH-5Y5Y neuroblastoma cell lines. NFN1 and nifurtimox IC50 values are shown in the presence and absence of 3 M Olaparib.
Fold difference in toxicity between the IC50 values obtained in the presence and absence of Olaparib are shown (data shown in Fig. 2). (C) IC50 values and fold differences
in sensitivity for NFN1 and hydrogen peroxide on mouse melanoma cells grown under different conditions to produce different levels of pigmentation. Fold differences in
compound IC50 are expressed relative to the IC50 for the heavily pigmented 15% CO2 culture (data shown in Fig. 3). All IC50 values, with 95% Condence Intervals shown
in parentheses, were calculated based upon two independent experiments using SigmaPlot 12.0 (Systat Software Inc.). Statistical signicance between IC50 values was
calculated using a t-test based upon the reported IC50 and SEM by SigmaPlot 12.0.
(A) ERCC1-procient and ERCC-decient mouse melanocytes

Cisplatin
Hydrogen peroxide
NFN1
Nifurtimox

IC50 (ERCC1+)

IC50 (ERCC1)

Fold

P-value

83.21 nM [73.9393.67]
0.44 mM [0.410.47]
0.08 M [0.070.09]
12.59 M [11.6113.65]

6.27 nM [5.656.96]
0.46 mM [0.440.49]
0.05 M [0.040.07]
18.90 M [17.5120.4]

13.3
1.0
1.6
0.7

<0.001
0.990
0.987
<0.001

(B) Human melanoma (A375) and neuroblastoma (SH-5Y5Y)

A375
NFN1
Nifurtimox
SH-5Y5Y
NFN1
Nifurtimox

IC50

IC50 (+3 M Olaparib)

Fold

P-value

0.59 M [0.152.36]
29.38 M [26.4432.65]

0.10 M [0.050.19]
18.83 M [16.0922.04]

5.9
1.6

0.029
<0.001

0.33 M [0.240.47]
58.04 M [36.1593.20]

0.18 M [0.130.26]
19.12 M [9.2939.35]

1.8
3.0

0.024
0.018

(C) Pigmented mouse melanoma

NFN1
15% CO2
5% CO2
5% CO2 + PTU
Hydrogen peroxide
15% CO2
5% CO2
5% CO2 + PTU

IC50

Fold

P-value

1.66 M [0.972.84]
0.54 M [0.390.74]
0.09 M [0.050.15]

3.1
18.4

<0.001
0.002

1.78 mM [1.582.01]
1.00 mM [0.941.06]
0.91 mM [0.791.07]

1.8
2.0

<0.001
<0.001

2.2. Compounds
Cisplatin (DBL ) was obtained from Faulding Pharmaceuticals.
NFN1 (BTB05727) was obtained from Maybridge and Olaparib
(S1060) was obtained from Stratech Scientic Ltd. Hydrogen peroxide (H1009), nifurtimox (N3415) and phenylthiourea (PTU) (P7629)
were obtained from SigmaAldrich.
2.3. Comet assays
Comet assays were performed as detailed by Olive et al. with
minor alterations [27]. Instead of water, agarose solutions were
prepared in PBS and slides were neutralised with 400 mM Tris
pH7.5. Comets were imaged using a 20 objective and analysed
using CometScoreTM Freeware v1.5 (TriTek Corp.).
3. Results and discussion

repair [28]. As expected, based upon the observed IC50s, ERCC1decient cells were 13-fold more sensitive to cisplatin than their
ERCC1-procient counterparts (see Table 1A). As the mechanism of
nitrofuran-induced DNA damage in eukaryotic cells is believed to
occur by superoxide formation, we utilised hydrogen peroxide to
induce ROS in our cells. As expected, in Fig. 1B, the ERCC1-decient
cells were no more sensitive to hydrogen peroxide than ERCC1procient cells as NER is not involved in the repair of most oxidative
lesions, which are dealt with instead by the BER/SSBR pathways.
To assess whether this was also the case for nitrofuran compounds
we show, in Fig. 1C and D, that the ERCC1-decient cell line was
not hypersensitive to NFN1 or nifurtimox when compared to the
ERCC1-procient line. We conclude that, contrary to the situation in
E. coli [17], the NER pathway is not required for repair of nitrofuraninduced DNA damage in mammalian cells. This could be due to
the different reactive intermediates generated by Type 1 and Type
2 nitroreductases [12,13], with Type 1 resulting in bulky lesions
requiring NER for repair, while Type 2 result in reactive oxygen
species that cause lesions that are substrates for BER/SSBR [12,13].

3.1. Nitrofuran-induced DNA damage is independent of

Nucleotide Excision Repair
While it is thought that the NER pathway repairs nitrofuraninduced DNA damage in E. coli, where both Type 1 and Type 2
nitroreductases are present [17], it is unknown whether this is
also the case in mammalian cells where the predominant mechanism of activation is by Type 2 nitroreductases. As the ERCC1-XPF
endonuclease is essential for NER [28], we investigated the NER
requirement in isogenic mouse melanocyte cell lines in which the
Ercc1 gene was either functional, or had previously been inactivated
by Cre-mediated recombination [24].
In Fig. 1A, we utilised cisplatin which causes mainly monofunctional adducts and intrastrand crosslinks that both require NER for

3.2. Nitrofurans for the treatment of melanoma and

neuroblastoma
As nifurtimox is showing clinical promise for the treatment of
neuroblastoma [2,3], and since melanoma and neuroblastoma arise
from a common cell lineage [4,7], we investigated whether nitrofurans would also show activity against melanoma and whether
NFN1 would show greater potency than nifurtimox against human
neuroblastoma and melanoma cell lines.
In Fig. 2A and B we show that NFN1 and nifurtimox have an IC50
of 0.59 M and 29.38 M, respectively, against the A375 human
malignant melanoma cell line. In Fig. 2C and D we show the IC50s

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Fig. 2. Enhancement of NFN1 and nifurtimox toxicity by Olaparib on human melanoma and neuroblastoma cell lines. (A) Showing the NFN1 toxicity on non-pigmented A375
human melanoma cells in the presence, and absence of 3 M Olaparib. In each case, growth is expressed relative to the respective 0 M NFN1 controls, so correcting for any
effect of Olaparib alone on cell growth or cell adhesion after plating. (B) Showing the nifurtimox toxicity on A375 human melanoma cells in the presence, and absence of 3 M
Olaparib. In each case, growth is expressed relative to the respective 0 M nifurtimox controls, so correcting for any effect of Olaparib alone. (C) Showing the NFN1 toxicity on
SH-5Y5Y human neuroblastoma cells [25] in the presence, and absence of 3 M Olaparib. (D) Showing the nifurtimox toxicity on SH-5Y5Y human neuroblastoma cells in the
presence, and absence of 3 M Olaparib. Growth assays were performed as detailed in Materials and methods. Cell growth after 5 (A375) or 6 days (SH-5Y5Y) was quantied
using a Sulphorhodamine B assay [26] and expressed as a percentage of the untreated control. Data from two independent experiments were analysed using SigmaPlot 12.0
(Systat Software Inc.) and IC50 values are shown in Table 1B. (E) Histogram of comet assay on A375 human melanoma cells showing the effect of compound treatment on
the Olive Tail Moment. Cells were treated as indicated with 10 M Olaparib, 200 M NFX, 80 M NFN1, or 100 M hydrogen peroxide for 1 h. Following treatment, Average
Olive Tail Moment values (and SEM), based upon greater than 50 individual comets, are shown. Statistical signicance was determined by t-test and signicance compared
to control is identied by white asterisks, and signicance comparisons to single agents are shown by black asterisks. *P < 0.05, **P < 0.01 and ***P < 0.001. (F) Representative
untreated control comet. (G) Representative hydrogen peroxide-treated comet. (H) Representative NFN1-treated comet. (I) Representative NFN1 and Olaparib-treated
comet.

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E.M. McNeil et al. / DNA Repair 12 (2013) 10001006

of NFN1 and nifurtimox for the human neuroblastoma SH-5Y5Y

cell line were 0.33 M and 58.04 M, respectively (see Table 1B).
These data show that NFN1 and nifurtomox have similar potencies
against neuroblastoma and melanoma cell lines (<2-fold difference), and that NFN1 is considerably (50- to 175-fold) more potent
than nifurtimox against both. As nitrofuran compounds have similarly toxicity to melanoma and neuroblastoma cell lines, and since
these compounds are known to result in in vivo melanotoxicity [8],
we suggest that nitrofuran compounds may also be benecial for
the treatment of malignant melanoma.
3.3. Nitrofuran compound toxicity is enhanced by Olaparib
In mammalian cells nitrofuran-induced DNA damage is thought
to be due to reactive oxygen species formed during Type 2 activation [29,30]. ROS can cause single-strand DNA breaks (ss breaks)
directly, and, if they also result in oxidised base damage, then ss
breaks will also arise during BER. As we have shown that NER is not
required for the repair of nitrofuran-induced DNA damage, we predict instead that the BER and/or SSBR pathways are responsible. If
this is the case, then the toxicity of NFN1 and nifurtimox should be
enhanced by the PARP inhibitor Olaparib [3133]. This is because
PARP is required for SSBR and, if PARP is inhibited by Olaparib, ss
intermediates trapped by PARP also block BER [33].
In Fig. 2, we show the toxicities of NFN1 and nifurtimox are
indeed enhanced by Olaparib in both A375 human melanoma and
SH-5Y5Y neuroblastoma cells. In A375, Olaparib combined with
NFN1 resulted in a 5.9-fold decrease in the NFN1 IC50 (Fig. 2A
and Table 1B), while combination with nifurtimox resulted in a
1.6-fold decrease in the nifurtimox IC50 (Fig. 2B and Table 1B). In
contrast, in SH-5Y5Y neuroblastoma cells, combination with Olaparib resulted in a 1.8-fold decrease in the NFN1 IC50 (Fig. 2C and
Table 1B) and a 3-fold decrease in the nifurtimox IC50 (Fig. 2D and
Table 1B). Although the reason for the differing Olaparib enhancements between compounds and cell lines is unexplained, the data
suggest that the BER and/or or SSBR pathways are responsible for
repair of NFN1- and nifurtimox-induced DNA damage in melanoma
and neuroblastoma cells.
To conrm this, we utilised an alkaline comet assay to detect
ss DNA breaks. In Fig. 2E, we show that upon hydrogen peroxide
treatment there was a 3.7-fold increase in comet tail moment relative to an untreated control. Upon nitrofuran treatment, a 1.7-fold
increase in tail moment was observed for nifurtimox, whereas no
signicant change was observed with NFN1. Furthermore, while
Olaparib treatment alone did not signicantly alter the tail moment
relative to the untreated control, in combination with nifurtimox a
1.2-fold increase in tail moment was observed and, when combined
with NFN1, a 2-fold increase in tail moment was observed relative
to either nitrofuran alone. This demonstrates that nitrofuran compounds do result in ss DNA breaks and that PARP is involved in their
repair.
Our observation suggests that combinatorial therapy of nitrofurans with Olaparib may present a novel approach to increasing
therapeutic efcacy in neuroblastoma and melanoma patients. Furthermore, it has recently been shown that T. cruzi is sensitive
to PARP inhibition by Olaparib [34], suggesting combination of
nifurtimox with Olaparib may also be of clinical advantage as an
anti-trypanosomal regime.
The comet assay result also provides information on the possible role of the ALDH2 enzyme in nitrofuran-induced DNA damage.
Aldehydes can cause interstrand DNA crosslinks (ICLs) and there is
considerable current interest in the genotoxic effects of endogenous aldehyde production in vivo. The repair pathway for this
particularly toxic form of DNA damage is defective in Fanconi
anaemia and Fancd2 knockout mice that also lack ALDH2 show a
haematopoietic stem cell depletion phenotype characteristic of the

human disease [35]. As nitrofurans act as competitive substrates for

the ALDH2 enzyme [8], this could result in increased levels of reactive aldehyde-induced ICLs. The presence of ICLs can be monitored
with the alkaline comet assay as shown in Supplementary Fig. 1. In
addition to causing mostly monofunctional adducts and intrastrand
crosslinks, cisplatin also causes some ICLs [28]. Until they have been
repaired, ICLs hold the DNA strands together and prevent comet
formation following ss DNA break induction with hydrogen peroxide. Since we have shown that nitrofuran treatments induce ss
breaks and comet formation, any ICLs also formed are evidently
insufcient to prevent comet formation, although this would not
prevent them contributing to nitrofuran toxicity. However, if this
were the case then we would expect ERCC1-decient cells to be
more sensitive to nitrofurans than ERCC1-procient cells, since
ERCC1 is also required for the removal of ICLs [28], and there was
no evidence for this (Fig. 1C and D). Another reason not to invoke
acetaldehyde-induced ICLs in nitrofuran toxicity is that chronic
exposure to alcohol, which is metabolised to acetaldehyde, results
in increased activation of nifurtimox by liver nitroreductases and
increased toxicity in male rats [36].
3.4. Melanin pigment is protective to nitrofuran-induced DNA
damage
The A375 cell line is sensitive to nitrofurans, but like many established human melanoma cell lines, it no longer makes melanin.
The ability of eumelanin to absorb ROS generated by UVA irradiation in the skin has been described [20,21]. In order to address
whether the presence of pigment in melanoma cell lines would
result in decreased sensitivity to nitrofuran compounds due to
melanin absorbing ROS formed by bioactivation, we rst determined the toxicity of the higher potency NFN1 on a panel of cell
lines. The amelanotic A375 melanoma line had an IC50 to NFN1
of 0.59 M (Table 1B), whereas the eumelanin-expressing HBL
melanoma cell line (Gentaur, Belgium) was 11-fold more resistant,
with an IC50 of 6.58 M (Supplementary Table 1). This pigmentrelated toxicity difference was replicated in mouse melanocyte cell
lines which showed a 23-fold difference between our amelanotic
Ercc1-procient melanocytes, with an IC50 of 0.08 M (Table 1A),
and the eumelanin-expressing Melan-a cell line [37] with an IC50
of 1.91 M (Supplementary Table 1).
While our data for the toxicity of nitrofurans in non-pigmented
cell lines are consistent with previous reports [9,11,38,39], the
increased resistance observed in the pigmented cell lines may be
as a result of other non-pigment related differences between the
lines. For instance, as nitrofuran compounds must be bioactivated
in cells, differences in the overall activity of bioactivating enzymes,
or in the activity of particular type 2 nitroreductases, or ALDH2,
could also be involved in the toxicity difference observed between
pigmented and non-pigmented melanoma and melanocyte cells.
To assess whether this differential toxicity was due to different levels of pigment, rather than non-pigment related differences
between cell lines, we utilised two methods to alter pigmentation levels in a recently isolated melanoma cell line. We used
eumelanin-expressing cells isolated from a mouse metastatic
melanoma on a C57BL/6 mouse lacking p16INK4a and with an
activating NRAS mutation [40]. When the tumour was isolated
to culture at 15% CO2 cells retained high levels of melanin. Upon
transferring to 5% CO2 , cells gradually lost pigment and loss
was exacerbated by the tyrosinase inhibitor phenylthiourea (PTU)
[41,42]. Bright eld and phase contrast images of the cultures are
shown in Fig. 3A.
We then assessed the relative toxicities of the three cultures to
NFN1. In Fig. 3B, we show that the highly pigmented melanoma
cells cultured at 15% CO2 have a NFN1 IC50 of 1.66 M, whereas
when pigmentation was reduced by lower CO2 concentration alone,

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Fig. 3. Effect of pigmentation on the toxicity of NFN1 and nifurtimox. (A) Bright eld and phase contrast images of the mouse melanoma cell line following culturing at
5% CO2 with 200 M PTU (low level of pigment), 5% CO2 (intermediate pigment level), or 15% CO2 (high pigment level). All images taken using a 10 objective lens. (B)
Toxicity of NFN1 on the mouse melanoma cell line following culturing at 5% CO2 with 200 M PTU, 5% CO2 or 15% CO2 . (C) Toxicity of nifurtimox on the mouse melanoma
cell line following culturing at 5% CO2 with 200 M PTU, 5% CO2 or 15% CO2 . Cell growth was quantied using a Sulphorhodamine B assay [26]. Data from two independent
experiments were analysed using SigmaPlot 12.0 (Systat Software Inc.) and IC50 values are shown in Table 1C. There were no noticeable effects of CO2 level on cell growth
and any minor growth differences between cultures at different CO2 levels were corrected for in the plots where growth curves for the various compounds were expressed
relative to control growth at each CO2 level.

the IC50 was reduced to 0.54 M. Upon further reduction of pigment levels by PTU, the NFN1 IC50 was reduced to 0.09 M. This
demonstrates directly that the presence of pigment in melanoma
cells can result in up to an 18-fold decrease in toxicity to NFN1 (see
Table 1C), analogous to the 11- to 23-fold difference we observed
between pigmented and non-pigmented human melanoma and
mouse melanocyte cell lines. These data are entirely consistent with
the known protective ability of eumelanin to absorb ROS [20,21].
This suggests that the therapeutic window for NFN1 in heavily
pigmented melanomas over non-tumour cells will be reduced. As
expected for another ROS-producer, a pigment-dependent IC50
shift was also observed with hydrogen peroxide, although the difference was smaller than that for NFN1 (Fig. 3C and Table 1C).
Further study of the therapeutic window for NFN1 and nifurtimox against melanoma will be required to determine which
drug would be most effective. An additional complication could
be that the new therapies for melanoma, involving BRAF and/or
MEK inhibitors, can lead to upregulation of MITF and differentiation

markers such as melanin levels [43,44]. By analogy with the relationship between levels of pigmentation and nitrofuran sensitivity
that we report here, this could render BRAF and/or MEK inhibitortreated melanomas more refractory to the nitrofuran therapy that
we have proposed.
3.5. Concluding remarks
We have shown that nitrofuran-induced DNA damage is consistent with the formation of reactive oxygen species generated
by Type 2 nitroreductases. While Nucleotide Excision Repair is not
required to protect against toxicity of NFN1 or nifurtimox in eukaryotic cells, toxicity of both compounds is enhanced by Olaparib,
indicating a role for Base Excision Repair and/or Single Strand Break
Repair. We have shown that the nitrofuran compound NFN1 is more
potent than nifurtimox on human neuroblastoma and melanoma
cell lines, and that combinatorial therapy of NFN1 or nifurtimox
with Olaparib may present a novel therapeutic strategy for both

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cancers. However, high levels of eumelanin pigment in melanoma

cells offer some protection against NFN1 toxicity.
Conict of interest statement
The authors declare that there are no conicts of interest.
Acknowledgements
We are grateful to Dr Elizabeth Patton (MRC Institute of Genetics
and Molecular Medicine, University of Edinburgh) for introducing us to nitrofurans through her work on zebrash. EMM was
supported by a PhD Scholarship from Cancer Research UK. ERCC1
research in DWMs laboratory is funded by the Scottish Chief Scientist Ofce and The Charon Fund.
Appendix A. Supplementary data
Supplementary data associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/
j.dnarep.2013.08.017.
References
[1] A. Rassi Jr., A. Rassi, J.A. Marin-Neto, Chagas disease, Lancet 375 (2010)
13881402.
[2] G.L. Saulnier Sholler, S. Kalkunte, C. Greenlaw, K. McCarten, E. Forman, Antitumor activity of nifurtimox observed in a patient with neuroblastoma, J. Pediatr.
Hematol. Oncol. 28 (2006) 693695.
[3] G.L. Saulnier Sholler, G.M. Bergendahl, L. Brard, A.P. Singh, B.W. Heath,
P.M. Bingham, et al., A phase 1 study of nifurtimox in patients with
relapsed/refractory neuroblastoma, J. Pediatr. Hematol. Oncol. 33 (2011)
2530.
[4] R.A. Ross, J.L. Biedler, Presence and regulation of tyrosinase activity in human
neuroblastoma cell variants in vitro, Cancer Res. 45 (1985) 16281632.
[5] K.D. Preter, J. Vandesompele, P. Heimann, N. Yigit, S. Beckman, A. Schramm,
et al., Human fetal neuroblast and neuroblastoma transcriptome analysis
conrms neuroblast origin and highlights neuroblastoma candidate genes,
Genome Biol. 7 (2006) R84.
[6] R. Shimono, S. Matsubara, H. Takamatsu, T. Fukushige, M. Ozawa, The expression of cadherins in human neuroblastoma cell lines and clinical tumors,
Anticancer Res. 20 (2000) 917923.
[7] L. Sommer, Generation of melanocytes from neural crest cells, Pigment Cell
Melanoma Res. 24 (2011) 411421.
[8] L. Zhou, H. Ishizaki, M. Spitzer, K.L. Taylor, N.D. Temperley, S.L. Johnson, et al.,
ALDH2 mediates 5-nitrofuran activity in multiple species, Chem. Biol. 19 (2012)
883892.
[9] L. Zhou, G. Stewart, E. Rideau, N.J. Westwood, T.K. Smith, A class of 5-nitro2-furancarboxylamides with potent trypanocidal activity against Trypanosoma
brucei in vitro, J. Med. Chem. 56 (2013) 796806.
[10] C. Bot, B.S. Hall, G. Alvarez, R. Di Maio, M. Gonzlez, H. Cerecetto, et al., Evaluating 5-nitrofurans as trypanocidal agents, Antimicrob. Agents Chemother. 57
(2013) 16381647.
[11] B.S. Hall, C. Bot, S.R. Wilkinson, Nifurtimox activation by trypanosomal type
I nitroreductases generates cytotoxic nitrile metabolites, J. Biol. Chem. 286
(2011) 1308813095.
[12] S.R. Wilkinson, M.C. Taylor, D. Horn, J.M. Kelly, I. Cheeseman, A mechanism for
cross-resistance to nifurtimox and benznidazole in trypanosomes, Proc. Natl.
Acad. Sci. U.S.A. 105 (2008) 50225027.
[13] I.M. Oliveira, D. Bonatto, J.A. Henriques, Nitroreductases: enzymes with environmental, biotechnological and clinical importance, Curr. Res. Technol. Educ.
Top. Appl. Microbiol. Microbial Biotechnol. 2 (2010) 10081019.
[14] S. Alsford, S. Eckert, N. Baker, L. Glover, A. Sanchez-Flores, K.F. Leung, et al.,
High-throughput decoding of antitrypanosomal drug efcacy and resistance,
Nature 482 (2012) 232236.
[15] X. Jin, S. Tang, Q. Chen, J. Zou, T. Zhang, F. Liu, et al., Furazolidone induced
oxidative DNA damage via up-regulating ROS that caused cell cycle arrest in
human hepatoma G2 cells, Toxicol. Lett. 201 (2011) 205212.
[16] C. Viod, N. Bettache, N. Cenas, R.L. Krauth-Siegel, G. Chauvire, N. Bakalara,
et al., Enzymatic reduction studies of nitroheterocycles, Biochem. Pharmacol.
57 (1999) 549557.
[17] K.R. Ona, C.T. Courcelle, J. Courcelle, Nucleotide excision repair is a predominant mechanism for processing nitrofurazone-induced DNA damage in
Escherichia coli, J. Bacteriol. 191 (2009) 49594965.

[18] C. Lu, D.R. McCalla, D.W. Bryant, Action of nitrofurans on E. coli: mutation and
induction and repair of daughter-strand gaps in DNA, Mutat. Res. 67 (1979)
133144.
[19] S. Raimondi, F. Sera, S. Gandini, S. Iodice, S. Caini, P. Maisonneuve, et al., MC1R
variants, melanoma and red hair color phenotype: a meta-analysis, Int. J. Cancer
122 (2008) 27532760.
[20] J.B. Nofsinger, Y. Liu, J.D. Simon, Aggregation of eumelanin mitigates photogeneration of reactive oxygen species, Free Radic. Biol. Med. 32 (2002)
720730.
[21] M. Tada, M. Kohno, Y. Niwano, Scavenging or quenching effect of melanin
on superoxide anion and singlet oxygen, J. Clin. Biochem. Nutr. 46 (2010)
224228.
[22] E. Wenczl, G.P. Van der Schans, L. Roza, R.M. Kolb, A.J. Timmerman, N.P. Smit,
et al., (Pheo)melanin photosensitizes UVA-induced DNA damage in cultured
human melanocytes, J. Invest. Dermatol. 111 (1998) 678682.
[23] K.A. Menear, C. Adcock, R. Boulter, X. Cockcroft, L. Copsey, A. Cranston,
et al., 4-[3-(4-Cyclopropanecarbonylpiperazine-1-carbonyl)-4-uorobenzyl]2H-phthalazin-1-one: a novel bioavailable inhibitor of poly(ADP-ribose)
polymerase-1, J. Med. Chem. 51 (2008) 65816591.
[24] L. Song, A.-M. Ritchie, E.M. McNeil, W. Li, D.W. Melton, Identication of DNA
repair gene Ercc1 as a novel target in melanoma, Pigment Cell Melanoma Res.
24 (2011) 966971.
[25] J.L. Biedler, S. Rofer-Tarlov, M. Schachner, L.S. Freedman, Multiple neurotransmitter synthesis by human neuroblastoma cell lines and clones, Cancer Res. 38
(1978) 37513757.
[26] P. Skehan, R. Storeng, D. Scudiero, A. Monks, J. McMahon, D. Vistica, et al., New
colorimetric cytotoxicity assay for anticancer-drug screening, J. Natl. Cancer
Inst. 82 (1990) 11071112.
[27] P.L. Olive, J.P. Banth, The comet assay: a method to measure DNA damage in
individual cells, Nat. Protocols 1 (2006) 2329.
[28] E.M. McNeil, D.W. Melton, DNA repair endonuclease ERCC1XPF as a novel
therapeutic target to overcome chemoresistance in cancer therapy, Nucl. Acids
Res. 40 (2012) 999010004.
[29] P.L. Olive, D.R. McCalla, Damage to mammalian cell DNA by nitrofurans, Cancer
Res. 35 (1975) 781784.
[30] J.I.G. Borroto, G.P. Machado, A. Creus, R. Marcos, Comparative genotoxic evaluation of 2-furylethylenes and 5-nitrofurans by using the comet assay in TK6
cells, Mutagenesis 20 (2005) 193197.
[31] F. Dantzer, V. Schreiber, C. Niedergang, C. Trucco, E. Flatter, G. De La Rubia, et al.,
Involvement of poly(ADP-ribose) polymerase in base excision repair, Biochimie
81 (1999) 6975.
[32] A. Masaoka, J.K. Horton, W.A. Beard, S.H. Wilson, DNA polymerase and PARP
activities in base excision repair in living cells, DNA Repair 8 (2009) 12901299.
[33] C.E. Strm, F. Johansson, M. Uhln, C.A.-K. Szigyarto, K. Erixon, T. Helleday,
Poly (ADP-ribose) polymerase (PARP) is not involved in base excision repair
but PARP inhibition traps a single-strand intermediate, Nucleic Acids Res. 39
(2011) 31663175.
[34] S.C. Vilchez Larrea, T. Haikarainen, M. Narwal, M. Schlesinger, H. Venkannagari,
M.M. Flawi, et al., Inhibition of poly(ADP-ribose) polymerase interferes with
Trypanosoma cruzi infection and proliferation of the parasite, PLoS ONE 7 (2012)
e46063.
[35] J.I. Garaycoechea, G.P. Crossan, F. Langevin, M. Daly, M.J. Arends, K.J. Patel, Genotoxic consequences of endogenous aldehydes on mouse haematopoietic stem
cell function, Nature 489 (2012) 571575.
[36] M. de Mecca, L.C. Bartel, J.A. Castro, Effect of chronic alcohol drinking on rat
liver microsomal nitroreductive metabolism of nifurtimox and benznidazole,
Hum. Exp. Toxicol. (2013), http://dx.doi.org/10.1177/0960327113485254.
[37] D.C. Bennett, P.J. Cooper, I.R. Hart, A line of non-tumorigenic mouse
melanocytes, syngeneic with the B16 melanoma and requiring a tumour promoter for growth, Int. J. Cancer 39 (1987) 414418.
[38] R.K. Singh, T.S. Lange, K.K. Kim, L. Brard, A coumarin derivative (RKS262) inhibits
cell-cycle progression, causes pro-apoptotic signaling and cytotoxicity in ovarian cancer cells, Invest. New Drugs 29 (2011) 6372.
[39] B.S. Hall, X. Wu, L. Hu, S.R. Wilkinson, Exploiting the drug-activating properties of a novel trypanosomal nitroreductase, Antimicrob. Agents Chemother.
54 (2010) 11931199.
[40] J. Ackermann, M. Frutschi, K. Kaloulis, T. McKee, A. Trumpp, F. Beermann, Metastasizing melanoma formation caused by expression of activated N-RasQ61K on
an INK4a-decient background, Cancer Res. 65 (2005) 40054011.
[41] A.M. Hall, S.J. Orlow, Degradation of tyrosinase induced by phenylthiourea
occurs following Golgi maturation, Pigment Cell Res. 18 (2005) 122129.
[42] E. Le Pape, K. Wakamatsu, S. Ito, R. Wolber, V.J. Hearing, Regulation of eumelanin/pheomelanin synthesis and visible pigmentation in melanocytes by
ligands of the melanocortin 1 receptor, Pigment Cell Melanoma Res. 21 (2008)
477486.
[43] W.D. Tap, K.-W. Gong, J. Dering, Y. Tseng, C. Ginther, G. Pauletti, et al., Pharmacodynamic characterization of the efcacy signals due to selective BRAF inhibition
with PLX4032 in malignant melanoma, Neoplasia 12 (2010) 637649.
[44] M.P. Smith, J. Ferguson, I. Arozarena, R. Hayward, R. Marais, A. Chapman, et al.,
Effect of SMURF2 targeting on susceptibility to MEK inhibitors in melanoma, J.
Natl. Cancer Inst. 105 (2013) 3346.