nitrofuran

DNA Repair 12 (2013) 1000 1006

Contents lists available at ScienceDirect

DNA Repair

j ourna l ho me p age : www.elsev ier .com/

Brief communication

The tox noneurob ndmelani

Ewan M.MRC Institute o it, WesEH4 2XU, UK

a r t i c l

Article history:Received 25 June 2013Received in revised form 29 August 2013Accepted 31 August 2013Available online 23 September 2013

Keywords:ERCC1-XPFMelaninMelanomaNeuroblastomNitrofuranPARP inhibitor

he trMore recently, following the fortuitous discovery that nifurtimox was clinically active against neurob-lastoma, nitrofuran compounds are being investigated for activity against cancer. Herein, we show thatnitrofuran 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 thannifurtimox against human melanoma and neuroblastoma cell lines. As nitrofuran compounds are known

1. Introdu

Nifurtimment of pathe Trypanositic diseaseinfected wirisk [1]. In 2also clinicalble importaPhase 1 clinwas well-toas a single o

CorresponE-mail add

1568-7864/$ http://dx.doi.oa

to act as pro-drugs, producing DNA-damaging reactive intermediates upon activation, we investigatedthe DNA repair pathways involved. We show that, contrary to research in Escherichia coli, the NucleotideExcision 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) poly-merase (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 activationproducing Reactive Oxygen Species which cause DNA damage that is repaired by the Single Strand BreakRepair and/or Base Excision Repair pathways, whereas in bacteria and trypanosomes, Type 1 nitrore-ductases are also utilised resulting in different DNA lesions. In addition we show that, consistent withReactive Oxygen Species being formed upon nitrofuran activation and the ability of melanin to absorbReactive Oxygen Species, production of melanin in melanoma cells offers some protection from NFN1-and hydrogen peroxide-induced toxicity. Our data suggest that combinations of Olaparib and nitrofu-ran compounds may be advantageous for the treatment of melanoma and neuroblastoma, but that theprotection offered to melanoma cells by their melanin pigment must be taken into account.

2013 Elsevier B.V. All rights reserved.

ction

ox is a nitrofuran compound widely used for the treat-rasitic infections including Chagas disease, caused bysoma cruzi parasite. Chagas disease is an endemic para-

in areas of Latin America where 7.7 million people areth T. cruzi, with a further 109 million people living at006, it was discovered fortuitously that nifurtimox wasly active against neuroblastoma [2], suggesting a possi-nt new role for this compound. It has since undergoneical trials for relapsed, or refractory neuroblastoma andlerated by paediatric patients, giving tumour responsesr combined agent [3]. Nifurtimox is currently in Phase 2

ding author. Tel.: +44 1314678449; fax: +44 1314678450.ress: [email protected] (D.W. Melton).

clinical trials for refractory, or relapsed neuroblastoma and medul-loblastoma.

Neuroblastoma is a cancer arising in neuroblasts that have adevelopmental origin in the neural crest [46]. As melanocytes alsoarise from the neural crest [7], it was hypothesised that melanomamay also be susceptible to nitrofuran therapy [8]. This notionwas supported by evidence that nifurtimox leads to melanocyto-toxicity in zebrash [8]. Furthermore, a more potent nitrofurancompound (NFN1) was identied with increased melanocytotox-icity in zebrash [8]. NFN1 has since been shown to have greaterpotency and a greater therapeutic index than nifurtimox against T.cruzi [9].

In vivo, nitrofurans act as pro-drugs requiring bioactivation bynitroreductases to form reactive intermediates. Type 1 nitrore-ductases, found predominantly in prokaryotes [10,11], are oxygeninsensitive and reduce the nitrofuran nitro-group to promoteDNA damage [1114]. Type 2 nitroreductases are oxygen sensitiveenzymes [1113] and act by reducing the nitro-group to generate

see front matter 2013 Elsevier B.V. All rights reserved.rg/10.1016/j.dnarep.2013.08.017icity of nitrofuran compounds on melalastoma cells is enhanced by Olaparib an pigment

McNeil, Ann-Marie Ritchie, David W. Melton

f Genetics and Molecular Medicine, University of Edinburgh, MRC Human Genetics Un

e i n f o a b s t r a c t

Nitrofurans are commonly used for tlocate /dnarepai r

ma and ameliorated by

tern General Hospital, Crewe Road, Edinburgh

eatment of trypanosomal diseases including Chagas disease.

E.M. McNeil et al. / DNA Repair 12 (2013) 1000 1006 1001

Fig. 1. Toxicit(C) NFN1 and (2. Cell growth were analysed

an unstablegoes futile cparent nitrorarely foundtion is throudiscovered capable of brans are a s

Nitrofurboth trypanmore poten[8]. In Eschecan occur, ipathway is [17], howev

Melanocradiation. Tlanin is usured-haired version doeincreased roccurs throing UV to ain cells upopheomelaning ROS in during nitry of nitrofuran compounds on immortalised ERCC1-procient and isogenic ERCC1-decienD) nifurtimox on non-pigmented ERCC1-procient and ERCC1-decient mouse melanocyafter 6 days was quantied using a Sulphorhodamine B assay [26] and expressed as percent

using SigmaPlot 12.0 (Systat Software Inc.) and IC50 values are shown in Table 1A.

nitro-radical which, in the presence of oxygen, under-ycling to generate superoxide (O2) and regenerate thefuran [12,13]. In eukaryotes Type 1 nitroreductases are, so the predominant mechanism of nitrofuran activa-gh Type 2 nitroreductases [11]. It has also recently beenthat the aldehyde dehydrogenase (ALDH) 2 enzyme isioactivating nitrofurans in zebrash and that nitrofu-

ubstrate for human ALDH2 [8].ans cause increased reactive oxygen species (ROS) inosomes and eukaryotic cells [12,15,16]. Similarly, thet NFN1 also results in ROS formation in melanoma cellsrichia coli, where both Type 1 and Type 2 bioactivationt is proposed that the Nucleotide Excision Repair (NER)required for repair of nitrofuran-induced DNA damageer this is controversial [18].ytes produce melanin to protect the skin from UVwo main forms of melanin exist: red/yellow pheome-ally converted to the brown/black eumelanin, but inindividuals with a mutation in the MC1R receptor, con-s not occur and such individuals have a 3- to 8-foldisk of developing melanoma [19]. Melanin protectionugh two distinct mechanisms, rstly by directly absorb-ct as a lter, and secondly by scavenging ROS formedn UV radiation [20,21]. While eumelanin absorbs ROS,in may actually act as a photosensitizer by produc-response to UV irradiation [22]. As ROS are producedofuran activation, we hypothesised that eumelanin

may protedamage.

In this sneuroblastoactive againwas considtect againstExcision ReIn supportOlaparib [2gesting a nooffer some

2. Materia

2.1. Cell cu

ERCC1-plines were [24]. The Aof Cell Culwere cultuwere perfocompound mouse melawell. Platescell growtht mouse melanocytes. Toxicity of (A) cisplatin, (B) hydrogen peroxide,te cell lines [24]. Growth assays were performed as detailed in Sectionage of the untreated control. Data from two independent experiments

ct melanoma cells against nitrofuran-induced DNA

tudy we investigate the sensitivity of melanoma andma to nitrofurans. NFN1 and nifurtimox were bothst melanoma and neuroblastoma cell lines, but NFN1erably more potent. NER was not required to pro-

nitrofuran toxicity, suggesting instead a role for Basepair (BER) and/or Single Strand Break Repair (SSBR)., the poly(ADP-ribose) polymerase (PARP) inhibitor,3], enhanced the toxicity of nitrofuran compounds, sug-vel combinatorial therapy. Finally, melanin did indeed

protection to melanoma cells against NFN1 toxicity.

ls and methods

lture

rocient and ERCC1-decient mouse melanocyte cellcultured in supplemented RPMI medium as described375 human melanoma cells (The European Collectiontures) and SH-5Y5Y human neuroblastoma cells [25]red in supplemented DMEM medium. Toxicity assaysrmed in 96-well plates by seeding cells directly intoat 1000 or 1500 (ERCC1-procient and ERCC1-decientnocytes) and 500 or 1500 (A375 and SH-5Y5Y) cells per

were incubated at 37 C and 5% CO2 for 5 or 6 days then was quantied using a Sulphorhodamine B assay [26].

1002 E.M. McNeil et al. / DNA Repair 12 (2013) 1000 1006

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 andnifurtimox 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 differencesin sensitivity for NFN1 and hydrogen peroxide on mouse melanoma cells grown under different conditions to produce different levels of pigmentation. Fold differences incompound IC5 ata shin parenthese .0 (Sycalculated usin

(A) ERCC1-p

RCC1Cisplatin M [5.6Hydrogen pe M [0.NFN1 M [0.0Nifurtimox M [17

(B) Human m

aparib

A375NFN1 0.19]Nifurtimo 922.0

SH-5Y5YNFN1 0.26]Nifurtimo 39.35

(C) Pigmente

NFN115% CO25% CO25% CO2 + P

Hydrogen pe15% CO25% CO25% CO2 + P

2.2. Compo

CisplatinNFN1 (BTB(S1060) waide (H1009)were obtain

2.3. Comet

Comet aminor alterprepared inpH7.5. Comusing Come

3. Results

3.1. NitrofuNucleotide E

While itinduced DNnitroreductalso the casanism of acendonuclearequiremenErcc1 gene wby Cre-med

In Fig. 1Ational addu

[28].nt ce-pro0 are expressed relative to the IC50 for the heavily pigmented 15% CO2 culture (ds, were calculated based upon two independent experiments using SigmaPlot 12g a t-test based upon the reported IC50 and SEM by SigmaPlot 12.0.

rocient and ERCC-decient mouse melanocytes

IC50 (ERCC1+) IC50 (E

83.21 nM [73.9393.67] 6.27 nroxide 0.44 mM [0.410.47] 0.46 m

0.08 M [0.070.09] 0.05 12.59 M [11.6113.65] 18.90

elanoma (A375) and neuroblastoma (SH-5Y5Y)

IC50 IC50 (+3 M Ol

0.59 M [0.152.36] 0.10 M [0.05x 29.38 M [26.4432.65] 18.83 M [16.0

0.33 M [0.240.47] 0.18 M [0.13x 58.04 M [36.1593.20] 19.12 M [9.29

d mouse melanoma

IC50 Fold

1.66 M [0.972.84]0.54 M [0.390.74] 3.1

TU 0.09 M [0.050.15] 18.4 roxide

1.78 mM [1.582.01]1.00 mM [0.941.06] 1.8

TU 0.91 mM [0.791.07] 2.0

unds

(DBL) was obtained from Faulding Pharmaceuticals.

repair decieERCC105727) was obtained from Maybridge and Olaparibs obtained from Stratech Scientic Ltd. Hydrogen perox-, nifurtimox (N3415) and phenylthiourea (PTU) (P7629)ed from SigmaAldrich.

assays

ssays were performed as detailed by Olive et al. withations [27]. Instead of water, agarose solutions were

PBS and slides were neutralised with 400 mM Trisets were imaged using a 20 objective and analysedtScoreTM Freeware v1.5 (TriTek Corp.).

and discussion

ran-induced DNA damage is independent ofxcision Repair

is thought that the NER pathway repairs nitrofuran-A damage in E. coli, where both Type 1 and Type 2ases are present [17], it is unknown whether this ise in mammalian cells where the predominant mech-tivation is by Type 2 nitroreductases. As the ERCC1-XPFse is essential for NER [28], we investigated the NERt in isogenic mouse melanocyte cell lines in which theas either functional, or had previously been inactivatediated recombination [24]., we utilised cisplatin which causes mainly monofunc-

cts and intrastrand crosslinks that both require NER for

nitrofuran-occur by suinduce ROScells were procient clesions, whTo assess wwe show, inot hyperseERCC1-proE. coli [17], tinduced DNthe differen2 nitroredurequiring Nspecies tha

3.2. Nitrofuneuroblasto

As nifurneuroblastofrom a comfurans wouNFN1 woulneuroblasto

In Fig. 2Aof 0.59 Mmalignant mown in Fig. 3). All IC50 values, with 95% Condence Intervals shownstat Software Inc.). Statistical signicance between IC50 values was

) Fold P-value

56.96] 13.3


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 A375human 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 anyeffect 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 MOlaparib. 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 onSH-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 thepresence, 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 quantiedusing 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 onthe 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, AverageOlive Tail Moment values (and SEM), based upon greater than 50 individual comets, are shown. Statistical signicance was determined by t-test and signicance comparedto 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) Representativeuntreated control comet. (G) Representative hydrogen peroxide-treated comet. (H) Representative NFN1-treated comet. (I) Representative NFN1 and Olaparib-treatedcomet.


of NFN1 and nifurtimox for the human neuroblastoma SH-5Y5Ycell line were 0.33 M and 58.04 M, respectively (see Table 1B).These data show that NFN1 and nifurtomox have similar potenciesagainst neuroblastoma and melanoma cell lines (


Fig. 3. Effect o5% CO2 with 2Toxicity of NFcell line followexperiments wand any minorrelative to con

the IC50 wment levelsdemonstratcells can resTable 1C), abetween pimouse melathe known This suggespigmented expected foshift was alference waFurther stutimox agaidrug wouldbe that theMEK inhibitf pigmentation on the toxicity of NFN1 and nifurtimox. (A) Bright eld and phase cont00 M PTU (low level of pigment), 5% CO2 (intermediate pigment level), or 15% CO2 (hN1 on the mouse melanoma cell line following culturing at 5% CO2 with 200 M PTU, 5%ing culturing at 5% CO2 with 200 M PTU, 5% CO2 or 15% CO2. Cell growth was quantieere analysed using SigmaPlot 12.0 (Systat Software Inc.) and IC50 values are shown in T

growth differences between cultures at different CO2 levels were corrected for in the pltrol growth at each CO2 level.

as reduced to 0.54 M. Upon further reduction of pig- by PTU, the NFN1 IC50 was reduced to 0.09 M. Thises directly that the presence of pigment in melanomault in up to an 18-fold decrease in toxicity to NFN1 (seenalogous to the 11- to 23-fold difference we observedgmented and non-pigmented human melanoma andnocyte cell lines. These data are entirely consistent withprotective ability of eumelanin to absorb ROS [20,21].ts that the therapeutic window for NFN1 in heavilymelanomas over non-tumour cells will be reduced. Asr another ROS-producer, a pigment-dependent IC50so observed with hydrogen peroxide, although the dif-s smaller than that for NFN1 (Fig. 3C and Table 1C).dy of the therapeutic window for NFN1 and nifur-nst melanoma will be required to determine which

be most effective. An additional complication could new therapies for melanoma, involving BRAF and/orors, can lead to upregulation of MITF and differentiation

markers sutionship bethat we reptreated mewe have pr

3.5. Conclu

We havsistent withby Type 2 nrequired tootic cells, tindicating aRepair. We potent thancell lines, awith Olaparast images of the mouse melanoma cell line following culturing atigh pigment level). All images taken using a 10 objective lens. (B)

CO2 or 15% CO2. (C) Toxicity of nifurtimox on the mouse melanomad using a Sulphorhodamine B assay [26]. Data from two independentable 1C. There were no noticeable effects of CO2 level on cell growthots where growth curves for the various compounds were expressed

ch as melanin levels [43,44]. By analogy with the rela-tween levels of pigmentation and nitrofuran sensitivityort here, this could render BRAF and/or MEK inhibitor-lanomas more refractory to the nitrofuran therapy thatoposed.

ding remarks

e shown that nitrofuran-induced DNA damage is con- the formation of reactive oxygen species generateditroreductases. While Nucleotide Excision Repair is not

protect against toxicity of NFN1 or nifurtimox in eukary-oxicity of both compounds is enhanced by Olaparib,

role for Base Excision Repair and/or Single Strand Breakhave shown that the nitrofuran compound NFN1 is more

nifurtimox on human neuroblastoma and melanomand that combinatorial therapy of NFN1 or nifurtimoxrib may present a novel therapeutic strategy for both


cancers. However, high levels of eumelanin pigment in melanomacells offer some protection against NFN1 toxicity.

Conict of interest statement

The authors declare that there are no conicts of interest.

Acknowled

We are gand Molecuing us to nsupported bresearch inentist Ofce

Appendix A

Supplemfound, in j.dnarep.20

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The toxicity of nitrofuran compounds on melanoma and neuroblastoma cells is enhanced by Olaparib and ameliorated by melani...1 Introduction2 Materials and methods2.1 Cell culture2.2 Compounds2.3 Comet assays

3 Results and discussion3.1 Nitrofuran-induced DNA damage is independent of Nucleotide Excision Repair3.2 Nitrofurans for the treatment of melanoma and neuroblastoma3.3 Nitrofuran compound toxicity is enhanced by Olaparib3.4 Melanin pigment is protective to nitrofuran-induced DNA damage3.5 Concluding remarks

Conflict of interest statementAcknowledgementsAppendix A Supplementary dataAppendix A Supplementary data

nitrofuran

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