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Research ArticleDNA Damage and Inhibition of Akt Pathway in MCF-7Cells and Ehrlich Tumor in Mice Treated with14-Naphthoquinones in Combination with Ascorbate
Fabiana Ourique1 Maicon R Kviecinski2 Karina B Felipe1
Joatildeo Francisco Gomes Correia1 Mirelle S Farias1 Luiza S E P W Castro1
Valdeluacutecia M A S Grinevicius1 Jaime Valderrama3 David Rios3 Julio Benites3
Pedro Buc Calderon4 and Rozangela Curi Pedrosa1
1Laboratorio de Bioquımica Experimental Departamento de Bioquımica Universidade Federal de Santa CatarinaFlorianopolis Brazil2Programa de Pos-Graduacao em Ciencias da Saude da Universidade do Sul de Santa Catarina (UNISUL) SC Brazil3Departamento de Ciencias Quımicas y Farmaceuticas Universidad Arturo Prat 2120 Avenida Arturo Prat Iquique Chile4Toxicology and Cancer Biology Research Group Louvain Drug Research Institute Universite Catholique de Louvain73 Avenue E Mounier GTOX 7309 1200 Brussels Belgium
Correspondence should be addressed to Rozangela Curi Pedrosa rozangelapedrosagmailcom
Received 29 November 2014 Revised 19 January 2015 Accepted 19 January 2015
Academic Editor Ryuichi Morishita
Copyright copy 2015 Fabiana Ourique et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited
The aim of this study was to enhance the understanding of the antitumor mechanism of 14-naphthoquinones and ascorbateJuglone phenylaminonaphthoquinone-7 and 9 (Q7Q9) were evaluated for effects on CT-DNA and DNA of cancer cellsEvaluations in MCF-7 cells are DNA damage ROS levels viability and proliferation Proteins from MCF-7 lysates wereimmunoblotted for verifying PARP integrity 120574H2AX and pAkt Antitumor activity was measured in Ehrlich ascites carcinoma-bearing mice The same markers of molecular toxicity were assessed in vivo The naphthoquinones intercalate into CT-DNAand caused oxidative cleavage which is increased in the presence of ascorbate Treatments caused DNA damage and reducedviability and proliferation ofMCF-7 cells Effects were potentiated by ascorbateNoPARP cleavagewas observedNaphthoquinonescombined with ascorbate caused phosphorylation of H2AX and inhibited pAkt ROS were enhanced in MCF-7 cells particularlyby the juglone andQ7 plus ascorbate Ehrlich carcinoma was inhibited by juglone Q7 or Q9 but the potentiating effect of ascorbatewas reproduced in vivo only in the cases of juglone and Q7 which caused up to 60 inhibition of tumor and the largest extensionof survival Juglone and Q7 plus ascorbate caused enhanced ROS and DNA damage and inhibited pAkt also in Ehrlich carcinomacells
1 Introduction
Current chemotherapy for cancer has limited efficacy andsafety It causes side effects and the tumor cells often becomeresistant [1] Studies dedicated to the development of novelagents for the treatment of cancer are highly encouragedQuinoid compounds have been widely studied for potentialcancer therapies [2ndash4] It is known that some quinoid com-pounds can induce oxidative stress lethal to cancer cells
Some quinoid compounds can also bind to DNA due to theirelectrical charge Doxorubicin is an example of that it canincrease the generation of reactive oxygen species (ROS) andbind to DNA [5]
DNA is the target for most anticancer drugs In cancercells the interactions between such drugs and DNA resultin cell damage block cell division and lead to cell death [6]Thesemolecules can bindDNAby externally interacting withtheminor ormajor groovesThey can also act as intercalating
Hindawi Publishing CorporationOxidative Medicine and Cellular LongevityVolume 2015 Article ID 495305 10 pageshttpdxdoiorg1011552015495305
2 Oxidative Medicine and Cellular Longevity
agents by inserting between the base-pairs and thus reducingthe helical twist and lengthening theDNA [7]The interactionof molecules with DNA has been applied in therapeuticapproaches that use themodulation of gene transcription andsuppression of the replication to kill tumor cells [8]
Naphthoquinones have been a subject of study because oftheir use in a variety of medical and biological applicationsAs part of our ongoing studies concerning the prepara-tion of potential biologically active compounds 14-naphtho-quinones such as juglone (5-hydroxy-14-naphthoquinone)2-(4-hydroxyaniline)-14-naphthoquinone (Q7) and 2-(4-methoxyaniline)-14-naphthoquinone (Q9) were screenedbecause they possess anticancer potential in vitro [3 4] It hasbeen shown that their anticancer effects can be potentiatedby combining themwith pharmacological doses of ascorbateThe use of ascorbate as an anticancer has been addressed bya large number of papers Pharmacological ascorbate itselfhas been already proposed as a pro-drug for the delivery ofH2O2to tumors [9 10] But data provide support for inves-
tigating the use of pharmacological ascorbate as an adjuvantbecause it was shown that ascorbate triggers synergism withchemotherapy agents such as gemcitabine for instance [11]
The activity of formulations including quinoid com-pounds and ascorbate has been traditionally attributed to theenhanced generation of cellular ROS to levels that are abovethe protection capacity of cancer cells [12] The current studyenhances the understanding of the antitumor mechanism of14-naphthoquinones and ascorbate The data highlight theeffects of these molecules on the DNA and shows that whenjuglone or Q7 are administered together with ascorbate avery damaging formulation is directed against the DNA ofcancer cells in vitro and in vivo The DNA of cancer cellsthus degrades due to an oxidative stress caused by moleculesthat bind and intercalate into its strands As a result cellproliferation and tumor growth are inhibited as shown in thefollowing in vitro and in vivo
2 Materials and Methods
21 Chemicals and Antibodies The 14-naphthoquinones Q7and Q9 were synthesized by amination of 14-naphtho-quinone with the respective arylamines under aerobic con-ditions using CeCl
3sdot7H2O as the Lewis acid catalyst as
previously described [13] Dulbeccorsquosmodified Eaglemedium(DMEM) fetal bovine serum (FBS) and antibiotics were pur-chased fromGibco (USA)The following stuff was purchasedfrom Sigma-Aldrich juglone sodium ascorbate calf thymusDNA (CT-DNA) agarose dimethyl sulfoxide (DMSO) 2101584071015840-dichlorofluorescein diacetate (DCFH-DA) 551015840-dithio-bis(2-nitrobenzoic acid) (DTNB) bovine serum albumin (BSA)ethidium bromide (EtdBr) thiobarbituric acid and the pro-tease inhibitor cocktail The phosphatase inhibitor cocktailwas fromCalbiochem (MerckBiosciences) Rabbit polyclonalantibodies against poly (ADP-ribose) polymerase (PARP)phosphorylated histone gamma H2AX (120574H2AX) and phos-phorylated Akt (pAkt) were from Santa Cruz Biotechnol-ogy Inc (USA) Mouse 120573-actin antibody the secondaryantibodies and the kit for chemiluminescence detection of
horseradish peroxidase- (HRP-) coupled antibodies werefromMillipore (USA)
22 Effects on CT-DNA In Vitro DNA intercalation wasexamined by fluorescence measurements using a TECANInfinity M200 microplate reader CT-DNA (10 120583M) wassaturated with ethidium bromide (3 120583M) in 50mM phos-phate buffer containing 01M NaCl (pH 74) Fluorescencetitrations were conducted by maintaining constant concen-trations of CT-DNA and ethidium bromide and varyingthe concentrations of 14-naphthoquinones (0ndash40 120583M) Theexcitationemission wavelengths were 492 nm and 620 nmrespectively [14]
Oxidative cleavage of CT-DNA was evaluated by themethod proposed by Jun et al [15] using 2-thiobarbituricacid CT-DNA (05mM) in 50mM phosphate buffer (pH 72)was exposed to 10 120583M of 14-naphthoquinones and ascorbate1mM and incubated at 37∘C for 2 h After incubation 2-thiobarbituric acid solution (1) in 50mMNaOH and glacialacetic acid were added (1 1 1) and incubated at 100∘Cfor 30min After cooling the absorbance was measured at532 nm Blanks contained all components except 14-naph-thoquinones and ascorbate The control had free radicalgenerators [Fe(EDTA)]2minus (100 120583M) and hydrogen peroxide(10mM)
23 Effects on the DNA of MCF-7 Cells In Vitro Humanbreast carcinoma MCF-7 cells were purchased from the Riode Janeiro cell bank Brazil Cells were cultured at 37∘C under5 CO
2atmosphere with 95 air humidity DMEM was
used supplemented with 10 FBS penicillin (100UmL)and streptomycin (100 120583gmL) The effects on the DNA ofMCF-7 cells were examined by the comet assay [16] and thephosphorylation on histone H2AX The occurrence of phos-phorylation on serine 139 of histone H2AX namely 120574H2AXhas been widely used as a sensitive marker of double-strandDNA breaks [17] Gamma-H2AX was measured in wholeMCF-7 cell homogenates through immunoelectrophoresisusing the method described in Section 25 PARP and pAktwere evaluated by the same method MCF-7 cells were usedprimarily because they allow studying the effects of com-pounds on the proliferation and they express H2AX and Akt[18 19]
24 The Comet Assay Treated cells were suspended in 075low-melting point agarose and then deposited on the surfaceof a slide containing a thin layer of 15 agarose and allowedto set for 10min at room temperature The slides weresubmerged for 2 h in a lysis solution (25MNaCl 10mMTris100mM EDTA 1 Triton X-100 10 DMSO and pH 100)and then subjected to horizontal electrophoresis at 300mA8∘C for 20min in a tank with buffer (300mM NaOH 1mMEDTA and pH 13) A neutralizing solution (04M Tris-HClpH 75) was added (3 times) followed bywashing inwater anddrying at 37∘C A fixing solution (15 trichloroacetic acid 5ZnSO
4 and 5 glycerol) was then added for 10min followed
bywashing and dryingThe slides were stainedwith ethidiumbromide (05mgmL) and analyzed under a fluorescence
Oxidative Medicine and Cellular Longevity 3
microscope Each nucleus received a fluorescence value inthe 0ndash4 range (arbitrary units 0mdashundamaged 4mdashmaximallydamaged) [20]
25 Immunoblotting Assays After treatment cells werewashed with phosphate buffered saline (PBS) and lysed inRIPA buffer (50mM Tris-Cl pH 74 150mM NaCl 1NP40 025 Na-deoxycholate and 1mM phenylmethylsul-fonyl fluoride) supplemented with 1 protease inhibitor and3 phosphatase inhibitor cocktails After denaturation inLaemmli buffer (60mM Tris-Cl pH 68 2 sodium dodecylsulfate (SDS) 10 glycerol 5 120573-mercaptoethanol and001 bromophenol blue) equal amounts of protein (30120583g)fromwhole cellular homogenates were subjected to polyacry-lamide gel electrophoresis (SDS-PAGE) followed by elec-troblotting to polyvinylidene fluoride (PVDF) membranesAfter blocking the membranes were incubated overnightwith the primary antibodies The membranes were washedand incubated with the secondary antibodies for 1 h Immun-odetection was performed using the enhanced chemilumi-nescence (ECL) detection kit (Millipore USA) for HRP-coupled secondary antibodies Beta-actin served as a loadingcontrol
26 Effects on MCF-7 Cell Viability and Proliferation Cyto-toxicity wasmeasured using the tetrazolium salt (MTT) assay[21] Briefly 104 cellswell were plated onto 96-well plates Atconfluence the cells were exposed to juglone Q7 and Q9(0ndash80120583M) in the absence or presence of ascorbate (1mM)for up to 24 h The cells were then washed twice with PBSand incubated for 2 h with MTT (05mgmL) The formazancrystals were solubilised by adding DMSO (100 120583Lwell) andthe colored solutionswere read at 550 nmThree independentexperiments were conducted and the results are presented asEC50values
The effects on cell proliferation were examined by thecolony formation assay according to Franken et al [22] Cells(500) were treated for 2 h with the compounds They werethen washed twice with warm PBS and fresh medium wasadded After 15 days the cells were stained by crystal violetand colonies with more than 50 cells were counted
27 Levels of MCF-7 Intracellular ROS Intracellular ROSwere measured as reported by Glorieux et al [23] Cells(15000) were loaded with 10 120583M DCFH-DA in Hankrsquos bal-anced salt solution (HBSS) at 37∘C and incubated for 30minExcess DCFH-DAwas removed by washing with fresh HBSSThe cells were incubated for 2 h with the test compoundswashed twice withHBSS and then 100120583L ofHBSSwas addedto each well The fluorescence intensity was measured witha TECAN Infinity M200 microplate reader at 485 nm forexcitation and 530 nm for emission
28 Antitumor Activity In Vivo Male BALBc inbred mice(20ndash22 g) received water and food ad libitum Procedureswere conducted in accordance with legal requirements andwith the approval of the local ethics committee (UFSCPP00784) Previous tests were conducted to select safe doses
of 14-naphthoquinones Ascorbatewas administered at doses100 times higher On day zero Ehrlich carcinoma cells (5 times106) were inoculated into the abdomen of mice from ninegroups (119899 = 12) Treatments were done via intraperitonealinjections every 24 h for 9 days The control group receivedsaline injections and the positive control group receiveddoxorubicin (12mgkg) Test groups received juglone Q7 orQ9 (1mgkg) andor ascorbate (100mgkg) After treatmentthe inhibition of tumor growth was measured based onchanges in the abdominal circumference [24]The percentageof increased life span was calculated by recording mortalityon a daily basis for 30 days according to themethod ofKaplanand Meier [25] Samples of ascitic fluid were collected 2 hafter the last dose and immediately processedThe moleculareffects in vivowere assessed again at level of DNA through thecomet assay and gamma-H2AX As done in vitrowithMCF-7cells also in vivoAkt-pathwaywas evaluated in Ehrlich tumorafter treatments through electrophoresis and immunoblot-ting as described in Sections 24 and 25 Lipid peroxidationwas estimated by measurement of malondialdehyde (MDA)formation using the thiobarbituric acid method [26]
29 Data Analysis In general the assays were performed intriplicate In vitro assays were repeated at least three timesThe results are presented as themeansplusmn standard deviation oras percentagesThe data were analyzed by the analysis of vari-ance (ANOVA) test followed by the Bonferroni test Com-parisons were performed using the GraphPad Prism software(San Diego USA) Values of 119875 lt 005 were consideredstatistically significant
3 Results and Discussion
Intercalation of juglone Q7 andQ9 into CT-DNAwas exam-ined by using the fluorescent intercalating agent ethidiumbromide Compounds that are able to intercalate into DNAcompete with ethidium bromide and reduce its fluorescencewhen read in a fluorimeter As show in Figure 1(a) whenDNA and ethidium bromide were incubated with jugloneQ7 or Q9 the fluorescence was reduced indicating that thecompounds can intercalate into CT-DNA The intercalatingcapacity of juglone Q7 and Q9 was always higher when theincubations were performed in the presence of ascorbate Asexpected doxorubicin which is a known intercalating agentalso reduced the fluorescence signal [27]
CT-DNA treated with juglone Q7 or Q9 underwentoxidative cleavage as shown by data in Figure 1(b) Freeradicals can attack DNA at C4 of desoxyribose causingcleavage and generation of products of degradation such asbase propenal This can be detected because base propenalreacts with 2-thiobarbituric acid producing color [28] Datain Figure 1(b) shows that the absorbance resulting from thio-barbituric acid species (TBARS) was increased in CT-DNAtreated with the free radical generators [Fe(EDTA)]2minusH
2O2
as well as it occurred in CT-DNA treated with juglone Q7or Q9 TBARS absorbance was always higher when CT-DNA was treated with the naphthoquinones combined withascorbate (Figure 1(b))
4 Oxidative Medicine and Cellular Longevity
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(c)
Figure 1 Continued
Oxidative Medicine and Cellular Longevity 5
120574H2AX
Control Asc Jug Q7 Q9JugAsc
Q7Asc
Q9Asc
1mM 20120583M 20120583M1mM
120573-actin
(d)
Figure 1The fluorescence of CT-DNA stained with ethidium bromide is reduced by doxorubicin and juglone Q7 or Q9 mainly in presenceof ascorbate (Asc) (a) Absorbance of thiobarbituric reactive species (TBARS) in CT-DNA treatedwith juglone Q7 orQ9 alone and combinedwith ascorbate Negative control (NC) phosphate buffer Positive control (PC) [Fe(EDTA)]2minusH
2O2(b) DNA damage index was determined
by the comet assay in MCF-7 cells treated for 24 h with juglone 75120583M Q7 50 120583M or Q9 50 120583Mwith or without ascorbate 1mM (c) 120574H2AXwas assessed by immunoelectrophoresis in MCF-7 cells treated for 2 h with juglone (Jug) Q7 or Q9 at 20 120583Mwith or without ascorbate (Asc)at 1mM (d) (lowastlowast) and (lowast lowast lowast) denote statistical differences at 119875 lt 001 and 119875 lt 0001 compared to indicated treatments
Table 1 EC50 values obtained by the MTT assay MCF-7 cells weretreated for 24 h with juglone Q7 or Q9 (5ndash80 120583M) with or withoutascorbate 1mM Data from three independent experiments
EC50 (120583M)Juglone Q7 Q9
61 416 50plus ascorbate 1mM 28 263 254
The effects on the DNA of MCF-7 cells were assessedby immunoelectrophoresis for 120574H2AX and the comet assayGamma-H2AX is required for many proteins during theDNA damage and repair response [17] Figure 1(d) showsthat individually neither ascorbate juglone Q7 nor Q9enhanced the phosphorylated protein band corresponding to120574H2AXThe level of 120574H2AXwas enhanced onlywhenMCF-7cells were treated with the compounds combined with ascor-bate (1mM) Consistent with these findings the comet assayshowed that juglone Q7 and Q9 induced the fragmentationof MCF-7 DNA on their own but the addition of ascorbateincreased DNA fragmentation by approximately 2-fold in thecase of juglone and Q7 (Figure 1(c))
The effects of juglone Q7 and Q9 with and withoutascorbate were then examined on viability and proliferationinMCF-7 cellsTheMTT assay showed that ascorbate (1mM)alone did not reduce the viability of the MCF-7 cells Thecytotoxicity of juglone Q7 and Q9 was determined showingEC50values of 61 416 and 50120583M respectively The addition
of ascorbate together with the naphthoquinones reduced theEC50values by approximately 2-fold (to 28 263 and 254 120583M
for juglone Q7 andQ9 resp) and thus enhanced cytotoxicity(Table 1)
Ascorbate activates the redox cycling of quinones gen-erating semiquinone radicals and ROS In T24 cells it waspreviously shown that ascorbate (1mM) administered withjuglone Q7 and Q9 caused the appearance of semiquinoneand elevated levels of intracellular ROS [3 4] If a similarphenomenon occurs in MCF7 cells DNA damage wouldresult from not only binding and intercalation but also froman oxidative attack Doxorubicin acts by such a mechanismand kills cancer cells through both DNA intercalation and
ROS induction [29] Figure 2(a) shows that the ROS levelsin MCF-7 cells were elevated by the compounds particularlyby juglone and Q7 ROS generation induced by juglone andQ7 was enhanced approximately 3-fold in the presence ofascorbate On the other hand ascorbate had less impact interms of ROS induction when administered together withQ9 on MCF-7 cells Later the formulations containing Q9 orQ9 plus ascorbate were proven as poor inducers of oxidativestress in both MCF-7 cells (Figure 2(a)) and Ehrlich ascitescarcinoma in mice (Figure 3(c)) Ascorbate caused someincrease in terms of oxidative damage by Q9 only whenour treatments were done on CT-DNA a noncellular system(Figure 1(b))
Cell death was further studied based on PARP cleavageas shown in Figure 2(b) Compounds that are able to induceapoptosis cleave the PARP protein replacing the full-length116 kD protein with a cleaved fragment of approximately89 kD [30] Sanguinarine (5 120583M) a flavonoid known toinduce apoptosis was used as a control [31] PARP cleavagewas observed only in sanguinarine-treated cells and not incells treated with juglone Q7 or Q9 with or without ascor-bate (Figure 2(b))
The effect on MCF-7 cell proliferation is shown inFigure 2(c) JugloneQ7 andQ9 administered alone inhibitedcell proliferation as indicated by a reduction in the numberof colony-forming units in comparison with nontreated cellsIn all cases the inhibition of proliferation was increasedup to 3-fold when the naphthoquinones were administeredin combination with ascorbate (1mM) (Figure 2(c)) Thesedata corroborate literature when considered that DNA inter-calators can block cell division [6] To examine whetherthe PI3KAktmTOR signalling pathway was affected by thetreatments in MCF-7 cells the levels of the active phospho-rylated form of Akt (pAkt) were determined (Figure 2(d))The PI3KAkt pathway regulates several biological processesincluding cell survival proliferation and differentiationUpregulation of this pathway is observed in several types ofcancer and it can be associated with uncontrolled cell prolif-eration [32ndash34] As demonstrated in Figure 2(d) significantinhibition of the pAkt occurred in MCF-7 cells followingtreatment most notably with juglone or Q7 in the presenceof ascorbate
6 Oxidative Medicine and Cellular Longevity
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(d)
Figure 2 ROS were measured in MCF-7 cells treated for 2 h with juglone Q7 or Q9 at 10120583M with or without ascorbate 1mM (a) Integrityof PARP protein in MCF-7 cells treated with juglone (Jug) Q7 or Q9 at 20 120583M with or without ascorbate (Asc) 1mM for 24 h Sanguinarine(Sang) 5120583M was used as a positive control of apoptosis (b) Colony-forming units of MCF-7 cells treated with juglone Q7 or Q9 at 10 120583Mwithwithout ascorbate 1mM for 2 h (c) Phosphorylated Akt (pAkt) was assessed by immunoelectrophoresis in MCF-7 cells treated for 24 h(d) Data were obtained from three independent experiments (lowastlowast) and (lowast lowast lowast) denote statistical differences at 119875 lt 001 and 119875 lt 0001compared to nontreated control cells or between indicated treatments respectively
Following these in vitro assays some in vivo effects werestudied in Ehrlich ascites carcinoma-bearing mice Ehrlichascites carcinoma was chosen to be used in vivo initially toverify whether some effects observed in vitro were repro-ducible in vivo But also to evaluate whether the effectswere consistent only with MCF-7 cells in vitro or they couldbe repeated against a different tumor cell line Figure 3(a)presents the levels of tumor growth inhibition Some inhi-bition on Ehrlich carcinoma was caused in animals treatedwith juglone Q7 orQ9 But the formulations of juglone orQ7plus ascorbate had the most potent activity and achieved upto 60 of inhibition of tumor growth approaching the effectof doxorubicin which caused up to 90 inhibition Actuallyconsidering data related to tumor growth and survival it is
possible to suggest that the potentiating effect of the com-bined treatment with ascorbate was reproduced in vivo withstatistical difference only in the case of juglone and Q7 Theability to increase the duration of animal survival is one ofthe most reliable criteria for evaluating potential antitumordrugs [35] Figure 3(b) presents graphs relating the numberof days after tumor inoculation to the percentage of survivorsfollowing treatment an increase in the area under the curvesindicates an increase in survival The smallest area in thegraphs corresponds to the group of nontreated animals Ingeneral these animals started to die on day 10 and by day12 no survivors remained The survival time was extendedby 2 to 6 days when the animals received treatments withindividual compounds (juglone Q7 or Q9) These animals
Oxidative Medicine and Cellular Longevity 7
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8 Oxidative Medicine and Cellular Longevity
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Ctrl Asc Jug Q7 Q9JugAsc
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(e)
Figure 3 Ehrlich ascites carcinoma inhibition (a) and survival time (b) in animals treated with juglone Q7 or Q9 at 1mgkg alone or incombination with ascorbate (Asc) at 100mgkg Doxorubicin was administered at 12mgkg for the positive control Lipid peroxidation (c)DNA damage (d) and phosphorylated proteins Akt (pAkt) and H2AX (120574H2AX) in ascitic cells from mice (e) (lowastlowast) and (lowast lowast lowast) denotedifference at 119875 lt 001 and 119875 lt 0001 compared to the negative control or between indicated treatments
generally died before day 20The largest area under the curvescorresponds to the treatments conducted with juglone or Q7in combination with ascorbate (Figure 3(b)) The synergisticeffect between these drugs occurred also in vivo
Figures 3(c) 3(d) and 3(e) present data of moleculartoxicity on Ehrlich tumor in mice Figure 3(c) depicts levelsof MDA a biomarker of lipid peroxidationThis was the end-point measurement done to verify oxidative stress causedby the treatments in vivo MDA levels were increased sig-nificantly by the combined treatments done with juglone orQ7 and ascorbate Data in Figure 3(d) corroborate with datafrom Figure 3(c) because they show that DNA fragmenta-tion was increased significantly in vivo only when animalsreceived juglone or Q7 plus ascorbate Finally data shownin Figure 3(e) provide concise evidences which demonstratethat juglone and Q7 plus ascorbate can cause DNA damageand pAkt inhibition in Ehrlich tumor in mice Thus theeffects are not restricted to MCF-7 cells in vitro
Data from this study are summarized illustratively inFigure 4 Although all 14-naphthoquinones presented someactivity primarily juglone and Q7 administered in combina-tion with ascorbate trigger the generation of free radicals thesemiquinone formof the naphthoquinone and dehydroascor-bate The resulting semiquinone form intercalates into DNA
The radicals can attack DNA which is degraded In cancercells these events are accompanied by an inhibition on Aktpathway The formulation of juglone or Q7 with ascorbateshowed the most promising activity in vivo
4 Conclusion
Overall it is possible to conclude that primarily jugloneand Q7 combined with ascorbate have significant antitumoreffects against MCF-7 cells in vitro and Ehrlich-ascites carci-noma in mice The effects are the result of intercalation andoxidative attack on DNA of tumor cells and inhibition of Aktpathway
Conflict of Interests
The authors declare that they have no conflict of interests
Acknowledgments
Fabiana Ourique Luiza S E P W Castro and ValdeluciaM A S Grinevicius are recipients of research grants from
Oxidative Medicine and Cellular Longevity 9
Juglone Q7 Q9
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Figure 4 Antitumor actions of juglone Q7 and Q9 administered in combination with ascorbate against MCF-7 cells and Ehrlich ascitescarcinoma in mice The effects are the result of intercalation and oxidative attack on DNA of tumor cells and inhibition of Akt pathway
Coordenacao de Aperfeicoamento de Pessoal de Nıvel Supe-rior (CAPES Brazil) Karina B Felipe and Rozangela CuriPedrosa (Proc 3024042011-2) are recipients of researchgrants from Conselho Nacional de Pesquisa (CNPq) Brazil
References
[1] M M Gottesman ldquoMechanisms of cancer drug resistancerdquoAnnual Review of Medicine vol 53 pp 615ndash627 2002
[2] M S Farias C T Pich M R Kviecinski et al ldquoSubstituted3-acyl-2-phenylamino-14-naphthoquinones intercalate intoDNA and cause genotoxicity through the increased generationof reactive oxygen species culminating in cell deathrdquoMolecularMedicine Reports vol 10 no 1 pp 405ndash410 2014
[3] K B Felipe J Benites C Glorieux et al ldquoAntiproliferativeeffects of phenylaminonaphthoquinones are increased by ascor-bate and associated with the appearance of a senescent pheno-type in human bladder cancer cellsrdquo Biochemical and Biophysi-cal Research Communications vol 433 no 4 pp 573ndash578 2013
[4] M R Kviecinski R C Pedrosa K B Felipe et al ldquoInhibitionof cell proliferation and migration by oxidative stress fromascorbate-driven juglone redox cycling in human bladder-derived T24 cellsrdquo Biochemical and Biophysical Research Com-munications vol 421 no 2 pp 268ndash273 2012
[5] C F Thorn C Oshiro S Marsh et al ldquoDoxorubicin pathwayspharmacodynamics and adverse effectsrdquo Pharmacogenetics andGenomics vol 21 no 7 pp 440ndash446 2011
[6] G Bischoff and S Hoffman ldquoDNA-binding of drugs used inmedicinal therapiesrdquo Current Medicinal Chemistry vol 9 no3 pp 321ndash348 2002
[7] R Palchaudhuri and P J Hergenrother ldquoDNA as a target foranticancer compounds methods to determine the mode ofbinding and the mechanism of actionrdquo Current Opinion inBiotechnology vol 18 no 6 pp 497ndash503 2007
[8] M S Shahabuddin M Gopal and S C Raghavan ldquoIntercalat-ing and antitumor activity of 4 oxopyrimido[410158405101584045]thieno(23-b) quinoline-4(3H)-onerdquo Journal of Cancer Molecules vol 3pp 139ndash146 2007
[9] Q Chen M G Espey M C Krishna et al ldquoPharamacologicascorbic acid concentrations selectively kill cancer cells actionas a pro-drug to deliver hydrogen peroxide to tissuserdquo Proceed-ings of the National Academy of Sciences of the United States ofAmerica vol 102 no 38 pp 13604ndash13609 2005
[10] J Du J J Cullen and G R Buettner ldquoAscorbic acid chemistrybiology and the treatment of cancerrdquo Biochimica et BiophysicaActa vol 1826 no 2 pp 443ndash457 2012
[11] M G Espey P Chen B Chalmers et al ldquoPharmacologicascorbate synergizes with gemcitabine in preclinical models of
10 Oxidative Medicine and Cellular Longevity
pancreatic cancerrdquo Free Radical Biology and Medicine vol 50no 11 pp 1610ndash1619 2011
[12] J Verrax R Beck N Dejeans et al ldquoRedox-active quinonesand ascorbate an innovative cancer therapy that exploits thevulnerability of cancer cells to oxidative stressrdquo Anti-CancerAgents in Medicinal Chemistry vol 11 no 2 pp 213ndash221 2011
[13] J Benites J A Valderrama K Bettega R C Pedrosa PB Calderon and J Verrax ldquoBiological evaluation of donor-acceptor aminonaphthoquinones as antitumor agentsrdquo Euro-pean Journal of Medicinal Chemistry vol 45 no 12 pp 6052ndash6057 2010
[14] V C da Silveira H Benezra J S Luz R C Georg C COliveira and A M D C Ferreira ldquoBinding of oxindole-Schiffbase copper(II) complexes to DNA and its modulation by theligandrdquo Journal of Inorganic Biochemistry vol 105 no 12 pp1692ndash1703 2011
[15] T Jun W Bochu and Z Liancai ldquoHydrolytic cleavage of DNAby quercetin manganese(II) complexesrdquo Colloids and SurfacesB Biointerfaces vol 55 no 2 pp 149ndash152 2007
[16] N P Singh M T McCoy R R Tice and E L Schneider ldquoAsimple technique for quantitation of low levels of DNA damagein individual cellsrdquo Experimental Cell Research vol 175 no 1pp 184ndash191 1988
[17] J Yuan R Adamski and J Chen ldquoFocus on histone variantH2AX to be or not to berdquo FEBS Letters vol 584 no 17 pp 3717ndash3724 2010
[18] T T Paull E P Rogakou V Yamazaki C U Kirchgessner MGellert and W M Bonner ldquoA critical role for histone H2AX inrecruitment of repair factors to nuclear foci after DNAdamagerdquoCurrent Biology vol 10 no 15 pp 886ndash895 2000
[19] R Lopez A Arumugam R Joseph et al ldquoHyperglycemiaenhances the proliferation of non-tumorigenic and malignantmammary epithelial cells through increased leptinIGF1R sig-naling and activation of AKTmTORrdquo PLoS ONE vol 8 no 11Article ID e79708 2013
[20] G M Ross T J McMillan P Wilcox and A R Collins ldquoThesingle cell microgel electrophoresis assay (comet assay) techni-cal aspects and applications Report on the 5th LH Gray TrustWorkshop Institute of Cancer Research 1994rdquo MutationResearch vol 337 no 1 pp 57ndash60 1995
[21] T Mosmann ldquoRapid colorimetric assay for cellular growth andsurvival application to proliferation and cytotoxicity assaysrdquoJournal of Immunological Methods vol 65 no 1-2 pp 55ndash631983
[22] N A P Franken H M Rodermond J Stap J Haveman and Cvan Bree ldquoClonogenic assay of cells in vitrordquo Nature Protocolsvol 1 no 5 pp 2315ndash2319 2006
[23] C Glorieux N Dejeans B Sid R Beck P B Calderon andJ Verrax ldquoCatalase overexpression in mammary cancer cellsleads to a less aggressive phenotype and an altered response tochemotherapyrdquo Biochemical Pharmacology vol 82 no 10 pp1384ndash1390 2011
[24] M R Kviecinski P Benelli K B Felipe et al ldquoSFE from Bidenspilosa Linne to obtain extracts rich in cytotoxic polyacetyleneswith antitumor activityrdquo Journal of Supercritical Fluids vol 56no 3 pp 243ndash248 2011
[25] E L Kaplan and P Meier ldquoNonparametric estimation fromincomplete observationsrdquo Journal of the American StatisticalAssociation vol 53 pp 457ndash481 1958
[26] R P Bird and H H Draper ldquoComparative studies on differentmethods of malonaldehyde determinationrdquo Methods in Enzy-mology vol 105 pp 299ndash305 1984
[27] V G S Box ldquoThe intercalation of DNA double helices withdoxorubicin and nagalomycinrdquo Journal of Molecular Graphicsand Modelling vol 26 no 1 pp 14ndash19 2007
[28] Q Jiang N Xiao P Shi Y Zhu and Z Guo ldquoDesign of artifi-cial metallonucleases with oxidative mechanismrdquo CoordinationChemistry Reviews vol 251 no 15-16 pp 1951ndash1972 2007
[29] M U Rehman M Tahir A Q Khan et al ldquoD-limonene sup-presses doxorubicin-induced oxidative stress and inflammationvia repression of COX-2 iNOS and NF120581B in kidneys of Wistarratsrdquo Experimental Biology and Medicine vol 239 no 4 pp465ndash476 2014
[30] A H Boulares A G Yakovlev V Ivanova et al ldquoRole ofpoly(ADP-ribose) polymerase (PARP) cleavage in apoptosisCaspase 3-resistant PARP mutant increases rates of apoptosisin transfected cellsrdquo Journal of Biological Chemistry vol 274 no33 pp 22932ndash22940 1999
[31] N Ahmad S Gupta M M Husain K M Heiskanen and HMukhtar ldquoDifferential antiproliferative and apoptotic responseof sanguinarine for cancer cells versus normal cellsrdquo ClinicalCancer Research vol 6 no 4 pp 1524ndash1528 2000
[32] S J Lee H Ahn K W Nam K H Kim and W Mar ldquoEffectsof rutaecarpine on hydrogen peroxide-induced apoptosis inmurine hepa-1c1c7 cellsrdquo Biomolecules amp Therapeutics vol 20no 5 pp 487ndash491 2012
[33] E-S A Arafa Q Zhu Z I Shah et al ldquoThymoquinoneup-regulates PTEN expression and induces apoptosis indoxorubicin-resistant human breast cancer cellsrdquo MutationResearchmdashFundamental and Molecular Mechanisms of Mutage-nesis vol 706 no 1-2 pp 28ndash35 2011
[34] G Viglietto M L Motti P Bruni et al ldquoCytoplasmic relocal-ization and inhibition of the cyclin-dependent kinase inhibitorp271198961198941199011 by PKBAkt-mediated phosphorylation in breast can-cerrdquo Nature Medicine vol 8 no 10 pp 1136ndash1144 2002
[35] B D Clarkson and J H Burchenal ldquoPreliminary screening ofantineoplastic drugsrdquoProgress in Clinical Cancer vol 1 pp 625ndash629 1965
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
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Computational and Mathematical Methods in Medicine
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Research and TreatmentAIDS
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Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
2 Oxidative Medicine and Cellular Longevity
agents by inserting between the base-pairs and thus reducingthe helical twist and lengthening theDNA [7]The interactionof molecules with DNA has been applied in therapeuticapproaches that use themodulation of gene transcription andsuppression of the replication to kill tumor cells [8]
Naphthoquinones have been a subject of study because oftheir use in a variety of medical and biological applicationsAs part of our ongoing studies concerning the prepara-tion of potential biologically active compounds 14-naphtho-quinones such as juglone (5-hydroxy-14-naphthoquinone)2-(4-hydroxyaniline)-14-naphthoquinone (Q7) and 2-(4-methoxyaniline)-14-naphthoquinone (Q9) were screenedbecause they possess anticancer potential in vitro [3 4] It hasbeen shown that their anticancer effects can be potentiatedby combining themwith pharmacological doses of ascorbateThe use of ascorbate as an anticancer has been addressed bya large number of papers Pharmacological ascorbate itselfhas been already proposed as a pro-drug for the delivery ofH2O2to tumors [9 10] But data provide support for inves-
tigating the use of pharmacological ascorbate as an adjuvantbecause it was shown that ascorbate triggers synergism withchemotherapy agents such as gemcitabine for instance [11]
The activity of formulations including quinoid com-pounds and ascorbate has been traditionally attributed to theenhanced generation of cellular ROS to levels that are abovethe protection capacity of cancer cells [12] The current studyenhances the understanding of the antitumor mechanism of14-naphthoquinones and ascorbate The data highlight theeffects of these molecules on the DNA and shows that whenjuglone or Q7 are administered together with ascorbate avery damaging formulation is directed against the DNA ofcancer cells in vitro and in vivo The DNA of cancer cellsthus degrades due to an oxidative stress caused by moleculesthat bind and intercalate into its strands As a result cellproliferation and tumor growth are inhibited as shown in thefollowing in vitro and in vivo
2 Materials and Methods
21 Chemicals and Antibodies The 14-naphthoquinones Q7and Q9 were synthesized by amination of 14-naphtho-quinone with the respective arylamines under aerobic con-ditions using CeCl
3sdot7H2O as the Lewis acid catalyst as
previously described [13] Dulbeccorsquosmodified Eaglemedium(DMEM) fetal bovine serum (FBS) and antibiotics were pur-chased fromGibco (USA)The following stuff was purchasedfrom Sigma-Aldrich juglone sodium ascorbate calf thymusDNA (CT-DNA) agarose dimethyl sulfoxide (DMSO) 2101584071015840-dichlorofluorescein diacetate (DCFH-DA) 551015840-dithio-bis(2-nitrobenzoic acid) (DTNB) bovine serum albumin (BSA)ethidium bromide (EtdBr) thiobarbituric acid and the pro-tease inhibitor cocktail The phosphatase inhibitor cocktailwas fromCalbiochem (MerckBiosciences) Rabbit polyclonalantibodies against poly (ADP-ribose) polymerase (PARP)phosphorylated histone gamma H2AX (120574H2AX) and phos-phorylated Akt (pAkt) were from Santa Cruz Biotechnol-ogy Inc (USA) Mouse 120573-actin antibody the secondaryantibodies and the kit for chemiluminescence detection of
horseradish peroxidase- (HRP-) coupled antibodies werefromMillipore (USA)
22 Effects on CT-DNA In Vitro DNA intercalation wasexamined by fluorescence measurements using a TECANInfinity M200 microplate reader CT-DNA (10 120583M) wassaturated with ethidium bromide (3 120583M) in 50mM phos-phate buffer containing 01M NaCl (pH 74) Fluorescencetitrations were conducted by maintaining constant concen-trations of CT-DNA and ethidium bromide and varyingthe concentrations of 14-naphthoquinones (0ndash40 120583M) Theexcitationemission wavelengths were 492 nm and 620 nmrespectively [14]
Oxidative cleavage of CT-DNA was evaluated by themethod proposed by Jun et al [15] using 2-thiobarbituricacid CT-DNA (05mM) in 50mM phosphate buffer (pH 72)was exposed to 10 120583M of 14-naphthoquinones and ascorbate1mM and incubated at 37∘C for 2 h After incubation 2-thiobarbituric acid solution (1) in 50mMNaOH and glacialacetic acid were added (1 1 1) and incubated at 100∘Cfor 30min After cooling the absorbance was measured at532 nm Blanks contained all components except 14-naph-thoquinones and ascorbate The control had free radicalgenerators [Fe(EDTA)]2minus (100 120583M) and hydrogen peroxide(10mM)
23 Effects on the DNA of MCF-7 Cells In Vitro Humanbreast carcinoma MCF-7 cells were purchased from the Riode Janeiro cell bank Brazil Cells were cultured at 37∘C under5 CO
2atmosphere with 95 air humidity DMEM was
used supplemented with 10 FBS penicillin (100UmL)and streptomycin (100 120583gmL) The effects on the DNA ofMCF-7 cells were examined by the comet assay [16] and thephosphorylation on histone H2AX The occurrence of phos-phorylation on serine 139 of histone H2AX namely 120574H2AXhas been widely used as a sensitive marker of double-strandDNA breaks [17] Gamma-H2AX was measured in wholeMCF-7 cell homogenates through immunoelectrophoresisusing the method described in Section 25 PARP and pAktwere evaluated by the same method MCF-7 cells were usedprimarily because they allow studying the effects of com-pounds on the proliferation and they express H2AX and Akt[18 19]
24 The Comet Assay Treated cells were suspended in 075low-melting point agarose and then deposited on the surfaceof a slide containing a thin layer of 15 agarose and allowedto set for 10min at room temperature The slides weresubmerged for 2 h in a lysis solution (25MNaCl 10mMTris100mM EDTA 1 Triton X-100 10 DMSO and pH 100)and then subjected to horizontal electrophoresis at 300mA8∘C for 20min in a tank with buffer (300mM NaOH 1mMEDTA and pH 13) A neutralizing solution (04M Tris-HClpH 75) was added (3 times) followed bywashing inwater anddrying at 37∘C A fixing solution (15 trichloroacetic acid 5ZnSO
4 and 5 glycerol) was then added for 10min followed
bywashing and dryingThe slides were stainedwith ethidiumbromide (05mgmL) and analyzed under a fluorescence
Oxidative Medicine and Cellular Longevity 3
microscope Each nucleus received a fluorescence value inthe 0ndash4 range (arbitrary units 0mdashundamaged 4mdashmaximallydamaged) [20]
25 Immunoblotting Assays After treatment cells werewashed with phosphate buffered saline (PBS) and lysed inRIPA buffer (50mM Tris-Cl pH 74 150mM NaCl 1NP40 025 Na-deoxycholate and 1mM phenylmethylsul-fonyl fluoride) supplemented with 1 protease inhibitor and3 phosphatase inhibitor cocktails After denaturation inLaemmli buffer (60mM Tris-Cl pH 68 2 sodium dodecylsulfate (SDS) 10 glycerol 5 120573-mercaptoethanol and001 bromophenol blue) equal amounts of protein (30120583g)fromwhole cellular homogenates were subjected to polyacry-lamide gel electrophoresis (SDS-PAGE) followed by elec-troblotting to polyvinylidene fluoride (PVDF) membranesAfter blocking the membranes were incubated overnightwith the primary antibodies The membranes were washedand incubated with the secondary antibodies for 1 h Immun-odetection was performed using the enhanced chemilumi-nescence (ECL) detection kit (Millipore USA) for HRP-coupled secondary antibodies Beta-actin served as a loadingcontrol
26 Effects on MCF-7 Cell Viability and Proliferation Cyto-toxicity wasmeasured using the tetrazolium salt (MTT) assay[21] Briefly 104 cellswell were plated onto 96-well plates Atconfluence the cells were exposed to juglone Q7 and Q9(0ndash80120583M) in the absence or presence of ascorbate (1mM)for up to 24 h The cells were then washed twice with PBSand incubated for 2 h with MTT (05mgmL) The formazancrystals were solubilised by adding DMSO (100 120583Lwell) andthe colored solutionswere read at 550 nmThree independentexperiments were conducted and the results are presented asEC50values
The effects on cell proliferation were examined by thecolony formation assay according to Franken et al [22] Cells(500) were treated for 2 h with the compounds They werethen washed twice with warm PBS and fresh medium wasadded After 15 days the cells were stained by crystal violetand colonies with more than 50 cells were counted
27 Levels of MCF-7 Intracellular ROS Intracellular ROSwere measured as reported by Glorieux et al [23] Cells(15000) were loaded with 10 120583M DCFH-DA in Hankrsquos bal-anced salt solution (HBSS) at 37∘C and incubated for 30minExcess DCFH-DAwas removed by washing with fresh HBSSThe cells were incubated for 2 h with the test compoundswashed twice withHBSS and then 100120583L ofHBSSwas addedto each well The fluorescence intensity was measured witha TECAN Infinity M200 microplate reader at 485 nm forexcitation and 530 nm for emission
28 Antitumor Activity In Vivo Male BALBc inbred mice(20ndash22 g) received water and food ad libitum Procedureswere conducted in accordance with legal requirements andwith the approval of the local ethics committee (UFSCPP00784) Previous tests were conducted to select safe doses
of 14-naphthoquinones Ascorbatewas administered at doses100 times higher On day zero Ehrlich carcinoma cells (5 times106) were inoculated into the abdomen of mice from ninegroups (119899 = 12) Treatments were done via intraperitonealinjections every 24 h for 9 days The control group receivedsaline injections and the positive control group receiveddoxorubicin (12mgkg) Test groups received juglone Q7 orQ9 (1mgkg) andor ascorbate (100mgkg) After treatmentthe inhibition of tumor growth was measured based onchanges in the abdominal circumference [24]The percentageof increased life span was calculated by recording mortalityon a daily basis for 30 days according to themethod ofKaplanand Meier [25] Samples of ascitic fluid were collected 2 hafter the last dose and immediately processedThe moleculareffects in vivowere assessed again at level of DNA through thecomet assay and gamma-H2AX As done in vitrowithMCF-7cells also in vivoAkt-pathwaywas evaluated in Ehrlich tumorafter treatments through electrophoresis and immunoblot-ting as described in Sections 24 and 25 Lipid peroxidationwas estimated by measurement of malondialdehyde (MDA)formation using the thiobarbituric acid method [26]
29 Data Analysis In general the assays were performed intriplicate In vitro assays were repeated at least three timesThe results are presented as themeansplusmn standard deviation oras percentagesThe data were analyzed by the analysis of vari-ance (ANOVA) test followed by the Bonferroni test Com-parisons were performed using the GraphPad Prism software(San Diego USA) Values of 119875 lt 005 were consideredstatistically significant
3 Results and Discussion
Intercalation of juglone Q7 andQ9 into CT-DNAwas exam-ined by using the fluorescent intercalating agent ethidiumbromide Compounds that are able to intercalate into DNAcompete with ethidium bromide and reduce its fluorescencewhen read in a fluorimeter As show in Figure 1(a) whenDNA and ethidium bromide were incubated with jugloneQ7 or Q9 the fluorescence was reduced indicating that thecompounds can intercalate into CT-DNA The intercalatingcapacity of juglone Q7 and Q9 was always higher when theincubations were performed in the presence of ascorbate Asexpected doxorubicin which is a known intercalating agentalso reduced the fluorescence signal [27]
CT-DNA treated with juglone Q7 or Q9 underwentoxidative cleavage as shown by data in Figure 1(b) Freeradicals can attack DNA at C4 of desoxyribose causingcleavage and generation of products of degradation such asbase propenal This can be detected because base propenalreacts with 2-thiobarbituric acid producing color [28] Datain Figure 1(b) shows that the absorbance resulting from thio-barbituric acid species (TBARS) was increased in CT-DNAtreated with the free radical generators [Fe(EDTA)]2minusH
2O2
as well as it occurred in CT-DNA treated with juglone Q7or Q9 TBARS absorbance was always higher when CT-DNA was treated with the naphthoquinones combined withascorbate (Figure 1(b))
4 Oxidative Medicine and Cellular Longevity
0 10 20 30 407000
8000
9000
10000
Fluo
resc
ence
inte
nsity
EtdBrDoxorubicinAsc
Juglone
0 10 20 30 407000
8000
9000
10000
Fluo
resc
ence
inte
nsity
EtdBrDoxorubicinAsc
Q7Juglone + Asc Q7 + Asc
0 10 20 30 407000
8000
9000
10000
Fluo
resc
ence
inte
nsity
(120583M)
(120583M)(120583M)
EtdBrDoxorubicinAsc
Q9Q9 + Asc
(a)
000
002
004
006
008
010
TBA
RS ab
sorb
ance
NC PC Asc
Jugl
one
Q7
Q9
lowastlowast lowastlowastlowastlowastlowast
Jugl
one+
Asc
Q7+
Asc
Q9+
Asc
(b)
0
50
100
150
200
DN
A d
amag
e ind
ex
Con
trol
Asc
Jugl
one
Q7
Q9
Dox
orub
icin
lowastlowastlowast
lowastlowastlowast
Jugl
one+
Asc
Q7+
Asc
Q9+
Asc
(c)
Figure 1 Continued
Oxidative Medicine and Cellular Longevity 5
120574H2AX
Control Asc Jug Q7 Q9JugAsc
Q7Asc
Q9Asc
1mM 20120583M 20120583M1mM
120573-actin
(d)
Figure 1The fluorescence of CT-DNA stained with ethidium bromide is reduced by doxorubicin and juglone Q7 or Q9 mainly in presenceof ascorbate (Asc) (a) Absorbance of thiobarbituric reactive species (TBARS) in CT-DNA treatedwith juglone Q7 orQ9 alone and combinedwith ascorbate Negative control (NC) phosphate buffer Positive control (PC) [Fe(EDTA)]2minusH
2O2(b) DNA damage index was determined
by the comet assay in MCF-7 cells treated for 24 h with juglone 75120583M Q7 50 120583M or Q9 50 120583Mwith or without ascorbate 1mM (c) 120574H2AXwas assessed by immunoelectrophoresis in MCF-7 cells treated for 2 h with juglone (Jug) Q7 or Q9 at 20 120583Mwith or without ascorbate (Asc)at 1mM (d) (lowastlowast) and (lowast lowast lowast) denote statistical differences at 119875 lt 001 and 119875 lt 0001 compared to indicated treatments
Table 1 EC50 values obtained by the MTT assay MCF-7 cells weretreated for 24 h with juglone Q7 or Q9 (5ndash80 120583M) with or withoutascorbate 1mM Data from three independent experiments
EC50 (120583M)Juglone Q7 Q9
61 416 50plus ascorbate 1mM 28 263 254
The effects on the DNA of MCF-7 cells were assessedby immunoelectrophoresis for 120574H2AX and the comet assayGamma-H2AX is required for many proteins during theDNA damage and repair response [17] Figure 1(d) showsthat individually neither ascorbate juglone Q7 nor Q9enhanced the phosphorylated protein band corresponding to120574H2AXThe level of 120574H2AXwas enhanced onlywhenMCF-7cells were treated with the compounds combined with ascor-bate (1mM) Consistent with these findings the comet assayshowed that juglone Q7 and Q9 induced the fragmentationof MCF-7 DNA on their own but the addition of ascorbateincreased DNA fragmentation by approximately 2-fold in thecase of juglone and Q7 (Figure 1(c))
The effects of juglone Q7 and Q9 with and withoutascorbate were then examined on viability and proliferationinMCF-7 cellsTheMTT assay showed that ascorbate (1mM)alone did not reduce the viability of the MCF-7 cells Thecytotoxicity of juglone Q7 and Q9 was determined showingEC50values of 61 416 and 50120583M respectively The addition
of ascorbate together with the naphthoquinones reduced theEC50values by approximately 2-fold (to 28 263 and 254 120583M
for juglone Q7 andQ9 resp) and thus enhanced cytotoxicity(Table 1)
Ascorbate activates the redox cycling of quinones gen-erating semiquinone radicals and ROS In T24 cells it waspreviously shown that ascorbate (1mM) administered withjuglone Q7 and Q9 caused the appearance of semiquinoneand elevated levels of intracellular ROS [3 4] If a similarphenomenon occurs in MCF7 cells DNA damage wouldresult from not only binding and intercalation but also froman oxidative attack Doxorubicin acts by such a mechanismand kills cancer cells through both DNA intercalation and
ROS induction [29] Figure 2(a) shows that the ROS levelsin MCF-7 cells were elevated by the compounds particularlyby juglone and Q7 ROS generation induced by juglone andQ7 was enhanced approximately 3-fold in the presence ofascorbate On the other hand ascorbate had less impact interms of ROS induction when administered together withQ9 on MCF-7 cells Later the formulations containing Q9 orQ9 plus ascorbate were proven as poor inducers of oxidativestress in both MCF-7 cells (Figure 2(a)) and Ehrlich ascitescarcinoma in mice (Figure 3(c)) Ascorbate caused someincrease in terms of oxidative damage by Q9 only whenour treatments were done on CT-DNA a noncellular system(Figure 1(b))
Cell death was further studied based on PARP cleavageas shown in Figure 2(b) Compounds that are able to induceapoptosis cleave the PARP protein replacing the full-length116 kD protein with a cleaved fragment of approximately89 kD [30] Sanguinarine (5 120583M) a flavonoid known toinduce apoptosis was used as a control [31] PARP cleavagewas observed only in sanguinarine-treated cells and not incells treated with juglone Q7 or Q9 with or without ascor-bate (Figure 2(b))
The effect on MCF-7 cell proliferation is shown inFigure 2(c) JugloneQ7 andQ9 administered alone inhibitedcell proliferation as indicated by a reduction in the numberof colony-forming units in comparison with nontreated cellsIn all cases the inhibition of proliferation was increasedup to 3-fold when the naphthoquinones were administeredin combination with ascorbate (1mM) (Figure 2(c)) Thesedata corroborate literature when considered that DNA inter-calators can block cell division [6] To examine whetherthe PI3KAktmTOR signalling pathway was affected by thetreatments in MCF-7 cells the levels of the active phospho-rylated form of Akt (pAkt) were determined (Figure 2(d))The PI3KAkt pathway regulates several biological processesincluding cell survival proliferation and differentiationUpregulation of this pathway is observed in several types ofcancer and it can be associated with uncontrolled cell prolif-eration [32ndash34] As demonstrated in Figure 2(d) significantinhibition of the pAkt occurred in MCF-7 cells followingtreatment most notably with juglone or Q7 in the presenceof ascorbate
6 Oxidative Medicine and Cellular Longevity
0
1
2
3
4In
crea
sed
ROS
Asc
Jugl
one
Q7
Q9
lowastlowastlowast
lowastlowast
Jugl
one+
Asc
Q7+
Asc
Q9+
Asc
(a)
Control Sang
PARP
Asc Jug Q7 Q9JugAsc
Q7Asc
Q9Asc
120573-actin
(b)
0
50
100
150
Num
ber o
f col
onie
s
Con
trol
Asc
Jugl
one
Jugl
one +
Asc Q7
Q7
+ A
sc Q9
Q9
+ A
sc
lowastlowastlowast
lowastlowastlowast
lowastlowastlowast
(c)
Control
pAkt
Asc Jug Q7 Q9JugAsc
Q7Asc
Q9Asc
1mM 20120583M 20120583M1mM
120573-actin
(d)
Figure 2 ROS were measured in MCF-7 cells treated for 2 h with juglone Q7 or Q9 at 10120583M with or without ascorbate 1mM (a) Integrityof PARP protein in MCF-7 cells treated with juglone (Jug) Q7 or Q9 at 20 120583M with or without ascorbate (Asc) 1mM for 24 h Sanguinarine(Sang) 5120583M was used as a positive control of apoptosis (b) Colony-forming units of MCF-7 cells treated with juglone Q7 or Q9 at 10 120583Mwithwithout ascorbate 1mM for 2 h (c) Phosphorylated Akt (pAkt) was assessed by immunoelectrophoresis in MCF-7 cells treated for 24 h(d) Data were obtained from three independent experiments (lowastlowast) and (lowast lowast lowast) denote statistical differences at 119875 lt 001 and 119875 lt 0001compared to nontreated control cells or between indicated treatments respectively
Following these in vitro assays some in vivo effects werestudied in Ehrlich ascites carcinoma-bearing mice Ehrlichascites carcinoma was chosen to be used in vivo initially toverify whether some effects observed in vitro were repro-ducible in vivo But also to evaluate whether the effectswere consistent only with MCF-7 cells in vitro or they couldbe repeated against a different tumor cell line Figure 3(a)presents the levels of tumor growth inhibition Some inhi-bition on Ehrlich carcinoma was caused in animals treatedwith juglone Q7 orQ9 But the formulations of juglone orQ7plus ascorbate had the most potent activity and achieved upto 60 of inhibition of tumor growth approaching the effectof doxorubicin which caused up to 90 inhibition Actuallyconsidering data related to tumor growth and survival it is
possible to suggest that the potentiating effect of the com-bined treatment with ascorbate was reproduced in vivo withstatistical difference only in the case of juglone and Q7 Theability to increase the duration of animal survival is one ofthe most reliable criteria for evaluating potential antitumordrugs [35] Figure 3(b) presents graphs relating the numberof days after tumor inoculation to the percentage of survivorsfollowing treatment an increase in the area under the curvesindicates an increase in survival The smallest area in thegraphs corresponds to the group of nontreated animals Ingeneral these animals started to die on day 10 and by day12 no survivors remained The survival time was extendedby 2 to 6 days when the animals received treatments withindividual compounds (juglone Q7 or Q9) These animals
Oxidative Medicine and Cellular Longevity 7
0
20
40
60
80
100
Ehrli
ch ca
rcin
oma i
nhib
ition
()
Con
trol
Dox
orub
icin Asc
Jugl
one
Q7
Q9
lowastlowastlowastlowastlowast
Jugl
one+
Asc
Q7+
Asc
Q9+
Asc
(a)
0 10 20 300
50
100
DoxorubicinControlAscJuglone
Days
Surv
ival
()
Juglone + Asc
0 10 20 300
50
100
DoxorubicinControlAscQ7
Days
Surv
ival
()
Q7 + Asc
0 10 20 300
50
100
DoxorubicinControlAscQ9
Days
Surv
ival
()
Q9 + Asc
(b)
Figure 3 Continued
8 Oxidative Medicine and Cellular Longevity
000
001
002
003
004
005
MD
A n
mol
mg
prot
ein
Con
trol
Asc
Jugl
one
Q7
Q9
lowastlowast
lowastlowast
Jugl
one+
Asc
Q7+
Asc
Q9+
Asc
(c)
0
50
100
150
200
DN
A d
amag
e ind
ex
Con
trol
Asc
Jugl
one
Q7
Q9
lowastlowastlowast
lowastlowast
Jugl
one+
Asc
Q7+
Asc
Q9+
Asc
(d)
120574H2AX
pAkt
Ctrl Asc Jug Q7 Q9JugAsc
Q7Asc
Q9Asc
120573-actin
(e)
Figure 3 Ehrlich ascites carcinoma inhibition (a) and survival time (b) in animals treated with juglone Q7 or Q9 at 1mgkg alone or incombination with ascorbate (Asc) at 100mgkg Doxorubicin was administered at 12mgkg for the positive control Lipid peroxidation (c)DNA damage (d) and phosphorylated proteins Akt (pAkt) and H2AX (120574H2AX) in ascitic cells from mice (e) (lowastlowast) and (lowast lowast lowast) denotedifference at 119875 lt 001 and 119875 lt 0001 compared to the negative control or between indicated treatments
generally died before day 20The largest area under the curvescorresponds to the treatments conducted with juglone or Q7in combination with ascorbate (Figure 3(b)) The synergisticeffect between these drugs occurred also in vivo
Figures 3(c) 3(d) and 3(e) present data of moleculartoxicity on Ehrlich tumor in mice Figure 3(c) depicts levelsof MDA a biomarker of lipid peroxidationThis was the end-point measurement done to verify oxidative stress causedby the treatments in vivo MDA levels were increased sig-nificantly by the combined treatments done with juglone orQ7 and ascorbate Data in Figure 3(d) corroborate with datafrom Figure 3(c) because they show that DNA fragmenta-tion was increased significantly in vivo only when animalsreceived juglone or Q7 plus ascorbate Finally data shownin Figure 3(e) provide concise evidences which demonstratethat juglone and Q7 plus ascorbate can cause DNA damageand pAkt inhibition in Ehrlich tumor in mice Thus theeffects are not restricted to MCF-7 cells in vitro
Data from this study are summarized illustratively inFigure 4 Although all 14-naphthoquinones presented someactivity primarily juglone and Q7 administered in combina-tion with ascorbate trigger the generation of free radicals thesemiquinone formof the naphthoquinone and dehydroascor-bate The resulting semiquinone form intercalates into DNA
The radicals can attack DNA which is degraded In cancercells these events are accompanied by an inhibition on Aktpathway The formulation of juglone or Q7 with ascorbateshowed the most promising activity in vivo
4 Conclusion
Overall it is possible to conclude that primarily jugloneand Q7 combined with ascorbate have significant antitumoreffects against MCF-7 cells in vitro and Ehrlich-ascites carci-noma in mice The effects are the result of intercalation andoxidative attack on DNA of tumor cells and inhibition of Aktpathway
Conflict of Interests
The authors declare that they have no conflict of interests
Acknowledgments
Fabiana Ourique Luiza S E P W Castro and ValdeluciaM A S Grinevicius are recipients of research grants from
Oxidative Medicine and Cellular Longevity 9
Juglone Q7 Q9
OH
OH
H H
H
OO
H
H
PmTOR
Growth and proliferation
Cytosol
AktP P
PKB
P
Growth factor
Tyrosine kinase-like receptor
Plasma membranePI-3K
OH
OH
OH
OH
HO
HO
HO
O O
O O
O
OR1
R1
R2
R2
R1
R2
Ominus
∙O
O∙
O2∙minus
O2
H∙∙∙N H
∙∙∙N OCH3
Figure 4 Antitumor actions of juglone Q7 and Q9 administered in combination with ascorbate against MCF-7 cells and Ehrlich ascitescarcinoma in mice The effects are the result of intercalation and oxidative attack on DNA of tumor cells and inhibition of Akt pathway
Coordenacao de Aperfeicoamento de Pessoal de Nıvel Supe-rior (CAPES Brazil) Karina B Felipe and Rozangela CuriPedrosa (Proc 3024042011-2) are recipients of researchgrants from Conselho Nacional de Pesquisa (CNPq) Brazil
References
[1] M M Gottesman ldquoMechanisms of cancer drug resistancerdquoAnnual Review of Medicine vol 53 pp 615ndash627 2002
[2] M S Farias C T Pich M R Kviecinski et al ldquoSubstituted3-acyl-2-phenylamino-14-naphthoquinones intercalate intoDNA and cause genotoxicity through the increased generationof reactive oxygen species culminating in cell deathrdquoMolecularMedicine Reports vol 10 no 1 pp 405ndash410 2014
[3] K B Felipe J Benites C Glorieux et al ldquoAntiproliferativeeffects of phenylaminonaphthoquinones are increased by ascor-bate and associated with the appearance of a senescent pheno-type in human bladder cancer cellsrdquo Biochemical and Biophysi-cal Research Communications vol 433 no 4 pp 573ndash578 2013
[4] M R Kviecinski R C Pedrosa K B Felipe et al ldquoInhibitionof cell proliferation and migration by oxidative stress fromascorbate-driven juglone redox cycling in human bladder-derived T24 cellsrdquo Biochemical and Biophysical Research Com-munications vol 421 no 2 pp 268ndash273 2012
[5] C F Thorn C Oshiro S Marsh et al ldquoDoxorubicin pathwayspharmacodynamics and adverse effectsrdquo Pharmacogenetics andGenomics vol 21 no 7 pp 440ndash446 2011
[6] G Bischoff and S Hoffman ldquoDNA-binding of drugs used inmedicinal therapiesrdquo Current Medicinal Chemistry vol 9 no3 pp 321ndash348 2002
[7] R Palchaudhuri and P J Hergenrother ldquoDNA as a target foranticancer compounds methods to determine the mode ofbinding and the mechanism of actionrdquo Current Opinion inBiotechnology vol 18 no 6 pp 497ndash503 2007
[8] M S Shahabuddin M Gopal and S C Raghavan ldquoIntercalat-ing and antitumor activity of 4 oxopyrimido[410158405101584045]thieno(23-b) quinoline-4(3H)-onerdquo Journal of Cancer Molecules vol 3pp 139ndash146 2007
[9] Q Chen M G Espey M C Krishna et al ldquoPharamacologicascorbic acid concentrations selectively kill cancer cells actionas a pro-drug to deliver hydrogen peroxide to tissuserdquo Proceed-ings of the National Academy of Sciences of the United States ofAmerica vol 102 no 38 pp 13604ndash13609 2005
[10] J Du J J Cullen and G R Buettner ldquoAscorbic acid chemistrybiology and the treatment of cancerrdquo Biochimica et BiophysicaActa vol 1826 no 2 pp 443ndash457 2012
[11] M G Espey P Chen B Chalmers et al ldquoPharmacologicascorbate synergizes with gemcitabine in preclinical models of
10 Oxidative Medicine and Cellular Longevity
pancreatic cancerrdquo Free Radical Biology and Medicine vol 50no 11 pp 1610ndash1619 2011
[12] J Verrax R Beck N Dejeans et al ldquoRedox-active quinonesand ascorbate an innovative cancer therapy that exploits thevulnerability of cancer cells to oxidative stressrdquo Anti-CancerAgents in Medicinal Chemistry vol 11 no 2 pp 213ndash221 2011
[13] J Benites J A Valderrama K Bettega R C Pedrosa PB Calderon and J Verrax ldquoBiological evaluation of donor-acceptor aminonaphthoquinones as antitumor agentsrdquo Euro-pean Journal of Medicinal Chemistry vol 45 no 12 pp 6052ndash6057 2010
[14] V C da Silveira H Benezra J S Luz R C Georg C COliveira and A M D C Ferreira ldquoBinding of oxindole-Schiffbase copper(II) complexes to DNA and its modulation by theligandrdquo Journal of Inorganic Biochemistry vol 105 no 12 pp1692ndash1703 2011
[15] T Jun W Bochu and Z Liancai ldquoHydrolytic cleavage of DNAby quercetin manganese(II) complexesrdquo Colloids and SurfacesB Biointerfaces vol 55 no 2 pp 149ndash152 2007
[16] N P Singh M T McCoy R R Tice and E L Schneider ldquoAsimple technique for quantitation of low levels of DNA damagein individual cellsrdquo Experimental Cell Research vol 175 no 1pp 184ndash191 1988
[17] J Yuan R Adamski and J Chen ldquoFocus on histone variantH2AX to be or not to berdquo FEBS Letters vol 584 no 17 pp 3717ndash3724 2010
[18] T T Paull E P Rogakou V Yamazaki C U Kirchgessner MGellert and W M Bonner ldquoA critical role for histone H2AX inrecruitment of repair factors to nuclear foci after DNAdamagerdquoCurrent Biology vol 10 no 15 pp 886ndash895 2000
[19] R Lopez A Arumugam R Joseph et al ldquoHyperglycemiaenhances the proliferation of non-tumorigenic and malignantmammary epithelial cells through increased leptinIGF1R sig-naling and activation of AKTmTORrdquo PLoS ONE vol 8 no 11Article ID e79708 2013
[20] G M Ross T J McMillan P Wilcox and A R Collins ldquoThesingle cell microgel electrophoresis assay (comet assay) techni-cal aspects and applications Report on the 5th LH Gray TrustWorkshop Institute of Cancer Research 1994rdquo MutationResearch vol 337 no 1 pp 57ndash60 1995
[21] T Mosmann ldquoRapid colorimetric assay for cellular growth andsurvival application to proliferation and cytotoxicity assaysrdquoJournal of Immunological Methods vol 65 no 1-2 pp 55ndash631983
[22] N A P Franken H M Rodermond J Stap J Haveman and Cvan Bree ldquoClonogenic assay of cells in vitrordquo Nature Protocolsvol 1 no 5 pp 2315ndash2319 2006
[23] C Glorieux N Dejeans B Sid R Beck P B Calderon andJ Verrax ldquoCatalase overexpression in mammary cancer cellsleads to a less aggressive phenotype and an altered response tochemotherapyrdquo Biochemical Pharmacology vol 82 no 10 pp1384ndash1390 2011
[24] M R Kviecinski P Benelli K B Felipe et al ldquoSFE from Bidenspilosa Linne to obtain extracts rich in cytotoxic polyacetyleneswith antitumor activityrdquo Journal of Supercritical Fluids vol 56no 3 pp 243ndash248 2011
[25] E L Kaplan and P Meier ldquoNonparametric estimation fromincomplete observationsrdquo Journal of the American StatisticalAssociation vol 53 pp 457ndash481 1958
[26] R P Bird and H H Draper ldquoComparative studies on differentmethods of malonaldehyde determinationrdquo Methods in Enzy-mology vol 105 pp 299ndash305 1984
[27] V G S Box ldquoThe intercalation of DNA double helices withdoxorubicin and nagalomycinrdquo Journal of Molecular Graphicsand Modelling vol 26 no 1 pp 14ndash19 2007
[28] Q Jiang N Xiao P Shi Y Zhu and Z Guo ldquoDesign of artifi-cial metallonucleases with oxidative mechanismrdquo CoordinationChemistry Reviews vol 251 no 15-16 pp 1951ndash1972 2007
[29] M U Rehman M Tahir A Q Khan et al ldquoD-limonene sup-presses doxorubicin-induced oxidative stress and inflammationvia repression of COX-2 iNOS and NF120581B in kidneys of Wistarratsrdquo Experimental Biology and Medicine vol 239 no 4 pp465ndash476 2014
[30] A H Boulares A G Yakovlev V Ivanova et al ldquoRole ofpoly(ADP-ribose) polymerase (PARP) cleavage in apoptosisCaspase 3-resistant PARP mutant increases rates of apoptosisin transfected cellsrdquo Journal of Biological Chemistry vol 274 no33 pp 22932ndash22940 1999
[31] N Ahmad S Gupta M M Husain K M Heiskanen and HMukhtar ldquoDifferential antiproliferative and apoptotic responseof sanguinarine for cancer cells versus normal cellsrdquo ClinicalCancer Research vol 6 no 4 pp 1524ndash1528 2000
[32] S J Lee H Ahn K W Nam K H Kim and W Mar ldquoEffectsof rutaecarpine on hydrogen peroxide-induced apoptosis inmurine hepa-1c1c7 cellsrdquo Biomolecules amp Therapeutics vol 20no 5 pp 487ndash491 2012
[33] E-S A Arafa Q Zhu Z I Shah et al ldquoThymoquinoneup-regulates PTEN expression and induces apoptosis indoxorubicin-resistant human breast cancer cellsrdquo MutationResearchmdashFundamental and Molecular Mechanisms of Mutage-nesis vol 706 no 1-2 pp 28ndash35 2011
[34] G Viglietto M L Motti P Bruni et al ldquoCytoplasmic relocal-ization and inhibition of the cyclin-dependent kinase inhibitorp271198961198941199011 by PKBAkt-mediated phosphorylation in breast can-cerrdquo Nature Medicine vol 8 no 10 pp 1136ndash1144 2002
[35] B D Clarkson and J H Burchenal ldquoPreliminary screening ofantineoplastic drugsrdquoProgress in Clinical Cancer vol 1 pp 625ndash629 1965
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
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Diabetes ResearchJournal of
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Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
Oxidative Medicine and Cellular Longevity 3
microscope Each nucleus received a fluorescence value inthe 0ndash4 range (arbitrary units 0mdashundamaged 4mdashmaximallydamaged) [20]
25 Immunoblotting Assays After treatment cells werewashed with phosphate buffered saline (PBS) and lysed inRIPA buffer (50mM Tris-Cl pH 74 150mM NaCl 1NP40 025 Na-deoxycholate and 1mM phenylmethylsul-fonyl fluoride) supplemented with 1 protease inhibitor and3 phosphatase inhibitor cocktails After denaturation inLaemmli buffer (60mM Tris-Cl pH 68 2 sodium dodecylsulfate (SDS) 10 glycerol 5 120573-mercaptoethanol and001 bromophenol blue) equal amounts of protein (30120583g)fromwhole cellular homogenates were subjected to polyacry-lamide gel electrophoresis (SDS-PAGE) followed by elec-troblotting to polyvinylidene fluoride (PVDF) membranesAfter blocking the membranes were incubated overnightwith the primary antibodies The membranes were washedand incubated with the secondary antibodies for 1 h Immun-odetection was performed using the enhanced chemilumi-nescence (ECL) detection kit (Millipore USA) for HRP-coupled secondary antibodies Beta-actin served as a loadingcontrol
26 Effects on MCF-7 Cell Viability and Proliferation Cyto-toxicity wasmeasured using the tetrazolium salt (MTT) assay[21] Briefly 104 cellswell were plated onto 96-well plates Atconfluence the cells were exposed to juglone Q7 and Q9(0ndash80120583M) in the absence or presence of ascorbate (1mM)for up to 24 h The cells were then washed twice with PBSand incubated for 2 h with MTT (05mgmL) The formazancrystals were solubilised by adding DMSO (100 120583Lwell) andthe colored solutionswere read at 550 nmThree independentexperiments were conducted and the results are presented asEC50values
The effects on cell proliferation were examined by thecolony formation assay according to Franken et al [22] Cells(500) were treated for 2 h with the compounds They werethen washed twice with warm PBS and fresh medium wasadded After 15 days the cells were stained by crystal violetand colonies with more than 50 cells were counted
27 Levels of MCF-7 Intracellular ROS Intracellular ROSwere measured as reported by Glorieux et al [23] Cells(15000) were loaded with 10 120583M DCFH-DA in Hankrsquos bal-anced salt solution (HBSS) at 37∘C and incubated for 30minExcess DCFH-DAwas removed by washing with fresh HBSSThe cells were incubated for 2 h with the test compoundswashed twice withHBSS and then 100120583L ofHBSSwas addedto each well The fluorescence intensity was measured witha TECAN Infinity M200 microplate reader at 485 nm forexcitation and 530 nm for emission
28 Antitumor Activity In Vivo Male BALBc inbred mice(20ndash22 g) received water and food ad libitum Procedureswere conducted in accordance with legal requirements andwith the approval of the local ethics committee (UFSCPP00784) Previous tests were conducted to select safe doses
of 14-naphthoquinones Ascorbatewas administered at doses100 times higher On day zero Ehrlich carcinoma cells (5 times106) were inoculated into the abdomen of mice from ninegroups (119899 = 12) Treatments were done via intraperitonealinjections every 24 h for 9 days The control group receivedsaline injections and the positive control group receiveddoxorubicin (12mgkg) Test groups received juglone Q7 orQ9 (1mgkg) andor ascorbate (100mgkg) After treatmentthe inhibition of tumor growth was measured based onchanges in the abdominal circumference [24]The percentageof increased life span was calculated by recording mortalityon a daily basis for 30 days according to themethod ofKaplanand Meier [25] Samples of ascitic fluid were collected 2 hafter the last dose and immediately processedThe moleculareffects in vivowere assessed again at level of DNA through thecomet assay and gamma-H2AX As done in vitrowithMCF-7cells also in vivoAkt-pathwaywas evaluated in Ehrlich tumorafter treatments through electrophoresis and immunoblot-ting as described in Sections 24 and 25 Lipid peroxidationwas estimated by measurement of malondialdehyde (MDA)formation using the thiobarbituric acid method [26]
29 Data Analysis In general the assays were performed intriplicate In vitro assays were repeated at least three timesThe results are presented as themeansplusmn standard deviation oras percentagesThe data were analyzed by the analysis of vari-ance (ANOVA) test followed by the Bonferroni test Com-parisons were performed using the GraphPad Prism software(San Diego USA) Values of 119875 lt 005 were consideredstatistically significant
3 Results and Discussion
Intercalation of juglone Q7 andQ9 into CT-DNAwas exam-ined by using the fluorescent intercalating agent ethidiumbromide Compounds that are able to intercalate into DNAcompete with ethidium bromide and reduce its fluorescencewhen read in a fluorimeter As show in Figure 1(a) whenDNA and ethidium bromide were incubated with jugloneQ7 or Q9 the fluorescence was reduced indicating that thecompounds can intercalate into CT-DNA The intercalatingcapacity of juglone Q7 and Q9 was always higher when theincubations were performed in the presence of ascorbate Asexpected doxorubicin which is a known intercalating agentalso reduced the fluorescence signal [27]
CT-DNA treated with juglone Q7 or Q9 underwentoxidative cleavage as shown by data in Figure 1(b) Freeradicals can attack DNA at C4 of desoxyribose causingcleavage and generation of products of degradation such asbase propenal This can be detected because base propenalreacts with 2-thiobarbituric acid producing color [28] Datain Figure 1(b) shows that the absorbance resulting from thio-barbituric acid species (TBARS) was increased in CT-DNAtreated with the free radical generators [Fe(EDTA)]2minusH
2O2
as well as it occurred in CT-DNA treated with juglone Q7or Q9 TBARS absorbance was always higher when CT-DNA was treated with the naphthoquinones combined withascorbate (Figure 1(b))
4 Oxidative Medicine and Cellular Longevity
0 10 20 30 407000
8000
9000
10000
Fluo
resc
ence
inte
nsity
EtdBrDoxorubicinAsc
Juglone
0 10 20 30 407000
8000
9000
10000
Fluo
resc
ence
inte
nsity
EtdBrDoxorubicinAsc
Q7Juglone + Asc Q7 + Asc
0 10 20 30 407000
8000
9000
10000
Fluo
resc
ence
inte
nsity
(120583M)
(120583M)(120583M)
EtdBrDoxorubicinAsc
Q9Q9 + Asc
(a)
000
002
004
006
008
010
TBA
RS ab
sorb
ance
NC PC Asc
Jugl
one
Q7
Q9
lowastlowast lowastlowastlowastlowastlowast
Jugl
one+
Asc
Q7+
Asc
Q9+
Asc
(b)
0
50
100
150
200
DN
A d
amag
e ind
ex
Con
trol
Asc
Jugl
one
Q7
Q9
Dox
orub
icin
lowastlowastlowast
lowastlowastlowast
Jugl
one+
Asc
Q7+
Asc
Q9+
Asc
(c)
Figure 1 Continued
Oxidative Medicine and Cellular Longevity 5
120574H2AX
Control Asc Jug Q7 Q9JugAsc
Q7Asc
Q9Asc
1mM 20120583M 20120583M1mM
120573-actin
(d)
Figure 1The fluorescence of CT-DNA stained with ethidium bromide is reduced by doxorubicin and juglone Q7 or Q9 mainly in presenceof ascorbate (Asc) (a) Absorbance of thiobarbituric reactive species (TBARS) in CT-DNA treatedwith juglone Q7 orQ9 alone and combinedwith ascorbate Negative control (NC) phosphate buffer Positive control (PC) [Fe(EDTA)]2minusH
2O2(b) DNA damage index was determined
by the comet assay in MCF-7 cells treated for 24 h with juglone 75120583M Q7 50 120583M or Q9 50 120583Mwith or without ascorbate 1mM (c) 120574H2AXwas assessed by immunoelectrophoresis in MCF-7 cells treated for 2 h with juglone (Jug) Q7 or Q9 at 20 120583Mwith or without ascorbate (Asc)at 1mM (d) (lowastlowast) and (lowast lowast lowast) denote statistical differences at 119875 lt 001 and 119875 lt 0001 compared to indicated treatments
Table 1 EC50 values obtained by the MTT assay MCF-7 cells weretreated for 24 h with juglone Q7 or Q9 (5ndash80 120583M) with or withoutascorbate 1mM Data from three independent experiments
EC50 (120583M)Juglone Q7 Q9
61 416 50plus ascorbate 1mM 28 263 254
The effects on the DNA of MCF-7 cells were assessedby immunoelectrophoresis for 120574H2AX and the comet assayGamma-H2AX is required for many proteins during theDNA damage and repair response [17] Figure 1(d) showsthat individually neither ascorbate juglone Q7 nor Q9enhanced the phosphorylated protein band corresponding to120574H2AXThe level of 120574H2AXwas enhanced onlywhenMCF-7cells were treated with the compounds combined with ascor-bate (1mM) Consistent with these findings the comet assayshowed that juglone Q7 and Q9 induced the fragmentationof MCF-7 DNA on their own but the addition of ascorbateincreased DNA fragmentation by approximately 2-fold in thecase of juglone and Q7 (Figure 1(c))
The effects of juglone Q7 and Q9 with and withoutascorbate were then examined on viability and proliferationinMCF-7 cellsTheMTT assay showed that ascorbate (1mM)alone did not reduce the viability of the MCF-7 cells Thecytotoxicity of juglone Q7 and Q9 was determined showingEC50values of 61 416 and 50120583M respectively The addition
of ascorbate together with the naphthoquinones reduced theEC50values by approximately 2-fold (to 28 263 and 254 120583M
for juglone Q7 andQ9 resp) and thus enhanced cytotoxicity(Table 1)
Ascorbate activates the redox cycling of quinones gen-erating semiquinone radicals and ROS In T24 cells it waspreviously shown that ascorbate (1mM) administered withjuglone Q7 and Q9 caused the appearance of semiquinoneand elevated levels of intracellular ROS [3 4] If a similarphenomenon occurs in MCF7 cells DNA damage wouldresult from not only binding and intercalation but also froman oxidative attack Doxorubicin acts by such a mechanismand kills cancer cells through both DNA intercalation and
ROS induction [29] Figure 2(a) shows that the ROS levelsin MCF-7 cells were elevated by the compounds particularlyby juglone and Q7 ROS generation induced by juglone andQ7 was enhanced approximately 3-fold in the presence ofascorbate On the other hand ascorbate had less impact interms of ROS induction when administered together withQ9 on MCF-7 cells Later the formulations containing Q9 orQ9 plus ascorbate were proven as poor inducers of oxidativestress in both MCF-7 cells (Figure 2(a)) and Ehrlich ascitescarcinoma in mice (Figure 3(c)) Ascorbate caused someincrease in terms of oxidative damage by Q9 only whenour treatments were done on CT-DNA a noncellular system(Figure 1(b))
Cell death was further studied based on PARP cleavageas shown in Figure 2(b) Compounds that are able to induceapoptosis cleave the PARP protein replacing the full-length116 kD protein with a cleaved fragment of approximately89 kD [30] Sanguinarine (5 120583M) a flavonoid known toinduce apoptosis was used as a control [31] PARP cleavagewas observed only in sanguinarine-treated cells and not incells treated with juglone Q7 or Q9 with or without ascor-bate (Figure 2(b))
The effect on MCF-7 cell proliferation is shown inFigure 2(c) JugloneQ7 andQ9 administered alone inhibitedcell proliferation as indicated by a reduction in the numberof colony-forming units in comparison with nontreated cellsIn all cases the inhibition of proliferation was increasedup to 3-fold when the naphthoquinones were administeredin combination with ascorbate (1mM) (Figure 2(c)) Thesedata corroborate literature when considered that DNA inter-calators can block cell division [6] To examine whetherthe PI3KAktmTOR signalling pathway was affected by thetreatments in MCF-7 cells the levels of the active phospho-rylated form of Akt (pAkt) were determined (Figure 2(d))The PI3KAkt pathway regulates several biological processesincluding cell survival proliferation and differentiationUpregulation of this pathway is observed in several types ofcancer and it can be associated with uncontrolled cell prolif-eration [32ndash34] As demonstrated in Figure 2(d) significantinhibition of the pAkt occurred in MCF-7 cells followingtreatment most notably with juglone or Q7 in the presenceof ascorbate
6 Oxidative Medicine and Cellular Longevity
0
1
2
3
4In
crea
sed
ROS
Asc
Jugl
one
Q7
Q9
lowastlowastlowast
lowastlowast
Jugl
one+
Asc
Q7+
Asc
Q9+
Asc
(a)
Control Sang
PARP
Asc Jug Q7 Q9JugAsc
Q7Asc
Q9Asc
120573-actin
(b)
0
50
100
150
Num
ber o
f col
onie
s
Con
trol
Asc
Jugl
one
Jugl
one +
Asc Q7
Q7
+ A
sc Q9
Q9
+ A
sc
lowastlowastlowast
lowastlowastlowast
lowastlowastlowast
(c)
Control
pAkt
Asc Jug Q7 Q9JugAsc
Q7Asc
Q9Asc
1mM 20120583M 20120583M1mM
120573-actin
(d)
Figure 2 ROS were measured in MCF-7 cells treated for 2 h with juglone Q7 or Q9 at 10120583M with or without ascorbate 1mM (a) Integrityof PARP protein in MCF-7 cells treated with juglone (Jug) Q7 or Q9 at 20 120583M with or without ascorbate (Asc) 1mM for 24 h Sanguinarine(Sang) 5120583M was used as a positive control of apoptosis (b) Colony-forming units of MCF-7 cells treated with juglone Q7 or Q9 at 10 120583Mwithwithout ascorbate 1mM for 2 h (c) Phosphorylated Akt (pAkt) was assessed by immunoelectrophoresis in MCF-7 cells treated for 24 h(d) Data were obtained from three independent experiments (lowastlowast) and (lowast lowast lowast) denote statistical differences at 119875 lt 001 and 119875 lt 0001compared to nontreated control cells or between indicated treatments respectively
Following these in vitro assays some in vivo effects werestudied in Ehrlich ascites carcinoma-bearing mice Ehrlichascites carcinoma was chosen to be used in vivo initially toverify whether some effects observed in vitro were repro-ducible in vivo But also to evaluate whether the effectswere consistent only with MCF-7 cells in vitro or they couldbe repeated against a different tumor cell line Figure 3(a)presents the levels of tumor growth inhibition Some inhi-bition on Ehrlich carcinoma was caused in animals treatedwith juglone Q7 orQ9 But the formulations of juglone orQ7plus ascorbate had the most potent activity and achieved upto 60 of inhibition of tumor growth approaching the effectof doxorubicin which caused up to 90 inhibition Actuallyconsidering data related to tumor growth and survival it is
possible to suggest that the potentiating effect of the com-bined treatment with ascorbate was reproduced in vivo withstatistical difference only in the case of juglone and Q7 Theability to increase the duration of animal survival is one ofthe most reliable criteria for evaluating potential antitumordrugs [35] Figure 3(b) presents graphs relating the numberof days after tumor inoculation to the percentage of survivorsfollowing treatment an increase in the area under the curvesindicates an increase in survival The smallest area in thegraphs corresponds to the group of nontreated animals Ingeneral these animals started to die on day 10 and by day12 no survivors remained The survival time was extendedby 2 to 6 days when the animals received treatments withindividual compounds (juglone Q7 or Q9) These animals
Oxidative Medicine and Cellular Longevity 7
0
20
40
60
80
100
Ehrli
ch ca
rcin
oma i
nhib
ition
()
Con
trol
Dox
orub
icin Asc
Jugl
one
Q7
Q9
lowastlowastlowastlowastlowast
Jugl
one+
Asc
Q7+
Asc
Q9+
Asc
(a)
0 10 20 300
50
100
DoxorubicinControlAscJuglone
Days
Surv
ival
()
Juglone + Asc
0 10 20 300
50
100
DoxorubicinControlAscQ7
Days
Surv
ival
()
Q7 + Asc
0 10 20 300
50
100
DoxorubicinControlAscQ9
Days
Surv
ival
()
Q9 + Asc
(b)
Figure 3 Continued
8 Oxidative Medicine and Cellular Longevity
000
001
002
003
004
005
MD
A n
mol
mg
prot
ein
Con
trol
Asc
Jugl
one
Q7
Q9
lowastlowast
lowastlowast
Jugl
one+
Asc
Q7+
Asc
Q9+
Asc
(c)
0
50
100
150
200
DN
A d
amag
e ind
ex
Con
trol
Asc
Jugl
one
Q7
Q9
lowastlowastlowast
lowastlowast
Jugl
one+
Asc
Q7+
Asc
Q9+
Asc
(d)
120574H2AX
pAkt
Ctrl Asc Jug Q7 Q9JugAsc
Q7Asc
Q9Asc
120573-actin
(e)
Figure 3 Ehrlich ascites carcinoma inhibition (a) and survival time (b) in animals treated with juglone Q7 or Q9 at 1mgkg alone or incombination with ascorbate (Asc) at 100mgkg Doxorubicin was administered at 12mgkg for the positive control Lipid peroxidation (c)DNA damage (d) and phosphorylated proteins Akt (pAkt) and H2AX (120574H2AX) in ascitic cells from mice (e) (lowastlowast) and (lowast lowast lowast) denotedifference at 119875 lt 001 and 119875 lt 0001 compared to the negative control or between indicated treatments
generally died before day 20The largest area under the curvescorresponds to the treatments conducted with juglone or Q7in combination with ascorbate (Figure 3(b)) The synergisticeffect between these drugs occurred also in vivo
Figures 3(c) 3(d) and 3(e) present data of moleculartoxicity on Ehrlich tumor in mice Figure 3(c) depicts levelsof MDA a biomarker of lipid peroxidationThis was the end-point measurement done to verify oxidative stress causedby the treatments in vivo MDA levels were increased sig-nificantly by the combined treatments done with juglone orQ7 and ascorbate Data in Figure 3(d) corroborate with datafrom Figure 3(c) because they show that DNA fragmenta-tion was increased significantly in vivo only when animalsreceived juglone or Q7 plus ascorbate Finally data shownin Figure 3(e) provide concise evidences which demonstratethat juglone and Q7 plus ascorbate can cause DNA damageand pAkt inhibition in Ehrlich tumor in mice Thus theeffects are not restricted to MCF-7 cells in vitro
Data from this study are summarized illustratively inFigure 4 Although all 14-naphthoquinones presented someactivity primarily juglone and Q7 administered in combina-tion with ascorbate trigger the generation of free radicals thesemiquinone formof the naphthoquinone and dehydroascor-bate The resulting semiquinone form intercalates into DNA
The radicals can attack DNA which is degraded In cancercells these events are accompanied by an inhibition on Aktpathway The formulation of juglone or Q7 with ascorbateshowed the most promising activity in vivo
4 Conclusion
Overall it is possible to conclude that primarily jugloneand Q7 combined with ascorbate have significant antitumoreffects against MCF-7 cells in vitro and Ehrlich-ascites carci-noma in mice The effects are the result of intercalation andoxidative attack on DNA of tumor cells and inhibition of Aktpathway
Conflict of Interests
The authors declare that they have no conflict of interests
Acknowledgments
Fabiana Ourique Luiza S E P W Castro and ValdeluciaM A S Grinevicius are recipients of research grants from
Oxidative Medicine and Cellular Longevity 9
Juglone Q7 Q9
OH
OH
H H
H
OO
H
H
PmTOR
Growth and proliferation
Cytosol
AktP P
PKB
P
Growth factor
Tyrosine kinase-like receptor
Plasma membranePI-3K
OH
OH
OH
OH
HO
HO
HO
O O
O O
O
OR1
R1
R2
R2
R1
R2
Ominus
∙O
O∙
O2∙minus
O2
H∙∙∙N H
∙∙∙N OCH3
Figure 4 Antitumor actions of juglone Q7 and Q9 administered in combination with ascorbate against MCF-7 cells and Ehrlich ascitescarcinoma in mice The effects are the result of intercalation and oxidative attack on DNA of tumor cells and inhibition of Akt pathway
Coordenacao de Aperfeicoamento de Pessoal de Nıvel Supe-rior (CAPES Brazil) Karina B Felipe and Rozangela CuriPedrosa (Proc 3024042011-2) are recipients of researchgrants from Conselho Nacional de Pesquisa (CNPq) Brazil
References
[1] M M Gottesman ldquoMechanisms of cancer drug resistancerdquoAnnual Review of Medicine vol 53 pp 615ndash627 2002
[2] M S Farias C T Pich M R Kviecinski et al ldquoSubstituted3-acyl-2-phenylamino-14-naphthoquinones intercalate intoDNA and cause genotoxicity through the increased generationof reactive oxygen species culminating in cell deathrdquoMolecularMedicine Reports vol 10 no 1 pp 405ndash410 2014
[3] K B Felipe J Benites C Glorieux et al ldquoAntiproliferativeeffects of phenylaminonaphthoquinones are increased by ascor-bate and associated with the appearance of a senescent pheno-type in human bladder cancer cellsrdquo Biochemical and Biophysi-cal Research Communications vol 433 no 4 pp 573ndash578 2013
[4] M R Kviecinski R C Pedrosa K B Felipe et al ldquoInhibitionof cell proliferation and migration by oxidative stress fromascorbate-driven juglone redox cycling in human bladder-derived T24 cellsrdquo Biochemical and Biophysical Research Com-munications vol 421 no 2 pp 268ndash273 2012
[5] C F Thorn C Oshiro S Marsh et al ldquoDoxorubicin pathwayspharmacodynamics and adverse effectsrdquo Pharmacogenetics andGenomics vol 21 no 7 pp 440ndash446 2011
[6] G Bischoff and S Hoffman ldquoDNA-binding of drugs used inmedicinal therapiesrdquo Current Medicinal Chemistry vol 9 no3 pp 321ndash348 2002
[7] R Palchaudhuri and P J Hergenrother ldquoDNA as a target foranticancer compounds methods to determine the mode ofbinding and the mechanism of actionrdquo Current Opinion inBiotechnology vol 18 no 6 pp 497ndash503 2007
[8] M S Shahabuddin M Gopal and S C Raghavan ldquoIntercalat-ing and antitumor activity of 4 oxopyrimido[410158405101584045]thieno(23-b) quinoline-4(3H)-onerdquo Journal of Cancer Molecules vol 3pp 139ndash146 2007
[9] Q Chen M G Espey M C Krishna et al ldquoPharamacologicascorbic acid concentrations selectively kill cancer cells actionas a pro-drug to deliver hydrogen peroxide to tissuserdquo Proceed-ings of the National Academy of Sciences of the United States ofAmerica vol 102 no 38 pp 13604ndash13609 2005
[10] J Du J J Cullen and G R Buettner ldquoAscorbic acid chemistrybiology and the treatment of cancerrdquo Biochimica et BiophysicaActa vol 1826 no 2 pp 443ndash457 2012
[11] M G Espey P Chen B Chalmers et al ldquoPharmacologicascorbate synergizes with gemcitabine in preclinical models of
10 Oxidative Medicine and Cellular Longevity
pancreatic cancerrdquo Free Radical Biology and Medicine vol 50no 11 pp 1610ndash1619 2011
[12] J Verrax R Beck N Dejeans et al ldquoRedox-active quinonesand ascorbate an innovative cancer therapy that exploits thevulnerability of cancer cells to oxidative stressrdquo Anti-CancerAgents in Medicinal Chemistry vol 11 no 2 pp 213ndash221 2011
[13] J Benites J A Valderrama K Bettega R C Pedrosa PB Calderon and J Verrax ldquoBiological evaluation of donor-acceptor aminonaphthoquinones as antitumor agentsrdquo Euro-pean Journal of Medicinal Chemistry vol 45 no 12 pp 6052ndash6057 2010
[14] V C da Silveira H Benezra J S Luz R C Georg C COliveira and A M D C Ferreira ldquoBinding of oxindole-Schiffbase copper(II) complexes to DNA and its modulation by theligandrdquo Journal of Inorganic Biochemistry vol 105 no 12 pp1692ndash1703 2011
[15] T Jun W Bochu and Z Liancai ldquoHydrolytic cleavage of DNAby quercetin manganese(II) complexesrdquo Colloids and SurfacesB Biointerfaces vol 55 no 2 pp 149ndash152 2007
[16] N P Singh M T McCoy R R Tice and E L Schneider ldquoAsimple technique for quantitation of low levels of DNA damagein individual cellsrdquo Experimental Cell Research vol 175 no 1pp 184ndash191 1988
[17] J Yuan R Adamski and J Chen ldquoFocus on histone variantH2AX to be or not to berdquo FEBS Letters vol 584 no 17 pp 3717ndash3724 2010
[18] T T Paull E P Rogakou V Yamazaki C U Kirchgessner MGellert and W M Bonner ldquoA critical role for histone H2AX inrecruitment of repair factors to nuclear foci after DNAdamagerdquoCurrent Biology vol 10 no 15 pp 886ndash895 2000
[19] R Lopez A Arumugam R Joseph et al ldquoHyperglycemiaenhances the proliferation of non-tumorigenic and malignantmammary epithelial cells through increased leptinIGF1R sig-naling and activation of AKTmTORrdquo PLoS ONE vol 8 no 11Article ID e79708 2013
[20] G M Ross T J McMillan P Wilcox and A R Collins ldquoThesingle cell microgel electrophoresis assay (comet assay) techni-cal aspects and applications Report on the 5th LH Gray TrustWorkshop Institute of Cancer Research 1994rdquo MutationResearch vol 337 no 1 pp 57ndash60 1995
[21] T Mosmann ldquoRapid colorimetric assay for cellular growth andsurvival application to proliferation and cytotoxicity assaysrdquoJournal of Immunological Methods vol 65 no 1-2 pp 55ndash631983
[22] N A P Franken H M Rodermond J Stap J Haveman and Cvan Bree ldquoClonogenic assay of cells in vitrordquo Nature Protocolsvol 1 no 5 pp 2315ndash2319 2006
[23] C Glorieux N Dejeans B Sid R Beck P B Calderon andJ Verrax ldquoCatalase overexpression in mammary cancer cellsleads to a less aggressive phenotype and an altered response tochemotherapyrdquo Biochemical Pharmacology vol 82 no 10 pp1384ndash1390 2011
[24] M R Kviecinski P Benelli K B Felipe et al ldquoSFE from Bidenspilosa Linne to obtain extracts rich in cytotoxic polyacetyleneswith antitumor activityrdquo Journal of Supercritical Fluids vol 56no 3 pp 243ndash248 2011
[25] E L Kaplan and P Meier ldquoNonparametric estimation fromincomplete observationsrdquo Journal of the American StatisticalAssociation vol 53 pp 457ndash481 1958
[26] R P Bird and H H Draper ldquoComparative studies on differentmethods of malonaldehyde determinationrdquo Methods in Enzy-mology vol 105 pp 299ndash305 1984
[27] V G S Box ldquoThe intercalation of DNA double helices withdoxorubicin and nagalomycinrdquo Journal of Molecular Graphicsand Modelling vol 26 no 1 pp 14ndash19 2007
[28] Q Jiang N Xiao P Shi Y Zhu and Z Guo ldquoDesign of artifi-cial metallonucleases with oxidative mechanismrdquo CoordinationChemistry Reviews vol 251 no 15-16 pp 1951ndash1972 2007
[29] M U Rehman M Tahir A Q Khan et al ldquoD-limonene sup-presses doxorubicin-induced oxidative stress and inflammationvia repression of COX-2 iNOS and NF120581B in kidneys of Wistarratsrdquo Experimental Biology and Medicine vol 239 no 4 pp465ndash476 2014
[30] A H Boulares A G Yakovlev V Ivanova et al ldquoRole ofpoly(ADP-ribose) polymerase (PARP) cleavage in apoptosisCaspase 3-resistant PARP mutant increases rates of apoptosisin transfected cellsrdquo Journal of Biological Chemistry vol 274 no33 pp 22932ndash22940 1999
[31] N Ahmad S Gupta M M Husain K M Heiskanen and HMukhtar ldquoDifferential antiproliferative and apoptotic responseof sanguinarine for cancer cells versus normal cellsrdquo ClinicalCancer Research vol 6 no 4 pp 1524ndash1528 2000
[32] S J Lee H Ahn K W Nam K H Kim and W Mar ldquoEffectsof rutaecarpine on hydrogen peroxide-induced apoptosis inmurine hepa-1c1c7 cellsrdquo Biomolecules amp Therapeutics vol 20no 5 pp 487ndash491 2012
[33] E-S A Arafa Q Zhu Z I Shah et al ldquoThymoquinoneup-regulates PTEN expression and induces apoptosis indoxorubicin-resistant human breast cancer cellsrdquo MutationResearchmdashFundamental and Molecular Mechanisms of Mutage-nesis vol 706 no 1-2 pp 28ndash35 2011
[34] G Viglietto M L Motti P Bruni et al ldquoCytoplasmic relocal-ization and inhibition of the cyclin-dependent kinase inhibitorp271198961198941199011 by PKBAkt-mediated phosphorylation in breast can-cerrdquo Nature Medicine vol 8 no 10 pp 1136ndash1144 2002
[35] B D Clarkson and J H Burchenal ldquoPreliminary screening ofantineoplastic drugsrdquoProgress in Clinical Cancer vol 1 pp 625ndash629 1965
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
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Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
4 Oxidative Medicine and Cellular Longevity
0 10 20 30 407000
8000
9000
10000
Fluo
resc
ence
inte
nsity
EtdBrDoxorubicinAsc
Juglone
0 10 20 30 407000
8000
9000
10000
Fluo
resc
ence
inte
nsity
EtdBrDoxorubicinAsc
Q7Juglone + Asc Q7 + Asc
0 10 20 30 407000
8000
9000
10000
Fluo
resc
ence
inte
nsity
(120583M)
(120583M)(120583M)
EtdBrDoxorubicinAsc
Q9Q9 + Asc
(a)
000
002
004
006
008
010
TBA
RS ab
sorb
ance
NC PC Asc
Jugl
one
Q7
Q9
lowastlowast lowastlowastlowastlowastlowast
Jugl
one+
Asc
Q7+
Asc
Q9+
Asc
(b)
0
50
100
150
200
DN
A d
amag
e ind
ex
Con
trol
Asc
Jugl
one
Q7
Q9
Dox
orub
icin
lowastlowastlowast
lowastlowastlowast
Jugl
one+
Asc
Q7+
Asc
Q9+
Asc
(c)
Figure 1 Continued
Oxidative Medicine and Cellular Longevity 5
120574H2AX
Control Asc Jug Q7 Q9JugAsc
Q7Asc
Q9Asc
1mM 20120583M 20120583M1mM
120573-actin
(d)
Figure 1The fluorescence of CT-DNA stained with ethidium bromide is reduced by doxorubicin and juglone Q7 or Q9 mainly in presenceof ascorbate (Asc) (a) Absorbance of thiobarbituric reactive species (TBARS) in CT-DNA treatedwith juglone Q7 orQ9 alone and combinedwith ascorbate Negative control (NC) phosphate buffer Positive control (PC) [Fe(EDTA)]2minusH
2O2(b) DNA damage index was determined
by the comet assay in MCF-7 cells treated for 24 h with juglone 75120583M Q7 50 120583M or Q9 50 120583Mwith or without ascorbate 1mM (c) 120574H2AXwas assessed by immunoelectrophoresis in MCF-7 cells treated for 2 h with juglone (Jug) Q7 or Q9 at 20 120583Mwith or without ascorbate (Asc)at 1mM (d) (lowastlowast) and (lowast lowast lowast) denote statistical differences at 119875 lt 001 and 119875 lt 0001 compared to indicated treatments
Table 1 EC50 values obtained by the MTT assay MCF-7 cells weretreated for 24 h with juglone Q7 or Q9 (5ndash80 120583M) with or withoutascorbate 1mM Data from three independent experiments
EC50 (120583M)Juglone Q7 Q9
61 416 50plus ascorbate 1mM 28 263 254
The effects on the DNA of MCF-7 cells were assessedby immunoelectrophoresis for 120574H2AX and the comet assayGamma-H2AX is required for many proteins during theDNA damage and repair response [17] Figure 1(d) showsthat individually neither ascorbate juglone Q7 nor Q9enhanced the phosphorylated protein band corresponding to120574H2AXThe level of 120574H2AXwas enhanced onlywhenMCF-7cells were treated with the compounds combined with ascor-bate (1mM) Consistent with these findings the comet assayshowed that juglone Q7 and Q9 induced the fragmentationof MCF-7 DNA on their own but the addition of ascorbateincreased DNA fragmentation by approximately 2-fold in thecase of juglone and Q7 (Figure 1(c))
The effects of juglone Q7 and Q9 with and withoutascorbate were then examined on viability and proliferationinMCF-7 cellsTheMTT assay showed that ascorbate (1mM)alone did not reduce the viability of the MCF-7 cells Thecytotoxicity of juglone Q7 and Q9 was determined showingEC50values of 61 416 and 50120583M respectively The addition
of ascorbate together with the naphthoquinones reduced theEC50values by approximately 2-fold (to 28 263 and 254 120583M
for juglone Q7 andQ9 resp) and thus enhanced cytotoxicity(Table 1)
Ascorbate activates the redox cycling of quinones gen-erating semiquinone radicals and ROS In T24 cells it waspreviously shown that ascorbate (1mM) administered withjuglone Q7 and Q9 caused the appearance of semiquinoneand elevated levels of intracellular ROS [3 4] If a similarphenomenon occurs in MCF7 cells DNA damage wouldresult from not only binding and intercalation but also froman oxidative attack Doxorubicin acts by such a mechanismand kills cancer cells through both DNA intercalation and
ROS induction [29] Figure 2(a) shows that the ROS levelsin MCF-7 cells were elevated by the compounds particularlyby juglone and Q7 ROS generation induced by juglone andQ7 was enhanced approximately 3-fold in the presence ofascorbate On the other hand ascorbate had less impact interms of ROS induction when administered together withQ9 on MCF-7 cells Later the formulations containing Q9 orQ9 plus ascorbate were proven as poor inducers of oxidativestress in both MCF-7 cells (Figure 2(a)) and Ehrlich ascitescarcinoma in mice (Figure 3(c)) Ascorbate caused someincrease in terms of oxidative damage by Q9 only whenour treatments were done on CT-DNA a noncellular system(Figure 1(b))
Cell death was further studied based on PARP cleavageas shown in Figure 2(b) Compounds that are able to induceapoptosis cleave the PARP protein replacing the full-length116 kD protein with a cleaved fragment of approximately89 kD [30] Sanguinarine (5 120583M) a flavonoid known toinduce apoptosis was used as a control [31] PARP cleavagewas observed only in sanguinarine-treated cells and not incells treated with juglone Q7 or Q9 with or without ascor-bate (Figure 2(b))
The effect on MCF-7 cell proliferation is shown inFigure 2(c) JugloneQ7 andQ9 administered alone inhibitedcell proliferation as indicated by a reduction in the numberof colony-forming units in comparison with nontreated cellsIn all cases the inhibition of proliferation was increasedup to 3-fold when the naphthoquinones were administeredin combination with ascorbate (1mM) (Figure 2(c)) Thesedata corroborate literature when considered that DNA inter-calators can block cell division [6] To examine whetherthe PI3KAktmTOR signalling pathway was affected by thetreatments in MCF-7 cells the levels of the active phospho-rylated form of Akt (pAkt) were determined (Figure 2(d))The PI3KAkt pathway regulates several biological processesincluding cell survival proliferation and differentiationUpregulation of this pathway is observed in several types ofcancer and it can be associated with uncontrolled cell prolif-eration [32ndash34] As demonstrated in Figure 2(d) significantinhibition of the pAkt occurred in MCF-7 cells followingtreatment most notably with juglone or Q7 in the presenceof ascorbate
6 Oxidative Medicine and Cellular Longevity
0
1
2
3
4In
crea
sed
ROS
Asc
Jugl
one
Q7
Q9
lowastlowastlowast
lowastlowast
Jugl
one+
Asc
Q7+
Asc
Q9+
Asc
(a)
Control Sang
PARP
Asc Jug Q7 Q9JugAsc
Q7Asc
Q9Asc
120573-actin
(b)
0
50
100
150
Num
ber o
f col
onie
s
Con
trol
Asc
Jugl
one
Jugl
one +
Asc Q7
Q7
+ A
sc Q9
Q9
+ A
sc
lowastlowastlowast
lowastlowastlowast
lowastlowastlowast
(c)
Control
pAkt
Asc Jug Q7 Q9JugAsc
Q7Asc
Q9Asc
1mM 20120583M 20120583M1mM
120573-actin
(d)
Figure 2 ROS were measured in MCF-7 cells treated for 2 h with juglone Q7 or Q9 at 10120583M with or without ascorbate 1mM (a) Integrityof PARP protein in MCF-7 cells treated with juglone (Jug) Q7 or Q9 at 20 120583M with or without ascorbate (Asc) 1mM for 24 h Sanguinarine(Sang) 5120583M was used as a positive control of apoptosis (b) Colony-forming units of MCF-7 cells treated with juglone Q7 or Q9 at 10 120583Mwithwithout ascorbate 1mM for 2 h (c) Phosphorylated Akt (pAkt) was assessed by immunoelectrophoresis in MCF-7 cells treated for 24 h(d) Data were obtained from three independent experiments (lowastlowast) and (lowast lowast lowast) denote statistical differences at 119875 lt 001 and 119875 lt 0001compared to nontreated control cells or between indicated treatments respectively
Following these in vitro assays some in vivo effects werestudied in Ehrlich ascites carcinoma-bearing mice Ehrlichascites carcinoma was chosen to be used in vivo initially toverify whether some effects observed in vitro were repro-ducible in vivo But also to evaluate whether the effectswere consistent only with MCF-7 cells in vitro or they couldbe repeated against a different tumor cell line Figure 3(a)presents the levels of tumor growth inhibition Some inhi-bition on Ehrlich carcinoma was caused in animals treatedwith juglone Q7 orQ9 But the formulations of juglone orQ7plus ascorbate had the most potent activity and achieved upto 60 of inhibition of tumor growth approaching the effectof doxorubicin which caused up to 90 inhibition Actuallyconsidering data related to tumor growth and survival it is
possible to suggest that the potentiating effect of the com-bined treatment with ascorbate was reproduced in vivo withstatistical difference only in the case of juglone and Q7 Theability to increase the duration of animal survival is one ofthe most reliable criteria for evaluating potential antitumordrugs [35] Figure 3(b) presents graphs relating the numberof days after tumor inoculation to the percentage of survivorsfollowing treatment an increase in the area under the curvesindicates an increase in survival The smallest area in thegraphs corresponds to the group of nontreated animals Ingeneral these animals started to die on day 10 and by day12 no survivors remained The survival time was extendedby 2 to 6 days when the animals received treatments withindividual compounds (juglone Q7 or Q9) These animals
Oxidative Medicine and Cellular Longevity 7
0
20
40
60
80
100
Ehrli
ch ca
rcin
oma i
nhib
ition
()
Con
trol
Dox
orub
icin Asc
Jugl
one
Q7
Q9
lowastlowastlowastlowastlowast
Jugl
one+
Asc
Q7+
Asc
Q9+
Asc
(a)
0 10 20 300
50
100
DoxorubicinControlAscJuglone
Days
Surv
ival
()
Juglone + Asc
0 10 20 300
50
100
DoxorubicinControlAscQ7
Days
Surv
ival
()
Q7 + Asc
0 10 20 300
50
100
DoxorubicinControlAscQ9
Days
Surv
ival
()
Q9 + Asc
(b)
Figure 3 Continued
8 Oxidative Medicine and Cellular Longevity
000
001
002
003
004
005
MD
A n
mol
mg
prot
ein
Con
trol
Asc
Jugl
one
Q7
Q9
lowastlowast
lowastlowast
Jugl
one+
Asc
Q7+
Asc
Q9+
Asc
(c)
0
50
100
150
200
DN
A d
amag
e ind
ex
Con
trol
Asc
Jugl
one
Q7
Q9
lowastlowastlowast
lowastlowast
Jugl
one+
Asc
Q7+
Asc
Q9+
Asc
(d)
120574H2AX
pAkt
Ctrl Asc Jug Q7 Q9JugAsc
Q7Asc
Q9Asc
120573-actin
(e)
Figure 3 Ehrlich ascites carcinoma inhibition (a) and survival time (b) in animals treated with juglone Q7 or Q9 at 1mgkg alone or incombination with ascorbate (Asc) at 100mgkg Doxorubicin was administered at 12mgkg for the positive control Lipid peroxidation (c)DNA damage (d) and phosphorylated proteins Akt (pAkt) and H2AX (120574H2AX) in ascitic cells from mice (e) (lowastlowast) and (lowast lowast lowast) denotedifference at 119875 lt 001 and 119875 lt 0001 compared to the negative control or between indicated treatments
generally died before day 20The largest area under the curvescorresponds to the treatments conducted with juglone or Q7in combination with ascorbate (Figure 3(b)) The synergisticeffect between these drugs occurred also in vivo
Figures 3(c) 3(d) and 3(e) present data of moleculartoxicity on Ehrlich tumor in mice Figure 3(c) depicts levelsof MDA a biomarker of lipid peroxidationThis was the end-point measurement done to verify oxidative stress causedby the treatments in vivo MDA levels were increased sig-nificantly by the combined treatments done with juglone orQ7 and ascorbate Data in Figure 3(d) corroborate with datafrom Figure 3(c) because they show that DNA fragmenta-tion was increased significantly in vivo only when animalsreceived juglone or Q7 plus ascorbate Finally data shownin Figure 3(e) provide concise evidences which demonstratethat juglone and Q7 plus ascorbate can cause DNA damageand pAkt inhibition in Ehrlich tumor in mice Thus theeffects are not restricted to MCF-7 cells in vitro
Data from this study are summarized illustratively inFigure 4 Although all 14-naphthoquinones presented someactivity primarily juglone and Q7 administered in combina-tion with ascorbate trigger the generation of free radicals thesemiquinone formof the naphthoquinone and dehydroascor-bate The resulting semiquinone form intercalates into DNA
The radicals can attack DNA which is degraded In cancercells these events are accompanied by an inhibition on Aktpathway The formulation of juglone or Q7 with ascorbateshowed the most promising activity in vivo
4 Conclusion
Overall it is possible to conclude that primarily jugloneand Q7 combined with ascorbate have significant antitumoreffects against MCF-7 cells in vitro and Ehrlich-ascites carci-noma in mice The effects are the result of intercalation andoxidative attack on DNA of tumor cells and inhibition of Aktpathway
Conflict of Interests
The authors declare that they have no conflict of interests
Acknowledgments
Fabiana Ourique Luiza S E P W Castro and ValdeluciaM A S Grinevicius are recipients of research grants from
Oxidative Medicine and Cellular Longevity 9
Juglone Q7 Q9
OH
OH
H H
H
OO
H
H
PmTOR
Growth and proliferation
Cytosol
AktP P
PKB
P
Growth factor
Tyrosine kinase-like receptor
Plasma membranePI-3K
OH
OH
OH
OH
HO
HO
HO
O O
O O
O
OR1
R1
R2
R2
R1
R2
Ominus
∙O
O∙
O2∙minus
O2
H∙∙∙N H
∙∙∙N OCH3
Figure 4 Antitumor actions of juglone Q7 and Q9 administered in combination with ascorbate against MCF-7 cells and Ehrlich ascitescarcinoma in mice The effects are the result of intercalation and oxidative attack on DNA of tumor cells and inhibition of Akt pathway
Coordenacao de Aperfeicoamento de Pessoal de Nıvel Supe-rior (CAPES Brazil) Karina B Felipe and Rozangela CuriPedrosa (Proc 3024042011-2) are recipients of researchgrants from Conselho Nacional de Pesquisa (CNPq) Brazil
References
[1] M M Gottesman ldquoMechanisms of cancer drug resistancerdquoAnnual Review of Medicine vol 53 pp 615ndash627 2002
[2] M S Farias C T Pich M R Kviecinski et al ldquoSubstituted3-acyl-2-phenylamino-14-naphthoquinones intercalate intoDNA and cause genotoxicity through the increased generationof reactive oxygen species culminating in cell deathrdquoMolecularMedicine Reports vol 10 no 1 pp 405ndash410 2014
[3] K B Felipe J Benites C Glorieux et al ldquoAntiproliferativeeffects of phenylaminonaphthoquinones are increased by ascor-bate and associated with the appearance of a senescent pheno-type in human bladder cancer cellsrdquo Biochemical and Biophysi-cal Research Communications vol 433 no 4 pp 573ndash578 2013
[4] M R Kviecinski R C Pedrosa K B Felipe et al ldquoInhibitionof cell proliferation and migration by oxidative stress fromascorbate-driven juglone redox cycling in human bladder-derived T24 cellsrdquo Biochemical and Biophysical Research Com-munications vol 421 no 2 pp 268ndash273 2012
[5] C F Thorn C Oshiro S Marsh et al ldquoDoxorubicin pathwayspharmacodynamics and adverse effectsrdquo Pharmacogenetics andGenomics vol 21 no 7 pp 440ndash446 2011
[6] G Bischoff and S Hoffman ldquoDNA-binding of drugs used inmedicinal therapiesrdquo Current Medicinal Chemistry vol 9 no3 pp 321ndash348 2002
[7] R Palchaudhuri and P J Hergenrother ldquoDNA as a target foranticancer compounds methods to determine the mode ofbinding and the mechanism of actionrdquo Current Opinion inBiotechnology vol 18 no 6 pp 497ndash503 2007
[8] M S Shahabuddin M Gopal and S C Raghavan ldquoIntercalat-ing and antitumor activity of 4 oxopyrimido[410158405101584045]thieno(23-b) quinoline-4(3H)-onerdquo Journal of Cancer Molecules vol 3pp 139ndash146 2007
[9] Q Chen M G Espey M C Krishna et al ldquoPharamacologicascorbic acid concentrations selectively kill cancer cells actionas a pro-drug to deliver hydrogen peroxide to tissuserdquo Proceed-ings of the National Academy of Sciences of the United States ofAmerica vol 102 no 38 pp 13604ndash13609 2005
[10] J Du J J Cullen and G R Buettner ldquoAscorbic acid chemistrybiology and the treatment of cancerrdquo Biochimica et BiophysicaActa vol 1826 no 2 pp 443ndash457 2012
[11] M G Espey P Chen B Chalmers et al ldquoPharmacologicascorbate synergizes with gemcitabine in preclinical models of
10 Oxidative Medicine and Cellular Longevity
pancreatic cancerrdquo Free Radical Biology and Medicine vol 50no 11 pp 1610ndash1619 2011
[12] J Verrax R Beck N Dejeans et al ldquoRedox-active quinonesand ascorbate an innovative cancer therapy that exploits thevulnerability of cancer cells to oxidative stressrdquo Anti-CancerAgents in Medicinal Chemistry vol 11 no 2 pp 213ndash221 2011
[13] J Benites J A Valderrama K Bettega R C Pedrosa PB Calderon and J Verrax ldquoBiological evaluation of donor-acceptor aminonaphthoquinones as antitumor agentsrdquo Euro-pean Journal of Medicinal Chemistry vol 45 no 12 pp 6052ndash6057 2010
[14] V C da Silveira H Benezra J S Luz R C Georg C COliveira and A M D C Ferreira ldquoBinding of oxindole-Schiffbase copper(II) complexes to DNA and its modulation by theligandrdquo Journal of Inorganic Biochemistry vol 105 no 12 pp1692ndash1703 2011
[15] T Jun W Bochu and Z Liancai ldquoHydrolytic cleavage of DNAby quercetin manganese(II) complexesrdquo Colloids and SurfacesB Biointerfaces vol 55 no 2 pp 149ndash152 2007
[16] N P Singh M T McCoy R R Tice and E L Schneider ldquoAsimple technique for quantitation of low levels of DNA damagein individual cellsrdquo Experimental Cell Research vol 175 no 1pp 184ndash191 1988
[17] J Yuan R Adamski and J Chen ldquoFocus on histone variantH2AX to be or not to berdquo FEBS Letters vol 584 no 17 pp 3717ndash3724 2010
[18] T T Paull E P Rogakou V Yamazaki C U Kirchgessner MGellert and W M Bonner ldquoA critical role for histone H2AX inrecruitment of repair factors to nuclear foci after DNAdamagerdquoCurrent Biology vol 10 no 15 pp 886ndash895 2000
[19] R Lopez A Arumugam R Joseph et al ldquoHyperglycemiaenhances the proliferation of non-tumorigenic and malignantmammary epithelial cells through increased leptinIGF1R sig-naling and activation of AKTmTORrdquo PLoS ONE vol 8 no 11Article ID e79708 2013
[20] G M Ross T J McMillan P Wilcox and A R Collins ldquoThesingle cell microgel electrophoresis assay (comet assay) techni-cal aspects and applications Report on the 5th LH Gray TrustWorkshop Institute of Cancer Research 1994rdquo MutationResearch vol 337 no 1 pp 57ndash60 1995
[21] T Mosmann ldquoRapid colorimetric assay for cellular growth andsurvival application to proliferation and cytotoxicity assaysrdquoJournal of Immunological Methods vol 65 no 1-2 pp 55ndash631983
[22] N A P Franken H M Rodermond J Stap J Haveman and Cvan Bree ldquoClonogenic assay of cells in vitrordquo Nature Protocolsvol 1 no 5 pp 2315ndash2319 2006
[23] C Glorieux N Dejeans B Sid R Beck P B Calderon andJ Verrax ldquoCatalase overexpression in mammary cancer cellsleads to a less aggressive phenotype and an altered response tochemotherapyrdquo Biochemical Pharmacology vol 82 no 10 pp1384ndash1390 2011
[24] M R Kviecinski P Benelli K B Felipe et al ldquoSFE from Bidenspilosa Linne to obtain extracts rich in cytotoxic polyacetyleneswith antitumor activityrdquo Journal of Supercritical Fluids vol 56no 3 pp 243ndash248 2011
[25] E L Kaplan and P Meier ldquoNonparametric estimation fromincomplete observationsrdquo Journal of the American StatisticalAssociation vol 53 pp 457ndash481 1958
[26] R P Bird and H H Draper ldquoComparative studies on differentmethods of malonaldehyde determinationrdquo Methods in Enzy-mology vol 105 pp 299ndash305 1984
[27] V G S Box ldquoThe intercalation of DNA double helices withdoxorubicin and nagalomycinrdquo Journal of Molecular Graphicsand Modelling vol 26 no 1 pp 14ndash19 2007
[28] Q Jiang N Xiao P Shi Y Zhu and Z Guo ldquoDesign of artifi-cial metallonucleases with oxidative mechanismrdquo CoordinationChemistry Reviews vol 251 no 15-16 pp 1951ndash1972 2007
[29] M U Rehman M Tahir A Q Khan et al ldquoD-limonene sup-presses doxorubicin-induced oxidative stress and inflammationvia repression of COX-2 iNOS and NF120581B in kidneys of Wistarratsrdquo Experimental Biology and Medicine vol 239 no 4 pp465ndash476 2014
[30] A H Boulares A G Yakovlev V Ivanova et al ldquoRole ofpoly(ADP-ribose) polymerase (PARP) cleavage in apoptosisCaspase 3-resistant PARP mutant increases rates of apoptosisin transfected cellsrdquo Journal of Biological Chemistry vol 274 no33 pp 22932ndash22940 1999
[31] N Ahmad S Gupta M M Husain K M Heiskanen and HMukhtar ldquoDifferential antiproliferative and apoptotic responseof sanguinarine for cancer cells versus normal cellsrdquo ClinicalCancer Research vol 6 no 4 pp 1524ndash1528 2000
[32] S J Lee H Ahn K W Nam K H Kim and W Mar ldquoEffectsof rutaecarpine on hydrogen peroxide-induced apoptosis inmurine hepa-1c1c7 cellsrdquo Biomolecules amp Therapeutics vol 20no 5 pp 487ndash491 2012
[33] E-S A Arafa Q Zhu Z I Shah et al ldquoThymoquinoneup-regulates PTEN expression and induces apoptosis indoxorubicin-resistant human breast cancer cellsrdquo MutationResearchmdashFundamental and Molecular Mechanisms of Mutage-nesis vol 706 no 1-2 pp 28ndash35 2011
[34] G Viglietto M L Motti P Bruni et al ldquoCytoplasmic relocal-ization and inhibition of the cyclin-dependent kinase inhibitorp271198961198941199011 by PKBAkt-mediated phosphorylation in breast can-cerrdquo Nature Medicine vol 8 no 10 pp 1136ndash1144 2002
[35] B D Clarkson and J H Burchenal ldquoPreliminary screening ofantineoplastic drugsrdquoProgress in Clinical Cancer vol 1 pp 625ndash629 1965
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
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Diabetes ResearchJournal of
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
Oxidative Medicine and Cellular Longevity 5
120574H2AX
Control Asc Jug Q7 Q9JugAsc
Q7Asc
Q9Asc
1mM 20120583M 20120583M1mM
120573-actin
(d)
Figure 1The fluorescence of CT-DNA stained with ethidium bromide is reduced by doxorubicin and juglone Q7 or Q9 mainly in presenceof ascorbate (Asc) (a) Absorbance of thiobarbituric reactive species (TBARS) in CT-DNA treatedwith juglone Q7 orQ9 alone and combinedwith ascorbate Negative control (NC) phosphate buffer Positive control (PC) [Fe(EDTA)]2minusH
2O2(b) DNA damage index was determined
by the comet assay in MCF-7 cells treated for 24 h with juglone 75120583M Q7 50 120583M or Q9 50 120583Mwith or without ascorbate 1mM (c) 120574H2AXwas assessed by immunoelectrophoresis in MCF-7 cells treated for 2 h with juglone (Jug) Q7 or Q9 at 20 120583Mwith or without ascorbate (Asc)at 1mM (d) (lowastlowast) and (lowast lowast lowast) denote statistical differences at 119875 lt 001 and 119875 lt 0001 compared to indicated treatments
Table 1 EC50 values obtained by the MTT assay MCF-7 cells weretreated for 24 h with juglone Q7 or Q9 (5ndash80 120583M) with or withoutascorbate 1mM Data from three independent experiments
EC50 (120583M)Juglone Q7 Q9
61 416 50plus ascorbate 1mM 28 263 254
The effects on the DNA of MCF-7 cells were assessedby immunoelectrophoresis for 120574H2AX and the comet assayGamma-H2AX is required for many proteins during theDNA damage and repair response [17] Figure 1(d) showsthat individually neither ascorbate juglone Q7 nor Q9enhanced the phosphorylated protein band corresponding to120574H2AXThe level of 120574H2AXwas enhanced onlywhenMCF-7cells were treated with the compounds combined with ascor-bate (1mM) Consistent with these findings the comet assayshowed that juglone Q7 and Q9 induced the fragmentationof MCF-7 DNA on their own but the addition of ascorbateincreased DNA fragmentation by approximately 2-fold in thecase of juglone and Q7 (Figure 1(c))
The effects of juglone Q7 and Q9 with and withoutascorbate were then examined on viability and proliferationinMCF-7 cellsTheMTT assay showed that ascorbate (1mM)alone did not reduce the viability of the MCF-7 cells Thecytotoxicity of juglone Q7 and Q9 was determined showingEC50values of 61 416 and 50120583M respectively The addition
of ascorbate together with the naphthoquinones reduced theEC50values by approximately 2-fold (to 28 263 and 254 120583M
for juglone Q7 andQ9 resp) and thus enhanced cytotoxicity(Table 1)
Ascorbate activates the redox cycling of quinones gen-erating semiquinone radicals and ROS In T24 cells it waspreviously shown that ascorbate (1mM) administered withjuglone Q7 and Q9 caused the appearance of semiquinoneand elevated levels of intracellular ROS [3 4] If a similarphenomenon occurs in MCF7 cells DNA damage wouldresult from not only binding and intercalation but also froman oxidative attack Doxorubicin acts by such a mechanismand kills cancer cells through both DNA intercalation and
ROS induction [29] Figure 2(a) shows that the ROS levelsin MCF-7 cells were elevated by the compounds particularlyby juglone and Q7 ROS generation induced by juglone andQ7 was enhanced approximately 3-fold in the presence ofascorbate On the other hand ascorbate had less impact interms of ROS induction when administered together withQ9 on MCF-7 cells Later the formulations containing Q9 orQ9 plus ascorbate were proven as poor inducers of oxidativestress in both MCF-7 cells (Figure 2(a)) and Ehrlich ascitescarcinoma in mice (Figure 3(c)) Ascorbate caused someincrease in terms of oxidative damage by Q9 only whenour treatments were done on CT-DNA a noncellular system(Figure 1(b))
Cell death was further studied based on PARP cleavageas shown in Figure 2(b) Compounds that are able to induceapoptosis cleave the PARP protein replacing the full-length116 kD protein with a cleaved fragment of approximately89 kD [30] Sanguinarine (5 120583M) a flavonoid known toinduce apoptosis was used as a control [31] PARP cleavagewas observed only in sanguinarine-treated cells and not incells treated with juglone Q7 or Q9 with or without ascor-bate (Figure 2(b))
The effect on MCF-7 cell proliferation is shown inFigure 2(c) JugloneQ7 andQ9 administered alone inhibitedcell proliferation as indicated by a reduction in the numberof colony-forming units in comparison with nontreated cellsIn all cases the inhibition of proliferation was increasedup to 3-fold when the naphthoquinones were administeredin combination with ascorbate (1mM) (Figure 2(c)) Thesedata corroborate literature when considered that DNA inter-calators can block cell division [6] To examine whetherthe PI3KAktmTOR signalling pathway was affected by thetreatments in MCF-7 cells the levels of the active phospho-rylated form of Akt (pAkt) were determined (Figure 2(d))The PI3KAkt pathway regulates several biological processesincluding cell survival proliferation and differentiationUpregulation of this pathway is observed in several types ofcancer and it can be associated with uncontrolled cell prolif-eration [32ndash34] As demonstrated in Figure 2(d) significantinhibition of the pAkt occurred in MCF-7 cells followingtreatment most notably with juglone or Q7 in the presenceof ascorbate
6 Oxidative Medicine and Cellular Longevity
0
1
2
3
4In
crea
sed
ROS
Asc
Jugl
one
Q7
Q9
lowastlowastlowast
lowastlowast
Jugl
one+
Asc
Q7+
Asc
Q9+
Asc
(a)
Control Sang
PARP
Asc Jug Q7 Q9JugAsc
Q7Asc
Q9Asc
120573-actin
(b)
0
50
100
150
Num
ber o
f col
onie
s
Con
trol
Asc
Jugl
one
Jugl
one +
Asc Q7
Q7
+ A
sc Q9
Q9
+ A
sc
lowastlowastlowast
lowastlowastlowast
lowastlowastlowast
(c)
Control
pAkt
Asc Jug Q7 Q9JugAsc
Q7Asc
Q9Asc
1mM 20120583M 20120583M1mM
120573-actin
(d)
Figure 2 ROS were measured in MCF-7 cells treated for 2 h with juglone Q7 or Q9 at 10120583M with or without ascorbate 1mM (a) Integrityof PARP protein in MCF-7 cells treated with juglone (Jug) Q7 or Q9 at 20 120583M with or without ascorbate (Asc) 1mM for 24 h Sanguinarine(Sang) 5120583M was used as a positive control of apoptosis (b) Colony-forming units of MCF-7 cells treated with juglone Q7 or Q9 at 10 120583Mwithwithout ascorbate 1mM for 2 h (c) Phosphorylated Akt (pAkt) was assessed by immunoelectrophoresis in MCF-7 cells treated for 24 h(d) Data were obtained from three independent experiments (lowastlowast) and (lowast lowast lowast) denote statistical differences at 119875 lt 001 and 119875 lt 0001compared to nontreated control cells or between indicated treatments respectively
Following these in vitro assays some in vivo effects werestudied in Ehrlich ascites carcinoma-bearing mice Ehrlichascites carcinoma was chosen to be used in vivo initially toverify whether some effects observed in vitro were repro-ducible in vivo But also to evaluate whether the effectswere consistent only with MCF-7 cells in vitro or they couldbe repeated against a different tumor cell line Figure 3(a)presents the levels of tumor growth inhibition Some inhi-bition on Ehrlich carcinoma was caused in animals treatedwith juglone Q7 orQ9 But the formulations of juglone orQ7plus ascorbate had the most potent activity and achieved upto 60 of inhibition of tumor growth approaching the effectof doxorubicin which caused up to 90 inhibition Actuallyconsidering data related to tumor growth and survival it is
possible to suggest that the potentiating effect of the com-bined treatment with ascorbate was reproduced in vivo withstatistical difference only in the case of juglone and Q7 Theability to increase the duration of animal survival is one ofthe most reliable criteria for evaluating potential antitumordrugs [35] Figure 3(b) presents graphs relating the numberof days after tumor inoculation to the percentage of survivorsfollowing treatment an increase in the area under the curvesindicates an increase in survival The smallest area in thegraphs corresponds to the group of nontreated animals Ingeneral these animals started to die on day 10 and by day12 no survivors remained The survival time was extendedby 2 to 6 days when the animals received treatments withindividual compounds (juglone Q7 or Q9) These animals
Oxidative Medicine and Cellular Longevity 7
0
20
40
60
80
100
Ehrli
ch ca
rcin
oma i
nhib
ition
()
Con
trol
Dox
orub
icin Asc
Jugl
one
Q7
Q9
lowastlowastlowastlowastlowast
Jugl
one+
Asc
Q7+
Asc
Q9+
Asc
(a)
0 10 20 300
50
100
DoxorubicinControlAscJuglone
Days
Surv
ival
()
Juglone + Asc
0 10 20 300
50
100
DoxorubicinControlAscQ7
Days
Surv
ival
()
Q7 + Asc
0 10 20 300
50
100
DoxorubicinControlAscQ9
Days
Surv
ival
()
Q9 + Asc
(b)
Figure 3 Continued
8 Oxidative Medicine and Cellular Longevity
000
001
002
003
004
005
MD
A n
mol
mg
prot
ein
Con
trol
Asc
Jugl
one
Q7
Q9
lowastlowast
lowastlowast
Jugl
one+
Asc
Q7+
Asc
Q9+
Asc
(c)
0
50
100
150
200
DN
A d
amag
e ind
ex
Con
trol
Asc
Jugl
one
Q7
Q9
lowastlowastlowast
lowastlowast
Jugl
one+
Asc
Q7+
Asc
Q9+
Asc
(d)
120574H2AX
pAkt
Ctrl Asc Jug Q7 Q9JugAsc
Q7Asc
Q9Asc
120573-actin
(e)
Figure 3 Ehrlich ascites carcinoma inhibition (a) and survival time (b) in animals treated with juglone Q7 or Q9 at 1mgkg alone or incombination with ascorbate (Asc) at 100mgkg Doxorubicin was administered at 12mgkg for the positive control Lipid peroxidation (c)DNA damage (d) and phosphorylated proteins Akt (pAkt) and H2AX (120574H2AX) in ascitic cells from mice (e) (lowastlowast) and (lowast lowast lowast) denotedifference at 119875 lt 001 and 119875 lt 0001 compared to the negative control or between indicated treatments
generally died before day 20The largest area under the curvescorresponds to the treatments conducted with juglone or Q7in combination with ascorbate (Figure 3(b)) The synergisticeffect between these drugs occurred also in vivo
Figures 3(c) 3(d) and 3(e) present data of moleculartoxicity on Ehrlich tumor in mice Figure 3(c) depicts levelsof MDA a biomarker of lipid peroxidationThis was the end-point measurement done to verify oxidative stress causedby the treatments in vivo MDA levels were increased sig-nificantly by the combined treatments done with juglone orQ7 and ascorbate Data in Figure 3(d) corroborate with datafrom Figure 3(c) because they show that DNA fragmenta-tion was increased significantly in vivo only when animalsreceived juglone or Q7 plus ascorbate Finally data shownin Figure 3(e) provide concise evidences which demonstratethat juglone and Q7 plus ascorbate can cause DNA damageand pAkt inhibition in Ehrlich tumor in mice Thus theeffects are not restricted to MCF-7 cells in vitro
Data from this study are summarized illustratively inFigure 4 Although all 14-naphthoquinones presented someactivity primarily juglone and Q7 administered in combina-tion with ascorbate trigger the generation of free radicals thesemiquinone formof the naphthoquinone and dehydroascor-bate The resulting semiquinone form intercalates into DNA
The radicals can attack DNA which is degraded In cancercells these events are accompanied by an inhibition on Aktpathway The formulation of juglone or Q7 with ascorbateshowed the most promising activity in vivo
4 Conclusion
Overall it is possible to conclude that primarily jugloneand Q7 combined with ascorbate have significant antitumoreffects against MCF-7 cells in vitro and Ehrlich-ascites carci-noma in mice The effects are the result of intercalation andoxidative attack on DNA of tumor cells and inhibition of Aktpathway
Conflict of Interests
The authors declare that they have no conflict of interests
Acknowledgments
Fabiana Ourique Luiza S E P W Castro and ValdeluciaM A S Grinevicius are recipients of research grants from
Oxidative Medicine and Cellular Longevity 9
Juglone Q7 Q9
OH
OH
H H
H
OO
H
H
PmTOR
Growth and proliferation
Cytosol
AktP P
PKB
P
Growth factor
Tyrosine kinase-like receptor
Plasma membranePI-3K
OH
OH
OH
OH
HO
HO
HO
O O
O O
O
OR1
R1
R2
R2
R1
R2
Ominus
∙O
O∙
O2∙minus
O2
H∙∙∙N H
∙∙∙N OCH3
Figure 4 Antitumor actions of juglone Q7 and Q9 administered in combination with ascorbate against MCF-7 cells and Ehrlich ascitescarcinoma in mice The effects are the result of intercalation and oxidative attack on DNA of tumor cells and inhibition of Akt pathway
Coordenacao de Aperfeicoamento de Pessoal de Nıvel Supe-rior (CAPES Brazil) Karina B Felipe and Rozangela CuriPedrosa (Proc 3024042011-2) are recipients of researchgrants from Conselho Nacional de Pesquisa (CNPq) Brazil
References
[1] M M Gottesman ldquoMechanisms of cancer drug resistancerdquoAnnual Review of Medicine vol 53 pp 615ndash627 2002
[2] M S Farias C T Pich M R Kviecinski et al ldquoSubstituted3-acyl-2-phenylamino-14-naphthoquinones intercalate intoDNA and cause genotoxicity through the increased generationof reactive oxygen species culminating in cell deathrdquoMolecularMedicine Reports vol 10 no 1 pp 405ndash410 2014
[3] K B Felipe J Benites C Glorieux et al ldquoAntiproliferativeeffects of phenylaminonaphthoquinones are increased by ascor-bate and associated with the appearance of a senescent pheno-type in human bladder cancer cellsrdquo Biochemical and Biophysi-cal Research Communications vol 433 no 4 pp 573ndash578 2013
[4] M R Kviecinski R C Pedrosa K B Felipe et al ldquoInhibitionof cell proliferation and migration by oxidative stress fromascorbate-driven juglone redox cycling in human bladder-derived T24 cellsrdquo Biochemical and Biophysical Research Com-munications vol 421 no 2 pp 268ndash273 2012
[5] C F Thorn C Oshiro S Marsh et al ldquoDoxorubicin pathwayspharmacodynamics and adverse effectsrdquo Pharmacogenetics andGenomics vol 21 no 7 pp 440ndash446 2011
[6] G Bischoff and S Hoffman ldquoDNA-binding of drugs used inmedicinal therapiesrdquo Current Medicinal Chemistry vol 9 no3 pp 321ndash348 2002
[7] R Palchaudhuri and P J Hergenrother ldquoDNA as a target foranticancer compounds methods to determine the mode ofbinding and the mechanism of actionrdquo Current Opinion inBiotechnology vol 18 no 6 pp 497ndash503 2007
[8] M S Shahabuddin M Gopal and S C Raghavan ldquoIntercalat-ing and antitumor activity of 4 oxopyrimido[410158405101584045]thieno(23-b) quinoline-4(3H)-onerdquo Journal of Cancer Molecules vol 3pp 139ndash146 2007
[9] Q Chen M G Espey M C Krishna et al ldquoPharamacologicascorbic acid concentrations selectively kill cancer cells actionas a pro-drug to deliver hydrogen peroxide to tissuserdquo Proceed-ings of the National Academy of Sciences of the United States ofAmerica vol 102 no 38 pp 13604ndash13609 2005
[10] J Du J J Cullen and G R Buettner ldquoAscorbic acid chemistrybiology and the treatment of cancerrdquo Biochimica et BiophysicaActa vol 1826 no 2 pp 443ndash457 2012
[11] M G Espey P Chen B Chalmers et al ldquoPharmacologicascorbate synergizes with gemcitabine in preclinical models of
10 Oxidative Medicine and Cellular Longevity
pancreatic cancerrdquo Free Radical Biology and Medicine vol 50no 11 pp 1610ndash1619 2011
[12] J Verrax R Beck N Dejeans et al ldquoRedox-active quinonesand ascorbate an innovative cancer therapy that exploits thevulnerability of cancer cells to oxidative stressrdquo Anti-CancerAgents in Medicinal Chemistry vol 11 no 2 pp 213ndash221 2011
[13] J Benites J A Valderrama K Bettega R C Pedrosa PB Calderon and J Verrax ldquoBiological evaluation of donor-acceptor aminonaphthoquinones as antitumor agentsrdquo Euro-pean Journal of Medicinal Chemistry vol 45 no 12 pp 6052ndash6057 2010
[14] V C da Silveira H Benezra J S Luz R C Georg C COliveira and A M D C Ferreira ldquoBinding of oxindole-Schiffbase copper(II) complexes to DNA and its modulation by theligandrdquo Journal of Inorganic Biochemistry vol 105 no 12 pp1692ndash1703 2011
[15] T Jun W Bochu and Z Liancai ldquoHydrolytic cleavage of DNAby quercetin manganese(II) complexesrdquo Colloids and SurfacesB Biointerfaces vol 55 no 2 pp 149ndash152 2007
[16] N P Singh M T McCoy R R Tice and E L Schneider ldquoAsimple technique for quantitation of low levels of DNA damagein individual cellsrdquo Experimental Cell Research vol 175 no 1pp 184ndash191 1988
[17] J Yuan R Adamski and J Chen ldquoFocus on histone variantH2AX to be or not to berdquo FEBS Letters vol 584 no 17 pp 3717ndash3724 2010
[18] T T Paull E P Rogakou V Yamazaki C U Kirchgessner MGellert and W M Bonner ldquoA critical role for histone H2AX inrecruitment of repair factors to nuclear foci after DNAdamagerdquoCurrent Biology vol 10 no 15 pp 886ndash895 2000
[19] R Lopez A Arumugam R Joseph et al ldquoHyperglycemiaenhances the proliferation of non-tumorigenic and malignantmammary epithelial cells through increased leptinIGF1R sig-naling and activation of AKTmTORrdquo PLoS ONE vol 8 no 11Article ID e79708 2013
[20] G M Ross T J McMillan P Wilcox and A R Collins ldquoThesingle cell microgel electrophoresis assay (comet assay) techni-cal aspects and applications Report on the 5th LH Gray TrustWorkshop Institute of Cancer Research 1994rdquo MutationResearch vol 337 no 1 pp 57ndash60 1995
[21] T Mosmann ldquoRapid colorimetric assay for cellular growth andsurvival application to proliferation and cytotoxicity assaysrdquoJournal of Immunological Methods vol 65 no 1-2 pp 55ndash631983
[22] N A P Franken H M Rodermond J Stap J Haveman and Cvan Bree ldquoClonogenic assay of cells in vitrordquo Nature Protocolsvol 1 no 5 pp 2315ndash2319 2006
[23] C Glorieux N Dejeans B Sid R Beck P B Calderon andJ Verrax ldquoCatalase overexpression in mammary cancer cellsleads to a less aggressive phenotype and an altered response tochemotherapyrdquo Biochemical Pharmacology vol 82 no 10 pp1384ndash1390 2011
[24] M R Kviecinski P Benelli K B Felipe et al ldquoSFE from Bidenspilosa Linne to obtain extracts rich in cytotoxic polyacetyleneswith antitumor activityrdquo Journal of Supercritical Fluids vol 56no 3 pp 243ndash248 2011
[25] E L Kaplan and P Meier ldquoNonparametric estimation fromincomplete observationsrdquo Journal of the American StatisticalAssociation vol 53 pp 457ndash481 1958
[26] R P Bird and H H Draper ldquoComparative studies on differentmethods of malonaldehyde determinationrdquo Methods in Enzy-mology vol 105 pp 299ndash305 1984
[27] V G S Box ldquoThe intercalation of DNA double helices withdoxorubicin and nagalomycinrdquo Journal of Molecular Graphicsand Modelling vol 26 no 1 pp 14ndash19 2007
[28] Q Jiang N Xiao P Shi Y Zhu and Z Guo ldquoDesign of artifi-cial metallonucleases with oxidative mechanismrdquo CoordinationChemistry Reviews vol 251 no 15-16 pp 1951ndash1972 2007
[29] M U Rehman M Tahir A Q Khan et al ldquoD-limonene sup-presses doxorubicin-induced oxidative stress and inflammationvia repression of COX-2 iNOS and NF120581B in kidneys of Wistarratsrdquo Experimental Biology and Medicine vol 239 no 4 pp465ndash476 2014
[30] A H Boulares A G Yakovlev V Ivanova et al ldquoRole ofpoly(ADP-ribose) polymerase (PARP) cleavage in apoptosisCaspase 3-resistant PARP mutant increases rates of apoptosisin transfected cellsrdquo Journal of Biological Chemistry vol 274 no33 pp 22932ndash22940 1999
[31] N Ahmad S Gupta M M Husain K M Heiskanen and HMukhtar ldquoDifferential antiproliferative and apoptotic responseof sanguinarine for cancer cells versus normal cellsrdquo ClinicalCancer Research vol 6 no 4 pp 1524ndash1528 2000
[32] S J Lee H Ahn K W Nam K H Kim and W Mar ldquoEffectsof rutaecarpine on hydrogen peroxide-induced apoptosis inmurine hepa-1c1c7 cellsrdquo Biomolecules amp Therapeutics vol 20no 5 pp 487ndash491 2012
[33] E-S A Arafa Q Zhu Z I Shah et al ldquoThymoquinoneup-regulates PTEN expression and induces apoptosis indoxorubicin-resistant human breast cancer cellsrdquo MutationResearchmdashFundamental and Molecular Mechanisms of Mutage-nesis vol 706 no 1-2 pp 28ndash35 2011
[34] G Viglietto M L Motti P Bruni et al ldquoCytoplasmic relocal-ization and inhibition of the cyclin-dependent kinase inhibitorp271198961198941199011 by PKBAkt-mediated phosphorylation in breast can-cerrdquo Nature Medicine vol 8 no 10 pp 1136ndash1144 2002
[35] B D Clarkson and J H Burchenal ldquoPreliminary screening ofantineoplastic drugsrdquoProgress in Clinical Cancer vol 1 pp 625ndash629 1965
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
6 Oxidative Medicine and Cellular Longevity
0
1
2
3
4In
crea
sed
ROS
Asc
Jugl
one
Q7
Q9
lowastlowastlowast
lowastlowast
Jugl
one+
Asc
Q7+
Asc
Q9+
Asc
(a)
Control Sang
PARP
Asc Jug Q7 Q9JugAsc
Q7Asc
Q9Asc
120573-actin
(b)
0
50
100
150
Num
ber o
f col
onie
s
Con
trol
Asc
Jugl
one
Jugl
one +
Asc Q7
Q7
+ A
sc Q9
Q9
+ A
sc
lowastlowastlowast
lowastlowastlowast
lowastlowastlowast
(c)
Control
pAkt
Asc Jug Q7 Q9JugAsc
Q7Asc
Q9Asc
1mM 20120583M 20120583M1mM
120573-actin
(d)
Figure 2 ROS were measured in MCF-7 cells treated for 2 h with juglone Q7 or Q9 at 10120583M with or without ascorbate 1mM (a) Integrityof PARP protein in MCF-7 cells treated with juglone (Jug) Q7 or Q9 at 20 120583M with or without ascorbate (Asc) 1mM for 24 h Sanguinarine(Sang) 5120583M was used as a positive control of apoptosis (b) Colony-forming units of MCF-7 cells treated with juglone Q7 or Q9 at 10 120583Mwithwithout ascorbate 1mM for 2 h (c) Phosphorylated Akt (pAkt) was assessed by immunoelectrophoresis in MCF-7 cells treated for 24 h(d) Data were obtained from three independent experiments (lowastlowast) and (lowast lowast lowast) denote statistical differences at 119875 lt 001 and 119875 lt 0001compared to nontreated control cells or between indicated treatments respectively
Following these in vitro assays some in vivo effects werestudied in Ehrlich ascites carcinoma-bearing mice Ehrlichascites carcinoma was chosen to be used in vivo initially toverify whether some effects observed in vitro were repro-ducible in vivo But also to evaluate whether the effectswere consistent only with MCF-7 cells in vitro or they couldbe repeated against a different tumor cell line Figure 3(a)presents the levels of tumor growth inhibition Some inhi-bition on Ehrlich carcinoma was caused in animals treatedwith juglone Q7 orQ9 But the formulations of juglone orQ7plus ascorbate had the most potent activity and achieved upto 60 of inhibition of tumor growth approaching the effectof doxorubicin which caused up to 90 inhibition Actuallyconsidering data related to tumor growth and survival it is
possible to suggest that the potentiating effect of the com-bined treatment with ascorbate was reproduced in vivo withstatistical difference only in the case of juglone and Q7 Theability to increase the duration of animal survival is one ofthe most reliable criteria for evaluating potential antitumordrugs [35] Figure 3(b) presents graphs relating the numberof days after tumor inoculation to the percentage of survivorsfollowing treatment an increase in the area under the curvesindicates an increase in survival The smallest area in thegraphs corresponds to the group of nontreated animals Ingeneral these animals started to die on day 10 and by day12 no survivors remained The survival time was extendedby 2 to 6 days when the animals received treatments withindividual compounds (juglone Q7 or Q9) These animals
Oxidative Medicine and Cellular Longevity 7
0
20
40
60
80
100
Ehrli
ch ca
rcin
oma i
nhib
ition
()
Con
trol
Dox
orub
icin Asc
Jugl
one
Q7
Q9
lowastlowastlowastlowastlowast
Jugl
one+
Asc
Q7+
Asc
Q9+
Asc
(a)
0 10 20 300
50
100
DoxorubicinControlAscJuglone
Days
Surv
ival
()
Juglone + Asc
0 10 20 300
50
100
DoxorubicinControlAscQ7
Days
Surv
ival
()
Q7 + Asc
0 10 20 300
50
100
DoxorubicinControlAscQ9
Days
Surv
ival
()
Q9 + Asc
(b)
Figure 3 Continued
8 Oxidative Medicine and Cellular Longevity
000
001
002
003
004
005
MD
A n
mol
mg
prot
ein
Con
trol
Asc
Jugl
one
Q7
Q9
lowastlowast
lowastlowast
Jugl
one+
Asc
Q7+
Asc
Q9+
Asc
(c)
0
50
100
150
200
DN
A d
amag
e ind
ex
Con
trol
Asc
Jugl
one
Q7
Q9
lowastlowastlowast
lowastlowast
Jugl
one+
Asc
Q7+
Asc
Q9+
Asc
(d)
120574H2AX
pAkt
Ctrl Asc Jug Q7 Q9JugAsc
Q7Asc
Q9Asc
120573-actin
(e)
Figure 3 Ehrlich ascites carcinoma inhibition (a) and survival time (b) in animals treated with juglone Q7 or Q9 at 1mgkg alone or incombination with ascorbate (Asc) at 100mgkg Doxorubicin was administered at 12mgkg for the positive control Lipid peroxidation (c)DNA damage (d) and phosphorylated proteins Akt (pAkt) and H2AX (120574H2AX) in ascitic cells from mice (e) (lowastlowast) and (lowast lowast lowast) denotedifference at 119875 lt 001 and 119875 lt 0001 compared to the negative control or between indicated treatments
generally died before day 20The largest area under the curvescorresponds to the treatments conducted with juglone or Q7in combination with ascorbate (Figure 3(b)) The synergisticeffect between these drugs occurred also in vivo
Figures 3(c) 3(d) and 3(e) present data of moleculartoxicity on Ehrlich tumor in mice Figure 3(c) depicts levelsof MDA a biomarker of lipid peroxidationThis was the end-point measurement done to verify oxidative stress causedby the treatments in vivo MDA levels were increased sig-nificantly by the combined treatments done with juglone orQ7 and ascorbate Data in Figure 3(d) corroborate with datafrom Figure 3(c) because they show that DNA fragmenta-tion was increased significantly in vivo only when animalsreceived juglone or Q7 plus ascorbate Finally data shownin Figure 3(e) provide concise evidences which demonstratethat juglone and Q7 plus ascorbate can cause DNA damageand pAkt inhibition in Ehrlich tumor in mice Thus theeffects are not restricted to MCF-7 cells in vitro
Data from this study are summarized illustratively inFigure 4 Although all 14-naphthoquinones presented someactivity primarily juglone and Q7 administered in combina-tion with ascorbate trigger the generation of free radicals thesemiquinone formof the naphthoquinone and dehydroascor-bate The resulting semiquinone form intercalates into DNA
The radicals can attack DNA which is degraded In cancercells these events are accompanied by an inhibition on Aktpathway The formulation of juglone or Q7 with ascorbateshowed the most promising activity in vivo
4 Conclusion
Overall it is possible to conclude that primarily jugloneand Q7 combined with ascorbate have significant antitumoreffects against MCF-7 cells in vitro and Ehrlich-ascites carci-noma in mice The effects are the result of intercalation andoxidative attack on DNA of tumor cells and inhibition of Aktpathway
Conflict of Interests
The authors declare that they have no conflict of interests
Acknowledgments
Fabiana Ourique Luiza S E P W Castro and ValdeluciaM A S Grinevicius are recipients of research grants from
Oxidative Medicine and Cellular Longevity 9
Juglone Q7 Q9
OH
OH
H H
H
OO
H
H
PmTOR
Growth and proliferation
Cytosol
AktP P
PKB
P
Growth factor
Tyrosine kinase-like receptor
Plasma membranePI-3K
OH
OH
OH
OH
HO
HO
HO
O O
O O
O
OR1
R1
R2
R2
R1
R2
Ominus
∙O
O∙
O2∙minus
O2
H∙∙∙N H
∙∙∙N OCH3
Figure 4 Antitumor actions of juglone Q7 and Q9 administered in combination with ascorbate against MCF-7 cells and Ehrlich ascitescarcinoma in mice The effects are the result of intercalation and oxidative attack on DNA of tumor cells and inhibition of Akt pathway
Coordenacao de Aperfeicoamento de Pessoal de Nıvel Supe-rior (CAPES Brazil) Karina B Felipe and Rozangela CuriPedrosa (Proc 3024042011-2) are recipients of researchgrants from Conselho Nacional de Pesquisa (CNPq) Brazil
References
[1] M M Gottesman ldquoMechanisms of cancer drug resistancerdquoAnnual Review of Medicine vol 53 pp 615ndash627 2002
[2] M S Farias C T Pich M R Kviecinski et al ldquoSubstituted3-acyl-2-phenylamino-14-naphthoquinones intercalate intoDNA and cause genotoxicity through the increased generationof reactive oxygen species culminating in cell deathrdquoMolecularMedicine Reports vol 10 no 1 pp 405ndash410 2014
[3] K B Felipe J Benites C Glorieux et al ldquoAntiproliferativeeffects of phenylaminonaphthoquinones are increased by ascor-bate and associated with the appearance of a senescent pheno-type in human bladder cancer cellsrdquo Biochemical and Biophysi-cal Research Communications vol 433 no 4 pp 573ndash578 2013
[4] M R Kviecinski R C Pedrosa K B Felipe et al ldquoInhibitionof cell proliferation and migration by oxidative stress fromascorbate-driven juglone redox cycling in human bladder-derived T24 cellsrdquo Biochemical and Biophysical Research Com-munications vol 421 no 2 pp 268ndash273 2012
[5] C F Thorn C Oshiro S Marsh et al ldquoDoxorubicin pathwayspharmacodynamics and adverse effectsrdquo Pharmacogenetics andGenomics vol 21 no 7 pp 440ndash446 2011
[6] G Bischoff and S Hoffman ldquoDNA-binding of drugs used inmedicinal therapiesrdquo Current Medicinal Chemistry vol 9 no3 pp 321ndash348 2002
[7] R Palchaudhuri and P J Hergenrother ldquoDNA as a target foranticancer compounds methods to determine the mode ofbinding and the mechanism of actionrdquo Current Opinion inBiotechnology vol 18 no 6 pp 497ndash503 2007
[8] M S Shahabuddin M Gopal and S C Raghavan ldquoIntercalat-ing and antitumor activity of 4 oxopyrimido[410158405101584045]thieno(23-b) quinoline-4(3H)-onerdquo Journal of Cancer Molecules vol 3pp 139ndash146 2007
[9] Q Chen M G Espey M C Krishna et al ldquoPharamacologicascorbic acid concentrations selectively kill cancer cells actionas a pro-drug to deliver hydrogen peroxide to tissuserdquo Proceed-ings of the National Academy of Sciences of the United States ofAmerica vol 102 no 38 pp 13604ndash13609 2005
[10] J Du J J Cullen and G R Buettner ldquoAscorbic acid chemistrybiology and the treatment of cancerrdquo Biochimica et BiophysicaActa vol 1826 no 2 pp 443ndash457 2012
[11] M G Espey P Chen B Chalmers et al ldquoPharmacologicascorbate synergizes with gemcitabine in preclinical models of
10 Oxidative Medicine and Cellular Longevity
pancreatic cancerrdquo Free Radical Biology and Medicine vol 50no 11 pp 1610ndash1619 2011
[12] J Verrax R Beck N Dejeans et al ldquoRedox-active quinonesand ascorbate an innovative cancer therapy that exploits thevulnerability of cancer cells to oxidative stressrdquo Anti-CancerAgents in Medicinal Chemistry vol 11 no 2 pp 213ndash221 2011
[13] J Benites J A Valderrama K Bettega R C Pedrosa PB Calderon and J Verrax ldquoBiological evaluation of donor-acceptor aminonaphthoquinones as antitumor agentsrdquo Euro-pean Journal of Medicinal Chemistry vol 45 no 12 pp 6052ndash6057 2010
[14] V C da Silveira H Benezra J S Luz R C Georg C COliveira and A M D C Ferreira ldquoBinding of oxindole-Schiffbase copper(II) complexes to DNA and its modulation by theligandrdquo Journal of Inorganic Biochemistry vol 105 no 12 pp1692ndash1703 2011
[15] T Jun W Bochu and Z Liancai ldquoHydrolytic cleavage of DNAby quercetin manganese(II) complexesrdquo Colloids and SurfacesB Biointerfaces vol 55 no 2 pp 149ndash152 2007
[16] N P Singh M T McCoy R R Tice and E L Schneider ldquoAsimple technique for quantitation of low levels of DNA damagein individual cellsrdquo Experimental Cell Research vol 175 no 1pp 184ndash191 1988
[17] J Yuan R Adamski and J Chen ldquoFocus on histone variantH2AX to be or not to berdquo FEBS Letters vol 584 no 17 pp 3717ndash3724 2010
[18] T T Paull E P Rogakou V Yamazaki C U Kirchgessner MGellert and W M Bonner ldquoA critical role for histone H2AX inrecruitment of repair factors to nuclear foci after DNAdamagerdquoCurrent Biology vol 10 no 15 pp 886ndash895 2000
[19] R Lopez A Arumugam R Joseph et al ldquoHyperglycemiaenhances the proliferation of non-tumorigenic and malignantmammary epithelial cells through increased leptinIGF1R sig-naling and activation of AKTmTORrdquo PLoS ONE vol 8 no 11Article ID e79708 2013
[20] G M Ross T J McMillan P Wilcox and A R Collins ldquoThesingle cell microgel electrophoresis assay (comet assay) techni-cal aspects and applications Report on the 5th LH Gray TrustWorkshop Institute of Cancer Research 1994rdquo MutationResearch vol 337 no 1 pp 57ndash60 1995
[21] T Mosmann ldquoRapid colorimetric assay for cellular growth andsurvival application to proliferation and cytotoxicity assaysrdquoJournal of Immunological Methods vol 65 no 1-2 pp 55ndash631983
[22] N A P Franken H M Rodermond J Stap J Haveman and Cvan Bree ldquoClonogenic assay of cells in vitrordquo Nature Protocolsvol 1 no 5 pp 2315ndash2319 2006
[23] C Glorieux N Dejeans B Sid R Beck P B Calderon andJ Verrax ldquoCatalase overexpression in mammary cancer cellsleads to a less aggressive phenotype and an altered response tochemotherapyrdquo Biochemical Pharmacology vol 82 no 10 pp1384ndash1390 2011
[24] M R Kviecinski P Benelli K B Felipe et al ldquoSFE from Bidenspilosa Linne to obtain extracts rich in cytotoxic polyacetyleneswith antitumor activityrdquo Journal of Supercritical Fluids vol 56no 3 pp 243ndash248 2011
[25] E L Kaplan and P Meier ldquoNonparametric estimation fromincomplete observationsrdquo Journal of the American StatisticalAssociation vol 53 pp 457ndash481 1958
[26] R P Bird and H H Draper ldquoComparative studies on differentmethods of malonaldehyde determinationrdquo Methods in Enzy-mology vol 105 pp 299ndash305 1984
[27] V G S Box ldquoThe intercalation of DNA double helices withdoxorubicin and nagalomycinrdquo Journal of Molecular Graphicsand Modelling vol 26 no 1 pp 14ndash19 2007
[28] Q Jiang N Xiao P Shi Y Zhu and Z Guo ldquoDesign of artifi-cial metallonucleases with oxidative mechanismrdquo CoordinationChemistry Reviews vol 251 no 15-16 pp 1951ndash1972 2007
[29] M U Rehman M Tahir A Q Khan et al ldquoD-limonene sup-presses doxorubicin-induced oxidative stress and inflammationvia repression of COX-2 iNOS and NF120581B in kidneys of Wistarratsrdquo Experimental Biology and Medicine vol 239 no 4 pp465ndash476 2014
[30] A H Boulares A G Yakovlev V Ivanova et al ldquoRole ofpoly(ADP-ribose) polymerase (PARP) cleavage in apoptosisCaspase 3-resistant PARP mutant increases rates of apoptosisin transfected cellsrdquo Journal of Biological Chemistry vol 274 no33 pp 22932ndash22940 1999
[31] N Ahmad S Gupta M M Husain K M Heiskanen and HMukhtar ldquoDifferential antiproliferative and apoptotic responseof sanguinarine for cancer cells versus normal cellsrdquo ClinicalCancer Research vol 6 no 4 pp 1524ndash1528 2000
[32] S J Lee H Ahn K W Nam K H Kim and W Mar ldquoEffectsof rutaecarpine on hydrogen peroxide-induced apoptosis inmurine hepa-1c1c7 cellsrdquo Biomolecules amp Therapeutics vol 20no 5 pp 487ndash491 2012
[33] E-S A Arafa Q Zhu Z I Shah et al ldquoThymoquinoneup-regulates PTEN expression and induces apoptosis indoxorubicin-resistant human breast cancer cellsrdquo MutationResearchmdashFundamental and Molecular Mechanisms of Mutage-nesis vol 706 no 1-2 pp 28ndash35 2011
[34] G Viglietto M L Motti P Bruni et al ldquoCytoplasmic relocal-ization and inhibition of the cyclin-dependent kinase inhibitorp271198961198941199011 by PKBAkt-mediated phosphorylation in breast can-cerrdquo Nature Medicine vol 8 no 10 pp 1136ndash1144 2002
[35] B D Clarkson and J H Burchenal ldquoPreliminary screening ofantineoplastic drugsrdquoProgress in Clinical Cancer vol 1 pp 625ndash629 1965
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
Oxidative Medicine and Cellular Longevity 7
0
20
40
60
80
100
Ehrli
ch ca
rcin
oma i
nhib
ition
()
Con
trol
Dox
orub
icin Asc
Jugl
one
Q7
Q9
lowastlowastlowastlowastlowast
Jugl
one+
Asc
Q7+
Asc
Q9+
Asc
(a)
0 10 20 300
50
100
DoxorubicinControlAscJuglone
Days
Surv
ival
()
Juglone + Asc
0 10 20 300
50
100
DoxorubicinControlAscQ7
Days
Surv
ival
()
Q7 + Asc
0 10 20 300
50
100
DoxorubicinControlAscQ9
Days
Surv
ival
()
Q9 + Asc
(b)
Figure 3 Continued
8 Oxidative Medicine and Cellular Longevity
000
001
002
003
004
005
MD
A n
mol
mg
prot
ein
Con
trol
Asc
Jugl
one
Q7
Q9
lowastlowast
lowastlowast
Jugl
one+
Asc
Q7+
Asc
Q9+
Asc
(c)
0
50
100
150
200
DN
A d
amag
e ind
ex
Con
trol
Asc
Jugl
one
Q7
Q9
lowastlowastlowast
lowastlowast
Jugl
one+
Asc
Q7+
Asc
Q9+
Asc
(d)
120574H2AX
pAkt
Ctrl Asc Jug Q7 Q9JugAsc
Q7Asc
Q9Asc
120573-actin
(e)
Figure 3 Ehrlich ascites carcinoma inhibition (a) and survival time (b) in animals treated with juglone Q7 or Q9 at 1mgkg alone or incombination with ascorbate (Asc) at 100mgkg Doxorubicin was administered at 12mgkg for the positive control Lipid peroxidation (c)DNA damage (d) and phosphorylated proteins Akt (pAkt) and H2AX (120574H2AX) in ascitic cells from mice (e) (lowastlowast) and (lowast lowast lowast) denotedifference at 119875 lt 001 and 119875 lt 0001 compared to the negative control or between indicated treatments
generally died before day 20The largest area under the curvescorresponds to the treatments conducted with juglone or Q7in combination with ascorbate (Figure 3(b)) The synergisticeffect between these drugs occurred also in vivo
Figures 3(c) 3(d) and 3(e) present data of moleculartoxicity on Ehrlich tumor in mice Figure 3(c) depicts levelsof MDA a biomarker of lipid peroxidationThis was the end-point measurement done to verify oxidative stress causedby the treatments in vivo MDA levels were increased sig-nificantly by the combined treatments done with juglone orQ7 and ascorbate Data in Figure 3(d) corroborate with datafrom Figure 3(c) because they show that DNA fragmenta-tion was increased significantly in vivo only when animalsreceived juglone or Q7 plus ascorbate Finally data shownin Figure 3(e) provide concise evidences which demonstratethat juglone and Q7 plus ascorbate can cause DNA damageand pAkt inhibition in Ehrlich tumor in mice Thus theeffects are not restricted to MCF-7 cells in vitro
Data from this study are summarized illustratively inFigure 4 Although all 14-naphthoquinones presented someactivity primarily juglone and Q7 administered in combina-tion with ascorbate trigger the generation of free radicals thesemiquinone formof the naphthoquinone and dehydroascor-bate The resulting semiquinone form intercalates into DNA
The radicals can attack DNA which is degraded In cancercells these events are accompanied by an inhibition on Aktpathway The formulation of juglone or Q7 with ascorbateshowed the most promising activity in vivo
4 Conclusion
Overall it is possible to conclude that primarily jugloneand Q7 combined with ascorbate have significant antitumoreffects against MCF-7 cells in vitro and Ehrlich-ascites carci-noma in mice The effects are the result of intercalation andoxidative attack on DNA of tumor cells and inhibition of Aktpathway
Conflict of Interests
The authors declare that they have no conflict of interests
Acknowledgments
Fabiana Ourique Luiza S E P W Castro and ValdeluciaM A S Grinevicius are recipients of research grants from
Oxidative Medicine and Cellular Longevity 9
Juglone Q7 Q9
OH
OH
H H
H
OO
H
H
PmTOR
Growth and proliferation
Cytosol
AktP P
PKB
P
Growth factor
Tyrosine kinase-like receptor
Plasma membranePI-3K
OH
OH
OH
OH
HO
HO
HO
O O
O O
O
OR1
R1
R2
R2
R1
R2
Ominus
∙O
O∙
O2∙minus
O2
H∙∙∙N H
∙∙∙N OCH3
Figure 4 Antitumor actions of juglone Q7 and Q9 administered in combination with ascorbate against MCF-7 cells and Ehrlich ascitescarcinoma in mice The effects are the result of intercalation and oxidative attack on DNA of tumor cells and inhibition of Akt pathway
Coordenacao de Aperfeicoamento de Pessoal de Nıvel Supe-rior (CAPES Brazil) Karina B Felipe and Rozangela CuriPedrosa (Proc 3024042011-2) are recipients of researchgrants from Conselho Nacional de Pesquisa (CNPq) Brazil
References
[1] M M Gottesman ldquoMechanisms of cancer drug resistancerdquoAnnual Review of Medicine vol 53 pp 615ndash627 2002
[2] M S Farias C T Pich M R Kviecinski et al ldquoSubstituted3-acyl-2-phenylamino-14-naphthoquinones intercalate intoDNA and cause genotoxicity through the increased generationof reactive oxygen species culminating in cell deathrdquoMolecularMedicine Reports vol 10 no 1 pp 405ndash410 2014
[3] K B Felipe J Benites C Glorieux et al ldquoAntiproliferativeeffects of phenylaminonaphthoquinones are increased by ascor-bate and associated with the appearance of a senescent pheno-type in human bladder cancer cellsrdquo Biochemical and Biophysi-cal Research Communications vol 433 no 4 pp 573ndash578 2013
[4] M R Kviecinski R C Pedrosa K B Felipe et al ldquoInhibitionof cell proliferation and migration by oxidative stress fromascorbate-driven juglone redox cycling in human bladder-derived T24 cellsrdquo Biochemical and Biophysical Research Com-munications vol 421 no 2 pp 268ndash273 2012
[5] C F Thorn C Oshiro S Marsh et al ldquoDoxorubicin pathwayspharmacodynamics and adverse effectsrdquo Pharmacogenetics andGenomics vol 21 no 7 pp 440ndash446 2011
[6] G Bischoff and S Hoffman ldquoDNA-binding of drugs used inmedicinal therapiesrdquo Current Medicinal Chemistry vol 9 no3 pp 321ndash348 2002
[7] R Palchaudhuri and P J Hergenrother ldquoDNA as a target foranticancer compounds methods to determine the mode ofbinding and the mechanism of actionrdquo Current Opinion inBiotechnology vol 18 no 6 pp 497ndash503 2007
[8] M S Shahabuddin M Gopal and S C Raghavan ldquoIntercalat-ing and antitumor activity of 4 oxopyrimido[410158405101584045]thieno(23-b) quinoline-4(3H)-onerdquo Journal of Cancer Molecules vol 3pp 139ndash146 2007
[9] Q Chen M G Espey M C Krishna et al ldquoPharamacologicascorbic acid concentrations selectively kill cancer cells actionas a pro-drug to deliver hydrogen peroxide to tissuserdquo Proceed-ings of the National Academy of Sciences of the United States ofAmerica vol 102 no 38 pp 13604ndash13609 2005
[10] J Du J J Cullen and G R Buettner ldquoAscorbic acid chemistrybiology and the treatment of cancerrdquo Biochimica et BiophysicaActa vol 1826 no 2 pp 443ndash457 2012
[11] M G Espey P Chen B Chalmers et al ldquoPharmacologicascorbate synergizes with gemcitabine in preclinical models of
10 Oxidative Medicine and Cellular Longevity
pancreatic cancerrdquo Free Radical Biology and Medicine vol 50no 11 pp 1610ndash1619 2011
[12] J Verrax R Beck N Dejeans et al ldquoRedox-active quinonesand ascorbate an innovative cancer therapy that exploits thevulnerability of cancer cells to oxidative stressrdquo Anti-CancerAgents in Medicinal Chemistry vol 11 no 2 pp 213ndash221 2011
[13] J Benites J A Valderrama K Bettega R C Pedrosa PB Calderon and J Verrax ldquoBiological evaluation of donor-acceptor aminonaphthoquinones as antitumor agentsrdquo Euro-pean Journal of Medicinal Chemistry vol 45 no 12 pp 6052ndash6057 2010
[14] V C da Silveira H Benezra J S Luz R C Georg C COliveira and A M D C Ferreira ldquoBinding of oxindole-Schiffbase copper(II) complexes to DNA and its modulation by theligandrdquo Journal of Inorganic Biochemistry vol 105 no 12 pp1692ndash1703 2011
[15] T Jun W Bochu and Z Liancai ldquoHydrolytic cleavage of DNAby quercetin manganese(II) complexesrdquo Colloids and SurfacesB Biointerfaces vol 55 no 2 pp 149ndash152 2007
[16] N P Singh M T McCoy R R Tice and E L Schneider ldquoAsimple technique for quantitation of low levels of DNA damagein individual cellsrdquo Experimental Cell Research vol 175 no 1pp 184ndash191 1988
[17] J Yuan R Adamski and J Chen ldquoFocus on histone variantH2AX to be or not to berdquo FEBS Letters vol 584 no 17 pp 3717ndash3724 2010
[18] T T Paull E P Rogakou V Yamazaki C U Kirchgessner MGellert and W M Bonner ldquoA critical role for histone H2AX inrecruitment of repair factors to nuclear foci after DNAdamagerdquoCurrent Biology vol 10 no 15 pp 886ndash895 2000
[19] R Lopez A Arumugam R Joseph et al ldquoHyperglycemiaenhances the proliferation of non-tumorigenic and malignantmammary epithelial cells through increased leptinIGF1R sig-naling and activation of AKTmTORrdquo PLoS ONE vol 8 no 11Article ID e79708 2013
[20] G M Ross T J McMillan P Wilcox and A R Collins ldquoThesingle cell microgel electrophoresis assay (comet assay) techni-cal aspects and applications Report on the 5th LH Gray TrustWorkshop Institute of Cancer Research 1994rdquo MutationResearch vol 337 no 1 pp 57ndash60 1995
[21] T Mosmann ldquoRapid colorimetric assay for cellular growth andsurvival application to proliferation and cytotoxicity assaysrdquoJournal of Immunological Methods vol 65 no 1-2 pp 55ndash631983
[22] N A P Franken H M Rodermond J Stap J Haveman and Cvan Bree ldquoClonogenic assay of cells in vitrordquo Nature Protocolsvol 1 no 5 pp 2315ndash2319 2006
[23] C Glorieux N Dejeans B Sid R Beck P B Calderon andJ Verrax ldquoCatalase overexpression in mammary cancer cellsleads to a less aggressive phenotype and an altered response tochemotherapyrdquo Biochemical Pharmacology vol 82 no 10 pp1384ndash1390 2011
[24] M R Kviecinski P Benelli K B Felipe et al ldquoSFE from Bidenspilosa Linne to obtain extracts rich in cytotoxic polyacetyleneswith antitumor activityrdquo Journal of Supercritical Fluids vol 56no 3 pp 243ndash248 2011
[25] E L Kaplan and P Meier ldquoNonparametric estimation fromincomplete observationsrdquo Journal of the American StatisticalAssociation vol 53 pp 457ndash481 1958
[26] R P Bird and H H Draper ldquoComparative studies on differentmethods of malonaldehyde determinationrdquo Methods in Enzy-mology vol 105 pp 299ndash305 1984
[27] V G S Box ldquoThe intercalation of DNA double helices withdoxorubicin and nagalomycinrdquo Journal of Molecular Graphicsand Modelling vol 26 no 1 pp 14ndash19 2007
[28] Q Jiang N Xiao P Shi Y Zhu and Z Guo ldquoDesign of artifi-cial metallonucleases with oxidative mechanismrdquo CoordinationChemistry Reviews vol 251 no 15-16 pp 1951ndash1972 2007
[29] M U Rehman M Tahir A Q Khan et al ldquoD-limonene sup-presses doxorubicin-induced oxidative stress and inflammationvia repression of COX-2 iNOS and NF120581B in kidneys of Wistarratsrdquo Experimental Biology and Medicine vol 239 no 4 pp465ndash476 2014
[30] A H Boulares A G Yakovlev V Ivanova et al ldquoRole ofpoly(ADP-ribose) polymerase (PARP) cleavage in apoptosisCaspase 3-resistant PARP mutant increases rates of apoptosisin transfected cellsrdquo Journal of Biological Chemistry vol 274 no33 pp 22932ndash22940 1999
[31] N Ahmad S Gupta M M Husain K M Heiskanen and HMukhtar ldquoDifferential antiproliferative and apoptotic responseof sanguinarine for cancer cells versus normal cellsrdquo ClinicalCancer Research vol 6 no 4 pp 1524ndash1528 2000
[32] S J Lee H Ahn K W Nam K H Kim and W Mar ldquoEffectsof rutaecarpine on hydrogen peroxide-induced apoptosis inmurine hepa-1c1c7 cellsrdquo Biomolecules amp Therapeutics vol 20no 5 pp 487ndash491 2012
[33] E-S A Arafa Q Zhu Z I Shah et al ldquoThymoquinoneup-regulates PTEN expression and induces apoptosis indoxorubicin-resistant human breast cancer cellsrdquo MutationResearchmdashFundamental and Molecular Mechanisms of Mutage-nesis vol 706 no 1-2 pp 28ndash35 2011
[34] G Viglietto M L Motti P Bruni et al ldquoCytoplasmic relocal-ization and inhibition of the cyclin-dependent kinase inhibitorp271198961198941199011 by PKBAkt-mediated phosphorylation in breast can-cerrdquo Nature Medicine vol 8 no 10 pp 1136ndash1144 2002
[35] B D Clarkson and J H Burchenal ldquoPreliminary screening ofantineoplastic drugsrdquoProgress in Clinical Cancer vol 1 pp 625ndash629 1965
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
8 Oxidative Medicine and Cellular Longevity
000
001
002
003
004
005
MD
A n
mol
mg
prot
ein
Con
trol
Asc
Jugl
one
Q7
Q9
lowastlowast
lowastlowast
Jugl
one+
Asc
Q7+
Asc
Q9+
Asc
(c)
0
50
100
150
200
DN
A d
amag
e ind
ex
Con
trol
Asc
Jugl
one
Q7
Q9
lowastlowastlowast
lowastlowast
Jugl
one+
Asc
Q7+
Asc
Q9+
Asc
(d)
120574H2AX
pAkt
Ctrl Asc Jug Q7 Q9JugAsc
Q7Asc
Q9Asc
120573-actin
(e)
Figure 3 Ehrlich ascites carcinoma inhibition (a) and survival time (b) in animals treated with juglone Q7 or Q9 at 1mgkg alone or incombination with ascorbate (Asc) at 100mgkg Doxorubicin was administered at 12mgkg for the positive control Lipid peroxidation (c)DNA damage (d) and phosphorylated proteins Akt (pAkt) and H2AX (120574H2AX) in ascitic cells from mice (e) (lowastlowast) and (lowast lowast lowast) denotedifference at 119875 lt 001 and 119875 lt 0001 compared to the negative control or between indicated treatments
generally died before day 20The largest area under the curvescorresponds to the treatments conducted with juglone or Q7in combination with ascorbate (Figure 3(b)) The synergisticeffect between these drugs occurred also in vivo
Figures 3(c) 3(d) and 3(e) present data of moleculartoxicity on Ehrlich tumor in mice Figure 3(c) depicts levelsof MDA a biomarker of lipid peroxidationThis was the end-point measurement done to verify oxidative stress causedby the treatments in vivo MDA levels were increased sig-nificantly by the combined treatments done with juglone orQ7 and ascorbate Data in Figure 3(d) corroborate with datafrom Figure 3(c) because they show that DNA fragmenta-tion was increased significantly in vivo only when animalsreceived juglone or Q7 plus ascorbate Finally data shownin Figure 3(e) provide concise evidences which demonstratethat juglone and Q7 plus ascorbate can cause DNA damageand pAkt inhibition in Ehrlich tumor in mice Thus theeffects are not restricted to MCF-7 cells in vitro
Data from this study are summarized illustratively inFigure 4 Although all 14-naphthoquinones presented someactivity primarily juglone and Q7 administered in combina-tion with ascorbate trigger the generation of free radicals thesemiquinone formof the naphthoquinone and dehydroascor-bate The resulting semiquinone form intercalates into DNA
The radicals can attack DNA which is degraded In cancercells these events are accompanied by an inhibition on Aktpathway The formulation of juglone or Q7 with ascorbateshowed the most promising activity in vivo
4 Conclusion
Overall it is possible to conclude that primarily jugloneand Q7 combined with ascorbate have significant antitumoreffects against MCF-7 cells in vitro and Ehrlich-ascites carci-noma in mice The effects are the result of intercalation andoxidative attack on DNA of tumor cells and inhibition of Aktpathway
Conflict of Interests
The authors declare that they have no conflict of interests
Acknowledgments
Fabiana Ourique Luiza S E P W Castro and ValdeluciaM A S Grinevicius are recipients of research grants from
Oxidative Medicine and Cellular Longevity 9
Juglone Q7 Q9
OH
OH
H H
H
OO
H
H
PmTOR
Growth and proliferation
Cytosol
AktP P
PKB
P
Growth factor
Tyrosine kinase-like receptor
Plasma membranePI-3K
OH
OH
OH
OH
HO
HO
HO
O O
O O
O
OR1
R1
R2
R2
R1
R2
Ominus
∙O
O∙
O2∙minus
O2
H∙∙∙N H
∙∙∙N OCH3
Figure 4 Antitumor actions of juglone Q7 and Q9 administered in combination with ascorbate against MCF-7 cells and Ehrlich ascitescarcinoma in mice The effects are the result of intercalation and oxidative attack on DNA of tumor cells and inhibition of Akt pathway
Coordenacao de Aperfeicoamento de Pessoal de Nıvel Supe-rior (CAPES Brazil) Karina B Felipe and Rozangela CuriPedrosa (Proc 3024042011-2) are recipients of researchgrants from Conselho Nacional de Pesquisa (CNPq) Brazil
References
[1] M M Gottesman ldquoMechanisms of cancer drug resistancerdquoAnnual Review of Medicine vol 53 pp 615ndash627 2002
[2] M S Farias C T Pich M R Kviecinski et al ldquoSubstituted3-acyl-2-phenylamino-14-naphthoquinones intercalate intoDNA and cause genotoxicity through the increased generationof reactive oxygen species culminating in cell deathrdquoMolecularMedicine Reports vol 10 no 1 pp 405ndash410 2014
[3] K B Felipe J Benites C Glorieux et al ldquoAntiproliferativeeffects of phenylaminonaphthoquinones are increased by ascor-bate and associated with the appearance of a senescent pheno-type in human bladder cancer cellsrdquo Biochemical and Biophysi-cal Research Communications vol 433 no 4 pp 573ndash578 2013
[4] M R Kviecinski R C Pedrosa K B Felipe et al ldquoInhibitionof cell proliferation and migration by oxidative stress fromascorbate-driven juglone redox cycling in human bladder-derived T24 cellsrdquo Biochemical and Biophysical Research Com-munications vol 421 no 2 pp 268ndash273 2012
[5] C F Thorn C Oshiro S Marsh et al ldquoDoxorubicin pathwayspharmacodynamics and adverse effectsrdquo Pharmacogenetics andGenomics vol 21 no 7 pp 440ndash446 2011
[6] G Bischoff and S Hoffman ldquoDNA-binding of drugs used inmedicinal therapiesrdquo Current Medicinal Chemistry vol 9 no3 pp 321ndash348 2002
[7] R Palchaudhuri and P J Hergenrother ldquoDNA as a target foranticancer compounds methods to determine the mode ofbinding and the mechanism of actionrdquo Current Opinion inBiotechnology vol 18 no 6 pp 497ndash503 2007
[8] M S Shahabuddin M Gopal and S C Raghavan ldquoIntercalat-ing and antitumor activity of 4 oxopyrimido[410158405101584045]thieno(23-b) quinoline-4(3H)-onerdquo Journal of Cancer Molecules vol 3pp 139ndash146 2007
[9] Q Chen M G Espey M C Krishna et al ldquoPharamacologicascorbic acid concentrations selectively kill cancer cells actionas a pro-drug to deliver hydrogen peroxide to tissuserdquo Proceed-ings of the National Academy of Sciences of the United States ofAmerica vol 102 no 38 pp 13604ndash13609 2005
[10] J Du J J Cullen and G R Buettner ldquoAscorbic acid chemistrybiology and the treatment of cancerrdquo Biochimica et BiophysicaActa vol 1826 no 2 pp 443ndash457 2012
[11] M G Espey P Chen B Chalmers et al ldquoPharmacologicascorbate synergizes with gemcitabine in preclinical models of
10 Oxidative Medicine and Cellular Longevity
pancreatic cancerrdquo Free Radical Biology and Medicine vol 50no 11 pp 1610ndash1619 2011
[12] J Verrax R Beck N Dejeans et al ldquoRedox-active quinonesand ascorbate an innovative cancer therapy that exploits thevulnerability of cancer cells to oxidative stressrdquo Anti-CancerAgents in Medicinal Chemistry vol 11 no 2 pp 213ndash221 2011
[13] J Benites J A Valderrama K Bettega R C Pedrosa PB Calderon and J Verrax ldquoBiological evaluation of donor-acceptor aminonaphthoquinones as antitumor agentsrdquo Euro-pean Journal of Medicinal Chemistry vol 45 no 12 pp 6052ndash6057 2010
[14] V C da Silveira H Benezra J S Luz R C Georg C COliveira and A M D C Ferreira ldquoBinding of oxindole-Schiffbase copper(II) complexes to DNA and its modulation by theligandrdquo Journal of Inorganic Biochemistry vol 105 no 12 pp1692ndash1703 2011
[15] T Jun W Bochu and Z Liancai ldquoHydrolytic cleavage of DNAby quercetin manganese(II) complexesrdquo Colloids and SurfacesB Biointerfaces vol 55 no 2 pp 149ndash152 2007
[16] N P Singh M T McCoy R R Tice and E L Schneider ldquoAsimple technique for quantitation of low levels of DNA damagein individual cellsrdquo Experimental Cell Research vol 175 no 1pp 184ndash191 1988
[17] J Yuan R Adamski and J Chen ldquoFocus on histone variantH2AX to be or not to berdquo FEBS Letters vol 584 no 17 pp 3717ndash3724 2010
[18] T T Paull E P Rogakou V Yamazaki C U Kirchgessner MGellert and W M Bonner ldquoA critical role for histone H2AX inrecruitment of repair factors to nuclear foci after DNAdamagerdquoCurrent Biology vol 10 no 15 pp 886ndash895 2000
[19] R Lopez A Arumugam R Joseph et al ldquoHyperglycemiaenhances the proliferation of non-tumorigenic and malignantmammary epithelial cells through increased leptinIGF1R sig-naling and activation of AKTmTORrdquo PLoS ONE vol 8 no 11Article ID e79708 2013
[20] G M Ross T J McMillan P Wilcox and A R Collins ldquoThesingle cell microgel electrophoresis assay (comet assay) techni-cal aspects and applications Report on the 5th LH Gray TrustWorkshop Institute of Cancer Research 1994rdquo MutationResearch vol 337 no 1 pp 57ndash60 1995
[21] T Mosmann ldquoRapid colorimetric assay for cellular growth andsurvival application to proliferation and cytotoxicity assaysrdquoJournal of Immunological Methods vol 65 no 1-2 pp 55ndash631983
[22] N A P Franken H M Rodermond J Stap J Haveman and Cvan Bree ldquoClonogenic assay of cells in vitrordquo Nature Protocolsvol 1 no 5 pp 2315ndash2319 2006
[23] C Glorieux N Dejeans B Sid R Beck P B Calderon andJ Verrax ldquoCatalase overexpression in mammary cancer cellsleads to a less aggressive phenotype and an altered response tochemotherapyrdquo Biochemical Pharmacology vol 82 no 10 pp1384ndash1390 2011
[24] M R Kviecinski P Benelli K B Felipe et al ldquoSFE from Bidenspilosa Linne to obtain extracts rich in cytotoxic polyacetyleneswith antitumor activityrdquo Journal of Supercritical Fluids vol 56no 3 pp 243ndash248 2011
[25] E L Kaplan and P Meier ldquoNonparametric estimation fromincomplete observationsrdquo Journal of the American StatisticalAssociation vol 53 pp 457ndash481 1958
[26] R P Bird and H H Draper ldquoComparative studies on differentmethods of malonaldehyde determinationrdquo Methods in Enzy-mology vol 105 pp 299ndash305 1984
[27] V G S Box ldquoThe intercalation of DNA double helices withdoxorubicin and nagalomycinrdquo Journal of Molecular Graphicsand Modelling vol 26 no 1 pp 14ndash19 2007
[28] Q Jiang N Xiao P Shi Y Zhu and Z Guo ldquoDesign of artifi-cial metallonucleases with oxidative mechanismrdquo CoordinationChemistry Reviews vol 251 no 15-16 pp 1951ndash1972 2007
[29] M U Rehman M Tahir A Q Khan et al ldquoD-limonene sup-presses doxorubicin-induced oxidative stress and inflammationvia repression of COX-2 iNOS and NF120581B in kidneys of Wistarratsrdquo Experimental Biology and Medicine vol 239 no 4 pp465ndash476 2014
[30] A H Boulares A G Yakovlev V Ivanova et al ldquoRole ofpoly(ADP-ribose) polymerase (PARP) cleavage in apoptosisCaspase 3-resistant PARP mutant increases rates of apoptosisin transfected cellsrdquo Journal of Biological Chemistry vol 274 no33 pp 22932ndash22940 1999
[31] N Ahmad S Gupta M M Husain K M Heiskanen and HMukhtar ldquoDifferential antiproliferative and apoptotic responseof sanguinarine for cancer cells versus normal cellsrdquo ClinicalCancer Research vol 6 no 4 pp 1524ndash1528 2000
[32] S J Lee H Ahn K W Nam K H Kim and W Mar ldquoEffectsof rutaecarpine on hydrogen peroxide-induced apoptosis inmurine hepa-1c1c7 cellsrdquo Biomolecules amp Therapeutics vol 20no 5 pp 487ndash491 2012
[33] E-S A Arafa Q Zhu Z I Shah et al ldquoThymoquinoneup-regulates PTEN expression and induces apoptosis indoxorubicin-resistant human breast cancer cellsrdquo MutationResearchmdashFundamental and Molecular Mechanisms of Mutage-nesis vol 706 no 1-2 pp 28ndash35 2011
[34] G Viglietto M L Motti P Bruni et al ldquoCytoplasmic relocal-ization and inhibition of the cyclin-dependent kinase inhibitorp271198961198941199011 by PKBAkt-mediated phosphorylation in breast can-cerrdquo Nature Medicine vol 8 no 10 pp 1136ndash1144 2002
[35] B D Clarkson and J H Burchenal ldquoPreliminary screening ofantineoplastic drugsrdquoProgress in Clinical Cancer vol 1 pp 625ndash629 1965
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
Oxidative Medicine and Cellular Longevity 9
Juglone Q7 Q9
OH
OH
H H
H
OO
H
H
PmTOR
Growth and proliferation
Cytosol
AktP P
PKB
P
Growth factor
Tyrosine kinase-like receptor
Plasma membranePI-3K
OH
OH
OH
OH
HO
HO
HO
O O
O O
O
OR1
R1
R2
R2
R1
R2
Ominus
∙O
O∙
O2∙minus
O2
H∙∙∙N H
∙∙∙N OCH3
Figure 4 Antitumor actions of juglone Q7 and Q9 administered in combination with ascorbate against MCF-7 cells and Ehrlich ascitescarcinoma in mice The effects are the result of intercalation and oxidative attack on DNA of tumor cells and inhibition of Akt pathway
Coordenacao de Aperfeicoamento de Pessoal de Nıvel Supe-rior (CAPES Brazil) Karina B Felipe and Rozangela CuriPedrosa (Proc 3024042011-2) are recipients of researchgrants from Conselho Nacional de Pesquisa (CNPq) Brazil
References
[1] M M Gottesman ldquoMechanisms of cancer drug resistancerdquoAnnual Review of Medicine vol 53 pp 615ndash627 2002
[2] M S Farias C T Pich M R Kviecinski et al ldquoSubstituted3-acyl-2-phenylamino-14-naphthoquinones intercalate intoDNA and cause genotoxicity through the increased generationof reactive oxygen species culminating in cell deathrdquoMolecularMedicine Reports vol 10 no 1 pp 405ndash410 2014
[3] K B Felipe J Benites C Glorieux et al ldquoAntiproliferativeeffects of phenylaminonaphthoquinones are increased by ascor-bate and associated with the appearance of a senescent pheno-type in human bladder cancer cellsrdquo Biochemical and Biophysi-cal Research Communications vol 433 no 4 pp 573ndash578 2013
[4] M R Kviecinski R C Pedrosa K B Felipe et al ldquoInhibitionof cell proliferation and migration by oxidative stress fromascorbate-driven juglone redox cycling in human bladder-derived T24 cellsrdquo Biochemical and Biophysical Research Com-munications vol 421 no 2 pp 268ndash273 2012
[5] C F Thorn C Oshiro S Marsh et al ldquoDoxorubicin pathwayspharmacodynamics and adverse effectsrdquo Pharmacogenetics andGenomics vol 21 no 7 pp 440ndash446 2011
[6] G Bischoff and S Hoffman ldquoDNA-binding of drugs used inmedicinal therapiesrdquo Current Medicinal Chemistry vol 9 no3 pp 321ndash348 2002
[7] R Palchaudhuri and P J Hergenrother ldquoDNA as a target foranticancer compounds methods to determine the mode ofbinding and the mechanism of actionrdquo Current Opinion inBiotechnology vol 18 no 6 pp 497ndash503 2007
[8] M S Shahabuddin M Gopal and S C Raghavan ldquoIntercalat-ing and antitumor activity of 4 oxopyrimido[410158405101584045]thieno(23-b) quinoline-4(3H)-onerdquo Journal of Cancer Molecules vol 3pp 139ndash146 2007
[9] Q Chen M G Espey M C Krishna et al ldquoPharamacologicascorbic acid concentrations selectively kill cancer cells actionas a pro-drug to deliver hydrogen peroxide to tissuserdquo Proceed-ings of the National Academy of Sciences of the United States ofAmerica vol 102 no 38 pp 13604ndash13609 2005
[10] J Du J J Cullen and G R Buettner ldquoAscorbic acid chemistrybiology and the treatment of cancerrdquo Biochimica et BiophysicaActa vol 1826 no 2 pp 443ndash457 2012
[11] M G Espey P Chen B Chalmers et al ldquoPharmacologicascorbate synergizes with gemcitabine in preclinical models of
10 Oxidative Medicine and Cellular Longevity
pancreatic cancerrdquo Free Radical Biology and Medicine vol 50no 11 pp 1610ndash1619 2011
[12] J Verrax R Beck N Dejeans et al ldquoRedox-active quinonesand ascorbate an innovative cancer therapy that exploits thevulnerability of cancer cells to oxidative stressrdquo Anti-CancerAgents in Medicinal Chemistry vol 11 no 2 pp 213ndash221 2011
[13] J Benites J A Valderrama K Bettega R C Pedrosa PB Calderon and J Verrax ldquoBiological evaluation of donor-acceptor aminonaphthoquinones as antitumor agentsrdquo Euro-pean Journal of Medicinal Chemistry vol 45 no 12 pp 6052ndash6057 2010
[14] V C da Silveira H Benezra J S Luz R C Georg C COliveira and A M D C Ferreira ldquoBinding of oxindole-Schiffbase copper(II) complexes to DNA and its modulation by theligandrdquo Journal of Inorganic Biochemistry vol 105 no 12 pp1692ndash1703 2011
[15] T Jun W Bochu and Z Liancai ldquoHydrolytic cleavage of DNAby quercetin manganese(II) complexesrdquo Colloids and SurfacesB Biointerfaces vol 55 no 2 pp 149ndash152 2007
[16] N P Singh M T McCoy R R Tice and E L Schneider ldquoAsimple technique for quantitation of low levels of DNA damagein individual cellsrdquo Experimental Cell Research vol 175 no 1pp 184ndash191 1988
[17] J Yuan R Adamski and J Chen ldquoFocus on histone variantH2AX to be or not to berdquo FEBS Letters vol 584 no 17 pp 3717ndash3724 2010
[18] T T Paull E P Rogakou V Yamazaki C U Kirchgessner MGellert and W M Bonner ldquoA critical role for histone H2AX inrecruitment of repair factors to nuclear foci after DNAdamagerdquoCurrent Biology vol 10 no 15 pp 886ndash895 2000
[19] R Lopez A Arumugam R Joseph et al ldquoHyperglycemiaenhances the proliferation of non-tumorigenic and malignantmammary epithelial cells through increased leptinIGF1R sig-naling and activation of AKTmTORrdquo PLoS ONE vol 8 no 11Article ID e79708 2013
[20] G M Ross T J McMillan P Wilcox and A R Collins ldquoThesingle cell microgel electrophoresis assay (comet assay) techni-cal aspects and applications Report on the 5th LH Gray TrustWorkshop Institute of Cancer Research 1994rdquo MutationResearch vol 337 no 1 pp 57ndash60 1995
[21] T Mosmann ldquoRapid colorimetric assay for cellular growth andsurvival application to proliferation and cytotoxicity assaysrdquoJournal of Immunological Methods vol 65 no 1-2 pp 55ndash631983
[22] N A P Franken H M Rodermond J Stap J Haveman and Cvan Bree ldquoClonogenic assay of cells in vitrordquo Nature Protocolsvol 1 no 5 pp 2315ndash2319 2006
[23] C Glorieux N Dejeans B Sid R Beck P B Calderon andJ Verrax ldquoCatalase overexpression in mammary cancer cellsleads to a less aggressive phenotype and an altered response tochemotherapyrdquo Biochemical Pharmacology vol 82 no 10 pp1384ndash1390 2011
[24] M R Kviecinski P Benelli K B Felipe et al ldquoSFE from Bidenspilosa Linne to obtain extracts rich in cytotoxic polyacetyleneswith antitumor activityrdquo Journal of Supercritical Fluids vol 56no 3 pp 243ndash248 2011
[25] E L Kaplan and P Meier ldquoNonparametric estimation fromincomplete observationsrdquo Journal of the American StatisticalAssociation vol 53 pp 457ndash481 1958
[26] R P Bird and H H Draper ldquoComparative studies on differentmethods of malonaldehyde determinationrdquo Methods in Enzy-mology vol 105 pp 299ndash305 1984
[27] V G S Box ldquoThe intercalation of DNA double helices withdoxorubicin and nagalomycinrdquo Journal of Molecular Graphicsand Modelling vol 26 no 1 pp 14ndash19 2007
[28] Q Jiang N Xiao P Shi Y Zhu and Z Guo ldquoDesign of artifi-cial metallonucleases with oxidative mechanismrdquo CoordinationChemistry Reviews vol 251 no 15-16 pp 1951ndash1972 2007
[29] M U Rehman M Tahir A Q Khan et al ldquoD-limonene sup-presses doxorubicin-induced oxidative stress and inflammationvia repression of COX-2 iNOS and NF120581B in kidneys of Wistarratsrdquo Experimental Biology and Medicine vol 239 no 4 pp465ndash476 2014
[30] A H Boulares A G Yakovlev V Ivanova et al ldquoRole ofpoly(ADP-ribose) polymerase (PARP) cleavage in apoptosisCaspase 3-resistant PARP mutant increases rates of apoptosisin transfected cellsrdquo Journal of Biological Chemistry vol 274 no33 pp 22932ndash22940 1999
[31] N Ahmad S Gupta M M Husain K M Heiskanen and HMukhtar ldquoDifferential antiproliferative and apoptotic responseof sanguinarine for cancer cells versus normal cellsrdquo ClinicalCancer Research vol 6 no 4 pp 1524ndash1528 2000
[32] S J Lee H Ahn K W Nam K H Kim and W Mar ldquoEffectsof rutaecarpine on hydrogen peroxide-induced apoptosis inmurine hepa-1c1c7 cellsrdquo Biomolecules amp Therapeutics vol 20no 5 pp 487ndash491 2012
[33] E-S A Arafa Q Zhu Z I Shah et al ldquoThymoquinoneup-regulates PTEN expression and induces apoptosis indoxorubicin-resistant human breast cancer cellsrdquo MutationResearchmdashFundamental and Molecular Mechanisms of Mutage-nesis vol 706 no 1-2 pp 28ndash35 2011
[34] G Viglietto M L Motti P Bruni et al ldquoCytoplasmic relocal-ization and inhibition of the cyclin-dependent kinase inhibitorp271198961198941199011 by PKBAkt-mediated phosphorylation in breast can-cerrdquo Nature Medicine vol 8 no 10 pp 1136ndash1144 2002
[35] B D Clarkson and J H Burchenal ldquoPreliminary screening ofantineoplastic drugsrdquoProgress in Clinical Cancer vol 1 pp 625ndash629 1965
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
10 Oxidative Medicine and Cellular Longevity
pancreatic cancerrdquo Free Radical Biology and Medicine vol 50no 11 pp 1610ndash1619 2011
[12] J Verrax R Beck N Dejeans et al ldquoRedox-active quinonesand ascorbate an innovative cancer therapy that exploits thevulnerability of cancer cells to oxidative stressrdquo Anti-CancerAgents in Medicinal Chemistry vol 11 no 2 pp 213ndash221 2011
[13] J Benites J A Valderrama K Bettega R C Pedrosa PB Calderon and J Verrax ldquoBiological evaluation of donor-acceptor aminonaphthoquinones as antitumor agentsrdquo Euro-pean Journal of Medicinal Chemistry vol 45 no 12 pp 6052ndash6057 2010
[14] V C da Silveira H Benezra J S Luz R C Georg C COliveira and A M D C Ferreira ldquoBinding of oxindole-Schiffbase copper(II) complexes to DNA and its modulation by theligandrdquo Journal of Inorganic Biochemistry vol 105 no 12 pp1692ndash1703 2011
[15] T Jun W Bochu and Z Liancai ldquoHydrolytic cleavage of DNAby quercetin manganese(II) complexesrdquo Colloids and SurfacesB Biointerfaces vol 55 no 2 pp 149ndash152 2007
[16] N P Singh M T McCoy R R Tice and E L Schneider ldquoAsimple technique for quantitation of low levels of DNA damagein individual cellsrdquo Experimental Cell Research vol 175 no 1pp 184ndash191 1988
[17] J Yuan R Adamski and J Chen ldquoFocus on histone variantH2AX to be or not to berdquo FEBS Letters vol 584 no 17 pp 3717ndash3724 2010
[18] T T Paull E P Rogakou V Yamazaki C U Kirchgessner MGellert and W M Bonner ldquoA critical role for histone H2AX inrecruitment of repair factors to nuclear foci after DNAdamagerdquoCurrent Biology vol 10 no 15 pp 886ndash895 2000
[19] R Lopez A Arumugam R Joseph et al ldquoHyperglycemiaenhances the proliferation of non-tumorigenic and malignantmammary epithelial cells through increased leptinIGF1R sig-naling and activation of AKTmTORrdquo PLoS ONE vol 8 no 11Article ID e79708 2013
[20] G M Ross T J McMillan P Wilcox and A R Collins ldquoThesingle cell microgel electrophoresis assay (comet assay) techni-cal aspects and applications Report on the 5th LH Gray TrustWorkshop Institute of Cancer Research 1994rdquo MutationResearch vol 337 no 1 pp 57ndash60 1995
[21] T Mosmann ldquoRapid colorimetric assay for cellular growth andsurvival application to proliferation and cytotoxicity assaysrdquoJournal of Immunological Methods vol 65 no 1-2 pp 55ndash631983
[22] N A P Franken H M Rodermond J Stap J Haveman and Cvan Bree ldquoClonogenic assay of cells in vitrordquo Nature Protocolsvol 1 no 5 pp 2315ndash2319 2006
[23] C Glorieux N Dejeans B Sid R Beck P B Calderon andJ Verrax ldquoCatalase overexpression in mammary cancer cellsleads to a less aggressive phenotype and an altered response tochemotherapyrdquo Biochemical Pharmacology vol 82 no 10 pp1384ndash1390 2011
[24] M R Kviecinski P Benelli K B Felipe et al ldquoSFE from Bidenspilosa Linne to obtain extracts rich in cytotoxic polyacetyleneswith antitumor activityrdquo Journal of Supercritical Fluids vol 56no 3 pp 243ndash248 2011
[25] E L Kaplan and P Meier ldquoNonparametric estimation fromincomplete observationsrdquo Journal of the American StatisticalAssociation vol 53 pp 457ndash481 1958
[26] R P Bird and H H Draper ldquoComparative studies on differentmethods of malonaldehyde determinationrdquo Methods in Enzy-mology vol 105 pp 299ndash305 1984
[27] V G S Box ldquoThe intercalation of DNA double helices withdoxorubicin and nagalomycinrdquo Journal of Molecular Graphicsand Modelling vol 26 no 1 pp 14ndash19 2007
[28] Q Jiang N Xiao P Shi Y Zhu and Z Guo ldquoDesign of artifi-cial metallonucleases with oxidative mechanismrdquo CoordinationChemistry Reviews vol 251 no 15-16 pp 1951ndash1972 2007
[29] M U Rehman M Tahir A Q Khan et al ldquoD-limonene sup-presses doxorubicin-induced oxidative stress and inflammationvia repression of COX-2 iNOS and NF120581B in kidneys of Wistarratsrdquo Experimental Biology and Medicine vol 239 no 4 pp465ndash476 2014
[30] A H Boulares A G Yakovlev V Ivanova et al ldquoRole ofpoly(ADP-ribose) polymerase (PARP) cleavage in apoptosisCaspase 3-resistant PARP mutant increases rates of apoptosisin transfected cellsrdquo Journal of Biological Chemistry vol 274 no33 pp 22932ndash22940 1999
[31] N Ahmad S Gupta M M Husain K M Heiskanen and HMukhtar ldquoDifferential antiproliferative and apoptotic responseof sanguinarine for cancer cells versus normal cellsrdquo ClinicalCancer Research vol 6 no 4 pp 1524ndash1528 2000
[32] S J Lee H Ahn K W Nam K H Kim and W Mar ldquoEffectsof rutaecarpine on hydrogen peroxide-induced apoptosis inmurine hepa-1c1c7 cellsrdquo Biomolecules amp Therapeutics vol 20no 5 pp 487ndash491 2012
[33] E-S A Arafa Q Zhu Z I Shah et al ldquoThymoquinoneup-regulates PTEN expression and induces apoptosis indoxorubicin-resistant human breast cancer cellsrdquo MutationResearchmdashFundamental and Molecular Mechanisms of Mutage-nesis vol 706 no 1-2 pp 28ndash35 2011
[34] G Viglietto M L Motti P Bruni et al ldquoCytoplasmic relocal-ization and inhibition of the cyclin-dependent kinase inhibitorp271198961198941199011 by PKBAkt-mediated phosphorylation in breast can-cerrdquo Nature Medicine vol 8 no 10 pp 1136ndash1144 2002
[35] B D Clarkson and J H Burchenal ldquoPreliminary screening ofantineoplastic drugsrdquoProgress in Clinical Cancer vol 1 pp 625ndash629 1965
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom