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Phytomedicine 21 (2014) 866–870

Contents lists available at ScienceDirect

Phytomedicine

jou rn al h om epage: www.elsev ier .de /phymed

ytotoxic activity of hirsutanone, a diarylheptanoid isolated fromlnus glutinosa leaves

.J. León-Gonzaleza, N. Acerob,∗, D. Munoz-Mingarroc, M. López-Lázaroa,

. Martín-Corderoa

Department of Pharmacology, Faculty of Pharmacy, Sevilla University, 41012 Sevilla, SpainDepartment of Pharmaceutical and Health Sciences, Faculty of Pharmacy, CEU San Pablo University, 28668 Madrid, SpainDepartment of Chemistry and Biochemistry, Faculty of Pharmacy, CEU San Pablo University, 28668 Madrid, Spain

r t i c l e i n f o

rticle history:eceived 6 September 2013eceived in revised form 29 October 2013ccepted 26 January 2014

eywords:lnus glutinosaiarylheptanoidytotoxicityirsutanoneopoisomerase IIeactive oxygen species

a b s t r a c t

Background: The low efficacy of cancer therapy for the treatment of patients with advanced disease makesthe development of new anticancer agents necessary. Because natural products are a significant sourceof anticancer drugs, it is important to explore cytotoxic activity of novel compounds from natural origin.Purpose: The aim of this work is to evaluate the cytotoxic capacity of hirsutanone, a diarylheptanoidisolated from Alnus glutinosa leaves. Hirsutanone cytotoxic way of action was also studied.Material and methods: The cytotoxic ability of Alnus glutinosa leaves ethyl acetate extract was studied overHeLa and PC-3 cell lines, with the MTT colorimetric assay. Hirsutanone was isolated from this extract usingchromatographic methods, and its structure elucidated by spectroscopic analysis. HT-29 cell viabilityafter hirsutanone treatment was determined using SRB assay. In order to understand hirsutanone wayof action, cytotoxicity was evaluated adding the diarylheptanoid and antioxidants. DNA topoisomerase II(topo II) poison activity, was also evaluated using purified topo II and a supercoiled form of DNA that bearsspecific topo II recognition and binding region; topo II poisons stabilize normally transient DNA-topo IIcleavage complexes, and lead an increased yield of linear form as a consequence of a lack of double-strandbreaks rejoining.Results: The diarylheptanoid hirsutanone was isolated from Alnus glutinosa (L.) Gaertn. (Betulaceae)leaves extract that showed cytotoxic activity against PC-3 and HeLa cell lines. Hirsutanone showed cyto-toxic activity against HT-29 human colon carcinoma cells. Pre-treatment with the antioxidants NAC(N-acetylcysteine) and MnTMPyP (Mn(III)tetrakis-(1-methyl-4-pyridyl)porthyrin) reduced this activity,suggesting that reactive oxygen species (ROS) participate in hirsutanone-induced cancer cell death. Using

human topo II and a DNA supercoiled form, hirsutanone was found to stabilize topo II-DNA cleavagecomplexes, acting as a topo II poison.Conclusion: Our data suggest that, like curcumin, an induction of oxidative stress and topo II-mediatedDNA damage may play a role in hirsutanone-induced cancer cell death. Since both compounds sharesimilar structure and cytotoxic profile, and curcumin is in clinical trials for the treatment of cancer, ourresults warrant further studies to evaluate the anticancer potential of hirsutanone.

© 2014 Elsevier GmbH. All rights reserved.

ntroduction

Cancer kills more than seven million people worldwide everyear (Jemal et al., 2011). The mortality rate of this disease has nothanged much in the past few decades even in developed countries

uch as the United States (Siegel et al., 2013). Although surgeryr radiotherapy as cancer therapy is effective when the disease isetected early, many cancers are still diagnosed when cells from

∗ Corresponding author. Tel.: +34 913724798; fax: +34 913510475.E-mail address: nacemes@ceu.es (N. Acero).

944-7113/$ – see front matter © 2014 Elsevier GmbH. All rights reserved.ttp://dx.doi.org/10.1016/j.phymed.2014.01.008

a primary tumor have already metastasized to other parts of thebody. The main form of treatment at this point is chemotherapy,which consists of delivering drugs systemically so they can reachand kill tumor cells.

Despite recent interest by pharmaceutical companies in molec-ular modeling, combinatorial chemistry and other syntheticchemistry techniques, natural products and medicinal plants havebeen found to be an important source of new drugs. Natural prod-

ucts are not only used as therapeutic agents, they also constitutea source of lead compounds that have provided the basis for newdrugs semisynthesis or total synthesis (Newman and Cragg, 2012;Cragg et al., 2009). The role of natural products in drug discovery

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A.J. León-Gonzalez et al. / Ph

s particularly important in oncology (Newman and Cragg, 2012).here are many mechanism of action of these drugs, as inhibi-ion of microtubule, inhibition of DNA topoisomerase II or DNAopoisomerase I inhibition (Cragg and Newman, 2005). Recentvidence suggests that the formation of reactive oxygen speciesROS) may also contribute to the cytotoxic effects of these drugsAlexandre et al., 2006, 2007; Gorman et al., 1997). The inductionf oxidative stress by pro-oxidant agents is indeed emerging as anttractive cytotoxic strategy (Pelicano et al., 2004; Renschler, 2004;chumacker, 2006; Lopez-Lazaro, 2007, 2010).

Alnus glutinosa (L.) Gaertn (alder), is a common Betulaceaeidely distributed in Europe. Alder bark and leaves are traditionallysed in folk medicine (Grieve, 1984). A decoction of leaves had beensed for treating several types of cancer (Hartwell, 1967–1971).ome of its constituents exhibit various biological properties,ncluding anti-inflammatory and cytotoxic activities (Acero and

unoz-Mingarro, 2012). Alnus genera phytoscreening had ledhe identification of numerous diarylheptanoids. Diarylheptanoids,uch as curcumin, belong to a phenolic class of natural prod-cts based on 1,7-diphenylheptane skeleton. In the last decade,umerous reports has been published on the cytotoxic activityf curcumin, and several clinical trials are currently ongoing orecruiting participants to evaluate the anticancer activity of thisatural product (Lopez-Lazaro, 2008; Hatcher et al., 2008). Cur-umin has been shown to interfere with multiple signal pathways,ncluding apoptosis, proliferation cell cycle, cell survival, inflam-

ation, invasion and metastasis (Bachmeier et al., 2010). One ofhe main molecular mechanisms involved in curcumin apoptoticnd antimetastasic effect is the inhibition of nuclear factor kappa

(NF-kappaB) transcription. Metastasis related proinflammatoryytokines CXCL-1 and -2 are also modulated as they are NF-kappaargets (Bachmeier et al., 2009). Evidence also suggests that theeneration of ROS and the induction of topoisomerase II-DNA com-lexes may play a role in curcumin-induced cancer cell deathKuo et al., 1996; Martin-Cordero et al., 2003; Lopez-Lazaro et al.,007).

In this work, we have isolated the diarylheptanoid hirsutanonerom a cytotoxic extract of Alnus glutinosa and shown that the for-

ation of ROS, and the inhibition of topo II may participate ints cytotoxic activity. Because curcumin and hirsutanone share aimilar structure and cytotoxic profile, our results warrant futuretudies to evaluate the anticancer potential of hirsutanone.

aterial and methods

ell culture and chemicals

The HT-29 human colon carcinoma cell line was obtained fromhe European Collection of Cell Cultures (ECACC, Ref. 91072201)nd was grown in McCoy’s 5a (Sigma), supplemented with 2 mMlutamine and 10% Fetal Bovine Serum (FBS). The human cervicalarcinoma HeLa cell line and the human prostate adenocarcinomaC-3 cell lines were obtained from the American Type Culture Col-ection (ATCC CCL-2 and CRL-1435 respectively). HeLa cells wererown in Eagle Minimal Essential Medium (EMEM), supplementedith 10% FBS, 1% non-essential amino acids (100×), 100 U/ml peni-

illin, 100 �g/ml streptomycin, and 2 mM l-glutamine. PC3 cellsere cultured in Coon’s modified Ham’s F12, supplemented with

5 mg/l ascorbic acid, 18 mg/l inositol, 2 mM l-glutamine, 100 U/mlenicillin, 100 �g/ml streptomycin, and 7% FBS. All cancer cell linesere cultured at 37 ◦C in a humidified 5% CO2 atmosphere.

Purified human topo II and supercoiled pRYG DNA wereurchased from TopoGen, Inc. (Columbus, OH). MnTMPyPMn(III)tetrakis-(1-methyl-4-pyridyl)porthyrin) was obtainedrom Biomol International. Other chemicals, including curcumin,

edicine 21 (2014) 866–870 867

etoposide, 5-fluorouracil, NAC (N-acetylcysteine), and proteinaseK, were obtained from Sigma.

Plant material and extract preparation

Alnus glutinosa (L.) Gaertn. (Betulaceae) was collected in July2007 in San Agustín de Guadalix (Madrid, Spain) (40◦41′ N 3◦36′

W). A voucher specimen (2642/09) was deposited in the Facultyof Pharmacy Herbarium, University San Pablo, CEU Madrid. Theleaves of this plant were dried at room temperature, powdered, andextracted with ethyl acetate under reflux for 2 h (Buchi, B-811). Theextract was concentrated to dryness under vacuum and stored at4 ◦C until use. The yield of extraction was 4.7%.

Extract fractionation and hirsutanone isolation

The ethyl acetate extract (2 g) was subjected to silica gel columnchromatography and eluted with a step gradient of CHCl2–MeOH(99:1, 95:5, 80:20, 70:30, 1:1) and MeOH 100% (v/v) to yield 258fractions of 20 ml each. Fractions 170–219 (343 mg) were againsubjected to silica gel column chromatography with CHCl2–MeOH(95:5) to give compound 1 (20 mg). This compound was character-ized by spectroscopic analyses, including 1H NMR, 13C NMR andEIMS, and identified as 1,7-di-(3′,4′-dihydroxyphenyl)-4-hepten-3-one (hirsutanone) (Martin-Cordero et al., 2001).

Cytotoxicity assays (MTT and SRB assay)

Cell viability was assessed using either the MTT (Twentymanand Luscombe, 1987) or the SRB assay (Vichai and Kirtikara, 2006).In MTT assay exponentially growing cells (HeLa and PC3) wereseeded in 96-well plates at a density of 2.5 × 103 cells/well. After24 h, cells were treated with the extract or reference compoundsand incubated for 72 h (MTT). After treatment, cells were washedtwice with PBS. Then, 50 �l/well of 3-(4,5-dimethylthiazol-2-yl)-2,5,diphenyl tetrazolium bromide (MTT) reagent (1 mg/1 ml inPBS), and 150 �l/well of prewarmed medium were added and cellswere incubated for 4 h. The medium was then aspirated, and theformazan product generated from the viable cells, was dissolved inDMSO before being measured at 570 nm using an automatic platereader (Opsys, MR). All experiments were performed at least threetimes. The percentage absorbance related to control was plottedagainst concentration. Concentration of extract required to inhibit50% of cell growth (IC50) was calculated. Data are expressed asmeans ± SEM.

In the SRB assay, exponentially growing HT-29 cells were seededin 96-well plates at a density of 10 × 103 cells/well. After 24 h, cellswere treated with the extract or isolated compounds and incubatedfor 48 h. Then cells were fixed by adding 50 �l/well of cold 50% (w/v)trichloroacetic acid (TCA) and incubated for 1 h at 4 ◦C. The super-natant was then discarded and plates were washed five times withdeionised water and dried. 100 �l/well of SRB (sulforhodamine B)solution (0.4%, w/v in 1% acetic acid) were added and plates wereincubated for 30 min at room temperature. Unbound SRB was thenremoved by washing five times with 1% acetic acid. The plates werethen air-dried and protein-bound dye was dissolved in 10 mM Trisbase solution for OD determination at 492 nm, using a microplatereader (Vichai and Kirtikara, 2006). Cell viability was expressed aspercentage in relation to controls. Data were averaged from at leastthree independent experiments and are expressed as means ± SEM.

To assess whether the cytotoxicity of hirsutanone was mediatedby a pro-oxidant mechanism, HT-29 cells were treated with 10 �M

hirsutanone, 10 �M curcumin, and 10 �M 5-fluorouracil for 48 h, inthe absence and presence of 5 mM NAC (N-acetyl-l-cysteine) and5 �M MnTMPyP (Mn(III)tetrakis-(1-methyl-4-pyridyl)porthyrin).The antioxidants NAC and MnTMPyP were added 0.5 h before

8 ytomedicine 21 (2014) 866–870

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these complexes into permanent DNA strand breaks that trigger celldeath (Fortune and Osheroff, 2000). Wang et al. (2001) showed thatthiol-reactive drugs containing an �,�-unsaturated ketone induced

Fig. 2. Involvement of ROS in the cytotoxic activity of hirsutanone and cur-cumin. HT-29 cells were treated with 10 �M hirsutanone (hir), 10 �M curcumin(cur), and 10 �M 5-fluorouracil (5-FU) for 48 h, in the absence and presence of5 mM NAC (N-acetyl-l-cysteine) and 5 �M MnTMPyP (Mn(III)tetrakis-(1-methyl-

68 A.J. León-Gonzalez et al. / Ph

irsutanone, curcumin, or 5-fluorouracil. Cell viability was esti-ated using the SRB assay. Statistical analysis had been done using

-test (paired, two-tailed) (p < 0.05 and p < 0.001).

n vitro DNA cleavage with topoisomerase II

This assay is based on the ability of topo II to break and rejoinouble-stranded pRYG DNA (supercoiled). Topo II poisons stabi-

ize normally transient DNA-topo II cleavage complexes and leado an increased yield of linear form as a consequence of a lack ofouble-strand breaks rejoining. These structures are evidenced by

proteolysis of DNA-linked topo II carried out by proteinase K.leavage buffer contained 30 mM Tris–HCl, pH 7.6, 60 mM NaCl,5 mM mercaptoethanol, 8 mM MgC12 and 3 mM ATP. Cleavageeaction (20 �l) contained water, cleavage buffer, drugs dissolvedn 2 �l of dimethyl sulfoxide/H2O (2.5%), pRYG DNA (0.25 �g in

�l of buffer), and 2 �l of topo II (4 units), which were mixed inhis order in ice/water. Reactions were carried out by incubationt 37 ◦C for 30 min and finished by additional 15 min incubationt 37 ◦C after 2 �l of 10% SDS and 1 �l of proteinase K (stockolution 20 �g/ml) addition. Afterward, samples were extractedith chloroform-isoamyl alcohol (24:1), and 2 �l of bromophenol

lue were added. Samples were loaded onto 1% agarose gels andlectrophoresed at 6 V/cm for 3 h in a Tris–acetate–EDTA buffer.els were then stained with ethidium bromide and washed withater. Band intensities were quantified with PCBAS software pro-

ram. The presence of linear DNA form, which indicates topo IIoisoning, was expressed as percentage of total DNA. Three inde-endent experiments were carried out and results are expressed aseans ± SEM.

esults and discussion

The cytotoxicity of an ethyl acetate extract of the leaves oflnus glutinosa (L.) Gaertn. (Betulaceae) was evaluated in humanervix carcinoma HeLa cells and human prostate carcinoma PC-3ells using the MTT assay. When these cells were exposed to thextract for 72 h, the IC50 values (�g/ml ± SEM) were 21.42 ± 2.83nd 35.59 ± 3.99, respectively. According to the American Nationalancer Institute (NCI), the criteria of cytotoxic activity for the crudextracts is IC50 < 30 �g/ml (Suffness and Pezzuto, 1990). Consider-ng HeLa IC50 value, we decided to continue with the extract study.he ethyl acetate extract of Alnus glutinosa was then fractionedsing silica gel column chromatography and a brown amorphousompound was isolated. The structure of this compound was ana-yzed by spectroscopic methods, including 1H NMR, 13C NMRnd EIMS, and was identified as 1,7-di-(3′,4′-dihydroxyphenyl)-4-epten-3-one (Martin-Cordero et al., 2001). This diarylheptanoidas been isolated in Viscum cruciatum Sieber (Martin-Cordero et al.,001), Alnus hirsuta Turcz (Jin et al., 2007), Alnus japonica Steud (Leet al., 2005) and Pinus flexilis (Lee et al., 1998). This compound isnown as hirsutanone or hirsutenone. Although both terms refero the keto group of this compound (-one), hirsutanone considershe plant where it was first isolated (Alnus hirsuta), and hirsutenoneonsiders its double bond at C4-C5 (Fig. 1).

The cytotoxic activity of hirsutanone was evaluated in HT-29uman colon carcinoma cells using the SRB assay. 5-fluorouracil5-FU), an anticancer drug widely used in the treatment of colonancers, was used as a positive control. The IC50 values forT-29 cells exposed for 48 h to several concentrations of hirsu-

anone and 5-FU were, respectively, 27.94 ± 1.58 and 9.32 ± 2.75

�g/ml ± SEM). Hirsutanone has been shown to induce cytotoxicffects in other cancer cell lines (Martin-Cordero et al., 2001).

Like curcumin, hirsutanone has an electrophilic �,�-nsaturated ketone in its structure, which can react with

HO OH

Fig. 1. Chemical structures of hirsutanona (hirsutenone) and curcumin.

nucleophilic groups through a reaction called Michael addi-tion. These �,�-unsaturated ketones can react covalently withthe thiol (SH) group of cysteine residues of peptides and proteins.Because some of these peptides and proteins have an antioxidantrole in the cell (e.g. glutathione), their interaction with drugscontaining an �,�-unsaturated ketone (e.g. curcumin) may causea pro-oxidant effect that may lead to cell death (Syng-Ai et al.,2004; Sandur et al., 2007). To assess whether the cytotoxicity ofhirsutanone was mediated by a pro-oxidant mechanism, HT-29cells were treated with hirsutanone in the presence and absenceof the antioxidants NAC and MnTMPyP, and cell viability wasestimated using SRB assay. NAC (a scavenger of hydroxyl radicaland hydrogen peroxide) and MnTMPyP (a superoxide dismu-tase/catalase mimetic) are antioxidants that are known to preventthe cytotoxicity induced by pro-oxidant agents (Li et al., 2007).Fig. 2 shows that both NAC and MnTMPyP reduced the cytotoxicactivity of hirsutanone in HT-29 cells. These two antioxidants alsoreduced the cytotoxic activity of curcumin, but did not modify theactivity of the anticancer drug 5-FU (Fig. 2). These data suggestthat the pro-oxidant effect play a role in the cytotoxic activity ofhirsutanone and curcumin in HT-29 human colon carcinoma cells.

The enzyme DNA topoisomerase II (topo II) is the target of sev-eral clinically useful anticancer drugs, including etoposide. Thesedrugs, known as topoisomerase poisons, induce topo-DNA cleav-age complexes with the enzyme; then cellular processing converts

4-pyridyl)porthyrin). The antioxidants NAC and MnTMPyP were added 0.5 h beforehirsutanone, curcumin, or 5-fluorouracil. Cell viability was estimated using the SRBassay. For statistical analysis a t-test (paired, two-tailed) had been used. p-Values<0.05 are considered statistically significant and are represented with an asterisk(*), p-values <0.001 are represented with three asterisks (***).

A.J. León-Gonzalez et al. / Phytom

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toposide (100 �M), which indicates topo II poisoning. Data are expressed aseans ± SEM.

opo II–DNA complexes via thiol alkylation of topo II. The authorsbserved that these topo II-DNA complexes were completely abol-shed in mutant yeast topo II with all cysteine residues replaced

ith alanine. They also showed that the potency of these drugs totimulate topo II cleavable complexes correlated with their abilityo undergo Michael addition (Wang et al., 2001). To assess whetherirsutanone could act as a topo II poison, we used human topo IInd a supercoiled form of DNA that bears a specific topo II recog-ition and binding region. Under normal conditions topo II breaksnd rejoins this double-stranded DNA. Topo II poisons stabilize theormally transient DNA-topo II cleavage complexes and lead ton increased yield of the linear form as a consequence of a lackf double-strand breaks rejoining. Three independent experimentshowed that hirsutanone induced linear DNA increased yield sim-lar to that of the positive control etoposide, acting therefore as aopo II poison. The percentage of linear DNA induced by 50 �M hir-utanone, 100 �M hirsutanone and 100 �M etoposide appears inig. 3. Results show dose-dependent hirsutanone effect. A repre-entative experiment is shown in Fig. 4.

Our data suggest that the generation of ROS and the inductionf topo II-mediated DNA damage may participate in hirsutanoneytotoxic activity against colon cancer cells. Previous reports havehown that, like curcumin (Lin et al., 2007; Li et al., 2004), hirsu-anone has anti-inflammatory properties which may be mediatedy the inhibition of NF-kappaB (Lee et al., 2005, 2009; Kim et al.,006). Since NF-kappaB plays a crucial role in cancer cells survivalKarin, 2006), its inhibition by hirsutanone and curcumin may alsolay a role in the cytotoxic activity of both compounds. Curcuminolecular mechanism also down-regulate CXCL-1 and -2 expres-

ions. Both cytokines are known to be associated with migrationf cancer cells, tumor growth and angiogenesis (Bachmeier et al.,009). These data indicate that both diarylheptanoids not onlyhare a similar structure, but also a similar cytotoxic profile. Asurcumin is in clinical trials for the treatment of cancer, our resultsuggest that hirsutanone should be more thoroughly tested as an

nticancer agent.

ig. 4. Evaluation of the topo II-DNA cleavable complexes induced by hirsutanone. 1.opo II + DNA (pRYG) + 50 �M hirsutanone. 2. Topo II + DNA + 100 �M hirsutanone.. Topo II + DNA + 100 �M etoposide. 4. Topo II + DNA. 5. DNA. 6. Linear DNA. An

ncrease in the yield of linear DNA is indicative of topo II poisoning.

edicine 21 (2014) 866–870 869

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