2009 coumarin aaa induces apoptosis like cell death

J BIOCHEM MOLECULAR TOXICOLOGYVolume 23, Number 4, 2009

Coumarin A/AA Induces Apoptosis-Like Cell Deathin HeLa Cells Mediated by the Releaseof Apoptosis-Inducing FactorCarolina Alvarez-Delgado,1 Ricardo Reyes-Chilpa,2 Elizabet Estrada-Muniz,2 C. AdrianaMendoza-Rodrıguez,1 Angelina Quintero-Ruiz,1 Jose Solano,1 and Marco A. Cerbon1

1Department of Biology, Faculty of Chemistry, National Autonomous University of Mexico, Coyoacan 04510, Mexico, D.F., Mexico;E-mail: [email protected] of Chemistry, National Autonomous University of Mexico, Mexico, D.F., Mexico

Received 21 August 2008; revised 8 January 2009; accepted 18 January 2009

ABSTRACT: It has been demonstrated that natu-rally occurring coumarins have strong biological ac-tivity against many cancer cell lines. In this study,we assessed the cytotoxicity induced by the natu-rally isolated coumarin A/AA in different cancer celllines (HeLa, Calo, SW480, and SW620) and in normalperipheral-blood mononuclear cells (PBMCs). Cyto-toxicity was evaluated using the MTT assay. The re-sults demonstrate that coumarin A/AA was cytotoxicin the four cancer cell lines tested and importantly wassignificantly less toxic in PBMCs isolated from healthydonors. The most sensitive cancer cell line to coumarinA/AA treatment was Hela. Thus, the programmed celldeath (PCD) mechanism induced by this coumarinwas further studied in this cell line. DNA fragmen-tation, histomorphology, cell cycle phases, and sub-cellular distribution of PCD proteins were assessed.The results demonstrated that DNA fragmentation,but not significant cell cycle disruptions, was part ofthe PCD activated by coumarin A/AA. Interestingly,it was found that apoptosis-inducing factor (AIF), aproapoptotic protein of the mitochondrial intermem-brane space, was released to the cytoplasm in treatedcells as detected by the western blot analysis in sub-cellular fractions. Nevertheless, the active form ofcaspase-3 was not detected. The overall results indicatethat coumarin A/AA induces a caspase-independentapoptotic-like cell death program in HeLa cells, me-diated by the early release of AIF and suggest thatthis compound may be helpful in clinical oncology.C© 2009 Wiley Periodicals, Inc. J Biochem Mol Toxicol

Correspondence to: Marco Cerbon.Contract Grant Sponsor: CONACyT.Contract Grant Numbers: 46759-Q and P47829-Q.Contract Grant Sponsor: UNAM.Contract Grant Numbers: PAPIIT IN207207 and PAIP 6190-08.

c© 2009 Wiley Periodicals, Inc.

23:263–272, 2009; Published online in Wiley InterScience(www.interscience.wiley.com). DOI 10:1002/jbt.20288

KEYWORDS: Apoptosis-inducing factor; Apoptosis-like programmed cell death; Caspase independent;Coumarin A/AA; HeLa

INTRODUCTION

Induction of the various forms of programmed celldeath (PCD) is one of the major modes of action of an-tineoplasic drugs. Even though activation of the classicapoptotic pathways has been implicated in many mod-els of malignant-cell death [1], it has become an increas-ingly known fact that tumor cells, as well as nonmalig-nant cells, can engage alternative pathways of cell deathin which different organelles are involved [2]. Althoughthe induction of the classic apoptotic pathway is one ofthe main targets in cancer chemotherapy, the activationof alternative cell death programs is important in thetreatment of neoplasic cells that carry mutations in celldeath related genes that render them resistant to clas-sic apoptosis activation [3]. In this context, the searchfor antineoplasic compounds that activate different celldeath programs is an important field of cancer research.

Cervical cancer is one of the deadliest malignan-cies for women in the third-world countries. One of themain problems with its treatment is the resistance toconventional chemotherapy [4]. Therefore, the activa-tion of nonconventional death pathways could be aninteresting approach for the treatment of this and otherresistant tumors.

Coumarins are a very common type of secondarymetabolite in higher plants. These natural compounds

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264 ALVAREZ-DELGADO ET AL. Volume 23, Number 4, 2009

have been used in the treatment of infectious diseases,and more recently it has been demonstrated that thesecompounds are capable of inducing apoptosis in dif-ferent types of cancer cells [5–7]. Interestingly, othergroups have reported that some coumarin derivativescan also trigger alternative, nonclassical PCD pathwaysthrough the generation of reactive oxygen species andmodulation of microtubule dynamics, even in the ab-sence of typical apoptotic mediators [8,9].

Nonclassical PCD pathways have been described,and one of the effectors involved is the highly con-served mitochondrial flavoprotein apoptosis-inducingfactor (AIF). This protein has been implicated in differ-ent models of caspase-independent cell death [10,11]and is an attractive target for the induction of PCDin malignant cells. AIF is found in the mitochondrialintermembrane space of healthy cells from higher eu-karyotes. It normally functions as an oxidoreductaseand has a possible role in the maintenance of the res-piratory complex I [11,12]. AIF is also essential for thenormal embryonic and morphological development inmammals, and recently it has been found to participatein different models of nonclassical PCD [10,11,13]. Thislatter function as an alternative cell death effector hasbeen explored in cells where classic-PCD proteins aremutated or absent [14]. It has also been demonstratedthat the dual-role (oxidoreductase-cell death effector)of AIF depends on its subcellular localization. Upon aproapoptotic stimulus, AIF is proteolytically processedin the mitochondrial intermembrane space where it be-comes a soluble protein [15,16]. After this mitochon-drial processing and the subsequent permeabilizationof the outer mitochondrial membrane, AIF can be re-leased from the mitochondria and translocated to thenucleus, where it participates in large-scale (50 Kbs)apoptotic chromatinolysis and could be involved in theactivation of other endonucleases that further degradeDNA [12,15]. In addition, the expression of AIF is posi-tively regulated by basal levels of the tumor suppressorp53. In this context, cells that express wild-type p53 andAIF can engage in either classic or alternative PCD pro-grammes [17].

In the present study, we report the induction ofan apoptosis-like PCD mediated by the early releaseof AIF in HeLa cells exposed to a naturally occurringcoumarin (A/AA; Figure 1) isolated from the fruit ofthe tropical tree Mammea americana.

MATERIALS AND METHODS

Cell Culture

HeLa and Calo (human cervical carcinoma), SW480(human colon adenocarcinoma), and SW620 (humancolorectal adenocarcinoma derived from lymph node

FIGURE 1. Structure of coumarin A/AA.

metastasis) cells were grown in DMEM supplementedwith 10% FBS (Invitrogen Corporation, Carlsbad, CA)and maintained in standard culture conditions (37◦C,95% humidified air, and 5% CO2). Cells were allowedto grow to a density of 80% and then were harvestedusing sterile PBS/EDTA (pH 7.4) before starting everyexperiment.

Cytotoxicity Assay (MTT)

Coumarin A/AA (406.47 MW) was isolated byDr. Reyes-Chilpa of Instituto de Quımica, National Au-tonomous University of Mexico, Mexico, as previouslydescribed [18]. For all experiments, coumarin A/AAwas dissolved in DMSO (J.T. Baker, Phillipsburg, NJ)and mixed with fresh DMEM to achieve various finalconcentrations. MTT (Sigma-Aldrich, St. Louis, MO)was diluted in PBS/EDTA to yield a stock solution of2.5 mg/mL.

HeLa, Calo, SW480, and SW620 cells were seededto a final density of 6000 cells/well in 96-well ELISAplates. The cultures were allowed to grow in standardculture conditions for 24 h and then were treated for 48and 72 h with coumarin A/AA, or 0.15% v/v DMSO(vehicle) or 0.25 μM Taxol (Sigma-Aldrich), as a posi-tive control. The final concentration of DMSO did notalter cell growth and cell cycle measurments whencompared with vehicle-free cultures. After exposure tocoumarin A/AA (final concentrations of 1, 5, 10, 20, 40,and 60 μM in each well) or the corresponding controls,cells were incubated with MTT for 4 h [19]. The for-mazan precipitate was dissolved in 250 μL DMSO, andthe absorbance at 550 nm was measured with an ELISAplate reader. The percentage of growth inhibition foreach cell line exposed to the different concentrationsof coumarin A/AA was calculated using the followingformula: percentage of inhibition = 100 – (100 × ob-served absorbance/negative control’s absorbance). TheIC50 value was obtained using the Software OriginPro7.0 (RockWare, Golden, CO). For all subsequent ex-periments, the final concentration of coumarin A/AAin each HeLa culture was 30 μM. This concentration

J Biochem Molecular Toxicology DOI 10:1002/jbt

Volume 23, Number 4, 2009 MECHANISM OF CYTOTOXICITY OF COUMARIN A/AA 265

was chosen because it induces a sustained cytotoxiceffect (determined by the IC50), and it is approximatelyhalf the IC50 at 48 h and twice the IC50 at 72 h of thetreatment.

Cytotoxicity was also assesed in peripheral bloodmononuclear cells (PBMC), obtained from peripheralblood of healthy adult donors. Briefly, 25–30 mL of pe-ripheral blood was obtained with a Vacutainer system(Becton Dickinson, Franklin Lakes, NJ) by venopunc-tion. Blood was then mixed with equal volumes of ster-ile PBS (pH 7.5) and transferred to a 15-mL polyestirenetube. After this, 3 mL of Histopaque-1077 (SigmaAldrich) was slowly added to the suspension drop bydrop. This suspension was centrifuged for 40 min at1200 rpm in a Heraeus Megafuge 1.0 general-purposecentrifuge (Thermo Scientific, Waltham, MA). PBMCswere collected from the phase between the Histopaque-1077 and the plasma phase. PBMCs were transferred toa sterile polyestirene tube and resuspended with 5 mLof PBS. This suspension was centrifuged for 10 min at1000 rpm. This procedure was repeated three times (thelast centrifugation lasted about 5 min). The cellularpellet was finally resuspended in 10 mL of RPMImedium (Invitrogen Corporation) supplementedwith 10% FBS. These PBMCs were counted and wereused for the cytotoxicity assays. 273,600 PBMCs wereseeded in each well and were supplemented withphytohemagglutinin (final concentration 10 μg/mL)and were cultured in standard conditions for 48 h priorto the cytotoxicity assays. PBMCs were then treated for48 h with different concentrations of coumarin A/AA(10, 30, 60, 100 μM). After this period, MTT assay wasperformed as described for tumor cells.

DNA Fragmentation (TUNEL Assay)

DNA fragmentation analysis was performed us-ing the in situ cell death detection kit-fluorescein (Roche,Basel, SW) with TdT enzyme (deoxynucleotidyl trans-ferase). HeLa cells were subcultured to a final densityof 400,000 cells in each well and were allowed to growin standard culture conditions for 24 h. 30 μM coumarinA/AA or 0.15% DMSO (vehicle) or 0.25 μM taxol (posi-tive control) was added to the culture. After 24, 48, and72 h of treatment, cells were fixed in 4% paraformalde-hyde for 1 h at room temperature and washed in coldPBS (pH 7.4). Cells were then permeabilized for 2 minin 0.1% Triton X-100 in 0.1% sodium citrate, washedwith PBS and incubated with the TUNEL reaction mix-ture for 1 h at 37◦C in the dark. Positive (cells treatedwith 1 μg/mL DNAse) and negative (reaction with-out TdT) controls were considered at this point. Cellswere washed twice in cold PBS, and the cover slideswere mounted using Dako mounting medium (Dako,Carpinteria, CA). DNA fragmentation was analyzed

with a Nikon Eclipse E600 fluorescence microscope(Nikon Corporation, Tokyo, Japan).

Cell Morphology (Hematoxylin–EosinStain)

HeLa cells were seeded to a final density of 400,000and were allowed to grow in standard culture condi-tions for 24 h. Cell cultures were treated for 24, 48, and72 h with 30 μM coumarin A/AA or 0.15% DMSO (ve-hicle) or 0.25 μM taxol (positive control). Cells werefixed in cover slides using 4% paraformaldehyde for1 h, washed twice with PBS and stained with hema-toxylin for 4 min and eosin for 3 min. Cells werethen dehydrated with increasing ethanol concentra-tions (40, 80, 96 100%, for 2 min each), washed with100% xylol and mounted for morphology analysis witha Nikon Eclipse E600 fluorescence microscope (NikonCorporation).

Cell Cycle Analysis (Flow Cytometry)

HeLa cells were subcultured to a final density of450,000. After a 24-h period in standard culture condi-tions, cells were treated with 30 μM coumarin A/AAor 0.15% DMSO (vehicle) or 0.25 μM taxol (positivecontrol) for 12, 24, 32, and 48 h. After these treatmentperiods, cells were harvested and centrifuged for 5 minat 1500 rpm. The pellet was resuspended in cold PBS(pH 7.4) and spinned for 5 min at 2000 rpm. Cells werefixed with 70% ethanol at −20◦C for at least 12 h. In-tracellular DNA was labeled with 5 mL of 0.02 mg/mLpropidium iodide (PI) solution (Sigma-Aldrich). Cellcycle analysis was made using a FACScan cytometer(Becton Dickinson) and CELLQuest software (BectonDickinson). The cell cycle profile was obtained by an-alyzing 10,000 cells using the ModFIT LT program(Becton Dickinson).

Subcellular Fractionation

HeLa cells were subcultured to a density of3.3 × 106 and treated for 12, 15, 20 and 24 h with 30 μMcoumarin A/AA or 200 nM staurosporine (for 24 h)dissolved in DMSO (Sigma-Aldrich) or 0.15% DMSO.Subcellular fractionation was performed as previouslydescribed [10,20], with minor modifications. All thesubfractionation and centrifugation steps were per-formed at 4◦C. Briefly, cells were harvested with coldPBS/EDTA (pH 7.4) at the indicated time points andwere spinned for 5 min at 200×g. Cells were thenfractionated by homogenization with a 27G syringe(35 passes) in isotonic buffer for mitochondria (pH 7.5)(210 mM mannitol, 70 mM sucrose, 1 mM EDTA, 10 mMHEPES, and complete protease inhibitor cocktail from



Roche, Basel, SW) and serial centrifugations. All cen-trifugations were performed in a Beckman GS-15Rcentrifuge, with a F2402H rotor (Beckman Coulter,Fullerton, CA), The cell homogenate was centrifugedat 1500×g for 10 min. The pellet (whole cells and nu-clei) was further homogenized twice as described ear-lier and was centrifuged at 1500×g, for 10 min. Thesupernatant of these centrifugations was collected, andthe pellet was discarded. This supernatant was cen-trifuged at 10,000×g for 15 min. The pellet correspondsto the “crude mitochondrial fraction” and was resus-pended in 1 mL washing buffer for mitochondria (pH 7.5)(10 mM Tris–HCl, 1 mM EDTA, 250 mM sucrose, andcomplete protease inhibitor cocktail from Roche) andwere spinned for 15 min at 10,000×g. The resultingsupernatant was kept as the cytosolic fraction and thepellet as the pure mitochondrial fraction. Both fractionswere stored at −20◦C. The mitochondrial pellet was re-suspended with a 27G syringe in lysis buffer (1 mMDTT, 10 mM Tris–HCl, 30% glycerol, 1 mM EDTA, 1%Triton X-100, 5 μg/μL leupeptin, 5 μg/μL aprotinin, 2μg/μL pesptatin, 1 mM PMSF, 1 mM sodium ortho-vanadate, and 15 mM sodium azide) and incubated onice for at least 30 min. This suspension was centrifugedat 14,300×g for 1 h, and the supernatant was storedas the total mitochondrial protein and was quantifiedby the Bradford method [21]. Protein subcellular lo-calization was analyzed by Western Blot as describedbelow.

Protein Expression (Western Blot)

HeLa cells were treated for 12, 15, 20, and 24 hwith 30 μM coumarin A/AA or 50 nM taxol (pos-itive control) or 0.15% DMSO (vehicle). Cells werethen harvested, lysed (1 mM DTT, 10 mM Tris–HCl,30% glycerol, 1 mM EDTA, 1% Triton X-100, 5 μg/mLleupeptin, 5 μg/mL aprotinin, 2 μg/mL pesptatin,1 mM PMSF, 1 mM sodium orthovanadate, and 15mM sodium azide) and were centrifuged for 1 hat 12,000 rpm (4◦C). The protein concentration wasdetermined by the Bradford method [21]. WesternBlot analysis was performed as previously described[22]. Proteins were separated in a 10% acrylamidegel, electrotransferred to a nitrocellulose membrane(Immobilon-P, Millipore, Billerica, MA) and probedwith the following primary antibodies: 1:200 anti-Bcl-2 (C-2), 1:500 anti-Bax (B-9), 1:150 anti-caspase-3 p20(N-19 and E-8), 1:200 anti-AIF (D-20 and E-1), 1:20,000anti-α-tubulin (B-7), and 1:200 anti-Cyt-c (7H8). Sec-ondary antibodies were goat–anti-mouse IgG-HRP(1:20,000), goat–anti-rabbit IgG-HRP (1:10,000), anddonkey–anti-goat IgG-HRP. All antibodies were pur-chased from Santa Cruz Biotechnology, Inc. (Santa

Cruz, CA) and were dissolved in TBS-0.1% tween(Sigma-Aldrich). Protein bands were detected by theECL chemiluminescent kit (Amersham Biosciences,Fairfield, CT).

Statistical Analysis

Statistically significant differences (P < 0.05) be-tween groups were determined by Student’s t-test usingPrism 3.0 (GraphPad Software, Inc., La Jolla, CA).

RESULTS

Coumarin A/AA Induces Cytotoxicity toHeLa and Other Cancer Cell Lines

The cytotoxic potential of coumarin A/AA wastested in four different cancer cell lines: HeLa and Calocervical cancer cell lines SW-480 and SW-620 colorectalcancer cell lines. The half maximal inhibitory concen-tration (IC50) was determined at 48 and 72 h of thetreatment for each cell line by the MTT method. Table 1shows the IC50 for each of the cell lines tested. As itcan be observed, HeLa was the most sensitive cell lineto the treatment with coumarin A/AA (IC50 of 65.6and 15.3 μM at 48 and 72 h, respectively). The cyto-toxic effect of coumarin A/AA was also examined inPBMCs isolated from healthy donors. The cytotoxicityof coumarin A/AA toward PBMCs was substantiallylower than for the cancer cell lines. In fact, IC50 val-ues were not achieved: even at the highest coumarinconcentration tested (100 μM), only 24.2% of inhibitionwas reached (data not shown).

Coumarin A/AA Causes Apoptotic-LikeMorphology Changes in HeLa Cells

Several distinctive features of PCD may be evi-denced in the morphology of a dying cell. As shownin Figure 2, coumarin A/AA treatment induces HeLacell shrinkage, chromatin condensation, and DNA

TABLE 1. Cytotoxic Effect of Coumarin A/AA in DifferentCancer Cell Lines

IC50

At 48 h At 72 h

Cancer Cell Line Mean ± SD (μM) Mean ± SD (μM)

HeLa 65 ± 2.8 13.3 ± 8Calo 65.6 ± 4.1 15.3 ± 5.7SW620 73.5 ± 5.2 65.4 ± 4.6SW480 75 ± 2.6 74 ± 9.2

The IC50 values for each cell line at 48 and 72 h is shown as the meanconcentration ± SD (μM) of three independent experiments.



FIGURE 2. Coumarin A/AA induces apoptosis-like morphology changes in HeLa cells. A representative hematoxylin–eosin staining is shown:(A–C) HeLa cells treated with 0.15% DMSO at 24, 48. and 72 h, respectively. (D–F) HeLa cells treated with 0.25 μM taxol at the same time points.(G–I) HeLa cells treated with 30 μM coumarin A/AA at the time-points indicated before. Bar = 100 μM.

hypercromicity after a 48-h exposure. As can be ob-served, the cells treated with taxol, a known inducerof apoptosis in this cancer cell line, presented thesesame characteristics after only 24 h of treatment. Theseresults suggest that coumarin A/AA could induce anapoptotic-like cell death in HeLa cells.

Nuclear-Apoptosis Occurs in a Two-StepManner in HeLa Cells Exposedto Coumarin A/AA

Figure 3 shows a different pattern of DNA fragmen-tation in HeLa cells treated with coumarin A/AA for24 h (Figure 3G) than that observed at 48 and 72 h of the

FIGURE 3. Nuclear-apoptosis occurs in a two-step manner in HeLa cells exposed to coumarin A/AA. A representative TUNEL assay is shown.(A) Technique’s negative control. (B) Technique’s positive control (cells treated with DNAse). (C): HeLa cells treated with vehicle (0.15% DMSO,48 h treatment). (D–F) HeLa cells treated with 0.25 μM taxol (24, 48, and 72 h treatments, respectively). (G–I): HeLa cells treated with 30 μMcoumarin A/AA at the same time points indicated previously. Arrows indicate “horseshoe-like” pattern of DNA fragmentation. Bar = 100 μM.



treatment (Figure 3H and 3I). Fragmented DNA wasnot detected by TUNEL staining in HeLa cells before20 h of coumarin A/AA exposure (data not shown).The first type of chromatin condensation was mainlyperipheral and can be observed as a “horseshoe-like”shape at 24 h posttreatment (Figure 3G, indicated byarrows). However, at a later time explored (48 and 72 htreatment), chromatin condensation appears more uni-form and some possible apoptotic-bodies can be seen(Figure 3H and 3I). In contrast, cells treated with taxolpresented uniform TUNEL staining since 24 h of treat-ment (Figure 3D).

Coumarin A/AA Induces Cell Deathin HeLa Cells without Disruptingthe Cell Cycle

HeLa cells were analyzed by flow cytometry toasses cell cycle disruptions. An intermediate 32 h treat-ment time point was included in these experimentssince we observed significant DNA fragmentation at48 h of treatment (Figure 3). No significant cell cyclealterations were found in cells treated with coumarinA/AA before 24 h (Figures 4D, 4G, and 5). Interest-ingly, at 32 h postexposure, HeLa cells show significantaccumulation of fragmented DNA (Figures 4J, 4M, and5, indicated by asterisks). Nevertheless, this subdiploidDNA accumulation does not coincide with any cell cy-cle arrest.

In contrast, cells treated with taxol show a markedcell cycle arrest at the G2-M phase as soon as 12 h oftreatment (Figure 4C), and this arrest precedes the sub-diploid DNA accumulation. According to these data,cell death induced by coumarin A/AA proceeds inde-pendently from cell cycle alterations.

Apopotis Inducing Factor Release from theMitochondria Is an Early Event in CellDeath Induced by Coumarin A/AA in HeLaCells and Occurs Independently ofCaspase-3 Activation

We analyzed the expression of some key pro-teins that are known to participate in many cell deathparadigms. It was found that the treatment withcoumarin A/AA causes an upregulation of Bax pro-tein 12 h posttreatment and also the downregulationof Bcl-2 protein after 24 h of coumarin A/AA ex-posure (Figure 6A), when compared with the cellstreated with DMSO. AIF expression was also studiedin isolated fractions of mitochondria and cytoplasmof coumarin A/AA-treated cells. It has been reportedthat staurosporin induces HeLa cell death by the re-lease of AIF from the mitochondria [23], so we used

FIGURE 4. Coumarin A/AA induces cell death in HeLa cells with-out disrupting the cell cycle. Flow cytometry of (A) nontreated cellsat time zero; (B,E,H,K) 0.15% DMSO-treated cells. (C,F,I,L) 0.25 μMtaxol treated cells; (D,G,J,M) 30 μM coumarin A/AA treated cells.The times of treatment are indicated in the figure. The arrows in Jand M show the subdiploid DNA. The data are representative resultsfrom three independent experiments.

staurosporin-treated HeLa cells as a positive control ofAIF release. Figure 6B clearly shows that in cells treatedwith coumarin A/AA, as well as in cells treated withstaurosporin (24 h treatment), AIF is released to the cy-toplasm (Figure 6B, middle panel). In contrast, in cellstreated with DMSO, AIF is primarily localized at themitochondria. In addition, the involvement of caspase-3 in the induction of PCD was studied. Figure 6C showsthat treatment with coumarin A/AA does not causean activation of caspase-3 at the time points studied.These results suggest that HeLa cell death triggeredby coumarin A/AA might be caspase-3 independentand rely predominantly in the release of AIF and itsfunction as a death effector outside the mitochondria.

DISCUSSION

In the present study, the cytotoxic activity ofcoumarin A/AA (Figure 1) was demonstrated in fourdifferent human cancer cell lines (colorectal cancercell lines SW480 and SW620 and cervical cancer celllines HeLa and Calo). Even though the cytotoxicity of



FIGURE 5. Distribution of the cell cycle phases. The graph showsthe distribution of the cell cycle phases for nontreated cells at timezero (T0). HeLa cells were treated with vehicle (0.15% DMSO) or0.25 μM taxol or 30 μM coumarin A/AA for the time-points indi-cated. The figure represents the mean values for three independentexperiments with their corresponding standard deviations. Statisti-cally significant differences vs. control group at the same time point(P < 0.05) are marked by asterisks.

coumarin A/AA and other coumarin compounds hasbeen reported by many investigators in different celllines [24–28], to our knowledge, there are no previousreports on the exact molecular mechanism by whichcoumarin A/AA exerts its cytotoxic effect. In contrastto the classic cell death mechanism initiated by the basiccoumarin 1,2 benzopyrone in HeLa cells [29], in thisstudy we show that coumarin A/AA induces a caspaseindependent PCD with the release of AIF from mito-chondria as an early event in cell death (Figure 6), andno cell cycle arrests preceding the cell death program(Figures 4 and 5).

In agreement with previous studies [7,24,26,30],we demonstrated that the complex coumarin A/AAhas a high cytotoxic activity against SW480, SW620,Calo, and HeLa malignant cell lines (Table 1), withIC50 values ranging from 13.3 to 74 μM for HeLa andSW480, respectively. As was expected from its chemi-cal structure, very low cytotoxicity was detected whencoumarin A/AA was tested against PBMCs, but fur-ther studies need to be conducted in vivo to test theselective nature of the compound.

In addition, it has been proven that the cytotoxicpotential of coumarins is due to their structure-basedability to induce apoptotic PCD, preceded by an arrestof the cell cycle and subdiploid DNA accumulation [5–7,29,31]. In this study, the typical morphological fea-tures of PCD were not noticeable until 48 h posttreat-ment (Figure 2). Interestingly, two DNA-fragmentationpatterns appear to be sequentially initiated. At 24 h,postexposure, some TUNEL-positive cells had periph-

FIGURE 6. Coumarin A/AA induces the release of AIF from themitochondria in HeLa cells. Protein expression and subcellular dis-tribution analysis by Western Blot. HeLa cells were treated with ve-hicle (0.15% DMSO) or 30 μM coumarin A/AA for the time-pointsindicated. Protein was extracted as described in the Materials andMethods section, and expression was evaluated. (A) Bax and Bcl-2protein expression at indicated times. α-tubulin was used as a load-ing control for 25 μg of total protein (lower panels). (B) Subcellularlocalization of AIF. Cytochrome-c (cyt-c) was used as an internal iso-lation control. HeLa cells treated with 200 nM staurosporine (ST)were used as a positive control for AIF release (middle-right panel,marked by asterisk). 15 μg of cytosolic and mitochondrial proteinwas used. (C) Active-caspase-3 expression in DMSO (lanes 2–5) andcoumarin A/AA (lanes 6–9) treated cells. Lane 1 shows a positivecontrol for active-caspase-3 expression. For this aim, 120 μg of totalprotein from rat uterus at 06:00 h of estrous day was used. For DMSOand coumarin A/AA treated cells, 25 μg of total protein was loaded.α tubulin was used as a loading control (lower panel).

eral DNA fragmentation and their chromatin was con-densed as a horseshoe-like structure (Figure 3G). At latertimes (48 h), chromatin condensation appeared morehomogeneous (Figure 3H). Different types of chro-matin condensation patterns associated with specificprotein activation have been previously described atdistinct stages of cell death [16,32]. Typically, it has beenreported that AIF protein causes large-scale (50 kb),type I nuclear apoptosis, and that caspase-activatedDNAses (CADs) initiate the oligonucleosomal, type IInuclear apoptosis [16] that can be evidenced as a lad-der pattern.In a similar setting, coumarin A/AA couldcause the sequential activation of type I and type IInuclear apoptosis in HeLa cells.



Several authors have demonstrated that in variouscancer cell lines PCD occurs after alterations or arrestof the cell cycle [6,7,26,29,33]. In this study, and in con-trast to what was expected, no arrest in any phase ofthe cell cycle was detected (Figures 4 and 5), althoughtreatment with coumarin A/AA did cause a significantaccumulation of subdiploid DNA after long exposureperiods (Figures 4J, 4M and 5). This indicates that thecytotoxicity of coumarin A/AA is not due to cell cyclealterations, at least at the concentrations tested. To ourknowledge, these results are a novel finding in the fieldof antitumor mechanisms for coumarin compounds, asno previous reports on the detailed mechanism of ac-tion of coumarin A/AA have been published.

To further test the possibility that an apoptotic-likeprogram could be activated by coumarin A/AA, Baxand Bcl-2 protein expression was studied. These twoproteins belong to the Bcl-2 family and are partially re-sponsible for regulating the status of the mitochondrialpermeability transition pore (mtPTP), thus allowing therelease and translocation of diverse proapoptotic pro-teins from the mitochondria to the cytoplasm and nu-cleus [33–35]. Bax and Bcl-2 can determine the proapop-totic balance of the cell. In the present work, we haveshown that 12 h of treatment with coumarin A/AA in-duces the overexpression of Bax protein and, 3 h laterthe inhibition of Bcl-2 protein expression (Figure 6A)was observed. These data suggest that coumarin A/AAcould induce a proapoptotic balance in the cell and thatthe release of proapoptotic proteins from the mitochon-drial intermembrane space is feasible.

The chromatin condensation pattern observed 24 hafter coumarin A/AA treatment is typical of a phase Inuclear apoptosis (Figure 3G) initiated by the release ofthe proapoptotic protein AIF [16]. This evidence, lead-ing to the description of a PCD pathway prompted bythe release of AIF, was further explored by the analysisof AIF expression in the mitochondrial and cytoplasmicfractions, as this protein only acts as a proapoptotic fac-tor when it is released to the cytosol and translocatedto the nucleus [36]. Indeed, the present work demon-strates that coumarin A/AA induces the release of AIFfrom mitochondria to the cytosol (Figure 6B). The re-lease of AIF could very likely be the reason for the“horseshoe-like” chromatin condensation observed 24h after the treatment (Figure 4G). The different con-densation patterns observed at 48 h after the treatmentcould be attributed to the late degradation action ofCADs.

Even though caspases are an integral part of theapoptotic machinery, cell death programs may be cas-pase independent and occur in the complete absenceof caspase activity [11,37]. Interestingly, in the presentstudy, the active subunits of caspase-3 were not de-tected (Figure 6C). This is in contrast to what has

been previously reported for other basic or complexcoumarins that induce cell death by activating the clas-sic, caspase-dependent death pathway [29,33,38]. Nev-ertheless, it is also possible that if the expression ofcaspase-3 had been examined at later times (48 and 72h of coumarin A/AA treatment), the active subunitsmight have been detected. For example, it is possi-ble that caspases could be active at prolonged timesof coumarin A/AA exposure and that AIF could act asan early initiator of cell death. This type of “sequenced”cell death program has already been reported by otherauthors in different models of coumarin-induced celldeath [39,40]. It has also been proven that AIF can actas a primary effector of cell death either with or withoutthe aid of caspases [10,11,13]. So it is very likely that inthis study, HeLa cell death induced by coumarin A/AAis initiated by the actions of AIF without the early par-ticipation of caspase-3. The PCD mechanism proposedin this work is outlined in Figure 7.

In conclusion, we demonstrated that coumarinA/AA is cytotoxic to HeLa and Calo cervical can-cer cell lines and SW480 and SW620 colorectal can-cer cell lines (Table 1). In HeLa cells, the cytotoxic-ity of coumarin A/AA is due to the activation of anapoptosis-like cell death program that begins as earlyas 12 h posttreatment, with the release of the proapop-totic protein AIF from the mitochondria to the cyto-plasm (Figure 6B). In this cell death paradigm, AIF actsas an early, caspase independent, cell death effectorthat prompts DNA fragmentation (Figures 3G–3I) andthe typical morphological changes of PCD (Figures 2Hand 2I), without disturbing the cell cycle’s distribu-tion (Figures 4 and 5). In addition to these findings,we observed that coumarin A/AA exerts a reduced cy-totoxic effect in normal PBMCs when compared with

FIGURE 7. Diagram of the possible mechanism of action of thePCD activated by coumarin A/AA in HeLa cells. Time-points af-ter coumarin A/AA treatment and relevant biochemical events andmorphological changes are indicated.



the important damage induced in HeLa cells. Thedata described suggest that coumarin A/AA couldbe an excellent candidate for a low-toxicity anticancertreatment.

ACKNOWLEDGMENTS

Alvarez-Delgado C. receieved a fellowship fromCONACyT. The author would like to thank Dr. J.J. Garcıa-Trejo for his kind help in the isolation ofmitochondria.

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