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Volume 23, Number 4, 2009 
Coumarin A/AA Induces Apoptosis-Like Cell Death 
in HeLa Cells ...
264 A´ LVAREZ-DELGADO ET AL. Volume 23, Number 4, 2009 
have been used in the treatment of infectious diseases, 
and more ...
was chosen because it induces a sustained cytoto...
266 A´ LVAREZ-DELGADO ET AL. Volume 23, Number 4, 2009 
Roche, Basel, SW) and serial centrifugations. All cen-trifugations...
FIGURE 2. Coumarin A/AA induces apoptosis-likemo...
268 A´ LVAREZ-DELGADO ET AL. Volume 23, Number 4, 2009 
treatment (Figure 3H and 3I). Fragmented DNA was 
not detected by ...
FIGURE 5. Distribution of the cell cycle phases....
270 A´ LVAREZ-DELGADO ET AL. Volume 23, Number 4, 2009 
Several authors have demonstrated that in various 
cancer cell lin...
the important damage induced in HeLa cells. The ...
272 A´ LVAREZ-DELGADO ET AL. Volume 23, Number 4, 2009 
28. Yang H, Protiva P, Gil RR, Jiang B, Baggett S, Basile MJ, 
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2009 coumarin aaa induces apoptosis like cell death

  1. 1. J BIOCHEM MOLECULAR TOXICOLOGY Volume 23, Number 4, 2009 Coumarin A/AA Induces Apoptosis-Like Cell Death in HeLa Cells Mediated by the Release of Apoptosis-Inducing Factor Carolina A´ lvarez-Delgado,1 Ricardo Reyes-Chilpa,2 Elizabet Estrada-Mun˜ iz,2 C. Adriana Mendoza-Rodr´ıguez,1 Angelina Quintero-Ruiz,1 Jos´e Solano,1 and Marco A. Cerb´on1 1Department of Biology, Faculty of Chemistry, National Autonomous University of Mexico, Coyoac´an 04510, Mexico, D.F., M´exico; E-mail: mcerbon85@yahoo.com.mx 2Institute of Chemistry, National Autonomous University of Mexico, Mexico, D.F., M´exico 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 cell lines (HeLa, Calo, SW480, and SW620) and in normal peripheral-blood mononuclear cells (PBMCs). Cyto-toxicity was evaluated using the MTT assay. The re-sults demonstrate that coumarin A/AA was cytotoxic in the four cancer cell lines tested and importantly was significantly less toxic in PBMCs isolated from healthy donors. The most sensitive cancer cell line to coumarin A/AA treatment was Hela. Thus, the programmed cell death (PCD) mechanism induced by this coumarin was 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 of the PCD activated by coumarin A/AA. Interestingly, it was found that apoptosis-inducing factor (AIF), a proapoptotic protein of the mitochondrial intermem-brane space, was released to the cytoplasm in treated cells as detected by the western blot analysis in sub-cellular fractions. Nevertheless, the active form of caspase-3was not detected. The overall results indicate that coumarin A/AA induces a caspase-independent apoptotic-like cell death program in HeLa cells, me-diated by the early release of AIF and suggest that this compound may be helpful in clinical oncology. C 2009 Wiley Periodicals, Inc. J Biochem Mol Toxicol Correspondence to: Marco Cerb´on. 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 2009Wiley Periodicals, Inc. 23:263–272, 2009; Published online inWiley 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 cell death (PCD) is one of the major modes of action of an-tineoplasic drugs. Even though activation of the classic apoptotic 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 death in which different organelles are involved [2].Although the induction of the classic apoptotic pathway is one of the main targets in cancer chemotherapy, the activation of alternative cell death programs is important in the treatment of neoplasic cells that carry mutations in cell death related genes that render them resistant to clas-sic apoptosis activation [3]. In this context, the search for antineoplasic compounds that activate different cell death programs is an important field of cancer research. Cervical cancer is one of the deadliest malignan-cies forwomen in the third-world countries. One of the main problems with its treatment is the resistance to conventional chemotherapy [4]. Therefore, the activa-tion of nonconventional death pathways could be an interesting approach for the treatment of this and other resistant tumors. Coumarins are a very common type of secondary metabolite in higher plants. These natural compounds 263
  2. 2. 264 A´ LVAREZ-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 these compounds are capable of inducing apoptosis in dif-ferent types of cancer cells [5–7]. Interestingly, other groups have reported that some coumarin derivatives can also trigger alternative, nonclassicalPCDpathways through the generation of reactive oxygen species and modulation 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-inducing factor (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 PCD in malignant cells. AIF is found in the mitochondrial intermembrane space of healthy cells from higher eu-karyotes. It normally functions as an oxidoreductase and has a possible role in the maintenance of the res-piratory complex I [11,12]. AIF is also essential for the normal embryonic and morphological development in mammals, and recently it has been found to participate in different models of nonclassical PCD [10,11,13]. This latter function as an alternative cell death effector has been explored in cells where classic-PCD proteins are mutated or absent [14]. It has also been demonstrated that the dual-role (oxidoreductase-cell death effector) of AIF depends on its subcellular localization. Upon a proapoptotic stimulus, AIF is proteolytically processed in themitochondrial intermembrane space where it be-comes a soluble protein [15,16]. After this mitochon-drial processing and the subsequent permeabilization of the outer mitochondrial membrane, AIF can be re-leased from the mitochondria and translocated to the nucleus, where it participates in large-scale (50 Kbs) apoptotic chromatinolysis and could be involved in the activation of other endonucleases that further degrade DNA [12,15]. In addition, the expression of AIF is posi-tively regulated by basal levels of the tumor suppressor p53. In this context, cells that expresswild-type p53 and AIF can engage in either classic or alternative PCD pro-grammes [17]. In the present study, we report the induction of an apoptosis-like PCD mediated by the early release of AIF in HeLa cells exposed to a naturally occurring coumarin (A/AA; Figure 1) isolated from the fruit of the tropical tree Mammea americana. MATERIALS AND METHODS Cell Culture HeLa andCalo (human cervical carcinoma), SW480 (human colon adenocarcinoma), and SW620 (human colorectal adenocarcinoma derived from lymph node FIGURE 1. Structure of coumarin A/AA. metastasis) cells were grown in DMEM supplemented with 10% FBS (Invitrogen Corporation, Carlsbad, CA) and maintained in standard culture conditions (37◦C, 95% humidified air, and 5% CO2). Cells were allowed to grow to a density of 80% and then were harvested using sterile PBS/EDTA (pH 7.4) before starting every experiment. Cytotoxicity Assay (MTT) Coumarin A/AA (406.47 MW) was isolated by Dr. Reyes-Chilpa of Instituto de Qu´ımica, National Au-tonomous University ofM´exico,M´exico, as previously described [18]. For all experiments, coumarin A/AA was dissolved in DMSO (J.T. Baker, Phillipsburg, NJ) and mixed with fresh DMEM to achieve various final concentrations. MTT (Sigma-Aldrich, St. Louis, MO) was diluted in PBS/EDTA to yield a stock solution of 2.5 mg/mL. HeLa, Calo, SW480, and SW620 cells were seeded to a final density of 6000 cells/well in 96-well ELISA plates. The cultures were allowed to grow in standard culture conditions for 24 h and then were treated for 48 and 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 not alter cell growth and cell cycle measurments when compared with vehicle-free cultures. After exposure to coumarin A/AA (final concentrations of 1, 5, 10, 20, 40, and 60 μMin 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, and the absorbance at 550 nm wasmeasuredwith an ELISA plate reader. The percentage of growth inhibition for each cell line exposed to the different concentrations of coumarin A/AA was calculated using the following formula: percentage of inhibition = 100 – (100 × ob-served absorbance/negative control’s absorbance). The IC50 value was obtained using the Software OriginPro 7.0 (RockWare, Golden, CO). For all subsequent ex-periments, the final concentration of coumarin A/AA in each HeLa culture was 30 μM. This concentration J Biochem Molecular Toxicology DOI 10:1002/jbt
  3. 3. Volume 23, Number 4, 2009 MECHANISM OF CYTOTOXICITY OF COUMARIN A/AA 265 was chosen because it induces a sustained cytotoxic effect (determined by the IC50), and it is approximately half the IC50 at 48 h and twice the IC50 at 72 h of the treatment. Cytotoxicity was also assesed in peripheral blood mononuclear cells (PBMC), obtained from peripheral blood 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 polyestirene tube. After this, 3 mL of Histopaque-1077 (Sigma Aldrich) was slowly added to the suspension drop by drop. This suspension was centrifuged for 40 min at 1200 rpm in a Heraeus Megafuge 1.0 general-purpose centrifuge (Thermo Scientific, Waltham, MA). PBMCs were collected fromthe phase between theHistopaque- 1077 and the plasma phase. PBMCs were transferred to a sterile polyestirene tube and resuspended with 5 mL of PBS. This suspension was centrifuged for 10 min at 1000 rpm. This procedure was repeated three times (the last centrifugation lasted about 5 min). The cellular pellet was finally resuspended in 10 mL of RPMI medium (Invitrogen Corporation) supplemented with 10% FBS. These PBMCs were counted and were used for the cytotoxicity assays. 273,600 PBMCs were seeded in each well and were supplemented with phytohemagglutinin (final concentration 10 μg/mL) and were cultured in standard conditions for 48 h prior to the cytotoxicity assays. PBMCs were then treated for 48 h with different concentrations of coumarin A/AA (10, 30, 60, 100 μM). After this period, MTT assay was performed 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 density of 400,000 cells in each well and were allowed to grow in standard culture conditions for 24 h. 30 μMcoumarin A/AA or 0.15% DMSO (vehicle) or 0.25 μMtaxol (posi-tive control) was added to the culture. After 24, 48, and 72 h of treatment, cells were fixed in 4% paraformalde-hyde for 1 h at room temperature and washed in cold PBS (pH 7.4). Cells were then permeabilized for 2 min in 0.1% Triton X-100 in 0.1% sodium citrate, washed with PBS and incubated with the TUNEL reactionmix-ture for 1 h at 37◦C in the dark. Positive (cells treated with 1 μg/mL DNAse) and negative (reaction with-out TdT) controls were considered at this point. Cells were washed twice in cold PBS, and the cover slides were 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–Eosin Stain) HeLa cells were seeded to a final density of 400,000 and were allowed to grow in standard culture condi-tions for 24 h. Cell cultures were treated for 24, 48, and 72 h with 30 μM coumarin A/AA or 0.15% DMSO (ve-hicle) or 0.25 μM taxol (positive control). Cells were fixed in cover slides using 4% paraformaldehyde for 1 h, washed twice with PBS and stained with hema-toxylin for 4 min and eosin for 3 min. Cells were then dehydrated with increasing ethanol concentra-tions (40, 80, 96 100%, for 2 min each), washed with 100% xylol and mounted for morphology analysis with a Nikon Eclipse E600 fluorescence microscope (Nikon Corporation). Cell Cycle Analysis (Flow Cytometry) HeLa cells were subcultured to a final density of 450,000. After a 24-h period in standard culture condi-tions, cells were treated with 30 μM coumarin A/AA or 0.15% DMSO (vehicle) or 0.25 μM taxol (positive control) for 12, 24, 32, and 48 h. After these treatment periods, cells were harvested and centrifuged for 5min at 1500 rpm. The pellet was resuspended in cold PBS (pH 7.4) and spinned for 5 min at 2000 rpm. Cells were fixed with 70% ethanol at −20◦C for at least 12 h. In-tracellular DNA was labeled with 5mL of 0.02 mg/mL propidium iodide (PI) solution (Sigma-Aldrich). Cell cycle analysis was made using a FACScan cytometer (Becton Dickinson) and CELLQuest software (Becton Dickinson). 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 of 3.3×106 and treated for 12, 15, 20 and 24 h with 30 μM coumarin A/AA or 200 nM staurosporine (for 24 h) dissolved in DMSO (Sigma-Aldrich) or 0.15% DMSO. Subcellular fractionation was performed as previously described [10,20], with minor modifications. All the subfractionation and centrifugation steps were per-formed at 4◦C. Briefly, cells were harvested with cold PBS/EDTA (pH 7.4) at the indicated time points and were spinned for 5 min at 200×g. Cells were then fractionated by homogenization with a 27G syringe (35 passes) in isotonic buffer for mitochondria (pH 7.5) (210mMmannitol, 70mMsucrose, 1mMEDTA, 10mM HEPES, and complete protease inhibitor cocktail from J Biochem Molecular Toxicology DOI 10:1002/jbt
  4. 4. 266 A´ LVAREZ-DELGADO ET AL. Volume 23, Number 4, 2009 Roche, Basel, SW) and serial centrifugations. All cen-trifugations were performed in a Beckman GS-15R centrifuge, with a F2402H rotor (Beckman Coulter, Fullerton, CA), The cell homogenate was centrifuged at 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. The supernatant of these centrifugations was collected, and the pellet was discarded. This supernatant was cen-trifuged at 10,000×g for 15min. The pellet corresponds to 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, and complete protease inhibitor cocktail from Roche) and were spinned for 15 min at 10,000×g. The resulting supernatant was kept as the cytosolic fraction and the pellet as the pure mitochondrial fraction. Both fractions were stored at −20◦C. Themitochondrial pellet was re-suspended with a 27G syringe in lysis buffer (1 mM DTT, 10 mM Tris–HCl, 30% glycerol, 1 mM EDTA, 1% Triton X-100, 5 μg/μL leupeptin, 5 μg/μL aprotinin, 2 μg/μL pesptatin, 1 mM PMSF, 1 mM sodium ortho-vanadate, and 15 mMsodium azide) and incubated on ice for at least 30min. This suspension was centrifuged at 14,300×g for 1 h, and the supernatant was stored as the total mitochondrial protein and was quantified by the Bradford method [21]. Protein subcellular lo-calization was analyzed by Western Blot as described below. Protein Expression (Western Blot) HeLa cells were treated for 12, 15, 20, and 24 h with 30 μM coumarin A/AA or 50 nM taxol (pos-itive control) or 0.15% DMSO (vehicle). Cells were then harvested, lysed (1 mM DTT, 10 mM Tris–HCl, 30% glycerol, 1 mM EDTA, 1% Triton X-100, 5 μg/mL leupeptin, 5 μg/mL aprotinin, 2 μg/mL pesptatin, 1 mM PMSF, 1 mM sodium orthovanadate, and 15 mM sodium azide) and were centrifuged for 1 h at 12,000 rpm (4◦C). The protein concentration was determined by the Bradford method [21]. Western Blot analysis was performed as previously described [22]. Proteins were separated in a 10% acrylamide gel, electrotransferred to a nitrocellulose membrane (Immobilon-P, Millipore, Billerica, MA) and probed with 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), 1:20,000 anti-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), and donkey–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 the ECL chemiluminescent kit (Amersham Biosciences, Fairfield, CT). Statistical Analysis Statistically significant differences (P 0.05) be-tween groups were determined by Student’s t-test using Prism 3.0 (GraphPad Software, Inc., La Jolla, CA). RESULTS Coumarin A/AA Induces Cytotoxicity to HeLa and Other Cancer Cell Lines The cytotoxic potential of coumarin A/AA was tested in four different cancer cell lines: HeLa and Calo cervical cancer cell lines SW-480 and SW-620 colorectal cancer cell lines. The half maximal inhibitory concen-tration (IC50) was determined at 48 and 72 h of the treatment for each cell line by the MTTmethod. Table 1 shows the IC50 for each of the cell lines tested. As it can be observed, HeLa was the most sensitive cell line to the treatment with coumarin A/AA (IC50 of 65.6 and 15.3 μM at 48 and 72 h, respectively). The cyto-toxic effect of coumarin A/AA was also examined in PBMCs isolated from healthy donors. The cytotoxicity of coumarin A/AA toward PBMCs was substantially lower than for the cancer cell lines. In fact, IC50 val-ues were not achieved: even at the highest coumarin concentration tested (100 μM), only 24.2% of inhibition was reached (data not shown). Coumarin A/AA Causes Apoptotic-Like Morphology Changes in HeLa Cells Several distinctive features of PCD may be evi-denced in the morphology of a dying cell. As shown in Figure 2, coumarin A/AA treatment induces HeLa cell shrinkage, chromatin condensation, and DNA TABLE 1. Cytotoxic Effect of Coumarin A/AA in Different Cancer Cell Lines IC50 At 48 h At 72 h Cancer Cell Line Mean±SD (μM) Mean±SD (μM) HeLa 65±2.8 13.3±8 Calo 65.6±4.1 15.3±5.7 SW620 73.5±5.2 65.4±4.6 SW480 75±2.6 74±9.2 The IC50 values for each cell line at 48 and 72 h is shown as the mean concentration±SD (μM) of three independent experiments. J Biochem Molecular Toxicology DOI 10:1002/jbt
  5. 5. Volume 23, Number 4, 2009 MECHANISM OF CYTOTOXICITY OF COUMARIN A/AA 267 FIGURE 2. Coumarin A/AA induces apoptosis-likemorphology changes inHeLa 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 μMtaxol 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 inducer of apoptosis in this cancer cell line, presented these same characteristics after only 24 h of treatment. These results suggest that coumarin A/AA could induce an apoptotic-like cell death in HeLa cells. Nuclear-Apoptosis Occurs in a Two-Step Manner in HeLa Cells Exposed to Coumarin A/AA Figure 3 shows a different pattern ofDNAfragmen-tation in HeLa cells treated with coumarin A/AA for 24 h (Figure 3G) than that observed at 48 and 72 h of the FIGURE 3. Nuclear-apoptosis occurs in a two-stepmanner 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 μM coumarin A/AA at the same time points indicated previously. Arrows indicate “horseshoe-like” pattern of DNA fragmentation. Bar = 100 μM. J Biochem Molecular Toxicology DOI 10:1002/jbt
  6. 6. 268 A´ LVAREZ-DELGADO ET AL. Volume 23, Number 4, 2009 treatment (Figure 3H and 3I). Fragmented DNA was not detected by TUNEL staining in HeLa cells before 20 h of coumarin A/AA exposure (data not shown). The first type of chromatin condensation was mainly peripheral and can be observed as a “horseshoe-like” shape at 24 h posttreatment (Figure 3G, indicated by arrows). However, at a later time explored (48 and 72 h treatment), chromatin condensation appears more uni-form and some possible apoptotic-bodies can be seen (Figure 3H and 3I). In contrast, cells treated with taxol presented uniform TUNEL staining since 24 h of treat-ment (Figure 3D). Coumarin A/AA Induces Cell Death in HeLa Cells without Disrupting the Cell Cycle HeLa cells were analyzed by flow cytometry to asses cell cycle disruptions. An intermediate 32 h treat-ment time point was included in these experiments since we observed significant DNA fragmentation at 48 h of treatment (Figure 3). No significant cell cycle alterations were found in cells treated with coumarin A/AA before 24 h (Figures 4D, 4G, and 5). Interest-ingly, at 32 h postexposure, HeLa cells show significant accumulation of fragmented DNA (Figures 4J, 4M, and 5, indicated by asterisks).Nevertheless, this subdiploid DNA accumulation does not coincide with any cell cy-cle arrest. In contrast, cells treated with taxol show a marked cell cycle arrest at the G2-M phase as soon as 12 h of treatment (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 the Mitochondria Is an Early Event in Cell Death Induced by Coumarin A/AA in HeLa Cells and Occurs Independently of Caspase-3 Activation We analyzed the expression of some key pro-teins that are known to participate in many cell death paradigms. It was found that the treatment with coumarin A/AA causes an upregulation of Bax pro-tein 12 h posttreatment and also the downregulation of Bcl-2 protein after 24 h of coumarin A/AA ex-posure (Figure 6A), when compared with the cells treated with DMSO. AIF expression was also studied in isolated fractions of mitochondria and cytoplasm of coumarin A/AA-treated cells. It has been reported that 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 cells at time zero; (B,E,H,K) 0.15% DMSO-treated cells. (C,F,I,L) 0.25 μM taxol 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 J andMshow the subdiploid DNA. The data are representative results from three independent experiments. staurosporin-treated HeLa cells as a positive control of AIF release. Figure 6B clearly shows that in cells treated with coumarin A/AA, as well as in cells treated with staurosporin (24 h treatment), AIF is released to the cy-toplasm (Figure 6B, middle panel). In contrast, in cells treated with DMSO, AIF is primarily localized at the mitochondria. In addition, the involvement of caspase- 3 in the induction of PCDwas studied. Figure 6Cshows that treatment with coumarin A/AA does not cause an activation of caspase-3 at the time points studied. These results suggest that HeLa cell death triggered by coumarin A/AA might be caspase-3 independent and rely predominantly in the release of AIF and its function as a death effector outside the mitochondria. DISCUSSION In the present study, the cytotoxic activity of coumarin A/AA (Figure 1) was demonstrated in four different human cancer cell lines (colorectal cancer cell lines SW480 and SW620 and cervical cancer cell lines HeLa and Calo). Even though the cytotoxicity of J Biochem Molecular Toxicology DOI 10:1002/jbt
  7. 7. Volume 23, Number 4, 2009 MECHANISM OF CYTOTOXICITY OF COUMARIN A/AA 269 FIGURE 5. Distribution of the cell cycle phases. The graph shows the distribution of the cell cycle phases for nontreated cells at time zero (T0). HeLa cells were treated with vehicle (0.15% DMSO) or 0.25 μM taxol or 30 μM coumarin A/AA for the time-points indi-cated. The figure represents the mean values for three independent experiments 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 has been reported by many investigators in different cell lines [24–28], to our knowledge, there are no previous reports on the exact molecular mechanism by which coumarin A/AA exerts its cytotoxic effect. In contrast to the classic cell death mechanism initiated by the basic coumarin 1,2 benzopyrone in HeLa cells [29], in this studywe show that coumarin A/AA induces a caspase independent PCD with the release of AIF from mito-chondria as an early event in cell death (Figure 6), and no 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/AA has a high cytotoxic activity against SW480, SW620, Calo, and HeLa malignant cell lines (Table 1), with IC50 values ranging from 13.3 to 74 μM for HeLa and SW480, respectively. As was expected from its chemi-cal structure, very low cytotoxicity was detected when coumarin A/AA was tested against PBMCs, but fur-ther studies need to be conducted in vivo to test the selective nature of the compound. In addition, it has been proven that the cytotoxic potential of coumarins is due to their structure-based ability to induce apoptotic PCD, preceded by an arrest of 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-fragmentation patterns 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 the mitochondria 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-points indicated. Protein was extracted as described in the Materials and Methods section, and expression was evaluated. (A) Bax and Bcl-2 protein expression at indicated times. α-tubulin was used as a load-ing control for 25 μg of total protein (lower panels). (B) Subcellular localization 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 protein was used. (C) Active-caspase-3 expression in DMSO (lanes 2–5) and coumarin A/AA (lanes 6–9) treated cells. Lane 1 shows a positive control for active-caspase-3 expression. For this aim, 120 μg of total protein fromrat uterus at 06:00 h of estrous daywas used. For DMSO and coumarin A/AA treated cells, 25 μg of total protein was loaded. αtubulin 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 later times (48 h), chromatin condensation appeared more homogeneous (Figure 3H). Different types of chro-matin condensation patterns associated with specific protein activation have been previously described at distinct stages of cell death [16,32]. Typically, it has been reported that AIF protein causes large-scale (50 kb), type I nuclear apoptosis, and that caspase-activated DNAses (CADs) initiate the oligonucleosomal, type II nuclear apoptosis [16] that can be evidenced as a lad-der pattern.In a similar setting, coumarin A/AA could cause the sequential activation of type I and type II nuclear apoptosis in HeLa cells. J Biochem Molecular Toxicology DOI 10:1002/jbt
  8. 8. 270 A´ LVAREZ-DELGADO ET AL. Volume 23, Number 4, 2009 Several authors have demonstrated that in various cancer cell lines PCD occurs after alterations or arrest of the cell cycle [6,7,26,29,33]. In this study, and in con-trast to what was expected, no arrest in any phase of the cell cycle was detected (Figures 4 and 5), although treatment with coumarin A/AA did cause a significant accumulation of subdiploid DNA after long exposure periods (Figures 4J, 4M and 5). This indicates that the cytotoxicity of coumarin A/AA is not due to cell cycle alterations, at least at the concentrations tested. To our knowledge, these results are a novel finding in the field of antitumor mechanisms for coumarin compounds, as no previous reports on the detailed mechanism of ac-tion of coumarin A/AA have been published. To further test the possibility that an apoptotic-like program could be activated by coumarin A/AA, Bax and Bcl-2 protein expression was studied. These two proteins belong to the Bcl-2 family and are partially re-sponsible for regulating the status of themitochondrial permeability transition pore (mtPTP), thus allowing the release 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 have shown that 12 h of treatment with coumarin A/AA in-duces the overexpression of Bax protein and, 3 h later the inhibition of Bcl-2 protein expression (Figure 6A) was observed. These data suggest that coumarinA/AA could induce a proapoptotic balance in the cell and that the release of proapoptotic proteins fromthe mitochon-drial intermembrane space is feasible. The chromatin condensation pattern observed 24 h after coumarin A/AA treatment is typical of a phase I nuclear apoptosis (Figure 3G) initiated by the release of the proapoptotic protein AIF [16]. This evidence, lead-ing to the description of a PCD pathway prompted by the release of AIF, was further explored by the analysis of AIF expression in themitochondrial and cytoplasmic fractions, as this protein only acts as a proapoptotic fac-tor when it is released to the cytosol and translocated to the nucleus [36]. Indeed, the present work demon-strates that coumarin A/AA induces the release of AIF from mitochondria to the cytosol (Figure 6B). The re-lease of AIF could very likely be the reason for the “horseshoe-like” chromatin condensation observed 24 h after the treatment (Figure 4G). The different con-densation patterns observed at 48 h after the treatment could be attributed to the late degradation action of CADs. Even though caspases are an integral part of the apoptotic machinery, cell death programs may be cas-pase independent and occur in the complete absence of caspase activity [11,37]. Interestingly, in the present study, 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 complex coumarins 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 of caspase-3 had been examined at later times (48 and 72 h of coumarin A/AA treatment), the active subunits might have been detected. For example, it is possi-ble that caspases could be active at prolonged times of coumarin A/AA exposure and that AIF could act as an early initiator of cell death. This type of “sequenced” cell death program has already been reported by other authors in different models of coumarin-induced cell death [39,40]. It has also been proven that AIF can act as a primary effector of cell death either with or without the aid of caspases [10,11,13]. So it is very likely that in this study, HeLa cell death induced by coumarinA/AA is initiated by the actions of AIF without the early par-ticipation of caspase-3. The PCD mechanism proposed in this work is outlined in Figure 7. In conclusion, we demonstrated that coumarin A/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 an apoptosis-like cell death program that begins as early as 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 acts as an early, caspase independent, cell death effector that prompts DNA fragmentation (Figures 3G–3I) and the typical morphological changes of PCD (Figures 2H and 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 the PCD activated by coumarin A/AA in HeLa cells. Time-points af-ter coumarin A/AA treatment and relevant biochemical events and morphological changes are indicated. J Biochem Molecular Toxicology DOI 10:1002/jbt
  9. 9. Volume 23, Number 4, 2009 MECHANISM OF CYTOTOXICITY OF COUMARIN A/AA 271 the important damage induced in HeLa cells. The data described suggest that coumarin A/AA could be an excellent candidate for a low-toxicity anticancer treatment. ACKNOWLEDGMENTS A´ lvarez-Delgado C. receieved a fellowship from CONACyT. The author would like to thank Dr. J. J. Garc´ıa-Trejo for his kind help in the isolation of mitochondria. REFERENCES 1. Kaufmann S, Earnshaw C. Induction of apoptosis by can-cer chemotherapy. Exp Cell Res 2000;256:42–49. 2. J¨a¨attel¨a M. Multiple cell death pathways as regula-tors of tumour initiation and progression. Oncogene 2004;23:2746–2756. 3. Ng CP, Bonavida B. A new challenge for successful immunotherapy by tumors that are resistant to apop-tosis: Two complementary signals to overcome cross-resistance. Adv Cancer Res 2002;85:145–174. 4. Ding Z, Yang X, Cherneko G, Tang SC, Pater A. 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