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
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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
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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
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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.
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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.
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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
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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.
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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.
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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.
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